U.S. patent application number 16/194899 was filed with the patent office on 2019-05-09 for regulatory b cells and their uses.
This patent application is currently assigned to Duke University. The applicant listed for this patent is Duke University. Invention is credited to Jean-David Bouaziz, Yohei Iwata, Takashi Matsushita, Thomas F. Tedder, Koichi Yanaba.
Application Number | 20190136185 16/194899 |
Document ID | / |
Family ID | 45560078 |
Filed Date | 2019-05-09 |
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United States Patent
Application |
20190136185 |
Kind Code |
A1 |
Tedder; Thomas F. ; et
al. |
May 9, 2019 |
REGULATORY B CELLS AND THEIR USES
Abstract
The present invention relates to a distinct B cell subset, B10
cells, that regulate T cell mediated inflammatory responses through
the secretion of interleukin-10 (IL-10). The invention also relates
to the use of B10 cells in the manipulation of immune and
inflammatory responses, and in the treatment of disease.
Therapeutic approaches involving adoptive transfer of B10 cells, or
expansion of their endogenous levels for controlling autoimmune or
inflammatory diseases and conditions are described. Ablation of B10
cells, or inhibition of their IL-10 production can be used to
upregulate immunodeficient conditions, ameliorate infectious
diseases and/or to treat tumors/cancer. Diagnostic applications are
also encompassed.
Inventors: |
Tedder; Thomas F.; (Durham,
NC) ; Matsushita; Takashi; (Kanazawa, JP) ;
Iwata; Yohei; (Magoya, JP) ; Yanaba; Koichi;
(Tokyo, JP) ; Bouaziz; Jean-David; (Maison-Alfort,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Assignee: |
Duke University
Durham
NC
|
Family ID: |
45560078 |
Appl. No.: |
16/194899 |
Filed: |
November 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13814165 |
Feb 4, 2013 |
10131875 |
|
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PCT/US2011/046643 |
Aug 4, 2011 |
|
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16194899 |
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61370677 |
Aug 4, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/388 20180101;
A61K 35/17 20130101; G01N 33/5052 20130101; C07K 16/2803 20130101;
A61K 2039/505 20130101; C12N 5/0635 20130101; Y02A 50/30 20180101;
Y02A 50/471 20180101; Y02A 50/484 20180101; G01N 33/6893 20130101;
G01N 2800/7095 20130101 |
International
Class: |
C12N 5/0781 20060101
C12N005/0781; G01N 33/50 20060101 G01N033/50; G01N 33/68 20060101
G01N033/68; A61K 35/17 20060101 A61K035/17; C07K 16/28 20060101
C07K016/28 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with United States government
support awarded by the National Institutes of Health grant number
National Cancer Institute grant numbers CA 105001, CA 96547, and
National Institute of Allergy and Infectious Disease grant number
AI 56363. The United States may have certain rights in this
invention.
Claims
1.-9. (canceled)
10. A pharmaceutical composition for treating disorders associated
with diminished levels of interleukin-10, comprising a cellular
composition comprising B lymphocyte cells wherein at least 50% of
the B lymphocyte cells are characterized as
CD24.sup.highCD27.sup.+, and a pharmaceutically acceptable
carrier.
11. The pharmaceutical composition of claim 10, wherein the cells
are producing IL-10.
12. A method for treating a disease or condition associated with
diminished levels of interleukin-10 or ameliorated by increasing
levels of IL-10 comprising administering a therapeutically
effective amount of the composition of claim 10 to a subject in
need of such treatment, wherein administration of the composition
ameliorates the disease or condition.
13. The method of claim 12, wherein the cells produce IL-10 in the
subject.
14.-18. (canceled)
19. The method of claim 12, in which the disease or condition is
inflammation.
20. The method of claim 12, in which the disease or condition is
autoimmune disease.
21. The method of claim 12, in which the subject in need of such
treatment is an organ transplant recipient.
22.-48. (canceled)
49. The pharmaceutical composition of claim 10, wherein the B
lymphocyte cells are further characterized by a
CD1d.sup.highCD5.sup.+ phenotype.
50. The pharmaceutical composition of claim 10, wherein the B
lymphocyte cells are further characterized by expression of a
marker selected from the group consisting of CD19, CD20, CD21,
CD22, CD23, CD25, CD38, CD48, and CD148.
51. The pharmaceutical composition of claim 10, wherein the B
lymphocyte cells in the composition are contacted ex vivo with a
mitogen, a cytokine, a growth factor, a antibody, a CD40 agonist or
a TLR agonist.
52. The pharmaceutical composition of claim 51, wherein the CD40
agonist is a CD40L (CD154), or an antibody agonist of CD40.
53. The pharmaceutical composition of claim 51, wherein the TLR
agonist is a TLR1 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7
agonist or a TLR9 agonist.
54. The pharmaceutical composition of claim 53, wherein the agonist
is selected from lipopolysaccharide, CpG oligodeoxynucleotides,
Pam3CSK4, Pam2CGDPKHPKSF, or Imiquimod.
55. The method of claim 12, wherein the B lymphocyte cells are
further characterized by a CD1d.sup.highCD5.sup.+ phenotype.
56. The method of claim 12, wherein the B lymphocyte cells are
further characterized by expression of a marker selected from the
group consisting of CD19, CD20, CD21, CD22, CD23, CD25, CD38, CD40,
CD48, CD72 and CD148.
57. The method of claim 12, wherein the B lymphocyte cells were
contacted ex vivo with a mitogen, a cytokine, a growth factor, a
antibody, a CD40 agonist or a TLR agonist.
58. The method of claim 57, wherein the CD40 agonist is a CD40L
(CD154), or an antibody agonist of CD40.
59. The method of claim 57, wherein the TLR agonist is a TLR1
agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist or a TLR9
agonist.
60. The method of claim 59, wherein the agonist is selected from
lipopolysaccharide, CpG oligodeoxynucleotides, Pam3CSK4,
Pam2CGDPKHPKSF, or Imiquimod.
61. The method of claim 12, wherein the B lymphocyte cells were
stimulated in vitro or ex vivo with PMA (phorbol 12-myristate
13-acetate) and ionomycin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 13/814,165, filed Feb. 4, 2013, which is a
national stage filing under 35 U.S.C. of International Application
No. PCT/US2011/046643 filed Aug. 4, 2011, which claims the benefit
of priority of U.S. Provisional Patent Application No. 61/370,677,
filed Aug. 4, 2010, all of which are incorporated herein by
reference in their entirety.
SEQUENCE LISTING
[0003] A Sequence Listing accompanies this application and is
incorporated herein by reference in its entirety. The Sequence
Listing was filed with the application as a text file on Aug. 4,
2011.
1. INTRODUCTION
[0004] The present invention relates to a distinct B cell subset,
B10 cells, that regulate T cell mediated inflammatory responses
through the secretion of interleukin-10 (IL-10). The invention also
relates to the use of B10 cells in the manipulation of immune and
inflammatory responses, and in the treatment of disease.
Therapeutic approaches involving adoptive transfer of B10 cells, or
expansion of their endogenous levels for controlling autoimmune or
inflammatory diseases and conditions are described. Ablation of B10
cells, or inhibition of their IL-10 production can be used to
upregulate immunodeficient conditions, and/or to treat
tumors/cancer. Diagnostic applications are also encompassed.
2. BACKGROUND
[0005] The immune response can loosely be divided into two
components: the humoral immune response which involves antibody
formation, and the cell-mediated immune response which involves the
activation of macrophages, natural killer (NK) cells,
antigen-specific cytotoxic T-lymphocytes, and the release of
various cytokines in response to antigen. Typically, B lymphocytes
(B cells) are characterized by their role in antibody production;
whereas T lymphocytes (T cells) are characterized by their role in
cell-mediated immunity. However, B cells possess additional immune
functions, including the production of cytokines, and the ability
to function as antigen presenting cells (APCs).
[0006] Once generated, immune responses need to be regulated to
prevent the responding effector cells from causing harmful effects.
Immunoregulation has traditionally been thought of as a function of
T cells. Functionally distinct regulatory T cell subsets have been
identified and clearly defined. For example, helper T cells that
up-regulate the immune response include T helper type 1 (Th1) cells
that regulate cell-mediated immune responses, and T helper type 2
(Th2) cells that regulate the humoral immune response. Suppressor T
cells crucial for the maintenance of immunological tolerance,
currently referred to as T regulatory cells, include
IL-10-producing T regulatory 1 (Tr1) cells, and
TGF-.beta.1-producing T helper type 3 (Th3) cells. Recent studies
of autoimmune conditions gave rise to the notion that B cells may
also participate in immunoregulation. However, regulatory B cell
subsets have not been clearly defined.
[0007] Multiple roles for B cells have been implicated in
autoimmune diseases. B cells, a major immune cell population, can
play a pathogenic role in acquired immune responses by producing
autoantibodies that drive the development of autoimmune diseases.
Certain therapies developed for treating autoimmunity are aimed at
depleting pathogenic B cells. However, the tools currently
available are not specific for the pathogenic B cells and deplete
most B cells. For example, B cell depletion in humans using CD20
monoclonal antibody (mAb) can be effective in treating patients
with various autoimmune disorders, such as rheumatoid arthritis and
lupus (Edwards et al., 2001, Rheumatol. 40:205-11; Edwards et al.,
2005, Rheumatol. 44:151-56; El Tal et al., 2006, J. Am. Acad.
Dermatol. 55:449-59; Anolik et al., 2004, Arth. Rheum. 50:3580-90;
Stasi et al., 2007, Blood 110:2924-30). CD20 is a B cell-specific
marker that is first expressed on the cell surface during the pre-B
to immature B cell transition, but is lost upon plasma cell
differentiation (Tedder & Engel, 1994, Immunol. Today
15:450-54; Uchida et al., 2004, Int. Immunol. 16:119-29). A recent
phase II trial using CD20 antibodies indicates clinical efficacy in
multiple sclerosis (MS) patients (Hauser et al., 2008, N. Engl. J.
Med. 358:676-88). However, the mechanisms underlying the effect of
B cell depletion on disease activity remains unknown. On the flip
side, B cell depletion may exacerbate disease. Indeed, B cell
depletion was recently found to exacerbate ulcerative colitis in
human clinical trials (Goetz et al., 2007, Inflamm Bowel Dis.
13:1365-8) and may contribute to the development of psoriasis (Dass
et al., 2007, Arthritis Rheum. 56:2715-8).
[0008] Over a decade ago, Janeway and colleagues (Wolf et al.,
1996, J. Exp. Med. 184: 2271-2278) described studies designed to
assess the role of B cells in the course of autoimmune disease by
inducing acute experimental autoimmune encephalomyelitis (EAE) in B
cell-deficient mice. EAE is an autoimmune disease of the central
nervous system (CNS) that models human multiple sclerosis. Results
showed that elimination of B cells did not prevent induction of
autoimmunity. Instead, the lack of B cells seemed to exacerbate
disease outcome, in that the B cell deficient mice did not fully
recover as compared to wild-type mice. Thus, while B cells supply
the autoantibodies thought to be responsible for disease, these
investigators concluded that B cells are not required for
activation of disease, and instead, that their presence is required
to enhance recovery. More recently, it was reported that B cell
IL-10 production correlated with recovery from EAE, a Th1-mediated
autoimmune disease (Fillatreau et al., 2002, Nature Immunol. 3:
944-950). IL-10 is an immunoregulatory cytokine produced by many
cell populations. IL-10 has been shown to suppress cell-mediated
immune and inflammatory responses.
[0009] Other recent studies in mouse models indicate that B cells
and IL-10 play a protective role in T cell-mediated inflammation,
e.g., in Th2-mediated inflammatory bowel disease (Mizoguchi et al.,
2002, Immunity 16:216-219), and in contact hypersensitivity (CHS)
responses--a cutaneous inflammatory immune reaction that is
mediated by T cells in sensitized individuals following subsequent
contact with the sensitizing antigen (Enk, 1997, Mol. Med. Today
3:423-8). In particular, mice with B cells deficient for CD19
expression (CD19.sup.-/-) have augmented CHS responses (Watanabe et
al., 2007, Am. J. Pathol. 171:560-70). IL-10 must be involved in
rotection since neutralizing IL-10 through mAb treatment enhances
CHS responses, while systemic IL-10 administration reduces CHS
responses (Ferguson et al., 1994, J. Exp. Med. 179:1597-1604;
Schwarz et al., 1994, J. Invest. Dermatol. 103:211-16).
[0010] On the basis of these and other studies, it has been
proposed that, like their T cell counterparts, B cells can be
divided into functionally distinct regulatory subsets capable of
inhibiting inflammatory responses and inducing immune tolerance by
mechanisms that include IL-10 and TGF-.beta. production, secondary
antigen presentation, and interaction with other immune cells
either directly or through secreted antibodies. (For reviews on the
subject, see Mauri & Ehrenstein, 2007, TRENDS in Immunol. 29:
34-40; and Mizoguchi & Bhan, 2006, J. Immunol.
176:705-710).
[0011] However, it remains unclear whether regulatory B cells
represent a unique regulatory lineage tasked with maintaining
self-tolerance the way that naturally occurring regulatory T cells
are. The generation of regulatory B cells has been reported in
multiple mouse models of chronic inflammation, although their
existence in normal mice remains unknown (Mizoguchi & Bhan,
2006, J. Immunol. 176:705-10). Despite the identification of a
regulatory B cell subset generated in these mouse models, no
definitive murine phenotype has been established and, in fact, only
a general list of cell-surface markers associated with regulatory B
cells exists (Mauri & Ehrenstein, 2007, Trends Immun.
29:34-40). Furthermore, the existence of regulatory B cells in
humans remains a matter of speculation, and the potential
phenotypic markers for human regulatory B cells are unknown (Mauri
& Ehrenstein, 2007, Trends Immun. 29:34-40). A role for CD40 in
the generation of regulatory B cells and the induction of IL-10
production by these cells has been postulated (Inoue et al., 2006
Cancer Res. 66:7741-7747). Nonetheless, it has yet to be proven
whether CD40 can be directly targeted, i.e., with CD40 antibodies,
as a means to generate regulatory B cells in vivo (Mauri &
Ehrenstein, 2007, Trends Immun. 29:34-40).
[0012] Further complicating these issues, during immune responses,
IL-10 is also secreted by multiple cell types, including T cells,
monocytes, macrophages, mast cells, eosinophils, and keratinocytes,
and can suppress both Th1 and Th2 polarization and inhibit antigen
presentation and proinflammatory cytokine production by monocytes
and macrophages (Asadullah et al., 2003, Pharmacol. Rev.
55:241-69). Clearly, it is unknown whether multiple B cell
populations or a novel B cell subset regulates inflammatory
responses, whether regulatory B cells produce IL-10 or other
cytokines directly, whether regulatory B cells have potent
activities in vivo, whether humans possess regulatory B cells, how
regulatory B cells can be activated and/or expanded, and the role
of regulatory B cells in disease. To advance therapeutic
application, subsets of immunoregulatory B cells need to be better
defined and their phenotype will need to be more closely correlated
with their function in vivo.
3. SUMMARY
[0013] The present invention relates to a distinct regulatory B
cell subset, termed B10 cells, that regulate T cell-mediated
inflammatory and immune responses through secretion of IL-10. The
invention also relates to harnessing this B10 cell subset for the
manipulation of immune and inflammatory responses in humans and
other mammals. Treatments for diseases associated with diminished
IL-10 levels, such as inflammatory and autoimmune diseases are
described, as well as treatments for diseases associated with
elevated IL-10 levels, such as immunosuppression, infectious
diseases and cancer.
[0014] Cellular compositions enriched for B10 cells, and methods
for their preparation are described. B10 cells are characterized by
increasing production of IL-10 after stimulation with a CD40
agonist or a TLR (Toll-like Receptor) agonist. For example, without
being bound by theory and without limitation, cellular compositions
enriched by selection using both CD1d.sup.high and CD5 as cellular
markers will contain a higher percentage of B10 cells than a
population enriched using only one of these markers. Additionally,
cellular compositions enriched by selection using both
CD24.sup.high and CD27 as cellular markers will contain a higher
percentage of B10 cells than a population enriched using only one
of these markers. Other markers that can be used in selection of
B10 cells include, without limitation, CD19, CD20, CD21, CD23,
CD25, CD38, CD48, and CD148. These cellular compositions can be
expanded and used to treat inflammatory and/or autoimmune
conditions or diseases by adoptive transfer.
[0015] In an alternative approach, therapeutic regimens designed to
expand the endogenous population of B10 cells in subjects in need
of such treatment can be used to treat inflammatory and/or
autoimmune conditions or diseases. This approach includes methods
of treating a disease or codition associated with diminished or
insufficient levels of IL-10 or a condition or disease ameliorated
by increasing levels of IL-10 or a disease or condition associated
with inflammation or immune hyperresponsiveness by administering to
a subject in need of such treatment a therapeutically effective
amount of an agent that stimulates the expansion of IL-10 producing
B10 cells or an agent that increases production of IL-10 by B cells
in the subject. The agent may be a TLR agonist or a CD40 agonist.
In an alternative approach, the agent may be antibodies that
activate and/or stimulate expansion of B10 cells, or increase their
production of IL-10.
[0016] In an alternative embodiment, methods of treating a disease
or condition associated with elevated levels of IL-10 or
insufficient or ineffective immune responsiveness by administering
to a subject in need of such treatment a therapeutically effective
amount of an agent that kills, abrogates, or inhibits the function,
localization, or expansion of B10 cells or an agent that inhibits
production of IL-10 by B10 cells in the subject. The methods are
suitable for treating diseases and/or conditions involving
immunosuppression, infectious diseases or cancer by depleting or
ablating B10 cells in subjects in need thereof. In this approach,
the agent may be antibodies that kill B10 cells, or inhibit their
function, proliferation or production of IL-10. In particular, the
agent may include antibodies that induce homotypic adhesion and
agents that selectively deplete or target B10 cells as opposed to
other types of B cells such as follicular B cells.
[0017] In yet another embodiment, methods for identifying B10 cells
in patients and/or patient samples are described for diagnosing the
immune status of affected individuals. The methods include assaying
for cells producing IL-10 or capable of producing IL-10 when
treated with a CD40 agonist or a TLR agonist. Methods for assessing
the number of B10 and B10pro cells are also encompassed.
[0018] In still another aspect, methods of generating an antibody
that preferentially or selectively depletes B10 cells is described.
The method includes selecting an antibody that binds to a marker
that is expressed by B10 cells, assaying the antibody for the
ability to induce homotypic adhesion of B cells, and assaying the
antibody for its ability to deplete the B10 cell population. In
some embodiments, the Fc portion of the antibody may be modified so
that the mechanism of B10 cell depletion by the antibody is
independent of the antibody's Fc region. The antibody may be
selected for its ability to deplete the B10 cells by a method that
is independent of antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC) and apoptosis.
[0019] In a further aspect, methods of selecting B10 cells is
provided. The method includes selecting B lymphocytes in a sample
from a subject, stimulating the B cells in vitro with PMA (phorbol
12-myristate 13-acetate) and ionomycin for five hours and selecting
IL-10 producing cells. The cells may also be selected by
stimulating them for at least 24 hours with a CD40 agonist or a TLR
agonist prior to the addition of PMA and ionomycin.
[0020] In a still further aspect, methods of inducing an IgG
antibody response to an antigen are described. The methods include
administering the antigen to a subject and administering an agent
that kills or inhibits the function, localization or expansion of
B10 cells or inhibits production of IL-10 by B10 cells to the
subject. The administration of the agent with the antigen increases
the antigen specific IgG antibody production in the subject as
compared to a subjet administered the antigen alone. The antigen
and the agent may be administered together, but need not be.
[0021] The invention is based, in part, on the identification of a
rare B10 cell subset that controls T cell-mediated immune and
inflammatory responses in vivo. The principles of the invention are
illustrated in animal models in the studies described in the
examples, infra, and resolve previously unexplained contradictions
reported in the literature for the role of B cells in disease
models such as EAE, arthritis, and inflammatory bowel disease. The
examples described infra demonstrate: [0022] a phenotypically
unique B cell subset, B10 cells, with potent regulatory activities
in vivo; [0023] a reliable method of intracellular cytokine
staining that clearly identifies B10 cells; [0024] adoptive
transfer of B10 cells has potent IL-10-dependent regulatory
functions during inflammation in vivo, which can apply to other T
cell-mediated inflammatory or autoimmune diseases; [0025] expansion
of B10 cells in human CD19 transgenic mice results in a decreased
inflammatory response; [0026] the absence of B10 cells in
CD19-deficient mice results in augmented T cell-mediated
inflammation; and [0027] the presence of B10 cells in healthy wild
type mice (1-2% of spleen B cells) and expansion of the population
during contact hypersensitivity responses.
4. DESCRIPTION OF THE FIGURES
[0028] FIG. 1A-D. CD22 mAb depletes B10 cells and increases IgG
responses. Eight week-old C57BL/6 mice were treated with CD22 mAb
(MB22-10; 250 .mu.g/mouse) or control mAb (B1; 250 .mu.g/mouse) 7
days before analysis. FIG. 1A shows representative CD1d and CD5
expression by CD19.sub.+ B cells. Splenocytes were stained with
CD1d, CD5, and CD19 mAbs with flow cytometry analysis of viable
cells. Results represent one mouse indicating the frequency of
CD1d.sup.hiCD5.sup.+ B cells among total B cells within the
indicated gates. Bar graphs indicate mean (.+-.SEM) percentages and
numbers of CD1d.sup.hiCD5.sup.+ B cells in one of two independent
experiments with three mice in each group. FIG. 1B shows IL-10
production by B cells. Splenocytes were cultured with LPS (10
.mu.g/ml), PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (2
.mu.M) for 5 h, then stained with B220 and CD19 mAb to identify B
cells, permeabilized, and stained using IL-10 mAb with flow
cytometry analysis of viable cells. Representative results
demonstrate the frequency of IL-10-producing cells among total
B220.sub.+ B cells within the indicated gates. Bar graphs indicate
mean (.+-.SEM) percentages and numbers of B cells that produced
IL-10 in one of two independent experiments with three mice in each
group. Leukocytes from IL-10.sup.-/- mice served as negative
controls to demonstrate specificity and to establish background
IL-10 staining levels. FIG. 1C and FIG. 1D show B10 cells regulate
IgG Ab responses. Wild-type mice were given CD22 or control mAb
(n=3 per group) on day 0, and immunized with DNP-KLH without
adjuvant on days 0 and 21. Serum DNP-specific Abs were quantified
by ELISA in FIG. 1C. The frequency of B cells secreting
DNP-specific IgG was determined by ELISPOT analysis of spleen cells
harvested on day 28 in FIG. 1D. In FIG. 1A-1D significant
differences between sample means are indicated: *, p<0.05; **,
p<0.01.
[0029] FIG. 2A-2G. Identification of human blood IL-10-competent
B10 and B 10pro cells. FIG. 2A shows frequencies of mouse blood B10
cells after stimulation with PIM, LPS+PIM, or CpG+PIM for 5 h as
described (see Yanaba et al., 2008, Immunity 28:639-650; Matsushita
et al., 2008, J. Clin. Invest. 118:3420-3430; and Yanaba et al.,
2009, J. Immunol. 182:7459-7472). Representative flow cytometry
histograms are shown for one mouse with values from 5 mice shown in
the scatter graph. FIG. 2B shows BFA treatment was optimal for
visualizing human blood B10 cell numbers. Purified blood
mononuclear cells were cultured for 5 h with L+PIB or LPS+PIM as in
FIG. 2A before immunofluorescence staining and flow cytometry
analysis. Bar graph values represent mean (.+-.SEM) B10 cell
frequencies from 3 individuals. FIG. 2C shows representative gating
strategy for identifying cytoplasmic IL-10.sup.+ human B cells by
flow cytometry. Blood mononuclear cells were cultured with LPS+PIB
for 5 h and stained for immunofluorescence analysis of viability
and cell surface CD19 expression. After membrane permeabilization,
the cells were stained with IL-10 mAb. For flow cytometry analysis,
single cells were identified by singlet gating using forward
scatter area (FSC-A) versus height (FSC-H) plots. The cells outside
or below the indicated gate (left panel) were excluded cell
doublets. The predominant lymphocyte population within the single
cell gate was identified by forward (FSC) and side (SSC-A) light
scatter properties. Within the single cell lymphocyte population,
the dead cells that were positive for Live/Dead staining were
excluded from the analysis. The initial gate for identifying B
cells was cell surface CD19 expression. Representative cytoplasmic
IL-10 staining by viable, single CD19.sup.+ B cells is shown in the
dot-plot histograms (right panels). Percentages indicate the
frequencies of cytoplasmic IL-10.sup.+ B cells within the indicated
gates among total CD19.sup.+ B cells. Blood mononuclear cells that
were cultured with BFA alone before immunofluorescence staining
served as negative controls, with background staining similar to
that obtained using isotype-matched control mAbs. FIG. 2D shows
representative IL-10 production by human blood B cells from a
normal individual with relatively high B10 cell frequencies. B10
cells were identified by cytoplasmic IL-10 expression after in
vitro culture with LPS, CpG, and/or PIB as indicated for 5 h. Blood
mononuclear cells cultured with BFA alone served as negative
controls for background IL-10 staining. Alternatively, B10 cell
frequencies were examined after in vitro B10pro cell maturation by
stimulation with LPS, CD40L+LPS, CpG, or CD40L+CpG, with PIB added
during the final 5 h of 48 h cultures. As negative controls for
IL-10 staining, the cells were also stimulated with CD40L+LPS or
CD40L+CpG for 48 h, with only BFA added during the final 5 h of
culture. Percentages indicate the frequencies of cytoplasmic
IL-10.sup.+ B cells within the indicated gates among total
CD19.sup.+ B cells. FIG. 2E shows frequencies of blood B10 cells in
individuals after stimulation with TLR agonists. Cell stimulation
and analysis was as described in FIG. 2C-2D. Dots represent results
from single individuals after 5 h culture with BFA alone, PIB, or
the indicated TLR agonist+PIB. Horizontal bars indicate means. FIG.
2F shows that CD40L was optimal for inducing B10+B10pro cell
maturation during 48 h in vitro cultures. Purified blood
mononuclear cells were cultured with either recombinant CD40L or
CD40 mAb, plus LPS for 48 h. PIB was added during the final 5 h of
culture. Bar graph values represent mean (.+-.SEM) from 5 different
individuals. Similar results were obtained in 2 independent
experiments. FIG. 2G shows representative human blood B10+B10pro
cell frequencies after in vitro maturation and stimulation. Blood
mononuclear cells were cultured for 48 h with media alone or media
containing CD40L, along with the indicated TLR agonists, with PIB
added during the last 5 h of each culture. (FIG. 2A, and FIG.
2E-2G) Significant differences between means of media controls and
individual stimuli are indicated: *p<0.05, **p<0.01.
[0030] FIG. 3A-3C. Human B10 and B10pro cells in cord blood, spleen
and tonsil. FIG. 3A shows the presence of B10 and B10pro cells in
human newborn blood. Mononuclear cells were cultured with the
indicated stimuli with PIB added during the last 5 h of culture.
Results from a representative cord blood sample are shown along
with graphs indicating IL-10.sup.+ B cell frequencies in individual
newborns. FIG. 3B shows representative cytoplasmic IL-10 expression
by human tissue B cells. B10 and B10pro cells were identified by
activation-induced cytoplasmic IL-10 expression as described in
FIG. 2C. Cells cultured with BFA alone served as negative controls
for background IL-10 staining. FIG. 3C shows human B10 cell
frequencies determined after 5 or 48 h in vitro cultures as
described in FIG. 2. Mouse spleen B10 and B10pro cell frequencies
are shown for comparison. Dots represent results from single
individuals or mice. Significant differences between means of BFA
or monensin (Mone) controls and individual stimuli are indicated:
*p<0.05, **p<0.01.
[0031] FIG. 4A-4E. Human B cell stimulation induces IL-10
transcription and secretion in vitro. FIG. 4A shows time course of
Il10 transcript induction. Purified blood CD19.sup.+ B cells were
cultured with media alone (open bars) or CD40L plus CpG (filled
bars) for the times indicated, with Il10 transcripts quantified by
real-time RT-PCR analysis. Bar graphs indicate mean IL-10 (.+-.SEM)
concentrations of six different individuals. Significant
differences between media alone and stimulated B cells are
indicated: *p<0.05. Similar results were obtained in 2
independent experiments. FIG. 4B shows that Il10 transcript
expression correlates with IL-10 secretion. Purified blood B cells
were cultured with PMA and ionomycin for 4 h before CD19 staining
and secreted IL-10 capture (left panel). Cell surface IL-10.sup.+
and IL-10.sup.- B cells were isolated using the indicated gates and
subsequently reassessed for IL-10 secretion (right panels) before
relative Il10 transcript levels were quantified by real-time RT-PCR
analysis. Mean fold-differences (.+-.SEM) for Il10 transcript
levels from 3 different individuals are shown, with transcript
levels normalized so that the relative mean IL-10.sup.- B cell
value is 1.0. Significant differences between means are indicated:
*p<0.05. Similar results were obtained in 2 independent
experiments. FIG. 4C shows the cell surface signals that regulate
cytoplasmic IL-10 expression. Blood B cells were cultured with CpG,
CD40L, and anti-IgM Ab (IgM) as indicated for 48 h with PIB added
during the final 5 h of culture. The B cells were then stained with
CD19 mAb, permeabilized, and stained using IL-10 mAb with flow
cytometry analysis. Representative frequencies of IL-10-producing
cells within the indicated gates are shown among total CD19.sup.+ B
cells. Bar graphs indicate mean (.+-.SEM) percentages of B cells
that produced IL-10 in 5 different individuals. Similar results
were obtained in 2 independent experiments. FIG. 4D shows TLR
agonists that induce B10 cell IL-10 secretion. Purified blood
CD19.sup.+ B cells were cultured with media alone, CD40L, or with
TLR agonists and CD40L as indicated for 48 (open bars) or 72
(filled bars) h. IL-10 secreted into the culture supernatant fluid
was quantified by ELISA. Bar graphs indicate mean IL-10 (.+-.SEM)
concentrations from .gtoreq.4 different individuals. Similar
results were obtained in 2 independent experiments. FIG. 4E shows
LPS and CpG induce mouse blood B10 cell IL-10 secretion. Purified
blood B cells were cultured with media alone or with TLR agonists
as indicated for 24 (open bars) or 72 (filled bars) h. IL-10
secreted into the culture supernatant fluid was quantified by
ELISA. Bar graphs indicate mean IL-10 (.+-.SEM) concentrations from
triplicate cultures that represent one of three independent
experiments. FIG. 4D-4E Significant differences between means of
cells cultured in media alone and TLR agonists are indicated:
*p<0.05, **, p<0.01.
[0032] FIG. 5A-5H. Representative phenotypes of human and mouse
blood and tissue B10 and B10pro cells. FIG. 5A shows the cell
surface phenotype of human blood B10 cells. Enriched B cells were
cultured with L+PIB for 5 h. FIG. 5B shows the cell surface
phenotype of human blood B10+B10pro cells after 48 h stimulation
with CD40L+LPS with PIE added during the final 5 h of culture. FIG.
5E shows the cell surface phenotype of mouse blood B10 cells.
Purified blood mononuclear cells were cultured with L+PIM for 5 h.
FIG. 5F shows the cell surface phenotype of mouse blood B10 plus
B10pro cells after 48 h stimulation with CD40 mAb with LPS, PMA,
ionomycin, and monensin added during the final 5 h of culture. FIG.
5C shows the cell surface phenotype of human spleen B10 cells.
Purified B cells were cultured with CpG+PIB for 5 h. FIG. 5D shows
the cell surface phenotype of human spleen B10+B10pro cells after
48 h stimulation with CD40L+CpG with PIB added during the final 5 h
of culture. FIG. 5G shows the cell surface phenotype of mouse
spleen B10 cells cultured with L+PIM for 5 h. FIG. 5H shows the
cell surface phenotype of mouse spleen B10+B10pro cells after 48 h
stimulation with CD40 mAb with L+PIM added during the final 5 h of
culture. In FIG. 5A-5H cultured cells were stained for viability
and cell surface molecule expression, permeabilized, stained with
anti-IL-10 mAb, and analyzed by flow cytometry. Representative cell
surface molecule expression by IL-10.sup.+ (thick line) and
IL10.sup.+ (thin line) CD19.sup.+ B cells from three individuals or
>3 mice is shown. Shaded histograms represent isotype-matched
control mAb staining.
[0033] FIG. 6A-6G. Human blood B10 and B10pro cells are
predominantly CD24.sup.hiCD27.sup.+. FIG. 6A shows that blood B10
cells were predominantly
CD24.sup.hiCD27.sup.+CD48.sup.hiCD148.sup.hi. Representative
phenotypes of purified blood B cells cultured with CpG+PIB for 5 h
before six-color immunofluorescence staining for viability, cell
surface molecule expression, and cytoplasmic IL-10. Subsequently,
CD24, CD27, CD48, and CD148 expression by IL-10.sup.+ (thick line)
and IL-10.sup.- (thin line) CD19.sup.+ cells was assessed by flow
cytometry. FIG. 6B shows that cell surface CD24, CD27, CD38, CD48,
or CD148 expression were not affected during IL-10 induction.
Representative phenotypes of CD19.sup.+ blood B cells cultured with
media on ice (thin line) or CpG+PIB (thick line) for 5 h before
immunofluorescence staining and flow cytometry analysis as in FIG.
6A. In FIG. 6A-6B the haded histograms represent isotype-matched
control mAb staining. Results represent those obtained for 3
individuals. FIG. 6C shows representative distributions of B10
cells within B cell subsets of three individuals as defined by
CD24, CD27, IgD/CD38, and IgD/CD27 expression. Purified blood B
cells were cultured with LPS+PIB for 5 h before immunofluorescence
staining and flow cytometry analysis as in FIG. 6A. The horizontal
and vertical lines on each contour plot are shown for reference,
with the lower left quadrants delineating the IgD.sup.-CD38.sup.-
and IgD.sup.-CD27.sup.- subsets as determined by control mAb
staining, respectively. Results represent those obtained for 5
individuals. FIG. 6D shows that blood B10 cells are predominantly
found within the CD24.sup.hiCD27.sup.+ B cell subset. Purified
blood B cells were cultured with LPS+PIB for 5 h before four-color
immunofluorescence staining for cell surface CD19, CD24, and CD27
expression and cytoplasmic IL-10 expression, with subsequent flow
cytometry analysis. FIG. 6E shows that B10pro cells derive from the
CD24.sup.hiCD27.sup.+ B cell subset. Purified blood B cells were
stained for CD19, CD24, and CD27 expression and sorted into the
CD24.sup.hiCD27.sup.+ and CD24.sup.lowCD27.sup.- B cell subsets as
indicated by the gates shown. Each purified subset was reanalyzed
by flow cytometry to determine purities, which were always >90%.
Subsequently, the purified B cells were cultured with CD40L plus
either LPS or CpG for 48 h, with PIB added during the final 5 h of
culture. The cultured cells were then stained for cell surface CD19
and intracellular IL-10 expression with the relative percentages of
IL-10.sup.+ B cells within the indicated gates determined. Similar
results were obtained in 2 independent experiments. FIG. 6F shows
clonal expansion of IL-10-producing B cells after CpG, but not LPS
or CD40L stimulation in vitro. Blood mononuclear cells were labeled
with CFSE and cultured with CD40L, and LPS or CPG for 48-96 h, with
PIB added for the last 5 h of culture. Histograms (right) represent
CFSE expression by the IL-10.sup.+ (thick line) or IL-10.sup.-
(thin line) B cell subsets. Results are representative of two
independent experiments. FIG. 6G shows that IL-10 is predominantly
secreted by CD24.sup.hiCD27.sup.+ B cells. Purified blood B cells
were sorted into the CD24.sup.hiCD27.sup.+ and
CD24.sup.lowCD27.sup.- B cell subsets as in FIG. 6E and cultured
with the indicated stimuli for 72 h. IL-10 secreted into the
culture supernatant fluid was quantified by ELISA. Bar graphs
indicate mean IL-10 (.+-.SEM) concentrations from triplicate ELISA
determinations. Significant differences between means from
CD24.sup.hiCD27.sup.+ and CD24.sup.lowCD27.sup.- B cells are
indicated: **, p<0.01. Significant differences between means
from cells cultured in media or with stimuli are indicated: ,
p<0.01.
[0034] FIG. 7A-7E. Blood B10 cell frequencies in patients with
autoimmune disease. FIG. 7A shows representative B cell cytoplasmic
IL-10 expression by control (Ctrl) individuals, and SLE, RA, SjS,
BD, and MS patients with relatively high B10 cell frequencies after
in vitro CpG plus PIB stimulation for 5 h. B10+B10pro cell
maturation was induced by 48 h CD40L plus CpG stimulation, with PIB
added during the final 5 h of culture. Percentages indicate
cytoplasmic IL-10.sup.+ B cell frequencies within the indicated
gates among total CD19.sup.+ B cells. FIG. 7B shows IL-10.sup.+ B
cell frequencies in control individuals and patients as represented
in FIG. 7A with each dot representing single individuals.
Horizontal bars indicate group means, the solid horizontal lines
indicate means+2 SD (95% confidence interval) for controls, while
dashed lines represent means+2 SD for all values. The patients are
described in Table 1. FIG. 7C shows the relationship between B10
and B10pro cell frequencies in control individuals and autoimmune
patients after in vitro culture with LPS or CpG plus PIB for 5 h.
B10+B10pro cells were identified by cytoplasmic IL-10 expression
after 48 h stimulation with CD40L plus LPS or CpG with PIB added
during the final 5 h of culture. FIG. 7D shows relative frequencies
of B10 cells and B10+B10pro cells identified for control
individuals and patients with autoimmune disease are compared after
CpG or LPS stimulation as shown in FIG. 7B with each dot
representing a single individual. FIG. 7E shows the relationship
between cytoplasmic IL-10 expression levels and B10pro cell
frequencies in control individuals and patients. Each dot
representing single individuals after stimulation with CD40L plus
CpG, with PIB added during the final 5 h of 48 h cultures. Linear
mean fluorescence intensities (MFI) for IL-10.sup.+ and IL-10.sup.-
B cells were determined using the gates indicated in FIG. 7AA with
the values shown representing a ratio of IL-10.sup.+ to IL-10.sup.-
MFIs. A linear regression line (.+-.95% prediction bands, dashed
lines) is shown for reference.
5. DETAILED DESCRIPTION
[0035] The present invention relates to a phenotypically distinct B
cell subset, B10 cells, that regulate T cell-mediated inflammatory
and immune responses through secretion of IL-10. The invention also
relates to harnessing B10 cells for the manipulation of the immune
and inflammatory responses, and for the treatment of diseases,
disorders and conditions associated with altered IL-10 levels,
including inflammatory and autoimmune diseases, as well as
immunosuppression, infectious diseases and cancer in humans and
other mammals.
[0036] Cellular compositions enriched for B10 cells, and methods
for their preparation are described. The B10 cells are
characterized by the ability to produce IL-10, in particular when
stimulated with a CD40 or TLR agonist. These cellular compositions
can be expanded and used in adoptive transfer therapies to treat
conditions associated with diminished IL-10 production or those
ameliorated by increased levels of IL-10, e.g., inflammatory and/or
autoimmune conditions or diseases. In an alternative approach,
therapeutic regimens designed to expand the endogenous population
of B10 cells, or increase their production of IL-10 can be used to
treat a disease or condition associated with diminished levels of
IL-10 or ameliorated by increased levels of IL-10 such as
inflammatory, immune hyperresponsive and/or autoimmune conditions
or diseases in subjects in need thereof. In this approach, agents
capable of activating or stimulating B10 cells are administered to
the subject in need of such treatment. The agent may be a TLR
agonist or a CD40 agonist. In an alternative approach, the agent
may be antibodies that activate and/or stimulate expansion of B10
cells, or increase their production of IL-10. Expansion can be
accomplished in vivo (e.g., by direct administration of the agent
such as an antibody or receptor agonist) or ex vivo (e.g., by
activating and/or expanding the cells obtained from the subject and
returning the activated cells to the subject).
[0037] Methods of treating a disease or condition associated with
elevated levels of IL-10 or insufficient or ineffective immune
responsiveness are also provided. These methods include
administering a therapeutically effective amount of an agent that
kills, abrogates, or inhibits the function, localization, or
expansion of B10 cells or an agent that inhibits production of
IL-10 by B10 cells to a subject in need of such treatment. The
methods are suitable for treating diseases and/or conditions
involving immunosuppression, infectious diseases or cancer by
depleting or ablating B10 cells in subjects in need thereof. In
this approach, the agent may be antibodies that kill B10 cells, or
inhibit their function, proliferation or production of IL-10. In
particular, the agent may include antibodies that induce homotypic
adhesion and agents that selectively deplete or target B10 cells as
opposed to other types of B cells such as follicular B cells.
[0038] Methods for identifying B10 cells in patients and/or patient
samples are described for diagnosing the immune status of affected
individuals. The methods include assaying for cells producing IL-10
or capable of producing IL-10 when treated with a CD40 agonist or a
TLR agonist. Methods for assessing the number of B10 and B10pro
cells in a subject are also encompassed. The immune status of the
individual may be useful in predicting the likelihood of
contracting a particular disease, such as an autoimmune disease, or
in predicting responsiveness to particular diseases or particular
therapeutics.
[0039] In another embodiment, a method for generating an antibody
that preferentially or selectively depletes B10 cells as compared
to other subsets of B cells is provided. The method comprises: (i)
selecting an antibody that binds to a marker that is expressed by
B10 cells including but not limited to, e.g. CD1d, CD5, CD19, CD20,
CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD40, CD48, CD72, and
CD148; (ii) assaying the antibody for the ability to induce
homotypic adhesion of B cells (Kansas G S, Wood G S, Tedder T F.
Expression, distribution and biochemistry of human CD39: Role in
activation-associated homotypic adhesion of lymphocytes. J Immunol.
1991; 146:2235-2244; Kansas G S, Tedder T F. Transmembrane signals
generated through MHC class II, CD19, CD20, CD39 and CD40 antigens
induce LFA-1-dependent and -independent adhesion in human B cells
through a tyrosine kinase-dependent pathway. J Immunol. 1991; 147:
4094-4102; Wagner N, Engel P, Vega M, Tedder T F. Ligation of MHC
class I and class II molecules leads to heterologous
desensitization of signal transduction pathways that regulate
homotypic adhesion in human lymphocytes. J Immunol. 1994;
152:5275-5287.); (iii) assaying the antibody for the ability to
deplete the B10 cell population. Optionally, the ability of the
antibody to deplete or avoid depletion of other B cell subsets may
also be assessed. In some embodiments, the Fc portion of the
antibody may be modified so that the mechanism of B10 cell
depletion by the antibody is independent of the antibody's Fc
region. The antibody may be selected for its ability to deplete the
B10 cells by a method that is independent of antibody-dependent
cellular cytotoxicity (ADCC), complement-dependent cytotoxicity
(CDC) and apoptosis.
[0040] Methods of selecting B10 cells are also provided. The method
includes selecting B lymphocytes in a sample from a subject,
stimulating the B cells in vitro with PMA and ionomycin for five
hours and selecting IL-10 producing cells. The cells may also be
selected by stimulating them for at least 24 hours with a CD40
agonist or a TLR agonist prior to the addition of PMA and
ionomycin. The cells may be further selected by screening for
markers of B10 cells including but not limited to expression of
CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38,
CD40, CD48, CD72, or CD148 or the relative levels of expression of
these markers on the cell surface.
[0041] Methods of inducing an IgG antibody response to an antigen,
such as in a vaccine or vaccination protocol are described. The
methods include administering the antigen to a subject and
administering an agent that kills or inhibits the function,
localization or expansion of B10 cells or inhibits production of
IL-10 by B10 cells to the subject. The administration of the agent
with the antigen increases the antigen specific IgG antibody
production in the subject as compared to a subjet administered the
antigen alone. The antigen and the agent may be administered
together, but need not be.
5.1 The B10 Cell Subset
[0042] The present invention relates to a regulatory subset of the
normal B cell population, B10 cells, with the ability to produce
IL-10. The invention also relates to therapeutic uses of B10
cells.
[0043] The phenotype of B10 cells can be determined by antibody
staining and flow cytometry, FACS, using antibodies to phenotypic
markers of B10 cells, and techniques known in the art, including
but not limited to those described in the examples, infra. See,
e.g., Section 6 et seq and U.S. Patent Publication No.
2011/0135666. The invention is based, in part, on the surprising
discovery that cellular compositions enriched by selection for B10
cell cellular markers (such as CD24.sup.highCD27.sup.+ or
CD1d.sup.highCD5.sup.+) will contain a high percentage of IL-10
producing B cells. It is also based on the discovery that these B10
cells can be selected based on their ability to produce IL-10 when
stimulated with CD40 agonists or TLR agonists.
[0044] The ability of the cells to produce IL-10 can be assessed by
measuring IL-10 production in naive cells and in cultured cells
stimulated with LPS (lipopolysaccharide), PMA (phorbol 12-myristate
13-acetate), ionomycin, CpG oligodeoxynucleotides or comparable
stimulatory Toll-like receptor (TLR) agonists, or with an agonist
of CD40 (e.g., using an antibody to CD40). Production of IL-10 by
the cells can be assessed by assaying for IL-10 in the cell culture
supernatant. In addition, production of IL-10 can be verified
directly by intracellular cytokine staining after additional
treatment with Brefeldin A or monensin. Standard immunoassays known
in the art can be used for such purpose. Examples of assays for
IL-10 production are described in Section 7, infra. While IL-10 is
produced at low levels in naive B10 cells, IL-10 production is
increased in response to stimulation.
5.1.1 Cellular Compositions Enriched In B10 Cells
[0045] The enriched, isolated and/or purified B10 cell subset
composition can comprise anywhere from 0.5% to 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 99%, or
100% B10 cells (as determined, e.g., by the assays described
above). In a preferred embodiment, the enriched/purified B10 cell
subset comprises greater than 50% B10 cells. In a more preferred
embodiment, the enriched/purified B10 cell subset comprises greater
than 75% B10 cells. In a still more preferred embodiment, the
enriched/purified B10 cell subset comprises greater than 90% B10
cells. In certain embodiments, the enriched, isolated and/or
purified B10 cells have a CD1d.sup.highCD5.sup.+ phenotype. In
certain embodiments, the enriched, isolated and/or purified B10
cells have a CD24.sup.hiCD27.sup.+ phenotype.
[0046] The enriched, isolated and/or purified B10 cells can be
obtained from a mammalian subject, including but not limited to
rodents, e.g. mice, rats; livestock, e.g. pigs, horses, cows, etc.,
pets, e.g. dogs, cats; and primates, e.g. humans. In one
embodiment, the subject is an animal model of an IL-10 associated
disease.
[0047] Alternatively, B10 cells may be enriched/purified from any
tissue where they reside including, but not limited to, blood
(including blood collected by blood banks and cord blood), spleen,
bone marrow, lymph nodes, tissues removed and/or exposed during
surgical procedures, and tissues obtained via biopsy procedures.
Tissues/organs from which the B10 cells are enriched, isolated,
and/or purified may be isolated from both living and non-living
subjects, wherein the non-living subjects include organ donors.
[0048] Methods for the isolation of B10 cells are based on
selecting cells having the B10 cell-specific markers. In a specific
embodiment, the B10 cell-specific markers comprise CD1d.sup.high
and CD5. In another specific embodiment, the B10 cell-specific
markers comprise CD24.sup.high and CD27. In certain embodiments,
additional B cell-specific markers can be used for selection
including, but not limited to, CD1d, CD19, CD20, CD21, CD23, CD24,
CD25, CD38, CD48, and CD148. Several of these markers or
combinations of these markers can be used to specifically select
B10 cells. In a particular aspect of this embodiment, a B10 cells
are enriched/purified by flow cytometry as demonstrated in the
examples described in Section 6 and 7, infra. However, a variety of
cell separation techniques known in the art can be used, including
but not limited to magnetic separation using antibody-coated
magnetic beads and/or particles, FACS, affinity chromatography,
affinity column separation, "panning" with antibody attached to a
solid matrix, density gradient centrifugation, and counter-flow
centrifugal elutriation. (See, e.g., Kumar and Lykke, 1984,
Pathology, 1:53-62).
[0049] According to these embodiments, a cellular composition
enriched for B10 cells that has been enriched by selection using
both CD1d.sup.high and CD5 as cellular markers or CD24.sup.high and
CD27 as cellular markers will contain a higher percentage of B10
cells than one enriched using only one of these markers. The use of
these markers to isolate/enrich/purify B10 cells has several
advantages. Using these cell surface markers, as opposed to
intracellular IL-10 as a marker, allows for the selection/sorting
of the IL-10 producing B cell population without permeabilizing the
cells, which would make them therapeutically useless. Once
enriched, the cells may be further purified or expanded by
stimulating or activating the cells with a CD40 agonist or a TLR
agonist. These agonists may be used in combination with PMA and
ionomycin.
[0050] B10 cells can also be isolated by negatively selecting
against cells that are not B10 cells. This can be accomplished by
performing a lineage depletion, wherein cells are labeled with
antibodies for particular lineages such as the T lineage, the
macrophage/monocyte lineage, the dendritic cell lineage, the
granulocyte lineages, the erythrocytes lineages, the megakaryocytes
lineages, and the like. Cells labeled with one or more lineage
specific antibodies can then be removed either by affinity column
processing (where the lineage marker positive cells are retained on
the column), by affinity magnetic beads or particles (where the
lineage marker positive cells are attracted to the separating
magnet), by "panning" (where the lineage marker positive cells
remain attached to the secondary antibody coated surface), or by
complement-mediated lysis (where the lineage marker positive cells
are lysed in the presence of complement by virtue of the antibodies
bound to their cell surface). Another lineage depletion strategy
involves tetrameric complex formation. Cells are isolated using
tetrameric anti-human antibody complexes (e.g., complexes specific
for multiple markers on multiple cell types that are not markers of
B10 cells, given in more detail infra) and magnetic colloid in
conjunction with StemSep columns (Stem Cell Technologies,
Vancouver, Canada). The cells can then optionally be subjected to
centrifugation to separate cells having tetrameric complexes bound
thereto from all other cells.
[0051] In a certain embodiment, the enriched/purified B10 cells can
be stored for a future use. In this regard, the B10 cells can be
stored by "cryopreservation." Cryopreservation is a process where
cells or whole tissues are preserved by cooling to low sub-zero
temperatures, such as 77 K or -196.degree. C. in the presence of a
cryoprotectant. At these low temperatures, any biological activity,
including the biochemical reactions that would lead to cell death,
is effectively stopped. Storage by cryopreservation includes, but
is not limited to, storage in liquid nitrogen, storage in freezers
maintained at a constant temperature of 0.degree. C., storage in
freezers maintained at a constant temperature of -20.degree. C.,
storage in freezers maintained at a constant temperature of
-80.degree. C., and storage in freezers maintained at a constant
temperature of lower than -80.degree. C. In one aspect of this
embodiment, the cells may be "flash-frozen," e.g., in ethanol/dry
ice or in liquid nitrogen prior to storage. In another aspect of
this embodiment, the cells can be preserved in medium comprising a
cryprotectant including, but not limited to dimethyl sulfoxide
(DMSO), glycerol, ethylene glycol, propylene glycol, sucrose, and
trehalose. Other methods of storing biological matter are well
known to those of skill in the art, such as "hibernation," wherein
cells are stored at temperatures above freezing or by preservation
of the cells in a "static" state, as described in U.S. patent
application publication No. 2007/0078113, incorporated herein by
reference in is entirety.
[0052] In certain embodiments, B10 cells can be obtained from a
subject in need of therapy or suffering from a disease associated
with elevated or diminished levels of IL-10. Alternatively, B10
cells can be obtained from a donor, preferably a histocompatibility
matched donor. B10 cells may be harvested from the peripheral
blood, bone marrow, spleen, or any other organ/tissue in which B10
cells reside in said subject or donor. In a further aspect, the B10
cells may be isolated from a pool of subjects and/or donors, or
from pooled blood.
[0053] When the population of B10 cells is obtained from a donor
distinct from the subject, the donor is preferably syngeneic, but
can also be allogeneic, or even xenogeneic, provided the cells
obtained are subject-compatible in that they can be introduced into
the subject. Allogeneic donor cells are preferably
human-leukocyte-antigen (HLA)-compatible, and are typically
administered in conjunction with immunosuppressive therapy. To be
rendered subject-compatible, xenogeneic cells may be subject to
gamma irradiation or PEN110 treatment as described (Fast et al.,
2004, Transfusion 44:282-5).
5.1.2. Enrichment of B10 Cells
[0054] B10 cells can be enriched by selecting cells having the
CD1d.sup.highCD5.sup.+ surface markers or the
CD24.sup.highCD27.sup.+ surface markers and separating using
automated cell sorting such as fluorescence-activated cell sorting
(FACS), solid-phase magnetic beads, etc. as demonstrated in
examples described in sections 6 and 7 infra. To enhance
enrichment, positive selection may be combined with negative
selection; i.e., by removing cells having surface markers specific
to non-B cells and/or those specific to non-B10 cells. Non-limiting
examples of methods of negative selection are described supra.
Exemplary surface markers specific to non-B10 cells include CD3,
CD4, CD7, CD8, CD15, CD16, CD34, CD56, CD57, CD64, CD94, CD116,
CD134, CD157, CD163, CD208, F4/80, Gr-1, and TCR.
[0055] The cells may be used to make pharmaceutical compositions.
Pharmaceutical compositions comprising the B10 cells described
herein and a pharmaceutically acceptable carrier are provided. A
pharmaceutically acceptable carrier is any carrier suitable for in
vivo administration. Examples of pharmaceutically acceptable
carriers suitable for use in the composition include, but are not
limited to, water, buffered solutions, glucose solutions, oil-based
or bacterial culture fluids. Additional components of the
compositions may suitably include, for example, excipients such as
stabilizers, preservatives, diluents, emulsifiers and lubricants.
Examples of pharmaceutically acceptable carriers or diluents
include stabilizers such as carbohydrates (e.g., sorbitol,
mannitol, starch, sucrose, glucose, dextran), proteins such as
albumin or casein, protein-containing agents such as bovine serum
or skimmed milk and buffers (e.g., phosphate buffer). Especially
when such stabilizers are added to the compositions, the
composition is suitable for freeze-drying or spray-drying. The
composition may also be emulsified.
5.2 Expansion of the B10 Cell Subset and/or Enhancing Their
Production of IL-10
[0056] In a particular embodiment, expansion of the B10 cell
population is achieved by contacting a population of B10 cells with
a stimulatory composition sufficient to cause an increase in the
number of B10 cells. This may be accomplished by contacting the
enriched, isolated and/or purified B10 cell subset with a mitogen,
cytokine, growth factor, antibody, CD40 agonist or TLR agonist. The
B10 cells are preferably expanded at least 10-fold and preferably
at least 50, 100, 200, 300, 500, 800, 1000, 10,000, or
100,000-fold. In a specific aspect of this embodiment, the expanded
B10 cell population retains all of the genotypic, phenotypic, and
functional characteristics of the original population. In another
embodiment, one or more of the characteristics of the B10 cell
population is lost or modified following expansion.
[0057] Levels of IL-10 produced by the B10 cell subset can be
increased by, e.g., administration of agonists to the B cell
surface receptor CD40. Non-limiting examples of CD40 agonists
include CD40 antibodies and fragments thereof, the CD40 ligand and
polypeptide fragments thereof, small molecules, synthetic drugs,
peptides (including cyclic peptides), polypeptides, proteins,
nucleic acids, synthetic or natural inorganic molecules, mimetic
agents, and synthetic or natural organic molecules.
[0058] In a certain embodiment, the CD40 agonist is a CD40
antibody. The CD40 antibodies of the invention can be of any form,
as disclosed above. Antibodies to CD40 are known in the art (see,
e.g., Buhtoiarov et al., 2005, J. Immunol. 174:6013-22; Francisco
et al., 2000, Cancer Res. 60:3225-31; Schwulst et al., 2006,
177:557-65, herein incorporated by reference in their
entireties).
[0059] Levels of IL-10 produced by the B10 cell subset can also be
increased by, e.g., administration of agonists to TLRs on the B10
cell surface. In particular embodiments the TLR agonist may be an
agonist of TLR1, TLR4, TLR6, TLR7, or TLR9. These agonists include
natural ligands of these receptors and non-natural ligands or
agonists. Non-limiting examples of TLR agonists include LPS, CpG
oligodeoxynucleotides, Pam3CSK4, Pam2CGDPKHPKSF, or Imiquimod and
also include variations of these molecules or other small
molecules, synthetic drugs, peptides (including cyclic peptides),
polypeptides, proteins, nucleic acids, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules.
[0060] Expansion of IL-10 production by the B10 cell subset can be
advantageously achieved ex vivo, e.g., by isolating the enriched
B10 cell population and contacting the cells with a CD40 agonist or
a TLR agonist. In an aspect of this embodiment, the cells are
contacted with a CD40 agonist or a TLR agonist and relevant
antigen(s). In a specific aspect of this embodiment, the cells are
contacted with a CD40 agonist, a TLR agonist and relevant
antigen(s).
5.3 Ablation of the B10 Cell Subset and/or Inhibiting their
Production of IL-10
[0061] The B10 cell subset can be ablated by, e.g., engaging a B
cell surface markers e.g., CD20 or CD22. Non-limiting examples of
compounds capable of engaging B cell surface markers to ablate the
B10 cell population include antibodies and fragments thereof, the
ligand for the cell surface marker and fragments thereof, ligand
mimetics, small molecules, synthetic drugs, peptides (including
cyclic peptides), polypeptides, proteins, nucleic acids, synthetic
or natural inorganic molecules, mimetic agents, and synthetic or
natural organic molecules. Antibodies to B cell surface markers are
known in the art (for CD22 see, e.g., Tuscano et al., 2003, Blood
101:3641-7; US 2004/0001828 A1; and US 2007/0264360, for CD20 see,
e.g., US 2009/0136516 incorporated by reference herein in their
entireties).
[0062] Alternatively, a bispecific antibody for CD1d and CD5 may be
used to target the B10 cell subset (these will be referred to
herein as bispecific "CD1d/CD5" antibodies). Bispecific antibodies
can be prepared from CD1d and CD5 antibodies using techniques that
are known in the art (see, e.g., U.S. Pat. Nos. 5,534,254,
5,837,242, 6,492,123; U.S. Patent application publication Nos.
20040058400 and 20030162709, which are all herein incorporated by
reference in their entireties). In another embodiment, a bispecific
antibody for CD24 and CD27 may be used to target the B10 cell
subset.
[0063] In order to kill or ablate the B10 cell subset, targeting
antibodies (e.g., CD20, CD22, bispecific CD1d/CD5, or bispecific
CD24/CD27) of an isotype that mediate ADCC (antibody-dependent and
mediated toxicity) or CDC (complement-dependent cytotoxicity) can
be used. Of the various human immunoglobulin classes, IgG1, IgG2,
IgG3, IgG4 and IgM are known to activate complement. Human IgG1 and
IgG3 are known to mediate ADCC. Antibodies to CD20 may be used to
deplete B10 cells selectively. The antibodies demonstrated to
target the IL-10 producing B10 cell subset were antibodies that
were not capable of inducing ADCC, CDC or apoptosis. Instead the
effective antibodies were those that induced homotypic adhesion.
Thus, antibodies to B cell surface markers with IgG3 or IgG2b Fc
regions which do not efficiently engage most Fcy receptors, but
induce homotypic adhesion are useful in the methods described
herein. Optionally the Fc portion of a known antibody may be
modified so that the mechanism of depletion of B10 cells is
independent of the antibody's Fc region.
[0064] Antibodies targeting the B10 cell subset can be further
conjugated to a cytotoxic agent, using methods known in the art
(see, e.g., DiJoseph et al., 2004, Clin. Cancer Res. 10:8620-9).
This may be preferred when using antibodies or antibody fragments
that do not mediate ADCC or CDC. Non-limiting examples of cytotoxic
agents include antimetabolites (e.g., cytosine arabinoside,
aminopterin, methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, and 5-fluorouracil decarbazine); alkylating agents
(e.g., mechlorethamine, thiotepa chlorambucil, melphalan,
carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiammine-platinum (II) (CDDP), and cisplatin); vinca
alkaloid; anthracyclines (e.g., daunorubicin (formerly daunomycin)
and doxorubicin); antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC));
calicheamicin; CC-1065 and derivatives thereof; auristatin
molecules (e.g., auristatin PHE, bryostatin-1, and dolastatin-10;
see Woyke et al., Antimicrob. Agents Chemother. 46:3802-8 (2002),
Woyke et al., Antimicrob. Agents Chemother. 45:3580-4 (2001),
Mohammad et al., Anticancer Drugs 12:735-40 (2001), Wall et al.,
Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad, et al.,
Int. J. Oncol. 15:367-72 (1999), all of which are incorporated by
reference herein in their entireties); DNA-repair enzyme inhibitors
(e.g., etoposide or topotecan); kinase inhibitors (e.g., compound
ST1571, imatinib mesylate (Kantarjian et al., Clin. Cancer Res.
8(7):2167-76 (2002)); demecolcine; and other cytotoxic agents
(e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracenedione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologues
thereof and those compounds disclosed in U.S. Pat. Nos. 6,245,759,
6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410,
6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376,
5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868,
5,648,239, 5,587,459, all of which are incorporated by reference
herein in their entirety); farnesyl transferase inhibitors (e.g.,
R115777, BMS-214662, and those disclosed by, for example, U.S. Pat.
Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959,
6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615,
6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487,
6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338,
6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786,
6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465,
6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853,
6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574,
and 6,040,305, all of which are herein incorporated by reference in
their entirety); topoisomerase inhibitors (e.g., camptothecin,
irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG211
(GI147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine, XR5000,
saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528,
ED-110, NB-506, ED-110, NB-506, and rebeccamycin); bulgarein; DNA
minor groove binders such as Hoechst dye 33342 and Hoechst dye
33258; nitidine; fagaronine; epiberberine; coralyne;
beta-lapachone; BC-4-1; antisense oligonucleotides (e.g., those
disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,
5,734,033, and 5,618,709, all of which are herein incorporated by
reference in their entirety); adenosine deaminase inhibitors (e.g.,
fludarabine phosphate and 2-chlorodeoxyadenosine); and
pharmaceutically acceptable salts, solvates, clathrates, and
prodrugs thereof.
[0065] In another embodiment, the targeting antibodies, such as a
CD20, CD22, bispecific CD1d/CD5, or bispecific CD24/CD27 antibody,
can be conjugated to a radioactive metal ion, such as the
alpha-emitters .sup.211astatine, .sup.212bismuth, .sup.213bismuth;
the beta-emitters .sup.131iodine, .sup.90yttrium, .sup.177lutetium,
.sup.153samarium, and .sup.109palladium; or macrocyclic chelators
useful for conjugating radiometal ions, including but not limited
to, 131indium, .sup.131L, .sup.131yttrium, .sup.131holmium,
.sup.131samarium, to polypeptides or any of those listed supra. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA), which can be attached to the antibody via a linker
molecule. Such linker molecules are commonly known in the art and
described in Denardo, et al., 1998, Clin Cancer Res 4(10):2483-90;
Peterson, et al., 1999, Bioconjug Chem 10(4):553-7; and Zimmerman,
et al., 1999, Nucl Med Biol 26(8):943-50, each incorporated by
reference herein in their entireties.
[0066] In still another embodiment, the targeting antibody, e.g.
the CD20, CD22, bispecific CD1d/CD5, or bispecific CD24/CD27
antibody is conjugated to a proteinaceous agent that modifies a
given biological response and leads to cytotoxicity. In one
embodiment, the antibody is conjugated to a plant-, fungus-, or
bacteria-derived toxin. Non-limiting examples of such toxins
include A chain toxins, ribosome inactivating proteins, ricin A,
deglycosylated ricin A chain, abrin, alpha sarcin, aspergillin,
restrictocin, ribonucleases, diphtheria toxin, bacterial endotoxin,
saporin toxin, Granzyme B or the lipid A moiety of bacterial
endotoxin, cholera toxin, or Pseudomonas exotoxin and derivatives
and variants thereof.
[0067] In another embodiment, an antagonist capable of engaging a B
cell surface marker such as CD20, CD22, CD 1d or CD5 may be used to
ablate the B10 cell population is a synthetic ligand targeted to a
B10 cell specific marker, such as that described in Collins et al.,
2006, J. Immunol. 5:2994-3003, incorporated herein by reference in
its entirety. In one aspect of this embodiment, the synthetic
ligand may be further conjugated to a toxin, such as the saporin
toxin.
[0068] Alternatively, a compound capable of engaging a marker or
markers on the B10 cell subset can inhibit the production of IL-10
by the B10 cells. Non-limiting examples of such compounds include
antibodies and fragments thereof, small molecules, synthetic drugs,
peptides (including cyclic peptides), polypeptides, proteins,
nucleic acids, synthetic or natural inorganic molecules, mimetic
agents, and synthetic or natural organic molecules. In one
embodiment, the compound engages CD22. In an aspect of this
embodiment, the compound is a CD22 antibody. In another aspect of
this embodiment, the compound engages CD5. In an aspect of this
embodiment, the compound is a CD5 antibody. In another aspect of
this embodiment, the compound engages CD1d. In an aspect of this
embodiment, the compound is a CD1d antibody. In still another
aspect of this embodiment, the compound is a bispecific CD1d/CD5
antibody. In another aspect of this embodiment, the compound
engages CD24. In an aspect of this embodiment, the compound is a
CD24 antibody. In another aspect of this embodiment, the compound
engages CD27. In an aspect of this embodiment, the compound is a
CD27 antibody. In still another aspect of this embodiment, the
compound is a bispecific a CD24/CD27 antibody. In yet another
aspect of this embodiment, the compound engages CD19. In an aspect
of this embodiment, the compound is a CD19 antibody. In one
embodiment, the compound engages CD20. In an aspect of this
embodiment, the compound is a CD20 antibody. Alternatively, the
compound may bind one or more of CD1d, CD5, CD19, CD20, CD21, CD22,
CD24, CD27, CD38, CD40, CD48, CD72, or CD148. In one aspect the
compound may be an antibody or a bispecifica antibody directed to
one or more of CD1d, CD5, CD19, CD20, CD21, CD22, CD24, CD27, CD38,
CD40, CD48, CD72, or CD148.
5.4 Production of Therapeutic Antibodies
[0069] Antibodies that target, activate, inhibit and/or kill the
B10 cell subset and which can be used in the therapeutic regimens
described herein can be made using techniques well known in the
art. The practice of the invention employs, unless otherwise
indicated, conventional techniques in molecular biology,
microbiology, genetic analysis, recombinant DNA, organic chemistry,
biochemistry, PCR, oligonucleotide synthesis and modification,
nucleic acid hybridization, and related fields within the skill of
the art. These techniques are described in the references cited
herein and are fully explained in the literature. See, e.g.,
Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel
et al, Current Protocols in Molecular Biology, John Wiley &
Sons (1987 and annual updates); Current Protocols in Immunology,
John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984)
Oligonucleotide Synthesis: A Practical Approach, IRL Press;
Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical
Approach, IRL Press; Birren et al (eds.) (1999) Genome Analysis: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, each of
which is incorporated by reference herein in its entirety.
[0070] Antibodies for use in the methods of the invention include,
but are not limited to, synthetic antibodies, monoclonal antibodies
(mAbs), recombinantly produced antibodies, multispecific antibodies
(including bi-specific antibodies), human antibodies, humanized
antibodies, chimeric antibodies, intrabodies, diabodies,
single-chain Fvs (scFv) (e.g., including monospecific, bispecific,
etc.), camelized antibodies, Fab fragments, F(ab') fragments,
disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies,
and epitope-binding fragments of any of the above.
[0071] In particular, antibodies to be used in the methods of the
invention include immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site that binds to a CD1d, CD5, CD22,
CD24, CD27 or CD40 antigen, or bispecifically to the CD1d and CD5
antigens or the CD24 and CD27 antigens. The immunoglobulin
molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and
IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule.
[0072] Variants and derivatives of antibodies include antibody
fragments that retain the ability to specifically bind to an
epitope. In certain embodiments, fragments include Fab fragments;
Fab'; F(ab').sub.2; a bispecific Fab; a single chain Fab chain
comprising a variable region, also known as, a sFv; a
disulfide-linked Fv, or dsFv; a camelized VH; a bispecific sFv; a
diabody; and a triabody. Derivatives of antibodies also include one
or more CDR sequences of an antibody combining site. In certain
embodiments, the antibody to be used with the invention comprises a
single-chain Fv ("scFv").
[0073] The antibodies used in the methods of the invention may be
from any animal origin including birds and mammals (e.g., human,
murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or
chicken).
[0074] In certain embodiments, the antibodies of the invention are
monoclonal antibodies (mAbs). Monoclonal antibodies can be prepared
using a wide variety of techniques known in the art including the
use of hybridoma, recombinant, and phage display technologies, or a
combination thereof. For example, mAbs can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681
(Elsevier, N.Y., 1981) (each of which is herein incorporated by
reference in their entireties).
[0075] Antibodies can also be generated using various phage display
methods. Examples of phage display methods that can be used to make
the antibodies include those disclosed in Brinkman et al., 1995, J.
Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.
24:952-958; Persic et al, 1997, Gene 187:9-18; Burton et al., 1994,
Advances in Immunology 57:191-280; PCT Application No. PCT/GB91/O1
134; International Publication Nos. WO 90/02809, WO 91/10737, WO
92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and
W097/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of
which is incorporated by reference herein in its entirety.
[0076] In certain embodiments, the antibodies of the invention are
chimeric antibodies or single chain antibodies. Techniques
developed for the production of "chimeric antibodies" (Morrison et
al., 1984, Proc Natl Acad Sci 81:851; Neuberger et al., 1984 Nature
312:604; Takeda et al., 1985, Nature 314:452, each incorporated by
reference herein in its entirety) and single chain antibodies (U.S.
Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al,
1988, Proc Natl Acad Sci USA 85:5879; and Ward et al, 1989, Nature
334:544, each incorporated by reference herein in its entirety) are
well known in the art.
[0077] In a certain embodiment, antibodies used in the methods of
the invention are humanized antibodies. Humanized antibodies can be
produced using a variety of techniques known in the art, including
but not limited to, CDR-grafting (European Patent No. EP 239,400;
International publication No. WO 91/09967; and U.S. Patent Nos.
5,225,539, 5,530,101, and 5,585,089, each of which is herein
incorporated by reference in its entirety), veneering or
resurfacing (European Patent Nos. EP 592,106 and EP 519,596;
Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et
al., 1994, Protein Engineering 7(6):805-814; and Roguska et al,
1994, PNAS 91:969-973, each of which is incorporated by reference
herein in its entirety), chain shuffling (U.S. Pat. No. 5,565,332,
herein incorporated by reference in its entirety), and techniques
disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No.
5,766,886, WO 9317105, Tan et al., 2002, J. Immunol. 169:1119 25,
Caldas et al., 2000 Protein Eng. 13(5):353-60, Morea et al., 2000,
Methods 20(3):267 79, Baca et al., 1997, J. Biol. Chem.
272(16):10678-84, Roguska et al., 1996, Protein Eng. 9(10):895 904,
Couto et al., 1995 Cancer Res. 55 (23 Supp):5973s-5977s, Couto et
al., 1995, Cancer Res. 55(8):1717-22, Sandhu J S, 1994, Gene
150(2):409-10, and Pedersen et al., 1994, J. Mol. Biol.
235(3):959-73 U.S. Patent Pub. No. US 2005/0042664 A1 (Feb. 24,
2005), each of which is incorporated by reference herein in its
entirety. Often, framework residues in the framework regions will
be substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; and Reichmann et al., 1988, Nature 332:323,
each of which is incorporated by reference herein in its
entirety).
[0078] Single domain antibodies can be produced by methods
well-known in the art. (See, e.g., Riechmann et al., 1999, J.
Immunol. 231:25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol.
1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302; U.S.
Patent No. 6,005,079; and International Publication Nos. WO
94/04678, WO 94/25591, and WO 01/44301, each of which is
incorporated herein by reference in its entirety).
[0079] Further, antibodies that bind to a desired antigen can, in
turn, be utilized to generate anti-idiotype antibodies that "mimic"
an antigen using techniques well known to those skilled in the art.
(See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and
Nissinoff, 1991, J. Immunol. 147(8):2429-2438, herein incorporated
by reference in their entireties).
[0080] Bispecific antibodies can be prepared using techniques that
are known in the art. (See, e.g., U.S. Pat. Nos. 5,534,254,
5,837,242, 6,492,123; U.S. patent application publication Nos.
20040058400 and 20030162709, which are all herein incorporated by
reference in their entireties).
[0081] The present invention contemplates the use of antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide. Fused
or conjugated antibodies of the present invention may be used for
ease in purification. For example, the antibodies or fragments
thereof for use in the present invention can be fused to marker
sequences, such as a peptide to facilitate purification. See e.g.,
PCT publication WO 93/21232; EP 439,095; Naramura et al., 1994,
Immunol Lett 39:91; U.S. Pat. No. 5,474,981; Gillies et al., 1992,
Proc Natl Acad Sci USA 89:1428; Fell et al., 1991, J Immunol
146:2446, which are herein incorporated by reference in their
entireties.
[0082] In certain aspects, the antibodies used in the present
invention can be produced intracellularly, in the periplasmic
space, or directly secreted into the medium. If the antibodies are
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, may be removed, for example,
by centrifugation or ultrafiltration.
[0083] Exemplary methods for the use of host cells and vectors in
the production of antibody can be found in U.S. Pat. Nos. 4,816,567
and 6,331,415 to Cabilly et al., each of which is incorporated by
reference herein in its entirety.
5.5 Therapeutic Applications of the B10 Cell Subset to Treat
Diseases and Disorders Associated with Diminished IL-10 Levels
[0084] Diseases or disorders associated with diminished levels of
IL-10 and elevated immune/inflammatory responses (particularly
inflammatory diseases and autoimmune diseases) can be treated in
accordance with the invention using different therapeutic
modalities designed to supply the B10 cell subset to an affected
subject (e.g., by adoptive transfer/transplant); expand the
endogenous B10 cell subset in an affected subject; and/or enhance
production of IL-10 by the B10 cell subset (either adoptively
transferred cells or the endogenous population) in the affected
subject.
[0085] In one approach, a cellular composition enriched for the B10
cell subset is administered to a subject in need thereof in amounts
effective to increase IL-10. The cellular composition can be
obtained from a histocompatibilty matched donor. Alternatively,
lymphocytes may be obtained from the subject to be treated,
enriched for the B10 cell subset and returned to the patient. In
either case the enriched cells can be exposed to an antigen of
interest prior to introduction into the subject to further
fine-tune the regulation of the immune response. The enriched or
selected B10 cells may also be expanded by stimulation in vitro
prior to introduction into the subject.
[0086] Alternatively, an effective amount of a therapeutic agent
capable of stimulating the proliferation of the endogenous B10 cell
subset, and/or increasing the amounts of IL-10 produced by the B10
cell subset can be administered to a subject in need thereof in
amounts effective to increase IL-10 levels in said subject. These
agents may be targeted to the B10 cell cell subset. Such agents
include CD40 agonists and TLR agonists.
5.5.1. Diseases and Disorders Associated with Reduced IL-10
Production that can Be Treated B10 Cell Subset
[0087] Diseases and conditions associated with diminished IL-10
levels can be treated in accordance with this aspect of the
invention. Decreased levels of IL-10 have been demonstrated in
autoimmune and inflammatory diseases including, but not limited to
psoriasis (Asadullah et al., 1998, J. Clin. Investig. 101:783-94,
Nickoloff et al., 1994, Clin. Immunol. Immunopathol., 73:63-8,
Mussi et al. 1994, J. Biol. Regul. Homeostatic Agents), rheumatoid
arthritis (Jenkins et al., 1994, Lymphokine Cytokine Res. 13:47-54;
Cush et al., 1995, Arthritis Rheum. 38:96-104; Al Janadi et al.,
1996, J. Clin. Immunol. 16:198-207), allergic contact dermatitis
(Kondo et al., 1994, J. Investig. Dermatol. 103:811-14; Schwarz et
al., 1994, J. Investig. Dermatol. 103:211-16), inflammatory bowel
disease (Kuhn et al., 1993, Cell 75:263-74; Lindsay and Hodgson,
2001, Aliment. Pharmacol. Ther. 15:1709-16) and multiple sclerosis
(Barrat et al., 2002, J. Exp. Med. 195:603-16; Cua et al., 2001, J.
Immunol. 166:602-8; Massey et al., 2002,Vet. Immunol. Immunopathol.
87:357-72; Link and Xiao, 2001, Immunol. Rev. 184:117-28).
[0088] Any type of autoimmune disease can be treated in accordance
with this method of the invention. Non-limiting examples of
autoimmune disorders include: alopecia areata, ankylosing
spondylitis, antiphospholipid syndrome, autoimmune Addison's
disease, autoimmune diseases of the adrenal gland, autoimmune
hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and
orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous
pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic
fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical
pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's
disease, discoid lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
neuropathy, juvenile arthritis, lichen planus, lupus erthematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, pemphigus vulgaris, pernicious anemia, polyarteritis
nodosa, polychrondritis, polyglandular syndromes, polymyalgia
rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal arteritis/ giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Examples of inflammatory disorders include, but are not limited to,
asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentiated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections. As described herein, some autoimmune disorders are
associated with an inflammatory condition. Thus, there is overlap
between what is considered an autoimmune disorder and an
inflammatory disorder. Therefore, some autoimmune disorders may
also be characterized as inflammatory disorders.
[0089] In an aspect of this embodiment, the methods of the
invention can be used to treat inflammatory diseases associated
with diminished IL-10 levels, but not autoimmune diseases.
[0090] In another aspect of this embodiment, the methods of the
invention can be used to treat autoimmune diseases associated with
diminished IL-10 levels, but not inflammatory diseases.
[0091] In yet another aspect of this embodiment, the methods of the
invention can be used to treat autoimmune diseases associated with
diminished IL-10 levels, wherein the autoimmune disease to be
treated is not systemic lupus erythematosus.
[0092] Any type of inflammatory disease can be treated in
accordance with this method of the invention. Non-limiting examples
of inflammatory diseases include, but are not limited to, asthma,
encephilitis, inflammatory bowel disease, chronic obstructive
pulmonary disease (COPD), allergic disorders, septic shock,
pulmonary fibrosis, undifferentiated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections.
[0093] In still another aspect of this embodiment, the methods of
the invention encompass therapies that are aimed at treating
diseases associated with a helper T (Th) 1-mediated inflammatory
response but not diseases associated with a Th2-mediated
inflammatory response.
[0094] In an alternative aspect of this embodiment, the methods of
the invention encompass therapies that are aimed at treating
diseases associated with a Th2-mediated inflammatory response but
not diseases associated with a Th1-mediated inflammatory
response.
[0095] IL-10 is capable of inhibiting ischemia/reperfusion injury
(Deng et al., 2001, Kidney Int. 60:2118-28), graft-versus-disease,
and transplant-related mortality (Baker et al., 1999, Bone Marrow
Transplant 23:1123-9; Holler et al., 2000, Bone Marrow Transplant
25:237-41). As such, one embodiment of the present invention
involves treating transplant-associated diseases/conditions by
increasing the level of IL-10 in a patient in need thereof.
[0096] In another embodiment, the levels of endogenous IL-10 are
increased in a subject receiving an organ transplant by
administration of a B10 cell subset. In one aspect of this
embodiment, the B10 cell population is isolated from the patient
themselves, i.e., the subject is the donor. In another aspect of
this embodiment, the B10 cell population is isolated from a donor
that is not the subject. The donor of the B10 cells may be the same
as the organ donor. In another embodiment, the B10 cell population
is pooled from several donors.
5.5.2. Therapeutic Modalities
[0097] In one embodiment, a subject suffering from an autoimmune
disease or an inflammatory disease associated with diminished
levels of IL-10 is administered a population of B10 cells. In one
aspect of this embodiment, the B10 cell population is isolated from
the patient themselves, i.e., the subject is the donor. In another
aspect of this embodiment, the B10 cell population is isolated from
a donor that is not the subject. In an aspect of this embodiment,
the B10 cell population is pooled from several donors. According to
this embodiment, administration of a B10 cell population to a
subject in need thereof results in an increased level of IL-10
production in the patient sufficient to control, reduce, or
eliminate symptoms of the disease being treated.
[0098] In one aspect of this embodiment, the therapeutic agent is
an antibody, in particular, a CD40 antibody or a CD40 agonist. In
other aspects, the therapeutic agent is a small molecule, a
polypeptide, DNA, or RNA that interacts with the B cell CD40
receptor. In another embodiment the therapeutic agent is a TLR
agonist, such as LPS or CpG. The therapeutic agent may be a
different TLR agonist or a small molecule, polypeptide, RNA, dsRNA,
or DNA capable of interacting with a TLR receptor.
[0099] In another embodiment, a subject suffering from an
inflammatory or autoimmune disease associated with diminished
levels of IL-10 is treated by administration of a therapeutic agent
capable of causing an increase in IL-10 production by the B10 cells
in the patient. In a specific aspect of this embodiment, the
therapeutic agent targets the B cell CD40 receptor or a TLR. In
another aspect of this embodiment, the therapeutic agent is a CD40
antibody a TLR agonist, a small molecule, a polypeptide, DNA, or
RNA that is capable of binding, targeting, and or modulating CD40
so as to result in an increase in IL-10 production by the B10 cells
in the subject.
[0100] An antibody according to these embodiments can be any type
of antibody or fragment thereof, as described above. According to
this embodiment administration of a CD40 antibody or fragment
thereof to a subject with an autoimmune disease or an inflammatory
disease associated with diminished levels of IL-10 results in an
upregulation of IL-10 production by the endogenous B10 cell
population in the subject.
[0101] In still another embodiment, a patient receiving a
transplant is administered a therapeutic agent capable of
increasing endogenous IL-10 production by the B10 cell subset of
that patient to increase the patient's tolerance to the transplant.
In yet another embodiment, a patient receiving a transplant is
administered a B10 cell subset to increase the patient's tolerance
to the transplant.
[0102] The subject is preferably a mammal such as non-primate
(e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate
(e.g., monkey, such as a cynomolgous monkey and a human). In a
preferred embodiment, the subject is a human.
5.5.2.1. B10 Cells As Therapeutic Agents
[0103] In one embodiment, adoptive transfer of B10 cells can be
effective to suppress a wide variety of diseases, including, but
not limited to any of those described above, i.e., autoimmune
diseases, inflammatory diseases, or any other disease which may be
treated by introduction of a B10 cell population into a subject.
Adoptive transfer of B10 cells can further be employed to minimize
the immune/inflammatory response associated with transplant of
cells and/or tissues.
[0104] In an exemplary adoptive cell transfer protocol, a mixed
population of B10 cells is initially extracted from a target donor.
The B10 cells isolated from the donor may be isolated from any
location in the donor in which they reside including, but not
limited to, the blood, spleen, lymph nodes, and/or bone marrow of
the donor. Depending on the application, the B 10 cells may be
extracted from a healthy donor; a donor suffering from a disease
that is in a period of remission or during active disease; or from
the organs, blood, or tissues of a donor that has died. In the case
of the latter, the donor is an organ donor. In yet another
embodiment, the B10 cells can be obtained from the subject,
expanded or activated and returned to the subject.
[0105] Harvested lymphocytes may be separated by flow cytometry or
other cell separation techniques based on B10 cell markers such as
those described herein (e.g., CD 1d, CD5, CD24, and CD27), and then
transfused to a recipient. Alternatively, the cells may be stored
for future use. In one aspect of this embodiment, the donor and the
recipient are the same subject. In another aspect of this
embodiment, the donor is a subject other than the recipient. In a
further aspect of this embodiment, the "donor" comprises multiple
donors other than the recipient, wherein the B10 cells from said
multiple donors are pooled.
[0106] In another embodiment, the B10 cell population obtained from
a donor can be expanded, enriched, or made to produce elevated
levels of IL-10, as described in sections 5.1 and 5.2, supra, prior
to being administered to a recipient.
[0107] In the adoptive transfer techniques contemplated herein,
wherein the donor is a subject other than the recipient, the
recipient and the donor are histocompatible. Histocompatibility is
the property of having the same, or mostly the same, alleles of a
set of genes called the major histocompatibility complex (MHC).
These genes are expressed in most tissues as antigens, to which the
immune system makes antibodies. When transplanted cells and/or
tissues are rejected by a recipient, the bulk of the immune system
response is to the MHC proteins. MHC proteins are involved in the
presentation of foreign antigens to T-cells, and receptors on the
surface of the T-cell are uniquely suited to recognition of
proteins of this type. MHC are highly variable between individuals,
and therefore the T-cells from the host recognize the foreign MHC
with a very high frequency leading to powerful immune responses
that cause rejection of transplanted tissue. As such, the chance of
rejection of the B10 cell population by the recipient is
minimized.
[0108] The amount of B10 cells which will be effective in the
treatment and/or suppression of a disease or disorder which may be
treated by introduction of a B10 cell population into a subject can
be determined by standard clinical techniques. The dosage will
depend on the type of disease to be treated, the severity and
course of the disease, the purpose of introducing the B10 cell
population, previous therapy the recipient has undertaken, the
recipient's clinical history, and the discretion of the attending
physician. The B10 cell population can be administered in treatment
regimes consistent with the disease, e.g., a single or a few doses
over one to several days to ameliorate a disease state or periodic
doses over an extended time to inhibit disease progression and
prevent disease recurrence. The precise dose to be employed in the
formulation will also depend on the route of administration, and
the seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each patient's
circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. Exemplary, non-limiting doses that could be used in the
treatment of human subjects range from at least 3.8.times.10.sup.4,
at least 3.8.times.10.sup.5, at least 3.8.times.10.sup.6, at least
3.8.times.10.sup.7, at least 3.8.times.10.sup.8, at least
3.8.times.10.sup.9, or at least 3.8.times.10.sup.10 B10
cells/m.sup.2. In a certain embodiment, the dose used in the
treatment of human subjects ranges from about 3.8.times.10.sup.9 to
about 3.8.times.10.sup.10 B 10 cells/m.sup.2.
[0109] In another aspect of this embodiment, the B10 cells obtained
from the donor can be introduced into a recipient at a desired
location, so as to specifically target the therapeutic effects of
the B10 cell population, i.e., IL-10 production. Such techniques
can be accomplished using implantable immune modulation devices,
e.g., virtual lymph nodes, such as those described in U.S. patent
application publication No. 2003/0118630; W01999/044583; and U.S.
Pat. No. 6,645,500, which are incorporated by reference herein in
their entireties. According to this embodiment, a B10 cell
population can be isolated from a donor as described above, added
to an implantable immune modulation device, and said device then
can be inplanted into a recipient at a location where the
therapeutic effects of the B10 cell population, i.e., IL-10
production, are needed.
5.5.2.2. Antigen-Specific B10 Cells
[0110] In another embodiment, the B10 cell population can be made
responsive to a certain antigen involved in a specific disease. In
an aspect of this embodiment, the B10 cell population, when
sensitized with a certain antigen, will produce therapeutic amounts
of IL-10 upon subsequent encounters with the antigen. In an aspect
of this embodiment, such an antigen-specific B10 cell population
may be used in an adoptive transfer technique, wherein a subject is
or has previously been immunized with a certain antigen and the
antigen-sensitized B10 cells from said subject are isolated and
transplanted to the same or another subject. In still another
aspect of this embodiment, a B10 cell population from a subject can
be isolated and subsequently can be sensitized with a
disease-specific antigen ex vivo or in vitro. The sensitized B10
cell population can then be transplanted into the original or
another subject by any method known in the art. In still another
aspect of this embodiment, the antigen-specific B10 cell population
can be added to an implantable immune modulation device, as
described above. According to this embodiment, the implanted B10
cell population will produce strategically localized IL-10 when
encountering antigen in the host. In a further aspect, the B10 cell
population and a disease-specific antigen can both be placed in an
implantable immune modulation device, and said device then can be
transplanted into a recipient at a location where the therapeutic
effects of the B10 cell population, i.e., IL-10 production, are
needed, thus resulting in an amplified response to the disease in
vivo.
[0111] In another aspect, a certain disease-specific antigen can be
administered in conjunction with a CD40 agonist or a TLR agonist.
In a certain aspect of this embodiment, the therapeutic agent is an
antibody, in particular, a CD40 antibody or LPS or CpG
oligodeoxynucleotides. In other aspects, the therapeutic agent is a
small molecule, a polypeptide, DNA, or RNA that interacts with the
B cell CD40 receptor or TLRs.
[0112] Any antigen from any disease, disorder, or condition may be
used in accordance with the methods of the invention. Exemplary
antigens include but are not limited to bacterial, viral,
parasitic, allergens, autoantigens and tumor-associated antigens.
If a DNA based vaccine is used the antigen will typically be
encoded by a sequence of the administered DNA construct.
Alternatively, if the antigen is administered as a conjugate the
antigen will typically be a protein comprised in the administered
conjugate. Particularly, the antigen can include protein antigens,
peptides, whole inactivated organisms, and the like.
[0113] Specific examples of antigens that can be used in the
invention include antigens from hepatitis A, B, C or D, influenza
virus, Listeria, Clostridium botulinum, tuberculosis, tularemia,
Variola major (smallpox), viral hemorrhagic fevers, Yersinia pestis
(plague), HIV, herpes, pappilloma virus, and other antigens
associated with infectious agents. Other antigens include antigens
associated with autoimmune conditions, inflammatory conditions,
allergy, and asthma. Non-limiting examples of autoimmune diseases
and inflammatory diseases are provided, supra.
[0114] In an aspect of this embodiment, a B10 cell population
sensitized with a disease-specific antigen can be administered
alone or in conjunction with a CD40 agonist or TLR, in particular,
a CD40 antibody, for use as a therapeutic or prophylactic vaccine
for conferring immunity against such disease conditions.
[0115] In another embodiment, a B10 cell subset may be sensitized
with antigen from a prospective transplant donor, so as to increase
the levels of IL-10 production by the B10 cells in a transplant
recipient. In an aspect of this embodiment, the increased IL-10
production by the B10 cell subset in the transplant recipient
results in a decreased immune/inflammatory response to the
transplant in the transplant recipient. The role of B10 cells in
transplants is described in section 5.5.2.3, infra.
5.5.2.3. B10 Cells in Organ Transplant Patients
[0116] In another embodiment, the levels of endogenous IL-10 are
increased in a subject receiving an organ transplant by
administration of a B10 cell subset. In one aspect of this
embodiment, the B10 cell population is isolated from the patient
themselves, i.e., the subject is the donor. In another aspect of
this embodiment, the B10 cell population is isolated from a donor
that is not the subject. In an aspect of this embodiment, the B10
cell population is pooled from several donors. In another aspect of
this embodiment, the B10 cell subset is isolated from a subject
that has died, wherein said subject is an organ donor. In
embodiments wherein the B10 cells are from a donor that is not the
subject, the subject and the donor are histocompatible.
[0117] In one aspect of this embodiment, the B10 cell subset is
administered prior to the transplant. According to this aspect, the
therapeutic agent can be administered at least 1 hour, at least 12
hours, at least 1 day, at least 2 days, at least 3 days, at least 4
days, at least 5 days, at least 6 days, at least 1 week, at least 2
weeks, at least 3 weeks, at least 4 weeks, or at least 1 month
prior to the transplant. Administration of the therapeutic agent
can be by any method known to those of skill in the art.
[0118] In another aspect of this embodiment, the B10 cell subset is
administered at the same time as the transplant.
[0119] In still another aspect of this embodiment, the B10 cell
subset is administered following the transplant.
[0120] In a certain aspect, the B10 cell subset is administered
before, during, and after the transplant. According to this aspect,
when the B10 cell subset is administered after the transplant, it
may be administered for at least 12 hours, at least 1 day, at least
2 days, at least 3 days, at least 4 days, at least 5 days, at least
6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at
least 4 weeks, least 1 month, or at least 1 year following the
transplant or for the duration of the patient's life.
[0121] As described in section 5.5.2.2, supra, in one embodiment, a
B10 cell subset administered to a patient that is receiving a
transplant can be sensitized with antigens specific to the
transplanted material. According to this embodiment, the transplant
recipient will have a decreased immune/inflammatory response to the
transplanted material and, as such, the likelihood of rejection of
the transplanted material is minimized.
[0122] In another embodiment, the levels of endogenous IL-10 are
increased in a subject receiving an organ transplant by
administration of a therapeutic agent capable of causing an
increase in IL-10 production by the B10 cells in the patient. The
therapeutic agent can be administered in vivo or ex vivo; i.e., the
B10 cell population can be isolated/enriched from the patient,
contacted with the therapeutic agent ex vivo, and the "activated"
population returned to the patient. In a specific aspect of this
embodiment, the therapeutic agent targets the B cell CD40 receptor
or a TLR. In another aspect of this embodiment, the therapeutic
agent is a CD40 antibody, a small molecule, a polypeptide, DNA, or
RNA that is capable of binding, targeting, and or modulating CD40
so as to result in increase in IL-10 production by the B10 cells in
the subject.
[0123] In one aspect of this embodiment, the therapeutic agent
capable of causing an increase in IL-10 production by the B10 cells
in the patient is administered prior to the transplant. According
to this aspect, the therapeutic agent can be administered at least
1 hour, at least 12 hours, at least 1 day, at least 2 days, at
least 3 days, at least 4 days, at least 5 days, at least 6 days, at
least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks,
or at least 1 month prior to the transplant. Administration of the
therapeutic agent can be by any method known to those of skill in
the art.
[0124] In another aspect of this embodiment, the therapeutic agent
capable of causing an increase in IL-10 production by the B10 cells
in the patient is administered at the same time as the
transplant.
[0125] In still another aspect of this embodiment, the therapeutic
agent capable of causing an increase in IL-10 production by the B10
cells in the patient is administered following the transplant.
[0126] In a certain aspect, the therapeutic agent capable of
causing an increase in IL-10 production by the B10 cells in the
patient is administered before, during, and after the transplant.
According to this aspect, when the therapeutic agent capable of
causing an increase in IL-10 production by the B10 cells in the
patient is administered after the transplant, it may be
administered for at least 12 hours, at least 1 day, at least 2
days, at least 3 days, at least 4 days, at least 5 days, at least 6
days, at least 1 week, at least 2 weeks, at least 3 weeks, at least
4 weeks, least 1 month, or at least 1 year following the transplant
or for the duration of the patient's life.
[0127] According to these embodiments, administration of a
therapeutic agent capable of causing an increase in IL-10
production by the B10 cells in the patient or administration of a
B10 cell subset results in a decreased immune response in the
patient receiving the transplant, wherein the decreased immune
response results in an increased likelihood that the transplant
will be accepted, an increased tolerance to the transplant, an
increased duration of time in which the transplant is accepted,
and/or an increased lifespan in the transplant recipient.
[0128] Any type of transplant can be performed according to these
methods.
5.6 Therapeutic Targeting of the B Cell Subset to Treat Diseases
and Disorders Associated with Enhanced IL-10 Levels
[0129] In another embodiment, the invention provides methods for
treating and/or managing a disease or disorder associated with a
decreased/depressed/impaired immune/inflammatory response,
particularly cancer or an infectious disease, by administrating to
a subject in need thereof a therapeutically or prophylactically
effective amount of a therapeutic agent capable of ablating the
population of B10 cells and/or the amounts of IL-10 being produced
by the B10 cell subset. In another embodiment, the invention
provides methods for the treatment of cancer by administrating to a
subject in need thereof a therapeutically or prophylactically
effective amount of a therapeutic agent capable of ablating the
population of B10 cells and/or the amounts of IL-10 being produced
by the B10 cell subset.
[0130] In an aspect of this embodiment, the therapeutic agent is an
antibody that mediates CDC or ADCC and kills target cells, or an
immunoconjugate that alters the function of or kills target cells
is used. In particular, a CD22 mAb that kills or inhibits the
proliferation of the B10 cell subset can be used. Alternatively, a
CD1d, a CD5, a CD24, or a CD27 antibody or a bispecific a CD1d/CD5
or a CD24/CD27 antibody can be used.
[0131] In another aspect of this embodiment, the therapeutic agent
is an antibody that does not utilize CDC or ADCC to kill the target
cells. In another aspect, the antibody does not kill the target
cells by apoptosis.
[0132] In another aspect of this embodiment, the therapeutic agent
is an antibody that does not utilize CDC, ADCC, or apoptosis as the
primary mechanism for killing target cells, i.e., the majority of
target cells are killed by a mechanism that is CDC-, ADCC-, and
apoptosis-independent.
[0133] In another aspect of this embodiment, the therapeutic agent
is a small molecule, a polypeptide, DNA, or RNA that interacts with
the B cell CD22 receptor or with CD1d, CD5, CD20, CD24, or
CD27.
[0134] The subject is preferably a mammal such as non-primate
(e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate
(e.g., monkey, such as a cynomolgous monkey and a human). In a
preferred embodiment, the subject is a human.
5.6.1 Diseases and Disorders Associated with Increased IL-10
Production
[0135] IL-10 has been shown to promote tumor growth and
overexpression of IL-10 has been demonstrated in certain cancers
(Matsuda et al., 1994, J. Exp. Med. 180:2371-6; S alazar-Onfray et
al., 1997, J. Immunol. 159:3195-3202; Hagenbaugh et al. 1997, J.
Exp. Med. 185:2101-110; Kruger-Kraskagakes et al. 1994, Br. J.
Cancer 70:1182-5, Dummer et al., 1996, Int. J. Cancer 66:607-10;
Kim et al., 1995, J. Immunol. 155:2240-47; Blay et al., 1993, Blood
82:2169-74; Asadullah et al., 2000, Exp. Dermatol. 9:71-6). As
such, one embodiment of the present invention involves treating
cancer by decreasing the level of IL-10 in a patient in need
thereof by ablation of the B10 cell subset and/or reducing the
amount of IL-10 produced by the B10 cell subset.
[0136] Any type of cancer can be treated in accordance with this
method of the invention. Non-limiting examples of cancers include:
leukemias, such as but not limited to, acute leukemia, acute
lymphocytic leukemia, acute myelocytic leukemias, such as,
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia leukemias and myelodysplastic syndrome; chronic
leukemias, such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including but not limited to ductal
carcinoma, adenocarcinoma, lobular (small cell) carcinoma,
intraductal carcinoma, medullary breast cancer, mucinous breast
cancer, tubular breast cancer, papillary breast cancer, Paget's
disease, and inflammatory breast cancer; adrenal cancer such as but
not limited to pheochromocytom and adrenocortical carcinoma;
thyroid cancer such as but not limited to papillary or follicular
thyroid cancer, medullary thyroid cancer and anaplastic thyroid
cancer; pancreatic cancer such as but not limited to, insulinoma,
gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and
carcinoid or islet cell tumor; pituitary cancers such as but
limited to Cushing's disease, prolactin-secreting tumor,
acromegaly, and diabetes insipius; eye cancers such as but not
limited to ocular melanoma such as iris melanoma, choroidal
melanoma, and cilliary body melanoma, and retinoblastoma; vaginal
cancers such as squamous cell carcinoma, adenocarcinoma, and
melanoma; vulvar cancer such as squamous cell carcinoma, melanoma,
adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;
cervical cancers such as but not limited to, squamous cell
carcinoma, and adenocarcinoma; uterine cancers such as but not
limited to endometrial carcinoma and uterine sarcoma; ovarian
cancers such as but not limited to, ovarian epithelial carcinoma,
borderline tumor, germ cell tumor, and stromal tumor; esophageal
cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungating (polypoid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma; gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to papillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, prostatic intraepithelial neoplasia, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
such as but not limited to squamous cell carcinoma; basal cancers;
salivary gland cancers such as but not limited to adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx
cancers such as but not limited to squamous cell cancer, and
verrucous; skin cancers such as but not limited to, basal cell
carcinoma, squamous cell carcinoma and melanoma, superficial
spreading melanoma, nodular melanoma, lentigo malignant melanoma,
acral lentiginous melanoma; kidney cancers such as but not limited
to renal cell carcinoma, adenocarcinoma, hypernephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/ or
uterer); Wilms' tumor; bladder cancers such as but not limited to
transitional cell carcinoma, squamous cell cancer, adenocarcinoma,
carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma,
mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma and papillary
adenocarcinomas (for a review of such disorders, see Fishman et al,
1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and
Murphy, 1997, Informed Decisions: The Complete Book of Cancer
Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books
U.S.A., Inc., United States of America, incorporated by reference
herein in its entirety).
[0137] Increased levels of IL-10 have been demonstrated in certain
autoimmune and inflammatory diseases including, but not limited to
systemic lupus erythematosus (Park et al., 1998, Clin. Exp.
Rheumatol. 16:283-88; Llorente et al., 1995, J. Exp. Med.
181:839-44), systemic sclerosis (Hasegawa et al., 1997, J.
Rheumatol. 24:328-32), Bullous Pemphigoid (Schmidt et al., 1996,
Arch. Dermatol. Res. 228:353-7; Giacalone et al., 1998, Exp.
Dermatol. 7:157-61), and atopic dermatitis (Ohmen et al., 1995, J.
Immunol. 154:1956-63; Asadullah et al., 1996, J. Investig.
Dermatol. 197:833-7). As such, one embodiment of the present
invention involves treating an autoimmune or inflammatory by
decreasing the level of IL-10 in a patient in need thereof by
ablation of the B10 cell subset and/or reducing the amount of IL-10
produced by the B10 cell subset.
[0138] Any type of autoimmune disease that is accompanied by
increased IL-10 production can be treated in accordance with this
method of the invention. A non-limiting list of autoimmune
disorders is provided above.
[0139] Any type of inflammatory disease that is accompanied by
increased IL-10 production can be treated in accordance with this
method of the invention. A non-limiting list of inflammatory
diseases is provided above.
[0140] In an aspect of this embodiment, the methods of the
invention can be used to treat inflammatory diseases associated
with diminished IL-10 levels, but not autoimmune diseases.
[0141] In another aspect of this embodiment, the methods of the
invention can be used to treat autoimmune diseases associated with
diminished IL-10 levels, but not inflammatory diseases.
[0142] In yet another aspect of this embodiment, the methods of the
invention can be used to treat autoimmune diseases associated with
diminished IL-10 levels, wherein the autoimmune disease to be
treated is not systemic lupus erythematosus.
[0143] In still another aspect of this embodiment, the methods of
the invention encompass therapies that are aimed at treating
diseases associated with a helper T (Th) 1-mediated inflammatory
response but not diseases associated with a Th2-mediated
inflammatory response.
[0144] In an alternative aspect of this embodiment, the methods of
the invention encompass therapies that are aimed at treating
diseases associated with a Th2-mediated inflammatory response but
not diseases associated with a Th1-mediated inflammatory
response.
[0145] IL-10 has also been shown to be associated with the
pathogenesis or ineffective immune response to many infectious
diseases as well. The infectious diseases may be mediated by
viruses, bacteria, yeast, parasites or prions. A non-limiting list
of infectious diseases that may benefit from decreasing or
inhibiting IL-10 production by the B10 cell subset includes, but is
not limited to Diphtheria, Tetanus, Pertussis, Haemophilus
influenzae type b, Anthrax, Measles, Rubella, Mumps, Botulism,
Chickenpox, Cholera, Hepatitis B, Influenza, Hepatitis A, Hepatitis
C , Rabies, Polio, Japanese Encephalitis Virus, Meningitis,
Typhoid, Pneumonia, Rocky Mountain Spotted Fever, Lyme Disease,
Smallpox, Tetanus, Mycobacterium, Malaria, HIV/AIDS, RSV,
Herpesviruses, and Yellow Fever.
5.6.2 Therapies
[0146] In one embodiment, a subject suffering from cancer who has
elevated levels of IL-10 is treated by administration of a
therapeutic agent capable of ablating the population of B10 cells
in the patient and/or reducing the amount of IL-10 produced by the
B10 cell population. In a specific aspect of this embodiment, the
therapeutic agent targets the B cell CD22 receptor. In another
aspect of this embodiment, the therapeutic agent is a CD22
antibody, a small molecule, a polypeptide, DNA, or RNA that is
capable of binding, targeting, and or modulating CD22 so as to
result in ablation of the B10 cell subset. In a specific aspect of
this embodiment, the therapeutic agent targets the B cell CD20
receptor. In another aspect of this embodiment, the therapeutic
agent is a CD20 antibody, a small molecule, a polypeptide, DNA, or
RNA that is capable of binding, targeting, and or modulating CD20
so as to result in ablation of the B10 cell subset.
[0147] In another embodiment, a subject suffering from an immune
deficiency disease associated with elevated levels of IL-10 is
treated by administration of a therapeutic agent capable of
ablating the population of B10 cells in the patient and thereby
reducing the amount of IL-10 produced by the B10 cell population.
In a specific aspect of this embodiment, the therapeutic agent
targets the B cell CD22 receptor. In another aspect of this
embodiment, the therapeutic agent is a CD22 antibody, a small
molecule, a polypeptide, DNA, or RNA that is capable of binding,
targeting, and or modulating CD22 so as to result in ablation of
the B10 cell subset.
[0148] In an alternative embodiment, a subject suffering from
cancer or an immune deficiency disease associated with elevated
levels of IL-10 is treated by administration of a CD1d, CD5, CD24,
CD27 antibody or a bispecific CD1d/CD5 or CD24/CD27 antibody
capable of ablating the population of B10 cells in the patient and
thereby reducing the amount of IL-10 produced.
[0149] In order to kill or ablate the B10 cell subset, targeting
antibodies of an isotype that mediate ADCC (antibody-dependent and
mediated toxicity) or CDC (complement-dependent cytotoxicity) can
be used. Of the various human immunoglobulin classes, IgG1, IgG2,
IgG3, IgG4 and IgM are known to activate complement. Human IgG1 and
IgG3 are known to mediate ADCC. Antibodies to CD20 may be used to
deplete B10 cells selectively. The antibodies demonstrated to
target the IL-10 producing B10 cell subset were antibodies that
were not capable of inducing ADCC, CDC or apoptosis. Instead the
effective antibodies were those that induced homotypic adhesion.
Thus, antibodies to B cell surface markers with IgG3 or IgG2b Fc
regions which do not efficiently engage most Fey receptors, but
induce homotypic adhesion are useful in the methods described
herein. Optionally the Fc portion of a known antibody may be
modified so that the mechanism of depletion of B10 cells is
independent of the antibody's Fc region.
[0150] Antibodies targeting the B10 cell subset can be further
conjugated to a cytotoxic agent, using methods known in the art
(see, e.g., DiJoseph et al., 2004, Clin. Cancer Res. 10:8620-9).
This may be preferred when using antibodies or antibody fragments
that do not mediate ADCC or CDC. Non-limiting examples of cytotoxic
agents include antimetabolites (e.g., cytosine arabinoside,
aminopterin, methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, and 5-fluorouracil decarbazine); alkylating agents
(e.g., mechlorethamine, thiotepa chlorambucil, melphalan,
carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiammine-platinum (II) (CDDP), and cisplatin); vinca
alkaloid; anthracyclines (e.g., daunorubicin (formerly daunomycin)
and doxorubicin); antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC));
calicheamicin; CC-1065 and derivatives thereof; auristatin
molecules (e.g., auristatin PHE, bryostatin-1, and dolastatin-10;
see Woyke et al., Antimicrob. Agents Chemother 46:3802-8 (2002),
Woyke et al., Antimicrob. Agents Chemother. 45:3580-4 (2001),
Mohammad et al., Anticancer Drugs 12:735-40 (2001), Wall et al.,
Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad, et al.,
Int. J. Oncol. 15:367-72 (1999), all of which are incorporated by
reference herein in their entireties); DNA-repair enzyme inhibitors
(e.g., etoposide or topotecan); kinase inhibitors (e.g., compound
ST1571, imatinib mesylate (Kantarjian et al., Clin. Cancer Res.
8(7):2167-76 (2002)); demecolcine; and other cytotoxic agents
(e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracenedione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologues
thereof and those compounds disclosed in U.S. Pat. Nos. 6,245,759,
6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410,
6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376,
5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868,
5,648,239, 5,587,459, all of which are incorporated by reference
herein in their entirety); farnesyl transferase inhibitors (e.g.,
R115777, BMS-214662, and those disclosed by, for example, U.S. Pat.
Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959,
6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615,
6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487,
6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338,
6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786,
6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465,
6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853,
6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574,
and 6,040,305, all of which are herein incorporated by reference in
their entirety); topoisomerase inhibitors (e.g., camptothecin,
irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG211
(GI147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine, XR5000,
saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528,
ED-110, NB-506, ED-110, NB-506, and rebeccamycin); bulgarein; DNA
minor groove binders such as Hoechst dye 33342 and Hoechst dye
33258; nitidine; fagaronine; epiberberine; coralyne;
beta-lapachone; BC-4-1; antisense oligonucleotides (e.g., those
disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,
5,734,033, and 5,618,709, all of which are herein incorporated by
reference in their entirety); adenosine deaminase inhibitors (e.g.,
fludarabine phosphate and 2-chlorodeoxyadenosine); and
pharmaceutically acceptable salts, solvates, clathrates, and
prodrugs thereof.
[0151] In another embodiment, the targeting antibody, such as a
CD20, CD22, bispecific CD1d/CD5, or bispecific CD24/CD27 antibody,
can be conjugated to a radioactive metal ion, such as the
alpha-emitters .sup.211astatine, .sup.212bismuth, .sup.213bismuth;
the beta-emitters .sup.131iodine, .sup.90yttrium, .sup.177lutetium,
.sup.153samarium, and .sup.109palladium; or macrocyclic chelators
useful for conjugating radiometal ions, including but not limited
to, .sup.131indium, .sup.131L, .sup.131yttrium, .sup.131holmium,
.sup.131samarium, to polypeptides or any of those listed supra. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA), which can be attached to the antibody via a linker
molecule. Such linker molecules are commonly known in the art and
described in Denardo, et al., 1998, Clin Cancer Res 4(10):2483-90;
Peterson, et al., 1999, Bioconjug Chem 10(4):553-7; and Zimmerman,
et al., 1999, Nucl Med Biol 26(8):943-50, each incorporated by
reference herein in their entireties.
[0152] In still another embodiment, the targeting antibody, such as
a CD20, CD22, bispecific CD1d/CDS, or bispecific CD24/CD27
antibody, can be conjugated to a proteinaceous agent that modifies
a given biological response and leads to cytotoxicity. In one
embodiment, the antibody is conjugated to a plant-, fungus-, or
bacteria-derived toxin. Non-limiting examples of such toxins
include A chain toxins, ribosome inactivating proteins, ricin A,
deglycosylated ricin A chain, abrin, alpha sarcin, aspergillin,
restrictocin, ribonucleases, diphtheria toxin, bacterial endotoxin,
saporin toxin, Granzyme B or the lipid A moiety of bacterial
endotoxin, cholera toxin, or Pseudomonas exotoxin and derivatives
and variants thereof.
[0153] In another embodiment, an antagonist capable of engaging a
B10 cell specific surface marker to ablate the B10 cell population
is a synthetic ligand specific for the marker, such as that
described in Collins et al., 2006, J. Immunol. 5:2994-3003,
incorporated herein by reference in its entirety. In one aspect of
this embodiment, the synthetic ligand may be further conjugated to
a toxin, such as the saporin toxin.
[0154] In an alternative embodiment, a subject suffering from
cancer, an infectious disease or an immune deficiency disease
associated with elevated levels of IL-10 is treated by
administration of a compound capable of engaging a marker or
markers on the B10 cell subset that can inhibit the production of
IL-10 by the B10 cells. Non-limiting examples of such compounds
include antibodies and fragments thereof, small molecules,
synthetic drugs, peptides (including cyclic peptides),
polypeptides, proteins, nucleic acids, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules. In one embodiment, the compound engages CD22. In
an aspect of this embodiment, the compound is a CD22 antibody. In
another aspect of this embodiment, the compound engages CDS. In an
aspect of this embodiment, the compound is a CD5 antibody. In
another aspect of this embodiment, the compound engages CD1d. In an
aspect of this embodiment, the compound is a CD1d antibody. In
still another aspect of this embodiment, the compound is a
bispecific CD1d/CD5 antibody. In another aspect of this embodiment,
the compound engages CD24. In an aspect of this embodiment, the
compound is a CD24 antibody. In another aspect of this embodiment,
the compound engages CD27. In an aspect of this embodiment, the
compound is a CD27 antibody. In still another aspect of this
embodiment, the compound is a bispecific CD24/CD27 antibody. In yet
another aspect of this embodiment, the compound engages CD19. In an
aspect of this embodiment, the compound is a CD19 antibody.
[0155] An antibody according to these embodiments can be any type
of antibody or fragment thereof, as described above. According to
this embodiment, administration of an antibody that targets the B10
cell population or fragment thereof, including a CD22 antibody or
fragment thereof to a patient with cancer, an infectious disease,
an autoimmune disease, or an inflammatory disease associated with
increased levels of IL-10 results in a downregulation of IL-10
production by the B10 cell population in the patient.
[0156] In another embodiment, a patient suffering from cancer, an
infectious disease or an immune deficiency disease associated with
elevated levels of IL-10 is treated by administration of an
antibody that binds to a B cell marker and selectively depletes the
B10 cell population in the patient. According to this embodiment,
the B cell marker can be any antigen that is presently known or
subsequently determined to be expressed by B10 cells including,
e.g. CD 1d, CD5, CD19, CD20, CD21, CD23, CD24, CD25, CD27, CD3,
CD48, and CD148. In one aspect of this embodiment, the antibody
that binds to a B cell marker and selectively depletes the B10 cell
population in the patient does not cause depletion of the B10 cell
population by an antibody-dependent cell-mediated cytotoxicity
(ADCC) mechanism, by complement-dependent cytotoxicity (CDC), or by
apoptosis. In another aspect, depletion of the B10 cell population
by the antibody is independent of the antibody's Fc region. In
another aspect of this embodiment, the antibody that binds to a B
cell marker and selectively depletes the B10 cell population
depletes splenic Marginal Zone B cells but does not substantially
deplete splenic Follicular B cells. In a specific aspect, the
antibody that binds to a B cell marker and selectively depletes the
B10 cell population is an IgG2b or an IgG3 isotype.
[0157] In another embodiment, the antibody for use in treating a
patient suffering from cancer or an immune deficiency disease
associated with elevated levels of IL-10 that binds to a B cell
marker and selectively depletes the B10 cell population comprises a
human IgG isotype or Fc region that does not activate complement or
lead to ADCC or kill cells by inducing apoptosis. Any human isotype
or Fc region that does not activate complement or lead to ADCC or
kill cells by inducing apoptosis can be used in accordance with
this embodiment. In one aspect, the isotype is IgG4.
[0158] In a specific embodiment, a patient suffering from cancer is
treated by administration of a CD20 antibody that selectively
depletes the B10 cell population in the patient, wherein the
depletion of the B10 cell population by the CD20 antibody is not
caused by ADCC, CDC, or apoptosis. In another aspect, depletion of
the B10 cell population by the antibody is independent of the
antibody's Fc region. In an aspect of this embodiment, the CD20
antibody depletes splenic Marginal Zone B cells but does not
substantially deplete splenic Follicular B cells. In a specific
aspect, the CD20 is an IgG2b or an IgG3 isotype. In another aspect,
the CD20 antibody comprises a human IgG isotype or Fc region that
does not activate complement or lead to ADCC or induce apoptosis.
Any human isotype or Fc region that does not activate complement or
lead to ADCC or kill cells by inducing apoptosis can be used in
accordance with this embodiment. In one aspect, the isotype is
IgG4.
[0159] In certain embodiments, the B10 cell population is depleted
by at least 1%, at least 1% to 5%, at least 1% to 10%, at least 1%
to 25%, at least 1% to 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 99%, or by 100% as measured by
assays known to one of skill in the art including those described
in the Examples infra, e.g., immunofluorescence staining with flow
cytometry analysis, ELISA assay for IL-10 secretion, or ELISpot
analysis for determining numbers of IL-10-secreting cells.
[0160] In certain embodiments, the antibodies described herein are
administered alone. In other embodiments, the antibodies described
herein are administered to patients as a front-line therapy. In
other embodiments, the antibodies described herein are administered
to patients as a secondary therapy. In certain embodiments, the
patient has not previously been treated for the cancer, infectious
disease or the immune deficiency disease. In other embodiments, the
patient is undergoing or has undergone treatment for the cancer or
the immune deficiency disease. In yet other embodiments, the
patient has failed treatment for the cancer or the immune
deficiency disease.
[0161] In certain embodiments, the antibodies described herein are
administered in combination with other therapeutic agents. Any
therapy that is useful, has been used, or is currently being used
for the prevention, treatment, and/or management of cancer,
infectious disease or an immune deficiency disease can be used in
compositions and methods of the invention. Such therapies include,
but are not limited to, peptides, polypeptides, antibodies,
conjugates, nucleic acid molecules, small molecules, mimetic
agents, synthetic drugs, inorganic molecules, and organic
molecules.
[0162] Non-limiting examples of cancer therapies include
chemotherapy, radiation therapy, hormonal therapy, surgery, small
molecule therapy, anti-angiogenic therapy, differentiation therapy,
epigenetic therapy, radioimmunotherapy, targeted therapy, and/or
biological therapy including immunotherapy including, but not
limited to acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; aminoglutethimide; amsacrine; anastrozole;
anthracyclin; anthramycin; asparaginase; asperlin; azacitidine
(Vidaza); azetepa; azotomycin; batimastat; benzodepa; bicalutamide;
bisantrene hydrochloride; bisnafide dimesylate; bisphosphonates
(e.g., pamidronate (Aredria), sodium clondronate (Bonefos),
zoledronic acid (Zometa), alendronate (Fosamax), etidronate,
ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;
bleomycin sulfate; brequinar sodium; bropirimine; busulfan;
cactinomycin; calusterone; caracemide; carbetimer; carboplatin;
carmustine; carubicin hydrochloride; carzelesin; cedefingol;
chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol
mesylate; cyclophosphamide; cytarabine (Ara-C); dacarbazine;
dactinomycin; daunorubicin hydrochloride; decitabine (Dacogen);
demethylation agents, dexormaplatin; dezaguanine; dezaguanine
mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin
hydrochloride; droloxifene; droloxifene citrate; dromostanolone
propionate; duazomycin; edatrexate; eflornithine hydrochloride;
EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;
epipropidine; epirubicin hydrochloride; erbulozole; esorubicin
hydrochloride; estramustine; estramustine phosphate sodium;
etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole
hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine
phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin
sodium; gemcitabine; histone deacetylase inhibitors (HDACs)
gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;
ifosfamide; ilmofosine; imatinib mesylate (Gleevec, Glivec);
interleukin II (including recombinant interleukin II, or rIL2),
interferon alpha-2a; interferon alpha-2b; interferon alpha-n1;
interferon alpha-n3; interferon beta-I a; interferon gamma-I b;
iproplatin; irinotecan hydrochloride; lanreotide acetate;
lenalidomide (Revlimid); letrozole; leuprolide acetate; liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; CD2 antibodies (e.g., siplizumab (MedImmune Inc.;
International Publication No. WO 02/098370, which is incorporated
herein by reference in its entirety)); megestrol acetate;
melengestrol acetate; melphalan; menogaril; mercaptopurine;
methotrexate; methotrexate sodium; metoprine; meturedepa;
mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;
mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;
mycophenolic acid; nocodazole; nogalamycin; ormaplatin;
oxaliplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; rogletimide; safingol; safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin;
streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan
sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride; 20-epi-1,25 dihydroxyvitamin
D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene;
adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;
altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;
amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;
angiogenesis inhibitors; antagonist D; antagonist G; antarelix;
anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic
carcinoma; antiestrogen; antineoplaston; antisense
oligonucleotides; aphidicolin glycinate; apoptosis gene modulators;
apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine
deaminase; asulacrine; atamestane; atrimustine; axinastatin 1;
axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine;
baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate;
[0163] bropirimine; budotitane; buthionine sulfoximine;
calcipotriol; calphostin C; camptothecin derivatives; canarypox
IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,
atorvastatin, cerivastatin, fluvastatin, lescol, lupitor,
lovastatin, rosuvastatin, and simvastatin); hepsulfam; heregulin;
hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin;
idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;
imiquimod; immunostimulant peptides; insulin-like growth factor-1
receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP
(Biogen, Cambridge, Mass.; International Publication No. WO 93/0686
and U.S. Pat. No. 6,162,432); liarozole; linear polyamine analogue;
lipophilic disaccharide peptide; lipophilic platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium
texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors; menogaril; merbarone; meterelin;
methioninase; metoclopramide; MIF inhibitor; mifepristone;
miltefosine; mirimostim; mismatched double stranded RNA;
mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin
fibroblast growth factor-saporin; mitoxantrone; mofarotene;
molgramostim; monoclonal antibody, human chorionic gonadotrophin;
monophosphoryl lipid A+myobacterium cell wall sk; mopidamol;
multiple drug resistance gene inhibitor; multiple tumor suppressor
1-based therapy; mustard anticancer agent; mycaperoxide B;
mycobacterial cell wall extract; myriaporone; N-acetyldinaline;
N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;
nilutamide; nisamycin; nitric oxide modulators; nitroxide
antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin;
oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; Ril retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;
tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase
inhibitors; temoporfin; temozolomide; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; totipotent stem cell factor; translation inhibitors;
tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived
growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin B; vector system, erythrocyte gene therapy;
thalidomide; velaresol; veramine; verdins; verteporfin;
vinorelbine; vinxaltine; VITAXIN.TM. (see U.S. Patent Pub. No. US
2002/0168360 A1, dated Nov. 14, 2002, entitled "Methods of
Preventing or Treating Inflammatory or Autoimmune Disorders by
Administering Integrin av.beta.3 Antagonists in Combination With
Other Prophylactic or Therapeutic Agents"); vorozole; zanoterone;
zeniplatin; zilascorb; and zinostatin stimalamer.
5.7 Vaccine Formulations
[0164] In another embodiment, a therapeutic agent capable of
ablating the B10 cell subset can be administered in conjunction
with a vaccine or other antigen in order to increase the immune
response directed against an infectious disease or
cancer-associated target, e.g., a tumor or antigen. In the methods,
the antigen is administered to the subject and an agent that kills
or inhibits the function, localization or expansion of the IL-10
producing B10 cells or an agent that inhibits production of IL-10
by B10 cells in subject is also administered to the subject. The
administration of the antigen and the agent increases or enhances
the immune response directed to the antigen after administration as
compared to the immune response directed to the antigen if
administered in the absence of the agent. In particular, the method
increases Ig class switching and in particular the levels of IgG
are enhanced. According to this embodiment, ablation of the B10
cell subset serves to decrease endogenous levels of IL-10 in the
subject being vaccinated or administered the antigen and thereby
boosts the immune response, in particular the IgG response,
directed to the infectious agent, infected cells, antigen or tumor
antigen. Any antigen, including antigens associated with any
infectious disease or malignant cell can be vaccinated against
according to this method of the invention.
[0165] A non-limiting list of FDA licensed vaccines (and associated
disease) that could be administered in accordance with the methods
of the invention includes: Acel-Immune (Diphtheria, tetanus,
pertussis), ActHIB (Haemophilus influenzae type b), Anthrax
vaccine, Attenuvax (Measles), Biavax II (Rubella, Mumps), Botox
(Botulism), Chickenpox vaccine, Cholera vaccine, Comvax
(Haemophilus influenzae type b, Hepatitis B), DTP (Diphtheria,
Tetanus, Pertussis), Diphtheria vaccine, Engerix-B (Hepatitis B),
Influenza vaccine, Fluvirin (Influenza), German Measles vaccine,
Havrix (Hepatitis A), HBIG (Hepatitis B), Hepatitis A vaccine,
Hepatitis B vaccine, Heptavax (Hepatitis B), HibTITER (Haemophilus
influenzae type b, Diphtheria) , Imovax Rabies vaccine, Infanrix
(Diphtheria, Tetanus, Pertussis), Ipol (Polio), JE-Vax (Japanese
Encephalitis Virus), Pedvax-HIB (Haemophilus influenzae type b,
Meningitis), Meningococcal polysaccharide vaccine (Meningitis),
Menomune-A/C/Y/W-135 (Meningitis), Meruvax-II (Rubella), M-M-R II
(Measles, Mumps, Rubella), M-R-VAX II (Measles, Mumps, Rubella),
Mumpsvax (Mumps), OmniHIB (Haemophilus influenzae type b,
Diphtheria), Orimune (Polio), Paratyphoid vaccine (Typhoid),
Pertussis vaccine, Plague vaccine, Pneumococcal vaccine
(Pneumonia), Pneumovax 23 (Pneumonia), Pne-Imune 23 (Pneumonia),
Polio vaccine, Recombivax HB (Hepatitis B), RhoGAM (Rhesus), Rocky
Mountain Spotted Fever vaccine, Rubella vaccine, Rubeola vaccine,
Smallpox vaccine, Tetanus vaccine, Tetramune (Diphtheria, Tetanus,
Pertussis, Haemophilus influenzae type b), Tice BCG USP
(Mycobacterium Bovis Infection), Tri-Immunol (Diphtheria, Tetanus,
Pertussis), Tripedia(Diphtheria, Tetanus, Pertussis), Typhim Vi
(Typhoid), Typhoid vaccine, Typhus vaccine, Vaqta (Hepatitis A),
Varicella vaccine, Varivax (Varicella), Vivotif Berna (Typhoid),
and Yellow Fever vaccine. Some infectious diseases and tumors have
been resistant to immunization protocols or have demonstrated
limited immunity or required multiple boosts to elicit an effective
immune response. The methods described herein may be combined with
known vaccines or vaccines that failed during development stages to
elicit a robust immune response to the antigen to reduce the number
of boosters required or to increase the immune response directed to
the antigen and increase the efficacy of the vaccine. As an
example, the Mycobacterium bovis BCG vaccine is known to only
provide partially protective immunity and thus is not used to
vaccinate against tuberculosis in much of the world. The BCG
vaccine could be administered with a an agent that inhibits the
function, localization or expansion of B10 cells or an agent that
inhibits IL-10 production to help elicit a more robust immune
response to the vaccine.
[0166] In one aspect of this embodiment, the therapeutic agent
capable of ablating the B10 cell subset and the vaccine or antigen
are administered concurrently. In another aspect of this
embodiment, the therapeutic agent capable of ablating the B10 cell
subset is administered prior to administration of the vaccine or
antigen. Alternatively, the therapeutic agent capable of ablating
the B10 cell subset can be administered following the
administration of the vaccine or antigen.
[0167] In another aspect of this embodiment, the therapeutic agent
capable of ablating the B10 cell subset and the vaccine are
administered in conjunction with an adjuvant. A non-limiting list
of adjuvants administered in accordance with the methods of the
invention includes: alum (e.g., aluminum hydroxide, aluminum
phosphate); Montanide ISA 720; MF-59; PROVAX; immunostimulatory
nucleic acids, such as CpG oligodeoxynucleotides; saponins purified
from the bark of the Q. saponaria tree, such as QS21;
poly[di(carboxylatophen- oxy)phosphazene, derivatives of
lipopolysaccharides (LPS), such as monophosphoryl lipid A, muramyl
dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi);
OM-174 ; Leishmania elongation factor; ISCOMs; SB-AS2; SB-AS4;
non-ionic block copolymers that form micelles such as CRL 1005;
Syntex Adjuvant Formulation CpG nucleic acids; Bacterial toxins,
e.g., Cholera toxin (CT), CT derivatives including but not limited
to CT B subunit (CTB); Zonula occludens toxin, zot; Escherichia
coli heat-labile enterotoxin; Labile Toxin (LT), LT derivatives
including but not limited to LT B subunit (LTB); Pertussis toxin,
PT; toxin derivatives; Lipid A derivatives (e.g., monophosphoryl
lipid A, MPL); bacterial outer membrane proteins (e.g., outer
surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer
membrane protein of Neisseria meningitidis).
[0168] In the methods described herein, the increase immune
response in response to administration of the antigen and an agent
that reduces B10 cell function, localization expansion or
production of IL-10 can be measured by methods known to those of
skill in the art including but not limited to ELISA, Western blot,
ELISpot, dot blot. The enhanced immune response may include
enhanced antigen specific antibody production and in particular
enhanced class switching, e.g. enhanced IgG production. As
described above the subjects include mammals, including
domesticated animals such as livestock, pets, and primates
including monkeys and humans.
5.8 Diagnostics
[0169] In another embodiment, methods are provided for diagnosing a
subject suffering from a disease that is associated with elevated
or diminished levels of IL-10 production. In another embodiment, a
subject with a predisposition to a certain disease can be
diagnosed. In an aspect of these embodiments, B 10 cells are
isolated from the subject and assayed for specificity to a certain
disease-specific antigen.
[0170] The B10 cells to be analyzed may be collected from any
location in which they reside in the subject including, but not
limited to, blood, spleen, thymus, lymph nodes, and bone marrow.
The isolated B10 cells may be analyzed intact, or lysates may be
prepared for analysis.
[0171] Methods for the quantitation of cells and detection of
antigenic specificity are known in the art, and may include
pre-labeling the sample directly or indirectly; adding a second
stage antibody that binds to the antibodies or to an indirect
label, e.g., labeled goat anti-human serum, rat anti-mouse, and the
like. For example, see U.S. Pat. No. 5,635,363. Generally, assays
will include various negative and positive controls, as known in
the art.
[0172] Various methods are used to determine the antigenic
specificity profile from a patient sample. The comparison of a
binding pattern obtained from a patient sample and a binding
pattern obtained from a control, or reference, sample is
accomplished by the use of suitable deduction protocols including,
but not limited to, AI systems, statistical comparisons, and
pattern recognition algorithms. Typically a data matrix is
generated, where each point of the data matrix corresponds to a
readout from a specific epitope. The information from reference
patterns can be used in analytical methods to determine relative
abundance, changes over time, and any other factors relevant to
analysis.
[0173] Any disease can be diagnosed according to these embodiments.
In particular, diseases associated with diminished levels of
endogenous IL-10, e.g., immune and inflammatory diseases, and
diseases associated with elevated levels of endogenous IL-10, e.g.,
cancer can be diagnosed based on isolation of B10 cells in a
subject with disease-specific antigen specificity.
[0174] In another embodiment, a subject diagnosed with a given
disease can be monitored for disease progression. Formats for
patient sampling include time courses that follow the progression
of disease, comparisons of different patients at similar disease
stages, e.g., early onset, acute stages, recovery stages; and
tracking a patient during the course of response to therapy. In an
aspect of this embodiment, the numbers of B10 cells having
specificity to a certain disease-specific antigen can be monitored
over the course of a given therapy. As a non-limiting example, a
therapy designed to expand the endogenous population of B10 cells
that respond to a given disease should result in an increase in the
numbers of B10 cells with specificity to a certain antigen
associated with said disease relative to the general population of
B10 cells.
6. EXAMPLE 1
CD22 Antibodies Deplete the Regulatory B Cell Population and
Depletion Enhanced Enhances IgG Production
[0175] Administration of CD22 mAbs to mice results in depletion of
the regulatory B cell population as evidenced by a decrease in
CD1d.sup.highCD5.sup.+ B cells (FIG. 1A) and a decrease in B cell
IL-10 production (FIG. 1B). Thus, CD22 mAb treatment efficiently
depleted spleen B10 cells while leaving most other spleen B cells
intact. Effective B10 cell depletion by MB22-10 mAb in wild-type
mice provides a system to investigate whether B10 cell depletion by
CD22 mAb would enhance the IgG responses in wild-type mice
immunized with a T cell dependent antigen in the absence of
adjuvant. DNP-KLH immunization induced significant primary and
secondary (day 21 boost) DNP-specific IgM responses in control
mAB-treated wild-type mice, while DNP-specific IgG responses rose
only slightly above background levels (FIG. 1C). By contrast, CD22
mAb-treated mice generated normal Ag-specific IgM responses and
robust DNP-specific IgG1, IgG2c, IgG2b, and IgG3 Ab responses that
remained high. CD22 mAb treatment also significantly expanded the
frequency of spleen IgG-secreting B cells in comparison with
control mAb-treated mice (FIG. 1D). Thereby B10 cell depletion
dramatically enhances IgG responses in wild-type mice.
7. EXAMPLE 2
Characterization of the Human B10 Cell Population
[0176] The results shown below identify IL-10 competent B10 and B
10pro cells in man that are comparable to those identified in mice,
and show that both adaptive and innate signals can drive human B10
cell maturation and IL-10 production.
7.1 Materials and Methods
7.1.1 Cell Preparation
[0177] Heparinized blood samples were obtained from healthy 22 to
53 year-old adult donors or from patients with autoimmune disease,
with B cells examined immediately thereafter. Tissue samples were
obtained anonymously at surgery or postmortem from individuals
without identifiable hematologic disorders, with the B cells
purified and immediately cryopreserved and then kept frozen in
liquid nitrogen until use. Tonsils were obtained from patients
undergoing a routine tonsillectomy. Umbilical cord blood samples
were obtained from frozen research units that were processed at the
Duke University Stem Cell Laboratory and the Carolinas Cord Blood
Bank.
7.1.2 Mice
[0178] C57BL/6 mice were from the Jackson Laboratory (Bar Harbor,
Me.). All mice were housed in a specific pathogen-free barrier
facility and used at 8-12 wk of age.
7.1.3 Antibodies
[0179] Anti-human mAbs included: IgD (IA6-2) from BD PharMingen
(San Diego, Calif.); CD21 (BU33), CD22 (RFB4), CD23 (D.6) from
Ancell (Bayport, Minn.); IgM (MHM-88), CD 1d (51.1), CD5 (UCHT2),
CD19 (HIB19), CD24 (ML5), CD25 (BC96), CD27 (0323), CD38 (HIT2),
CD40 (HB14), CD48 (BJ40), and CD148 (A3) mAbs from BioLegend (San
Diego, Calfi.). Anti-human IgM Ab was from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, Pa.). Phycoerythrin-conjugated
anti-human IL-10 mAb (JES3-19F1) was from BioLegend (San Diego,
Calif.).
[0180] Anti-mouse mAbs included: CD20 mAb (MB20-11; Uchida et al.,
2004. Int. Immunol. 16:119-129); B220 mAb RA3-6B2 (obtained from
Dr. Robert Coffman, DNAX Corp., Palo, Alto, Calif.); and CD19
(1D3), CD5 (53-7.3), CD 1d (1B1), CD21/35 (7G6), CD23 (B3B4), CD24
(M1/69), CD25 (PC61), and CD40 (3/23) mAbs from BD PharMingen (San
Diego, Calif.); CD27 (LG.3A10), CD38 (90) from BioLegend (San
Diego, Calif.); IgM (11/41) from eBioscience (San Diego, Calif.);
and IgD (11-26) from Southern Biotechnology Associates (Birmingham,
Ala.); FITC-conjugated anti-mouse CD22 N-terminus (Cy34, TIB163,
American Type Culture Collection). Phycoerythrin-conjugated
anti-mouse IL-10 mAb (JES5-16E3) was from eBioscience (San Diego,
Calif.).
[0181] 7.1.4. B Cell Isolation, Immunofluorescence Analysis and
Cell Sorting
[0182] Blood mononuclear cells were isolated from heparinized blood
after centrifugation over a discontinuous Lymphoprep (Axis-Shield
PoC As, Oslo, Norway) gradient. Single cell splenocyte, lymph node,
and tonsil suspensions were generated by gentle dissection with
>90% cell viability as determined by trypan blue exclusion. Cell
numbers were quantified using a hemocytometer, with relative
lymphocyte percentages among viable cells (based on scatter
properties) determined by flow cytometry analysis. In some
experiments, B cells were enriched using RosetteSep (STEMCELL
Technologies, Vancouver, BC, Canada) following the manufacturer's
protocols. CD19-mAb coated microbeads (Miltenyi Biotech) were used
to purify blood B cells by positive selection following the
manufacturer's instructions. When necessary, the cells were
enriched a second time using a fresh MACS column to obtain >99%
purities.
[0183] Single cell leukocyte suspensions were stained on ice using
predetermined optimal concentrations of each Ab for 20-60 min, and
fixed as described (Sato et al., 1996, J. Immunol. 157:4371-4378).
Cells with the light scatter properties of lymphocytes were
analyzed by 2-6 color immunofluorescence staining and FACScan or
FACSCalibur flow cytometers (Becton Dickinson, San Jose, Calif.).
Dead cells were excluded from the analysis based on their forward-
and side-light scatter properties and the use of LIVE/DEAD Fixable
Dead Cell Stain Kits (Invitrogen-Molecular Probes, Carlsbad,
Calif.). All histograms are shown on a 4-decade logarithmic scale,
with gates shown to indicate background isotype-matched control mAb
staining set with <2% of the cells being positive. Blood
CD24.sup.hiCD27.sup.+ and CD24.sup.1''.sup./CD27.sup.- B cells were
isolated using a FACSVantage SE flow cytometer (Becton Dickinson,
San Jose, Calif.) with 90-95% purities.
[0184] 7.1.5. Analysis of IL-10 Production
[0185] Intracellular IL-10 analysis by flow cytometry was as
described (Yanaba et al., 2008, Immunity 28:639-650). Briefly,
isolated mononuclear cells or purified B cells were resuspended
(2.times.10.sup.6 cells/ml) in complete medium [RPMI 1640 media
containing 10% FCS, 200 .mu.g/ml penicillin, 200 U/ml streptomycin,
and 4 mM L-Glutamine, with 5 x le M 2-mercaptoethanol in mice,
without 2-mercaptoethanol in human (all from Gibco, Carlsbad,
Calif.)]. The cells were stimulated with LPS (10 .mu.g/ml,
Escherichia coli serotype 0111: B4; Sigma), CpG (human ODN 2006,
mouse ODN 1826, 10 .mu.g/ml; Invivogen), or other TLR agonists
(TLR1, Pam3CSK4, 1 .mu.g/ml; TLR2, heat-killed Listeria
monocytogenes, 10.sup.8 cells/ml; TLR3, Poly(I:C), 10 .mu.g/ml;
TLR5, S. typhimurium flagellin, 1 .mu.g/ml; TLR6, Pam2CGDPKHPKSF, 1
.mu.g/ml; TLR7, Imiquimod, 1 .mu.g/ml; TLR8, ssRNA40, 1 .mu.g/ml;
all from Invivogen), CD40L (1 .mu.g/ml; R&D Systems,
Minneapolis, Minn.), anti-human CD40 mAb (1 .mu.g/mL; BioLegend),
anti-mouse CD40 mAb (1 .mu.g/mL; BD Pharmingen), PMA (50 ng/ml;
Sigma), ionomycin (human 1 .mu.g/ml, mouse 500 ng/ml; Sigma), BFA
(1.times. solution/ml; BioLegend), monensin (2 mM; eBioscience),
and anti-human IgM Ab (10 .mu.g/mL; Jackson ImmunoResearch
Laboratories) as indicated for 5 or 48 h, in 48-well flat-bottom
plates before staining and flow cytometry analysis. For analysis of
cell proliferation, lymphocytes were stained with CFSE Vybrant.TM.
CFDA SE fluorescent dye (5 .mu.M; Invitrogen-Molecular Probes)
according to the manufacturer's instructions. For IL-10 detection,
Fc receptors were blocked with mouse Fc receptor mAb (2.4G2; BD
PharMingen) or human Fc.gamma.R-Binding inhibitor (eBioscience)
with dead cells detected by using a LIVE/DEAD.RTM. Fixable Violet
Dead Cell Stain Kit (Invitrogen-Molecular Probes) before cell
surface staining. Stained cells were fixed and permeabilized using
a Cytofix/Cytoperm kit (BD PharMingen) according to the
manufacturer's instructions and stained with
phycoerythrin-conjugated anti-human or anti-mouse IL-10 mAb.
[0186] Secreted IL-10 was quantified by ELISA. Purified B cells
(4.times.10.sup.5) were cultured in 0.2 ml of complete medium in a
96-well flat-bottom tissue culture plates. Culture supernatant
fluid IL-10 concentrations for triplicate samples were quantified
using IL-10 OptEIA ELISA kits (BD PharMingen) following the
manufacturer's protocols.
7.1.6. B Cell IL10 Transcript Expression
[0187] In some experiments, IL-10-secreting blood B cells were
identified after 4 h of in vitro stimulation using an IL-10
secretion detection kit (Miltenyi Biotech, Auburn, Calif.) with
subsequent staining for CD19 expression before cell sorting into
IL-10.sup..+-.CD19.sup.+and IL-10.sup.-CD19.sup.+ populations.
Total RNA was extracted from the purified B cells using Qiagen
RNeasy spin columns (Qiagen Ltd., Crawley, UK). Random hexamer
primers (Promega, Madison, Wis.) and Superscript II RNase H Reverse
Transcriptase (Invitrogen, Carlsbad, Calif.) were used to generate
cDNA. IL-10 transcripts were quantified by real-time PCR analysis
using SYBR Green as the detection agent as described. The PCR was
performed with the iCycler iQ system (Bio-Rad, Hercules, Calif.).
All components of the PCR mix were purchased from Bio-Rad and used
according to the manufacturer instructions. The reaction conditions
were as follows: 2 min at 50.degree. C. (1 cycle), 10 min at
95.degree. C. (1 cycle), 15 s at 95.degree. C., and 1 min at
60.degree. C. (50 cycles). Specificity of the RT-PCR was controlled
by the generation of melting curves. Relative expression of PCR
products was determined using the .DELTA..DELTA.CT method. Briefly,
each set of samples was normalized using the difference in
threshold cycle (CT) between the target gene and housekeeping gene
(GAPDH): .DELTA.CT=(CT target gene-CT GAPDH). Relative mRNA levels
were calculated by the expression 2.sup.-.DELTA..DELTA.CT, where
.DELTA..DELTA.CT=.DELTA.CT sample-.DELTA.CT calibrator. For all
reactions, each condition was performed in triplicate. Data
analysis was performed using iQ Cycler analysis software. The sense
IL-10 primer was 5'-CTTCGAGATCTCCGAGATGCCTTC-3' (SEQ ID NO: 6) and
the antisense primer was 5'-ATTCTTCACCTGCTCCACGGCCTT-3' (SEQ ID NO:
7). The sense GAPDH primer was 5'-GCCACCCAGAAGACTGTGGATGGC-3' (SEQ
ID NO: 8) and the antisense primer was
5'-CATGTAGGCCATGAGGTCCACCAC-3' (SEQ ID NO: 9).
7.1.7. Patients
[0188] All subjects with rheumatoid arthritis (RA) met the American
College of Rheumatology 1987 revised criteria for classification
(Arnett et al., 1988, Arthritis Rheum. 31:315-324); subjects with
systemic lupus erythematosus (SLE) satisfied the 1982 revised
criteria for the classification (Tan et al., 1982, Arthritis Rheum.
25:1271-1277); and subjects with primary Sjogren's syndrome (SjS)
fulfilled the American-European consensus group revised criteria
for the classification (Vitali et al., 2002, Ann. Rheum. Dis.
61:554-558). Patients with autoimmune vesiculobullous skin disease
(BD) included bullous pemphigoid (BP), pemphigus foliaceus (PF),
and pemphigus vulgaris (PV). All patients had typical clinical and
histologic findings with diagnostic findings on direct
immunofluroesence of perilesional skin or oral mucosa (Yancey and
Egan, 2000, J. Amer. Med. Assoc. 284:350-356; Udey and Stanley,
1999, JAMA 282:572-576). Informed consent for multiple sclerosis
(MS) patient's blood samples was obtained in each instance
according to protocols approved by the Institutional Review Board
of St. Luke's-Roosevelt Hospital Center Institute for Health
Sciences. All patients with MS fulfilled 2005 revised McDonald
criteria for relapsing remitting or primary progressive MS (Polman
et al., 2005, Ann. Neurol. 58:840-846). Secondary progressive MS
was defined using the Lublin and Reingold criteria (Lublin and
Reingold, 1996, Neurology 46:907-911). Most patients were receiving
treatment with immunomodulatory drugs and/or low doses of
prednisone (see Table 1, below).
7.1.8. Statistical Analysis
[0189] All data are shown as means (.+-.SEM). Significant
differences between sample means were determined using the
Student's t test.
7.2 Results
7.2.1. Identification of Human IL-10-Producing B Cells
[0190] Mouse blood B10 cell frequencies were determined after
culturing the cells with media, PIM, L+PIM, or CpG oligonucleotides
plus PIM (CpG+PIM) for 5 h in vitro. Blood B cells did not express
detectable IL-10 without in vitro stimulation. However, IL-10
competent B10 cells represented <3% of mouse blood B cells, with
the combination of CpG+PIM inducing higher frequencies of B10 cells
than L+PIM or PIM alone (FIG. 2A). A similar strategy to that used
in mice was optimized to maximize human IL-10 competent B10 cell
enumeration. Brefeldin A (BFA) was used to block IL-10 golgi
transport rather than monensin since it optimized human B cell
cytoplasmic IL-10 expression (FIG. 2B). Optimal human B10 cell
numbers were observed after 5 h of PIB stimulation in vitro, with
overall B cell viability decreasing after this time point.
Background IL-10 mAb staining was reduced by the exclusion of all
cell doublets and dead cells from the flow cytometry analysis with
BFA cultures used as negative controls (FIG. 2C). These assay
conditions were then used to identify human IL-10-competent blood B
cells.
[0191] Human blood was found to contain a rare, but distinct subset
of IL-10-competent B10 cells that was detectable at low 0.25-2%
frequencies after in vitro stimulation. B cell activation with PMA,
ionomycin, and BFA (PIB) for 5 h induced 0.8.+-.0.1% of B cells on
average to express IL-10 (n=14, 1.9.+-.0.3.times.10.sup.-3 B10
cells/ml, FIG. 2D-2E). Some blood B cells may spontaneously express
IL-10, but their frequencies and levels of IL-10 expression were
below the 0.2% threshold of quantification by immunofluorescence
staining; which is similar to background cytoplasmic IL-10 staining
when using B cells from IL-10.sup.-/- mice (see Yanaba et al.,
2008, Immunity 28:639-650; Matsushita et al., 2008, J. Clin.
Invest. 118:3420-3430; and Yanaba et al., 2009, J. Immunol.
182:7459-7472). B cell stimulation using TLR agonists did not
substantially alter mean B10 cell numbers, although IL-10.sup.+ B
cell frequencies were enhanced in some individuals by adding either
CpG (TLR9 agonist) or LPS to the PIB cultures. Thus, blood B10
cells were rare, a trait shared by both healthy humans and
mice.
7.2.2. Human B10Pro Cell Identification
[0192] In mice, B10pro cell maturation into IL-10-competent B10
cells can be induced by 48 h stimulation with either LPS or
agonistic CD40 mAb (Yanaba et al., 2009, J. Immunol.
182:7459-7472). B10pro cells capable of maturing into IL-10
competent B10 cells after in vitro culture for 48 h were identified
in human blood by their ability to express cytoplasmic IL-10 after
5 h PIB stimulation (FIG. 2D). The total frequency of B10 and
B10pro cells (B10+B10pro) is quantified in this assay, as the B
cells that acquire IL-10 competence in vitro (e.g. matured B10pro
cells) cannot be differentiated from preexisting blood B10 cells.
Culturing human B cells with LPS, CpG, or recombinant CD40 ligand
(CD40L, CD154) alone, together, or in combination with BFA did not
induce detectable cytoplasmic IL-10 expression, while .about.0.2%
of B cells cultured in media alone with 5 h PIB stimulation during
the last 5 h of culture expressed cytoplasmic IL-10 (FIG. 2F-2G).
However, 1-4% of human blood B cells expressed IL-10 following TLR
agonist (48 h)+PIB (5 h) stimulation. B10+B10pro cell frequencies
increased to 0.6.+-.0.1, 1.9.+-.0.4, 0.8.+-.0.1, 1.2.+-.0.2, and
4.1.+-.1.0% following TLR1 agonist, LPS, TLR6 agonist, TLR7
agonist, and CpG stimulation, respectively.
[0193] The addition of recombinant CD40L alone to B cell cultures
did not induce B10pro cell maturation, while the addition of CD40L
to B cells stimulated with LPS induced higher frequencies of
B10+B10pro cells. CD40L induced 47% higher frequencies of
IL-10.sup.+ B10 cells than agonistic CD40 mAb (FIG. 2F). CD40L
stimulation also significantly enhanced mean B10+B10pro cell
frequencies when combined with TLR1 agonist (1.1.+-.0.2%), LPS
(3.4.+-.0.7%), TLR6 agonist (1.4.+-.0.2%), TLR7 agonist
(2.2.+-.0.4%), or CpG (7.0.+-.1.4%) stimulation (FIG. 2G right
panel). Thus, dual CD40 and TLR stimulation induced the highest
frequencies of human B10pro cells to become IL-10 competent B10
cells, with the highest numbers of B10+B10pro cells
(1.6.+-.0.3.times.10.sup.4 cells/ml, n=14) induced after 48 h of
CD40L plus CpG stimulation. Thereby, human blood B10 and B10pro
cells can be readily and reproducibly quantified after stimulation
or in vitro maturation with CD40 ligation in combination with
select TLR signals.
7.2.3. B10 Cell Numbers in Newborn Blood and Adult Lymphoid
Tissues
[0194] Newborn mice have higher spleen B10 and B10pro cell
frequencies than adult mice (Yanaba et al., 2009, J. Immunol.
182:7459-7472). Mean B10 cell frequencies in human newborn blood
after 5 h of CpG+PIB stimulation (0.45.+-.0.14%, n=8; FIG. 3A) were
42% lower than those observed for adult human blood (FIG. 2E),
although this may reflect donor pool diversity rather than
represent differences between newborns and adults. Nonetheless,
B10+B10pro cell frequencies were similar or higher in newborn blood
relative to adult human blood after culture with CD40L and TLR
agonists; TLR1 (2.6.+-.0.6%), LPS (7.6.+-.1.8%), TLR6
(4.2.+-.1.4%), or TLR9 (CpG, 9.6.+-.2.3%) agonists with PIE added
during the final 5 h of culture. Thus, newborn and adult blood
contained both B10 and B10pro cells.
[0195] B10 cells were also found within spleens (0.31.+-.0.06, n=4,
CpG+PIB) and tonsils (0.31.+-.0.11, n=3, CpG+PIB) of individuals
without known disease (FIG. 3B-3C). Stimulating spleen and tonsil B
cells with LPS or CpG in combination with CD40L also induced B10pro
cells to mature into IL-10 competent B10 cells, with B10+B10pro
cell frequencies ranging from <0.5% to almost 30% (FIG. 3B).
Human tonsil and spleen B10 cell frequencies were numerically
similar to those observed in blood, but spleen B10+B10pro cell
frequencies were 2.2-fold higher. In these samples, human spleen
and tonsil B10 cell frequencies were 62-85% lower than those
observed for mouse spleen (0.3% vs. 2.1%) and lymph node (0.3% vs.
0.8%), respectively, but B10+B10pro cell frequencies were higher
within human tissues. It was observed that CpG stimulation induced
human B10pro cell maturation, while CpG failed to induce mouse
B10pro cell maturation (FIG. 3C). Regardless, B10 cells represented
a small subset of human spleen and lymph node B cells.
7.2.4. Regulation of B10 Cell IL-10-Production and Secretion In
Vitro
[0196] The time course of B10 cell IL-10 induction was assessed in
vitro by quantifying IL-10 transcripts in cultured human blood B
cells stimulated with CD40L+CpG. By 12, 24, and 48 h, B cell
stimulation induced 6.8-, 24-, and 5.9-fold higher Il10 transcript
levels, respectively, than was observed for unstimulated B cells
(p<0.05; FIG. 4A). Human blood B10 cells that were actively
secreting IL-10 expressed Il10 transcripts at 19-fold higher levels
than IL-10.sup.- B cells after in vitro stimulation (FIG. 4B).
Thus, B10 cell IL-10 expression paralleled Il10 gene
transcription.
[0197] The response of human B10+B10pro cells to CD40L, CpG, and
antigen receptor generated signals was examined. In comparison with
CD40L alone, CpG alone induced the highest levels of B10pro cell
maturation into IL-10-competent B10 cells, which was further
increased when both CD40L and CpG were added to the cultures (FIG.
4C). By contrast, BCR ligation using mitogenic anti-IgM Ab did not
induce cytoplasmic IL-10 expression, but actually inhibited the B10
cell inducing effects of CpG+CD40L stimulation. In vitro BCR
signals also inhibit mouse B10pro cell maturation and cytoplasmic
IL-10 induction (Yanaba et al., 2009, J. Immunol. 182:7459-7472).
Among TLR agonists, LPS and CpG were also the most potent stimuli
for inducing IL-10 secretion by human blood B cells (FIG. 4D) and
mouse blood B cells (FIG. 4E). Thus, similar signals regulate human
and mouse B10 and B10pro cells to mature and express cytoplasmic
IL-10 in vitro.
7.2.5. Phenotypic Characterization of Blood and Spleen
IL-10-Competent B Cells
[0198] Whether human B10 cells represent a phenotypically defined B
cell subset was determined by immunofluorescence staining. B cells
that were either untreated, stimulated with PIB, L+PIB, or CpG+PIB
for 5 h, and/or permeabilized, were found to express identical cell
surface IgM, IgD, CD1d, CD5, CD10, CD19, CD21, CD22, CD23, CD24,
CD25, CD27, CD38, and CD40 densities. The transport of newly
synthesized proteins to the cell surface is also inhibited by BFA.
The use of ten-fold higher PMA concentrations did not alter B cell
surface phenotypes or survival during these 5 h assays, while the
use of ionomycin at ten-fold higher concentrations significantly
altered B cell surface phenotypes even in the presence of BFA due
to extensive cell death. Since these cell surface molecules were
not affected by the stimulation and/or cell permeabilization
protocols used to visualize cytoplasmic IL-10 expression, they were
used to categorize the phenotype of freshly isolated blood B10
cells. Half of blood B10 cells expressed high IgM levels and low
IgD levels (FIG. 5A). Both CD24 and CD27 expression were high on
the majority of B10 cells, while IL-10.sup.- B cells expressed
either high or low density CD24 and CD27. CD19 and CD25 expression
were also higher on B10 cells than IL-10.sup.- B cells. Otherwise,
the remaining cell surface markers were absent or expressed
similarly by both B10 cells and IL-10.sup.- B cells. The same
results were obtained following PIB, L+PIB, or CpG+PIB stimulation.
Thereby, freshly isolated human blood IL-10-competent B10 cells
were predominantly CD24.sup.hiCD27.sup.+ B cells.
[0199] The phenotype of blood B10+B10pro cells induced during 48 h
in vitro cultures was also assessed. In comparison with freshly
isolated B cells after 5 h of L+PIB stimulation (FIG. 5A),
prolonged cell culture and these stimulation conditions induced
significant changes in the cell surface phenotype of B10 and
non-B10 cells (FIG. 5B). For example, most B cells were induced to
express CD25 and CD38 at high densities. Nonetheless, B10+B10pro
cells on average expressed higher densities of CD1d, CD19, CD20,
CD21, CD23, CD24, CD25, CD27, and CD38 when compared with
IL-10.sup.- B cells, consistent with an activated phenotype. Spleen
B10 cells were also predominantly CD27.sup.+, although the
expression of most cell surface molecules was similar if not
identical for B10 cells and IL-10.sup.- B cells (FIG. 5C). Spleen
B10+B10pro cells and IL-10.sup.- B cells also had similar
phenotypes after 48 h of stimulation in vitro, with the exception
that IL-10.sup.+ B10 cells were predominantly IgM'' while
IL-10.sup.- B cells were predominantly IgM.sup.low (FIG. 5D).
Thereby, spleen B10 cells were also predominantly
CD24.sup.hiCD27.sup.+.
[0200] The phenotype of mouse blood B10 cells was also assessed for
comparison. Mouse blood B10 cells expressed higher levels of IgM
than IL-10.sup.- B cells, but most other cell surface molecules
were expressed at similar densities (FIG. 5E). CD 1d, CD5, CD19,
CD24, and CD38 expression were slightly higher on IL-10.sup.+ than
IL-10.sup.- B cells, while CD23 expression was heterogeneous in
comparison to IL-10.sup.- B cells. Mouse blood B10+B10pro cells
were predominantly IgM.sup.hiCD5'' CD19.sup.hiCD23.sup.low
CD24.sup.hiCD38.sup.hi after in vitro activation (FIG. 5F). Spleen
B10 cells were predominantly IgM.sup.hi IgD.sup.low CD1d.sup.hi
CD5.sup.+ CD19.sup.hiCD21.sup.hi/int CD23.sup.low
CD24.sup.hiCD38.sup.hi (FIG. 5G). Mouse spleen B10+B10pro cells
were more similar to IL-10.sup.- B cells after 48 h of culture
(FIG. 5H). Thereby, human blood B10 cells were predominantly
CD24.sup.hiCD27.sup.+, while mouse spleen B10+B10pro cells were
predominantly CD1d.sup.hiCD5.sup.+.
7.2.6. Blood B10 Cells are Enriched Within the
CD24.sup.hiCD27.sup.+ B Cell Subpopulation
[0201] When the spectrum of normal control blood donors was
compared, blood B10 cells were predominantly CD24.sup.hi and
CD27.sup.+ (FIG. 5A). Most B10 cells also expressed additional cell
surface markers of activation (CD48.sup.hi) and memory
(CD148.sup.hi) (FIG. 6A). Since cell surface CD24, CD27, CD38, and
CD48, and CD148 expression were not affected by the 5 h culture
conditions used to induce B cell cytoplasmic IL-10 expression (FIG.
6B), circulating B10 cells were predominantly CD24.sup.hi,
CD2.sup.+, CD48.sup.hi, and CD148.sup.hi. Cell surface CD27 and
CD38 expression profiles have been used frequently to define human
blood B cell subsets (see, e.g., Levesque and St. Clair, 2008, J.
Allergy Clin. Immunol. 121:13-21; and Sanz et al., 2008, Sem.
Immunol. 20:67-82). However, when blood B10 cells were analyzed
based on their CD38 versus IgD expression profiles, IL-10.sup.+ B10
cells from representative blood donors fell into both the
CD38.sup.hi and CD38.sup.lo populations (FIG. 6C). Similarly, when
blood B10 cells were analyzed based on CD27 versus IgD expression,
IL-10.sup.+ B cells from representative blood donors did not fall
into pre-defined subsets, but were predominantly CD27.sup.hi.
[0202] To determine whether B10 cells predominantly localize within
the CD24.sup.hiCD27.sup.+ subpopulation that represented 24.+-.5%
(n=7) of blood B cells, blood CD24.sup.hiCD27.sup.+ and
CD24.sup.lowCD27.sup.- subpopulations were first purified and then
cultured individually with L+PIB for 5 h to induce IL-10 expression
(FIG. 6D). Following cell permeabilization and cytoplasmic IL-10
staining, B10 cell frequencies were at least 10-fold higher within
the previously purified CD24.sup.hiCD27.sup.+ subpopulation when
compared with the isolated CD24.sup.lowCD27.sup.- B cells. Thus,
B10 cells were predominantly CD24.sup.hiCD27.sup.+ in vivo.
[0203] To determine whether B10pro cells also predominantly
localize within the CD24.sup.hiCD27.sup.+ subpopulation, blood
CD24.sup.hiCD27.sup.+ and CD24.sup.lowCD27.sup.- subpopulations
were purified and then cultured individually with CD40L, and LPS or
CpG for 48 h to induce B10pro cell maturation. Again, the frequency
of B10 cells was 10-fold higher within the CD24.sup.hiCD27.sup.+
subpopulation when compared with CD24.sup.lowCD27.sup.- cells (FIG.
6E). To eliminate the possibility that B10pro cell proliferation
during the 48 h cultures contributed to their expansion, the
purified blood B cells were labeled with CFSE before in vitro
stimulation. Regardless of whether the cells were stimulated with
LPS or CpG, there was no B cell division until 72-96 h of culture.
At this point, B10 cells exhibited a significant proliferative
capacity, while IL-10.sup.- B cells exhibited a modest
proliferative capacity (FIG. 6F). Thus, the preferential
localization of blood B10 and B10+B10pro cells within the
CD24.sup.hiCD27.sup.+ B cell subpopulation was not due to cell
proliferation during the 48 h culture period.
[0204] The capacity of freshly isolated CD24.sup.hiCD27.sup.+ and
CD24.sup.lowCD27.sup.- B cells to secrete IL-10 was also assessed.
CD40L, LPS, and LPS+CD40L stimulation for 72 h induced modestly
increased IL-10 production by the CD24.sup.hiCD27.sup.+ population,
but not CD24.sup.lowCD27.sup.- cells (FIG. 6G).
CD24.sup.hiCD27.sup.+ cells secreted >10-fold more IL-10 in
response to either CpG or CpG+CD40L stimulation when compared with
LPS stimulation alone. IL-10 secretion by CD24.sup.lowCD27.sup.- B
cells was 78% and 82% lower than for CD24.sup.hiCD27.sup.+ B cells
in response to CpG and CpG+CD40L, respectively (p<0.001). Thus,
blood B10 and B10+B10pro cells were predominantly a small subset of
the CD24.sup.hiCD27.sup.+ B cell subpopulation.
7.2.7. B10 Cell Development in Patients with Autoimmune Disease
[0205] To determine whether blood B10 cell numbers are altered in
patients with autoimmune disease, B10 and B10pro cells were
examined in healthy controls, and fifty-two patients with SLE, RA,
SjS, autoimmune BD, or MS. Most of the patients were undergoing
active treatment with immune modulatory agents when the blood was
taken (see Table 1, below). Nonetheless, some patients had high
blood B10 cell frequencies even though there were no significant
differences in mean B10 cell numbers between patient groups in
comparison with controls (FIG. 7A-7B). One BD patient that had not
been given immunosuppressive therapy had blood B10 cell frequencies
that were significantly higher than the population mean. Two SLE
patients and one RA patient also had blood B10 cell frequencies
that were significantly higher than the population mean, but
retrospective evaluation of their disease status, autoantibody
profile, and treatment regimen did not reveal obvious explanations
for why these individuals had higher blood B10 frequencies than
other patients. B10 cell frequencies were similar regardless of
whether LPS or CPG were added to the PIB-stimulated cultures (FIG.
7D, left panel). No patient groups were identified that expressed
significantly lower B10 cell frequencies relative to controls or
other patient groups.
[0206] Mean B10+B10pro cell frequencies from patients with SLE and
BD were significantly higher than controls following CD40L+LPS
stimulation, while mean B10+B10pro cell numbers in RA patient's
were significantly higher after CD40L+CpG stimulation when compared
with the control group (FIG. 7B). Notably, patients with high blood
B10 cell frequencies did not necessarily have high B10+B10pro cell
frequencies after either LPS or CpG stimulation (FIG. 7C). While
B10 cell frequencies were linearly correlated following either LPS
or CPG stimulation, the scatter of the results obtained for
B10+B10pro cells was broad, suggesting inherently different patient
sensitivities to LPS and CpG stimulation (FIG. 7D). Likewise, B10
and B10+B10pro cell frequencies did not correlate with CD27.sup.+ B
cell frequencies. Regardless, relative B10 and B10+B10pro cell
frequencies were significantly correlated with the intensity of
cytoplasmic IL-10 expression (FIG. 7E). One patient among the
entire group appeared to generate significantly higher (p<0.05)
cytoplasmic IL-10 expression levels on a per cell basis relative to
controls and other patients.
TABLE-US-00001 TABLE 1 Patient characteristics. Diagnosis Disease
Immunosuppressive Number Sex Age Duration (y) Autoantibody/Clinical
Status Therapy RA01.sup.1 M 54 11 RF = 600 IU/ml; anti-CCP = 68.5
U/ml MTX, ADA, Pred 5 mg/d RA02 F 44 4 RF = 352 IU/ml; anti-CCP
> 100 U/ml MTX RA03 F 54 14 RF = 146 IU/mL MTX, LEF RA04 F 85 18
RF = neg MTX, IFX RA05 M 69 19 RF = 208 IU/ml; anti-CCP > 100
U/ml MTX, Pred 5 mg/d RA06 F 71 8 RF = neg MTX RA07 F 67 13 RF =
333 IU/ml ETN RA08 F 58 25 RF = 53 IU/ml MTX, ETN, Pred 3 mg/d RA09
F 68 7 RF = 339 IU/ml MTX, Pred 3 mg/d RA10 M 75 13 RF = 420 IU/ml;
anti-CCP = 18.9 U/ml MTX, LEF, Pred 10 mg/d RA11 F 73 7 RF and
anti-CCP = neg MTX, LEF RA12 M 61 27 RF = 107 IU/ml MTX, LEF RA13 F
66 6 RF = pos ADA RAM F 84 11 RF = 275 IU/ml; anti-CCP = 28 U/ml
ETN RA15 F 52 3 RF = neg; anti-CCP > 100 U/ml ETN RA16 F 76 18
RF = pos MTX RA17 M 63 11 RF = neg; anti-CCP > 100 U/ml MTX,
SSZ, Pred 1 mg/d RA18 F 43 15 RF = 23 IU/ml MTX, IFX RA19 F 62 30
RF = 148 IU/ml; anti-CCP > 100 U/ml LEF; Pred 5 mg/d SLE01 F 65
11 ANA = 1:2560; IgG CL and anti-dsDNA = pos HCQ, LEF, Pred 5 mg/d
SLE02 M 31 3 ANA = 1:640; anti-RNP, anti-Sm, and anti-Ro = pos HCQ,
Pred 3 mg/d SLE03 M 63 32 ANA = 1:640; anti-dsDNA and IgG anti-CL =
pos HCQ, Pred 5 mg/d SLE04 F 37 5 ANA = 1:2560; anti-Ro = pos None
SLE05 F 43 15 ANA = 1:160; RF = 36 IU/ml HCQ, MMF SLE06 F 46 8 ANA
= pos; anti-Ro = pos MMF 2 g/d, Pred 10 mg/d SLE07 M 31 23 ANA =
1:160; anti-dsDNA and IgG anti-CL = pos HCQ SLE08 F 47 10 ANA =
1:2560; anti-dsDNA, anti-Ro and anti-La = pos; None RF = 103 IU/ml
SLE09 F 37 16 ANA = pos; anti-dsDNA, anti-IgM and IgG CL = pos HCQ;
Pred 10 mg/d SjS01 F 52 1 ANA = 1:2560; RF = 354 IU/ml; anti-Ro and
None anti-La = pos SjS02 F 65 15 ANA = 1:2560; RF = 22 IU/ml;
anti-Ro = pos HCQ SjS03 F 57 37 ANA = 1:160; anti-Ro = pos MMF,
Pred 40 mg/d SjS04 F 67 22 ANA = 1:2560; RF 110 IU/ml, anti-Ro and
anti-La = pos HCQ SjS05 F 60 9 ANA = 1:2560; RF = 126 IU/ml,
anti-Ro = pos HCQ SjS06 F 58 21 ANA = 1:2560; anti-Ro and anti-La =
pos None SjS07 F 41 13 ANA = 1:2560; anti-Ro and anti-La = pos HCQ
SjS08 F 59 8 ANA = 1:2560; RF = 508 IU/ml; anti-Ro = pos None SjS09
F 42 4 ANA = 1:2560; RF = 110 IU/ml; anti-Ro and HCQ anti-La = pos
SjS10 F 58 5 ANA = 1:2560; anti-Ro and anti-La = pos HCQ, Pred 3
mg/d SjS11 M 66 5 ANA = 1:2560; anti-Ro and anti-La = pos None
SjS12-46 F 76 13 ANA = 1:2560; anti-Ro = pos; RF = 32 IU/ml None
BP01 M 72 0.3 Anti-BP180 = 84 U/ml; anti-BP230 = 115 U/ml, no
clinical Pred 60 mg/d disease BP02 M 54 1.2 Anti-BP180 = 72 U/ml;
anti-BP230 = neg, no clinical disease MMF, Pred 12 mg/d BP03 F 56 2
Anti-BP180 = 51 U/ml; anti-BP230 = neg, no clinical disease Pred 20
mg/d BP04 M 75 4.3 Anti-BP1 80 = 45 U/ml; anti-BP230 = 3, minimal
disease None BP05 F 66 1.8 Anti-BP180 = 96 U/ml; anti-BP230 = 131,
severe disease None BP06 M 77 0.5 Anti-BP180 = 5 U/ml; anti-BP230 =
95, minimal disease None BP07 F 67 17 Anti-BP180 = 46 U/ml;
anti-BP230 = neg, trace disease RTX (20 mos earlier) PF01 M 54 8.6
Anti-DSG1 = 134 U/ml; anti-DSG3 = neg, minimal disease AZA activity
PF02 M 55 9.8 Anti-DSG1 = neg; anti-DSG3 = neg, minimal disease
activity RTX (30 mos earlier) PF03 M 46 6.6 Anti-DSG1 = neg;
anti-DSG3 = neg, minimal disease activity None PF04 M 50 5.3
Anti-DSG1 = neg; anti-DSG3 = neg, minimal disease activity Cellcept
3 g/d PF05 M 72 2.8 Anti-DSG1 = neg; anti-DSG3 = neg, no clinical
disease Dapsone 100 mg/d PV01 M 47 2.6 Anti-DSG 1 = 24 U/ml;
anti-DSG3 = 5171 U/ml, mild disease AZA PV02 M 43 3 Anti-DSG1 =
neg; anti-DSG3 = 213 U/ml, mild disease MMF, Pred 20 mg/d PV03 M 73
3.3 Anti-DSG1 = neg; anti-DSG3 = 948 U/ml, mild disease Pred 12
mg/d PV04 F 55 4.7 Anti-DSG1 = neg; anti-DSG3 = 406 U/ml, mild
disease RTX (15 mos earlier) PV05 M 59 8.3 Anti-DSG1 = neg;
anti-DSG3 = 50 U/ml, no disease activity AZA PV06 F 48 8.6
Anti-DSG1 = neg; anti-DSG3 = 25 U/ml, no disease activity None PV07
M 45 0.25 Anti-DSG1 = 968; anti-DSG3 = 735, severe disease, ~20%
MMF; Pred 80 mg/d body surface area involving ulcerations and
erosions PV08 M 84 0.2 Anti-DSG1 = 75; anti-DSG3 = 146 U/ml,
moderate disease None PV09 M 64 9.8 Anti-DSG1 = 14; anti-DSG3 = 115
U/ml, minimal disease Pred 20 mg/d; IFX activity PV10 M 59 3.8
Anti-DSG 1 = 1; anti-DSG3 = 49 U/ml, no disease activity AZA PV11 M
55 6 Anti-DSG1 = 34; anti-DSG3 = 35 U/ml, minimal disease Pred 20
mg/d; MMF 1000 activity mg/d PV12 F 58 9.5 Anti-DSG1 = 1; anti-DSG3
= 43 U/ml, trace disease AZA MS01 F 72 54 SPMS, EDSS 6.5, not
clinically active None MS02 M 62 24 RRMS, EDSS 6.5, clinically
active BIFN MS03 M 33 2 RRMS, EDSS 1.0, disease not clinically
active BIFN MS04 M 75 29 SPMS, EDSS 8.0, disease not clinically
active ITMTX MS05 M 52 24 PPMS, EDSS 6.5, disease clinically active
MMF, pulse steroids MS06 M 55 25 PPMS, EDSS 7.5, disease clinically
active ITMTX MS07 F 39 16 SPMS, EDSS 7.0, disease not clinically
active Natalizumab (2 mos prior) MS08 F 51 7 SPMS, EDSS 5.5,
disease not clinically active BIFN .sup.1Abbreviations: ANA,
antinuclear Ab; ADA, adalimumab; AZA, azathioprine; BIFN, beta
interferon; BP, bullous pemphigoid; CCP, cyclic citrullinated
peptide; CL, cardiolipin; DH, dermatitis herpetiformis; dsDNA,
double stranded DNA; DSG, desmoglein; EDSS, disability scale from 0
= normal to 10 = death; ETN, etanercept; HCQ, hydroxychloroquine;
IFX, infliximab; ITMTX, intrathecal methotrexate; LEF, leflunomide;
MMF, mycophenolate mofetil; MTX, methotrexate; PF, pemphigus
foliaceus; PPMS, primary progressive multiple sclerosis; Pred,
Prednisone; PV, pemphigus vulgaris; RA, rheumatoid arthritis; RF,
rheumatoid factor; RRMS, relapsing remitting multiple sclerosis;
RTX, rituximab; SjS, primary Sjogren's syndrome; SLE, lupus; SPMS,
secondary progressive multiple sclerosis; SSZ, sulfasalazine; y,
year. .sup.2Normal values: anti-BP = 180, anti-BP = 230, anti-DSG1
and anti-DSG3 antibodies < 9 IU/ml.
7.3 Discussion
[0207] This example demonstrates the existence of human
IL-10-competent B10 cells, which were readily identified by their
ability to express cytoplasmic IL-10 after appropriate in vitro
stimulation. Peripheral blood B10 cell frequencies were
characteristically low (0.6%) in most individuals, consistent with
their low frequencies in mice. Human B10pro cells were also
identified by their ability to express IL-10 after in vitro
maturation. Remarkably, the adaptive and innate activation pathways
that induced human B10 and B10pro cell generation, maturation,
cytoplasmic IL-10 expression, and IL-10 secretion were similar to
those used by mouse regulatory B10 cells. Human B10 cell
frequencies were also similar in the blood, spleen, and lymph nodes
(FIG. 2-3).
[0208] In this example, B10 cells were defined by their IL-10
production using optimized stimulation conditions similar to those
that have defined mouse B10 and B10pro cells. IL-10-competence
remains the best phenotypic marker for defining human B10 cells.
However, freshly isolated blood B10 and. B10pro cells were also
predominantly CD24.sup.hiCD27.sup.+, with .about.60% also
expressing CD38 at high levels (FIG. 6A, 6C). B10 cells were also
predominantly CD48.sup.hi and CD148.sup.hi(FIG. 6A). CD148 is
considered a marker for human splenic memory B cells (see Tangye et
al., 1988, J. Exp. Med. 188:1691-1703) and CD48 is upregulated on
activated B cells (see Yokoyama et al., 1991, J. Immunol.
146:2192-2200). By contrast, most CD24.sup.loCD27.sup.- B cells
were not IL-10 competent, even after 48 h of LPS or CpG stimulation
along with agonistic CD40 ligation. CD27 expression is a
well-characterized marker for memory B cells, although some memory
B cells may be CD27.sup.- (see Sanz et al., 2008, Seim. Immunol.
20:67-82; Klein et al., 1998, J. Exp. Med. 188:1679-1689; and
Agematsu et al., 2000, Immunol. Today 21:204-206). The CD27.sup.+ B
cell subpopulation can also expand during the course of
autoimmunity and may serve as a marker for disease activity (see
Sanz et al., 2008, Seim. Immunol. 20:67-82; and Agematsu et al.,
2000, Immunol. Today 21:204-206). However, B10 cell frequencies did
not parallel the size of the CD27.sup.+memory B cell pool in the
blood of normal donors or in patients with autoimmune diseases,
suggesting that these two subsets may be regulated independently.
Thus, the CD24.sup.hiCD27.sup.+ phenotype of B10 and B10pro cells
indicates that they may either be selected into the memory B cell
pool during their development or B10 and B10pro cells represent a
distinct B cell subset that shares common cell surface markers with
memory B cells. However, consistent with their memory phenotype,
the proliferative capacity of blood B10 cells in response to
mitogen stimulation was higher than that for other B cells (FIG.
6F), as is also seen in mice (see Yanaba et al., 2009, J. Immunol.
182:7459-7472). Human transitional B cells are also rare (2-3% of B
cells) in adult blood, and are generally
CD10.sup.+CD24.sup.hiCD38.sup.hi cells that are IgD.sup.+CD27.sup.-
(see Sims et al., 2005, Blood 105:4390-4398; and Cuss et al., 2006,
J. Immunol. 176:1506-1516). Given that CD10 expression is a
well-accepted marker for most cells within the transitional B cell
pool (see Wardemann et al, 2003, Science 301:1374-1377), its
absence on B10 cells suggests that these cells are not recent
emigrants from the bone marrow. Thereby, IL-10 competence and
elevated proliferative responses characterize the human blood B10
cell subset, with most blood B10 cells expressing a
CD24.sup.hiCD27.sup.+ phenotype.
[0209] While a small subset of blood B cells was inherently
competent to express IL-10, a subset of blood B cells also had the
capacity to acquire IL-10 competence following stimulation with
LPS, CpG, and other TLR agonists (FIG. 2D and 2G). Combining TLR
stimulation with prolonged CD40 stimulation facilitated the
acquisition of IL-10 competence. Since IL-10 is critical for B cell
regulatory activity in mice, the current studies demonstrate that
B10 cells in normal individuals and autoimmune disease patients are
functionally competent to express IL-10 (FIG. 7A-7B).
[0210] Mean blood B10 cell frequencies in most patients with SLE,
RA, SjS, autoimmune BD, and MS were not significantly different
from those observed in normal controls (FIG. 7B). However, four
patients did have significantly higher B10 or B10+B10pro cell
frequencies, including two SLE patients. This also included one PV
patient (PV07) with severe disease involving 20% of the skin (Table
1). Significantly increased B10+B10pro cell frequencies were also
found in an untreated SLE patient (SLE04). Interestingly, one
patient previously treated with rituximab had elevated B10+B10pro
cell frequencies (PF02). Thus, some patients may have elevated
B10/B10pro cell frequencies as found in some autoimmune prone mouse
strains (see Haas et al., 2010, J. Immunol. (in press); and Yanaba
et al., 2009, J. Immunol. 182:7459-7472) and during inflammation
(see Yanaba et al., 2008, Immunity 28:639-650; and Matsushita et
al., 2008, J. Clin. Invest. 118:3420-3430).
[0211] The current assessment of B10 and B10+B10pro cell
frequencies in patients provides a context for previous studies,
where spontaneous IL-10 production by resting blood B cells is
reported to be dramatically higher in untreated patients with RA,
SLE, and systemic sclerosis than in controls, as measured by RT-PCR
and ELISA assays (see Llorente et al., 1994, Arthritis &
Rheumatism 37:1647-1655). More recently, B cell cytoplasmic IL-10
production by blood mononuclear cells has been examined in patients
with SLE compared with normal controls after 24 h in culture with
or without PMA plus ionomycin, or LPS (see Amel Kashipaz et al.,
2003, Lupus 12:356-363). In that study, significantly more SLE B
cells spontaneously produced cytoplasmic IL-10 (1.1%) than controls
(0.6%). However, after stimulation by PMA plus ionomycin, the
number of IL-10.sup.+ B cells was no higher in the SLE patients
(1.3%) when compared with the unstimulated cultures, but was
slightly higher in the controls (1.5%). By contrast, LPS
stimulation failed in either case to increase intracellular
IL-10-producing B cell frequencies in comparison with unstimulated
cells. Furthermore, unstimulated and stimulated CD5.sup.+ B cells
from SLE patients were enriched for cells producing high levels of
IL-10, although stimulation of CD5.sup.- B cells from normal
controls induced more IL-10 production than stimulation of
CD5.sup.+ cells from normal controls. In other studies, 26% of SLE
patients' blood B cells spontaneously expressed cytoplasmic IL-10,
while 50% were. IL-10.sup.+ after mitogen activation (see
Diaz-Alderete et al., 2004, J. Autoimmun. 23:379-383). In this
case, IL-10 expression was confined to a CD154.sup.+ (CD40L.sup.+)
subset of B cells, but not to CD5.sup.+ B cells.
[0212] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
[0213] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 1
1
9120DNAArtificial SequenceSynthetic Sense IL-10 primer 1ggttgccaag
ccttatcgga 20220DNAArtificial SequenceSynthetic Antisense IL-10
primer 2acctgctcca ctgccttgct 20320DNAArtificial SequenceSynthetic
sense GAPDH primer 3ttcaccacca tggagaaggc 20420DNAArtificial
SequenceSynthetic antisense GAPDH primer 4ggcatggact gtggtcatga
20521PRTArtificial SequenceSynthetic MOG35-55 peptide 5Met Glu Val
Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu1 5 10 15Tyr Arg
Asn Gly Lys 20624DNAArtificial SequenceSynthetic Sense IL-10 primer
6cttcgagatc tccgagatgc cttc 24724DNAArtificial SequenceSynthetic
Antisense IL-10 primer 7attcttcacc tgctccacgg cctt
24824DNAArtificial SequenceSynthetic sense GAPDH primer 8gccacccaga
agactgtgga tggc 24924DNAArtificial SequenceSynthetic antisense
GAPDH primer 9catgtaggcc atgaggtcca ccac 24
* * * * *