U.S. patent application number 13/840377 was filed with the patent office on 2013-12-05 for antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance.
This patent application is currently assigned to GEORGIA HEALTH SCIENCES UNIVERSITY. The applicant listed for this patent is GEORGIA HEALTH SCIENCES UNIVERSITY. Invention is credited to JEFFREY ROBERTS LEE, ANDREW L. MELLOR, DAVID H. MUNN.
Application Number | 20130323832 13/840377 |
Document ID | / |
Family ID | 37567953 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323832 |
Kind Code |
A1 |
MUNN; DAVID H. ; et
al. |
December 5, 2013 |
ANTIGEN-PRESENTING CELL POPULATIONS AND THEIR USE AS REAGENTS FOR
ENHANCING OR REDUCING IMMUNE TOLERANCE
Abstract
The present invention is based on the discovery
antigen-presenting cells (APCs) may be generated to have
predetermined levels of expression of the intracellular enzyme,
indoleamine 2,3-dioxygenase (IDO). Because expression of high
levels of IDO is correlated with a reduced ability to stimulate T
cell responses and an enhanced ability to induce immunologic
tolerance, APCs having high levels of IDO may be used to increase
tolerance in the immune system, as for example in transplant
therapy or treatment of autoimmune disorders. For example, APCs
having high levels of IDO, and expressing or loaded with at least
one antigen from a donor tissue may be used to increase tolerance
of the recipient to the donor's tissue. Alternatively, APCs having
reduced levels of IDO expression and expressing or loaded with at
least one antigen from a cancer or infectious pathogen may be used
as vaccines to promote T cell responses and increase immunity.
Inventors: |
MUNN; DAVID H.; (AUGUST,
GA) ; MELLOR; ANDREW L.; (AUGUSTA, GA) ; LEE;
JEFFREY ROBERTS; (MARTINEZ, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GEORGIA HEALTH SCIENCES UNIVERSITY |
August |
GA |
US |
|
|
Assignee: |
GEORGIA HEALTH SCIENCES
UNIVERSITY
AUGUST
GA
|
Family ID: |
37567953 |
Appl. No.: |
13/840377 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13397152 |
Feb 15, 2012 |
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13840377 |
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12798307 |
Apr 1, 2010 |
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13397152 |
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11474144 |
Jun 23, 2006 |
7763251 |
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12798307 |
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10121909 |
Apr 12, 2002 |
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11474144 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/564 20130101; C12N 5/064 20130101; C12Q 1/6886 20130101;
C12Q 2600/118 20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 5/0784 20060101
C12N005/0784 |
Goverment Interests
FEDERAL SUPPORT
[0002] The studies described herein were supported at least in part
by Federal grants from the National Institutes of Health (NIH RO1
HL60137; NIH RO1 HL57930; NTH RO1 AI44219; NIH R21 AI49849; NIH R21
AI44759; and NIH K08 HL03395), the National Institutes of Health
and National Cancer Institute (NIH/NCI/RAID) and the Mason Trust
Foundation. Thus, the Federal government may have rights in this
invention.
Claims
1. An isolated population of immunosuppressive dendritic APCs
wherein said dendritic APCs express IDO, CD1c and CD123 wherein
said dendritic APCs are able to suppress T cell proliferation.
2. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express one or more of
CCR6, CD83, and CD11b.
3. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CCR6.
4. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CD83.
5. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CD11b.
6. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express two or more of
CCR6, CD83, and CD11b.
7. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CCR6 and
CD83.
8. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CCR6 and
CD11b.
9. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein said dendritic APCs further express CD83 and
CD11b.
10. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein the dendritic APCs do not express BDCA-2.
11. The isolated population of immunosuppressive dendritic APCs of
any one of claims 2-9 wherein the dendritic APCs do not express
BDCA-2.
12. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein the dendritic APCs do not express CD14.
13. The isolated population of immunosuppressive dendritic APCs of
any one of claims 2-9 wherein the dendritic APCs do not express
CD14.
14. The isolated population of immunosuppressive dendritic APCs of
claim 1 wherein the dendritic APCs do not express CD14 or
BDCA-2.
15. The isolated population of immunosuppressive dendritic APCs of
any one of claims 2-9 wherein the dendritic APCs do not express
CD14 or BDCA-2.
16. An isolated population of immunosuppressive dendritic APCs
wherein said dendritic APCs express IDO, CD11c, CD123, CD83, CCR6
and CD11b wherein said dendritic APCs do not express CD14, BDCA-2
and wherein said dendritic APCs are able to suppress T cell
proliferation.
Description
STATEMENT OF RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/474,144, filed Jun. 23, 2006, which is a
divisional of U.S. patent application Ser. No. 10/121,909, filed
Apr. 12, 2002, now abandoned. U.S. patent application Ser. Nos.
11/474,144 and 10/121,909 are incorporated by reference herein in
their entireties.
FIELD OF THE INVENTION
[0003] The invention relates to the use of cell-based
pharmaceuticals, and more specifically, to the use of
antigen-presenting cells (APCs) selected as comprising
immunosuppressive APCs for inducing tolerance, or immunostimulatory
APCs for inducing an increased immune response. As examples,
immunosuppressive APCs may be used as transplant therapeutics,
whereas preparations of immunostimulatory APCs may be used as
anti-cancer or anti-viral vaccines.
BACKGROUND OF THE INVENTION
[0004] Once established, human tumors are not rejected by the
immune system, a state of functional tolerance which eventually
proves fatal to the host (Smyth, M. J., et al., Nat. Immunol. 2,
293 (2001)). Evidence from murine models suggests that immunologic
unresponsiveness may arise when tumor-associated antigens are
presented by certain bone marrow-derived tolerogenic
(tolerance-producing) antigen-presenting cells (APCs) (Sotomayor,
E. M., et al., Blood, 98: 1070-1077 (2001); Doan, T., et al.,
Cancer Res., 60: 2810-2815 (2000)). In the setting of tissue
transplantation, it would be desirable to isolate and administer
such tolerogenic APCs. However, in humans and other mammals (other
than mice), the identity of these APCs, and the mechanisms they use
to induce tolerance, remain elusive.
[0005] In humans, "immature" myeloid dendritic cells (DCs) have
been postulated to function as tolerizing APCs based on findings
that these cells: (1) have a decreased ability to stimulate T cell
responses in vitro (Reddy, A., et al., Blood, 90: 3640-3646 (1997);
Jonuleit, H., et al., Eur. I Immunol., 27: 3135-3142 (1997)); (2)
may promote the function of immunosuppressive or "regulatory" T
cells following prolonged co-incubation (Jonuleit, H., et al.,
Trends Immunol., 22: 394-400 (2001)); and (3) have the ability to
abrogate antigen-specific T cell responses in vivo (Dhodapkar, M
N., et al., J. Exp. Med., 193: 233-238 (2001); see also U.S. Pat.
Nos. 5,871,728 and 6,224,859). However, the molecular mechanism
used by immature DCs or other putative tolerogenic APCs to suppress
T cell responses is unclear. Moreover, there is currently no way to
identify or isolate tolerogenic APCs in vitro or in vivo, and thus,
their use as therapeutic agents is still not available for most
applications.
[0006] More fundamentally, the supposition that immature DCs are
tolerogenic is based on an unproven and potentially flawed model of
how APCs regulate T cell activation. Thus, a prevailing model
teaches that T cells are rendered unresponsive (or "tolerized")
when they receive an activation signal (signal 1) via the T cell
antigen receptor (TCR) without receiving co-stimulatory signals
(e.g. from CD80 and CD86) delivered on APCs (signal 2). Immature
DCs express low levels of TCR ligands (such as MHC class II
antigens) and low levels of the putative costimulatory molecules.
Thus, the model teaches that immature of DCs are unable to activate
T cells because T cells receive signal 1 without adequate signal
2.
[0007] Other findings teach against the prevailing model, and
indicate that maturation of DCs is not necessarily associated with
abrogation of T cell suppression and/or tolerance (Albert, M. L.,
Nature Immunol., 2: 1010 (2001); Shortman, K. et al., Nature
Immunol., 2: 988-989 (2001); T. Bankenstein and T. Schuler, Trends
in Immunol., 23: 171-173 (2002)). Instead, there may be a third, as
yet undefined signal (signal 3) that acts after T cells have
received the signals of antigen presentation and co-stimulation
(i.e. signals 1 and 2) from a fully mature APC. The third signal
then diverts T cells to activation or tolerance. In this model, the
tolerogenic phenotype is independent of the maturation status of
the APC (in fact, maturation enhances tolerance induction) and
depends instead on an intrinsic attribute of the APC (i.e. whether
it expresses signal 3).
[0008] The inventors believe that most DC preparations are in fact
mixtures of immunizing (stimulatory) and tolerizing APCs. The
presence of a mixed population of DCs in such preparations would
explain why therapeutic immunization in cancer patients using DCs
remains problematic, with most studies having only limited success
(M. A. Morse and H. K. Lyerly, Curr. Opin. Mol. Ther., 2: 20
(2000)). For example, the preferred source and differentiation
status of DCs for clinical use remains controversial (Curiel T. J.,
and Curiel, D. T., J. Clin. Invest., 109: 311-312, 2002). Although
development of the field has been assisted by the recognition that
the maturation state of human DCs plays an important role in their
ability to stimulate effective immunity (Dhodapkar, M. V., et al.,
J. Clin. Invest., 105: R9-R14 (2000); Dhodapkar, M. V., et al., J.
Exp. Mea., 193: 233-238 (2001)), even using the best isolation and
maturation strategies and multiple tumor antigens, clinically
useful therapeutic immunization in patients with established tumors
has been only partially effective (Banchereau, I, et al., Cancer
Res., 61: 6451-6458 (2001)). Thus, it would be useful to develop
methods to isolate DCs which, rather than being a mixed population
of activating and suppressive DCs, comprise pure activating
DCs.
[0009] Conversely, these are some situations where increased
tolerance to foreign antigens is desired. In one approach, immature
dendritic cells (DCs) uncharacterized as suppressive or immunogenic
subsets are propagated in the presence of a cytokine regimen to
maintain the cells in an immature state. The immature cells are
administered to a host in advance of a transplant to enhance
tolerance (U.S. Pat. Nos. 5,871,728 and 6,224,859). However, this
approach inherently sacrifices efficient antigen presentation and
co-stimulation due to the immaturity of the APCs, and risks
delivering unwanted immunizing (non-tolerogenic) DCs as part of the
heterogeneous DC population. It would be helpful in transplant
therapeutics to be able to create well-characterized populations of
mature maximally effective tolerogenic APCs which present the
antigen subset of interest, but in a tolerizing
(tolerance-promoting) preparation.
[0010] What is needed is a way to separate tolerance-inducing APCs
from other (nontolerance-inducing) APCs. The tolerance-inducing
APCs can then be used in transplant procedures to promote tolerance
to specific donor antigens. The non-tolerance-inducing APCs can be
used in conjunction with undesirable foreign antigens (such as
tumor antigens) as a vaccine, to prime the recipient immune system
against the antigen in question.
SUMMARY OF THE INVENTION
[0011] The present invention relies on the discovery that
tolerance-inducing (suppressive) antigen-presenting cells (APCs)
exhibit high levels of expression of the intracellular enzyme
indoleamine-2,3-dioxygenase (IDO), and non-tolerance-inducing
(non-suppressive or T-cell activating) APCs exhibit low levels of
IDO expression. IDO is both a marker for the suppressive subset,
and also the causal mechanism of suppression. Thus, the present
invention describes the generation of enriched populations of
tolerance-inducing APCs and their use as therapeutics, and the
generation of enriched populations of non-suppressive APCs and
their use as therapeutics. For example, APCs having high levels of
IDO (IDO.sup.+), and exposed to antigens from a donor may be used
to increase tolerance of a transplant recipient to the donor's
tissue by presenting the donor's antigens on tolerance-inducing
APCs. Conversely, APCs having low levels of IDO) (IDO.sup.LO) may
be used to enhance responses to neo-antigens from tumors and
infectious agents.
[0012] Thus, in one aspect, the present invention comprises a
method of making antigen-presenting cells (APCs) for enhancing T
cell tolerance comprising the steps of:
[0013] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject; and
[0014] (b) treating the cells to select for tolerance-inducing APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity sufficient to suppress proliferation of T cells (IDO.sup.+
APCs).
[0015] In another aspect, the present invention comprises a method
for increasing the number of tolerance-inducing antigen-presenting
cells (APCs) in a subject comprising treating the subject to
increase the production of antigen-presenting cells (APCs) or their
precursors (APC progenitors) expressing levels of indoleamine
2,3-dioxygenase (IDO) enzyme activity sufficient to suppress
proliferation of T cells (IDO.sup.+ APCs).
[0016] In another aspect, the present invention comprises a method
for enhancing tolerance in a subject comprising the steps of:
[0017] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject;
[0018] (b) treating the cells to select for tolerance-inducing APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity sufficient to suppress proliferation of T cells (IDO.sup.+
APCs); and
[0019] (c) administering the treated cells of step (b) to the
original subject or to a second subject in an amount effective to
generate a tolerance-promoting immune response in the recipient
subject.
[0020] The present invention also provides compositions for
enhancing T cell tolerance comprising APCs that express high levels
of indoleamine 2,3-dioxygenase (IDO) enzyme activity (IDO.sup.+
APCs). Such tolerizing APCs may be used to promote acceptance of
graft or transplant tissue from a donor subject in a recipient.
IDO+ APCs may be made by the methods described herein, or by other
methods in the art. Thus, in one aspect, the present invention
comprises isolated antigen-presenting cells (APCs) selected as
comprising APCs expressing levels of indoleamine 2,3-dioxygenase
(IDO) enzyme activity sufficient to suppress proliferation of T
cells (IDO.sup.+ APCs). In another aspect, the present invention
comprises an isolated antigen-presenting cell selected as
comprising expression of indoleamine 2,3-dioxygenase (IDO) enzyme
activity at a level sufficient to suppress proliferation of T
cells. In yet another aspect, the present invention comprises
antigen-presenting cells comprising expression of indoleamine
2,3-dioxygenase (IDO) enzyme activity at a level sufficient to
suppress proliferation of T cells (IDO.sup.+ APCs) made by the
methods of the present invention.
[0021] Alternatively, the present invention describes the
generation of immunostimulatory (non-tolerance-inducing) APCs
having reduced IDO expression (IDO.sup.LO APCs). APCs having
reduced levels of IDO expression and exposed to antigens expressed
by a tumor or pathogen (such as HIV) may be used as vaccines, by
presenting cancer or pathogen antigens on APCs which contain fewer
tolerance-inducing APCs.
[0022] Thus, in this aspect, the present invention comprises a
method of making antigen-presenting cells (APCs) for enhancing T
cell dependent immunologic activation in a subject comprising the
steps of:
[0023] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a subject; and
[0024] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs).
[0025] In another aspect, the present invention comprises a method
for increasing the number of non-suppressive antigen-presenting
cells (APCs) in a subject comprising treating said subject to
increase the population of antigen-presenting cells (APCs) or their
precursors (APC progenitors) expressing levels of indoleamine
2,3-dioxygenase (IDO) enzyme activity not sufficient to cause
suppression of T cell proliferation (IDO.sup.LO APCs).
[0026] In another aspect, the present invention comprises a method
for increasing the protective immune response in a subject
comprising the steps of:
[0027] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject;
[0028] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs); and
[0029] (c) administering the treated cells from step (b) to the
subject in an amount effective to generate a protective immune
response in the subject.
[0030] The present invention also provides compositions for
increasing T cell activation. Such compositions may be used to
increase the T cell response to antigens in a subject. In this
aspect, the present invention comprises isolated antigen-presenting
cells (APCs) selected as comprising APCs expressing levels of
indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient to
cause suppression of T cell proliferation (IDO.sup.LO APCs). In
another aspect, the present invention comprises an isolated
antigen-presenting cell (APC) selected as comprising levels of
indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient to
cause suppression of T cell proliferation. In yet another aspect,
the present invention comprises antigen-presenting cells (APCs)
comprising levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO) APCs) made by the methods of the present
invention.
[0031] The present invention also describes methods to quantitate
the levels of immunosuppressive APCs in a population of APCs. Thus,
in one aspect, the present invention comprises a method to
determine the number of tolerance-inducing antigen-presenting cells
(APCs) in a cell population comprising measuring the number of
cells expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
sufficient to suppress proliferation of T cells (IDO.sup.+ APCs) in
the population. In another aspect, the present invention comprises
a kit for determining the number of tolerance-inducing
antigen-presenting cells (APCs) in a cell population comprising
reagents to measure levels of indoleamine 2,3-dioxygenase (IDO)
enzyme in the population of APCs, wherein the reagents are packaged
in at least one individual container.
[0032] The immunosuppressive APCs may also be quantified using a
biological assay. Thus, in another aspect, the present invention
comprises a method to quantify the ability of a population of cells
to suppress T cell proliferation comprising measuring the increase
in T cell proliferation in the presence of an IDO inhibitor as
compared to in the absence of an IDO inhibitor. The present
invention also comprises a kit for determining the ability of a
population of antigen-presenting cells to suppress T cell
proliferation comprising an IDO inhibitor packaged in at least one
individual container.
[0033] Additionally, the present invention provides for a
diagnostic assay, based on detection of IDO.sup.+ APCs and/or
mip-3.alpha. expression in tumors and tumor-draining lymph nodes.
In this aspect, the present invention comprises a method for
assessing the relative risk of tumor progression in a subject
comprising the steps of:
[0034] (a) assaying a sample of tissue from a tumor or tumor
draining lymph node from a subject for expression of the enzyme
indoleamine 2,3-dioxygenase (IDO); and
[0035] (b) correlating the risk of tumor progression to IDO
expression in the tissue sample, wherein IDO expression is
positively correlated with an increase in the risk of tumor
progression.
[0036] The present invention also comprises a method for assessing
the risk of tumor progression in a subject comprising the steps
of:
[0037] (a) assaying a sample of tissue from a tumor or tumor
draining lymph nodes from a subject for mip-3.alpha. expression;
and
[0038] (b) correlating the risk of tumor progression to
mip-3.alpha. expression in the tissue sample, wherein mip-3.alpha.
expression is positively correlated with an increase in the risk of
tumor progression.
[0039] The present invention also comprises kits for assessing the
relative risk of tumor progression in a subject. For example, in
one aspect, the present invention comprises a kit for assessing the
relative risk of tumor progression in a subject comprising reagents
for detection of the enzyme indoleamine 2,3-dioxygenase (IDO) in a
sample of tissue from a tumor or tumor draining lymph node from a
subject, wherein the reagents are packaged in at least one
individual container. In another aspect, the present invention
comprises a kit for assessing the relative risk of tumor
progression in a subject comprising reagents for detection of
relative levels of expression of mip-3.alpha. in a sample of tissue
from a tumor or tumor draining lymph node from a subject, wherein
the reagents are packaged in at least one individual container.
[0040] The foregoing focuses on the more important features of the
invention in order that the detailed description which follows may
be better understood and in order that the present contribution to
the art may be better appreciated. There are, of course, additional
features of the invention which will be described hereinafter and
which will form the subject matter of the claims appended hereto.
It is to be understood that the invention is not limited in its
application to the specific details as set forth in the following
description and figures. The invention is capable of other
embodiments and of being practiced or carried out in various
ways.
[0041] From the foregoing summary, it is apparent that an object of
the present invention is to provide methods and compositions for
enriching and isolating IDO.sup.+ antigen-presenting cells for use
in therapeutic applications such as the prevention of transplant
rejection. In addition, it is apparent that an object of the
present invention is to provide methods and compositions for
isolating antigen-presenting cells depleted of IDO.sup.+
antigen-presenting cells (i.e. IDO.sup.LO APCs) comprising reduced
suppression or tolerance, as for example in cancer prevention and
therapy. These, together with other objects of the present
invention, along with various features of novelty which
characterize the invention, are pointed out with particularity in
the claims and description provided herein.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 shows a schematic representation of a 3-step model
for the regulation of IDO during dendritic cell (DC)
differentiation in accordance with an embodiment of the present
invention.
[0043] FIG. 2 shows a schematic representation of methods to
generate subsets of peripheral blood-derived APCs which are either:
(1) enriched in tolerance-inducing APCs, as for example, for use in
transplant therapy; or (2) depleted of tolerance-inducing APCs, as
for example, for use in anti-cancer vaccines, in accordance with an
embodiment of the present invention.
[0044] FIG. 3 shows a schematic representation of
tolerance-inducing antigen-presenting cells (APCs) comprising
expression of intracellular indoleamine 2,3-dioxygenase (IDO), cell
surface markers CD123, CD11c, and the chemokine receptor CCR6
juxtaposed next to tumor cells that express mip-3.alpha., in
accordance with an embodiment of the present invention.
[0045] FIG. 4 shows the regulation of IDO during maturation in
accordance with an embodiment of the present invention wherein (A)
shows DCs cultured in GMCSF+IL4 for 7 days with and without the
addition of 1-methyl-(D)-tryptophan; (B) shows the same DCs matured
with a cocktail of cytokines (IL 1.beta., TNF.alpha., IL6,
prostaglandin E2 (PGE2)); and (C) shows the same DCs matured with
monocyte-conditioned medium. In all groups there is significant
IDO-mediated suppression.
[0046] FIG. 5 shows expression of CD123, the chemokine receptor
CCR6, and indoleamine 2,3-dioxygenase (IDO) by antigen-presenting
cells in accordance with an embodiment of the present invention. In
panels (A) and (B), human monocytes were cultured to produce
myeloid dendritic cells (A) or macrophages (B), and then both
groups received interferon-.gamma. during the final 18 hrs of
culture and harvested cells were triple-stained for CD123, CD11c
and IDO. In (A) and (B), panels on the right show expression of IDO
and CD123 in the gated CD11c.sup.+ population shown on the left. In
(C) myeloid dendritic cells, cultured as in panel (A), were
triple-stained for CD123, IDO, and the chemokine receptor CCM Both
panels show the entire (ungated) population. In (D), the adherent
(non-dendritic) population of APCs is shown, taken from a culture
similar to panel (A) but using serum-free conditions. Cells were
stained for IDO and CD123. Panel (E) compares IDO-mediated
suppression by DCs and non-dendritic APCs from the same culture
where IDO-mediated suppression is the difference in thymidine
incorporation in T cells in the absence (stippled bars) vs. the
presence (striped bars) of 1-methyl-(D,L)-tryptophan (1-MT).
[0047] FIG. 6 shows suppression of allogeneic T cell proliferation
by indoleamine 2,3-dioxygenase/CD123 expressing
(IDO.sup.+/CD123.sup.+) dendritic cells in accordance with an
embodiment of the present invention. Panel (A) shows myeloid
dendritic cells which were activated for 24 hrs with TNF.alpha.,
and labeled with anti-CD123 antibody and enriched by sorting
(CD123.sup.+) with goat anti-mouse secondary antibody conjugated to
magnetic beads (immunosorting), wherein the left panel shows the
population prior to enrichment and the right panel shows the
population after enrichment. Panel (B) shows a comparison of the
effect of CD123.sup.+ enriched and CD123.sup.+ depleted cells on
allogeneic T cell proliferation as measured in a mixed-leukocyte
reaction by thymidine incorporation in the absence (.cndot.) or the
presence (.quadrature.) of 1-methyl-(D,L)-tryptophan (1-MT; an
inhibitor of IDO). Panel (C) shows experiments similar to panel
(B), using 3 different pairs of donors, each allogeneic to the
other, and each pair pre-tested to produce an active allogenic
mixed leukocyte reaction (MLR) using sorted CD123.sup.+ cells
without (.box-solid.) or with (.quadrature.) 1-MT.
[0048] FIG. 7A-B shows that sorting to generate a population of
cells enriched for CD123 expression (CD123.sup.+) by immunosorting
results in APCs are enriched for cells having high levels of IDO
expression (IDO.sup.+ APCs) in accordance with an embodiment of the
present invention.
[0049] FIG. 8 shows detection of IDO-expressing (IDO.sup.+)
CD123.sup.+ dendritic cells in human tumors and draining lymph
nodes in accordance with an embodiment of the present invention.
Panel (A) shows a positive control for IDO (brown) in
syncytiotrophoblast cells of term human placenta (inset: the same
tissue, but with anti-IDO antibody neutralized by an excess of the
immunizing peptide and shown at half scale). Panel (B) shows a
malignant melanoma primary cutaneous tumor stained for IDO (arrows)
(Fast Red chromogen). Panel (C) shows a draining lymph node of a
malignant melanoma, showing accumulation of IDO-expressing cells
(red) in the lymphoid and perivascular regions of the node, but
sparing the macrophage-rich sinuses (asterisk). Panel (D) shows a
higher magnification of panel (C), with a characteristic collection
of IDO-expressing cells (dark signal) around a high-endothelial
venule (V). Panel (E) shows a low-power view of a draining lymph
node containing heavily pigmented metastatic melanoma cells
(endogenous melanin, black; darkest signal), with confluent
infiltration of IDO-expressing cell (red; next darkest signal)
around the tumor deposits. Panel (F) shows normal lymphoid tissue
with scattered IDO.sup.+ cells (red; scattered dark signals) in a
germinal center (GC) and T cell regions (T) of a human pharyngeal
tonsil from a routine tonsillectomy. Panels (G) and (H) (higher
magnification of the region in panel (G) indicated by the arrow)
shows co-localization of cells expressing IDO (brown; darkest
cytoplasmic signal) and mip-3.alpha. (red; next darkest cytoplasmic
signal) in the lamina propria of the small intestine, particularly
in the subepithelial areas overlying mucosal lymphoid aggregates
(LA). Panels (I) and (J) (higher magnification of the region in
panel (I) indicated by the arrow) shows expression of mip-3.alpha.
(red) by tumor cells in a lesion of malignant melanoma metastatic
to lymph node, such that the mip-3.alpha..sup.+ cells are scattered
throughout the tumor (arrow) (T), while the IDO.sup.+ (brown) cells
are congregated at the margins of the metastasis but confined to
the residual lymph node tissue (LN).
[0050] FIG. 9 shows expression of mip-3.alpha. mRNA by human tumors
in accordance with an embodiment of the present invention. RNA from
melanomas (M, n=18), renal cell carcinomas (R, n=19) or non-small
cell lung cancers (L, n=9) was analyzed for expression of
mip-3.alpha. by quantitative PCR calculated as the ratio of
mip-3.alpha. to the 25 GAPDH housekeeping gene in each sample.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention describes the isolation of
myeloid-derived antigen-presenting cells (APCs) which are enriched
for tolerance-inducing APCs, or depleted of tolerance-inducing
APCs, and the use of these cells for various therapeutic
applications. The present invention relies on the discovery that
antigen-presenting cells may be separated into a tolerance-inducing
population, which is associated with high levels of expression of
the enzyme indoleamine-2,3-dioxygenase (IDO), and a T cell
activating (non-tolerance-inducing) population, which is associated
with low levels of expression of IDO. For example, APCs having high
levels of IDO (IDO.sup.+ APCs), and constitutively expressing or
exposed to donor tissue antigens may be used to increase tolerance
of the recipient to the donor's tissue in transplant therapy by
presenting the antigens on tolerance-inducing APCs. Alternatively,
APCs having reduced levels of IDO expression (IDO.sup.LO APCs) and
exposed to antigens expressed by cancer tissue or virus may be used
as anti-cancer vaccines or anti-viral vaccines, respectively, by
presenting the antigens on APCs depleted on tolerance-inducing
cells.
[0052] Thus, the current invention teaches that conventional
preparations of human APCs can contain two independent subsets: an
IDO.sup.+ subset (comprising relatively high levels of IDO
expression); and an IDO.sup.LO subset (comprising little to no IDO
expression). Moreover, which of these two types of APC predominates
is highly (and in some cases unpredictably) dependent on the
culture conditions or other variables. In many applications, even a
minor contaminating admixture of the undesired type of APC (i.e.
IDO.sup.LO vs. IDO.sup.+) may render the APC population unusable,
or even harmful, for the desired application. For example, if the
goal is to generate tolerance toward donor histocompatability
antigens prior to organ transplantation, exposure to even a
minority of activating dendritic cells could promote worsened
rejection. Conversely, if the goal is to enhance responses to weak
tumor antigens, the presence of even a minor population of IDO+
tolerance-inducing cells may be enough to suppress the desired
response (see e.g., Grohmann, U., et al., J. Immunol. 167: 708-714
(2001), for studies in murine model).
[0053] Thus, in one aspect, the present invention describes a
method of making antigen-presenting cells (APCs) for enhancing T
cell tolerance comprising the steps of:
[0054] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject; and
[0055] (b) treating the isolated cells to select for
tolerance-inducing APCs expressing levels of indoleamine
2,3-dioxygenase (IDO) enzyme activity sufficient to suppress
proliferation of T cells (IDO.sup.+ APCs).
[0056] Preferably, the tolerance-inducing IDO.sup.+ APCs comprise
at least 90% of the APC population expressing IDO at levels of at
least 2-fold over background. More preferably, the
tolerance-inducing IDO.sup.+ APCs comprise at least 95% of the APC
population expressing IDO at levels of at least 2-fold over
background.
[0057] Alternatively, IDO.sup.+ APCs may be quantitated by
measuring the biological activity of the preparation. Thus, in an
embodiment, the tolerance-inducing IDO.sup.+ APCs comprise
suppressor activity, comprising an at least a 2-fold increase in T
cell proliferation in the presence of an IDO inhibitor as compared
to in the absence of an IDO inhibitor. Suppressor activity may be
measured using a mixed leukocyte reaction or similar assay of T
cell proliferation. Preferably, the IDO inhibitors comprise
1-methyl-(D,L)-tryptophan, .beta.-(3-benzofuranyl)-(D,L)-alanine,
.beta.-(3-benzo(b)thienyl)-(D,L)-alanine, or
6-nitro-(D,L)-tryptophan. More preferably, the IDO inhibitors
comprise 1-methyl-(D)-tryptophan or 6-nitro-(D)-tryptophan.
[0058] In an embodiment, the isolated APCs or APC progenitors
comprise mature blood-derived dendritic cells, mature tissue
dendritic cells, monocyte-derived macrophages, non-dendritic APCs,
B cells, plasma cells, or any mixture thereof. Preferably, the
isolated APCs or APC progenitors comprise a cell type bearing
markers of antigen presentation and costimulatory function. Also
preferably, the APCs or APC progenitors are isolated from
peripheral blood, bone marrow, lymph nodes or a solid organ from a
human or other mammal.
[0059] The treatment to select for IDO.sup.+ APCs may comprise
predetermined culture conditions or physical selection. Preferably,
step (b) comprises culturing the cells in medium which is
essentially free of serum. Also preferably, step (b) comprises
culturing the cells in the presence of granulocyte-macrophage
colony stimulating factor (GMCSF). Step (b) may also comprise
culturing the cells in the presence of macrophage colony
stimulating factor (MCSF). In addition, step (b) may comprise
culturing the cells in the presence of IL4. Step (b) may also
comprise culturing the cells in the presence of TGF.beta. and/or
IL10. For example, in an embodiment, a cytokine cocktail such as
those known in the art (Jonuleit, H., et al., Eur. J. Immunol., 27:
3135-3142 (1997)) may be employed.
[0060] In an embodiment, step (b) also comprises culturing the
cells with an agent to cause or regulate maturation of those APCs
that express high levels of IDO. Such maturation agents may
include, but are not limited to TNF.alpha., IL10, TGF.beta.,
CD40-ligand, activating anti-CD40 antibodies, cells engineered to
express cell-surface CD40-ligand, proinflammatory bacterial or
pathogen products, or any combination thereof. Thus, for selection
of IDO.sup.+ APCs these agents may be combined singly, or added
together with other agents used for the maturation of DCs
(Jonuleit, H., et al., Eur. J. Immunol. 27: 315-3142 (1997); Reddy,
A., et al., Blood 90: 3640-3646 (1997).
[0061] In an embodiment, step (b) may comprise genetically
modifying the APCs or APC progenitors such that the final
preparation comprises APCs expressing levels of indoleamine
2,3-dioxygenase (IDO) enzyme activity sufficient to suppress
proliferation 10 of T cells (IDO.sup.+ APCs). As an example,
transfection of the culture of APCs or APC progenitors with a gene
for a cytokine or ligand may be employed (Kikuchi, T., et al.,
Blood 98: 91-99 (2000); Gorckynski, R., et al., Transplantation
Proceedings 33: 15651566 (2001); Morita, Y., et al., J. Clin.
Invest., 107: 1275-1284 (2001)).
[0062] In an embodiment, the method utilizes cell surface proteins
or other markers for separation (enrichment or depletion) of
IDO.sup.+ cells from other APCs. Thus, in an embodiment, the method
includes measuring expression of at least one cell surface marker
that identifies the APCs as expressing levels of IDO sufficient to
suppress T cell proliferation (IDO.sup.+ APCs) or as expressing
levels of IDO not sufficient to suppress T cell proliferation
(IDO.sup.LO APCs). Preferably, the cell surface marker is used to
separate IDO.sup.+20 APCs from IDO.sup.LO APCs. The markers used
for differential selection of IDO.sup.+ cells from IDO.sup.LO cells
include, but are not limited to, CD123, CD11c, CCR6, CD14 or any
combination thereof. Alternatively, the method may include
differential adhesion to a substrate to separate APCs that
expressing levels of IDO sufficient to suppress T cell
proliferation (IDO.sup.+ APCs) from APCs expressing levels of IDO
not sufficient to suppress T cell proliferation (IDO.sup.LO
APCs).
[0063] One object of the present invention is to develop
tolerance-promoting APCs that present a specific subset of antigens
of interest. For example, tolerance-promoting ACPs that present
antigens from a donor may be administered to a transplant recipient
to promote acceptance of a graft or transplant. Thus, in an
embodiment, the subject from which the APCs or APC progenitors are
isolated comprises a tissue donor to a second subject. In another
embodiment, the APCs or APC progenitors are isolated from a subject
with an autoimmune disorder for subsequent preparation of IDO.sup.+
APCs for use in treating the disorder.
[0064] In addition, the treated APCs may be exposed to at least one
source of antigen after isolation from a subject and treatment to
select for IDO.sup.+ APCs. In an embodiment, the antigen comprises
a purified, or a synthetic or recombinant polypeptide representing
a specific antigen to which it is desired that tolerance be
induced, or a short synthetic polypeptide fragment derived from the
amino acid sequence of such an antigen. Preferably, the source of
antigen comprises antigens expressed by a donor tissue graft. Also
preferably, the source of antigen comprises protein or other
material to which a patient has an autoimmune disorder (see e.g.
Yoon, J.-W., et al., Science 284: 1183-1187 (1999) for examples of
such proteins). In yet another embodiment, the method comprises
transfecting or genetically engineering the IDO.sup.+ APCs to
express at least one antigenic polypeptide.
[0065] The tolerance-inducing APCs or their precursors (or
non-tolerance inducing APCs or their precursors) as defined by the
methods of the present invention may also be increased in number in
a subject by administering to the subject agents that increase the
number of the desired APCs. Numerous cytokines and other agents
have been shown to increase the number of one or more of different
types of APCs when administered in vivo. Examples of such agents
include MCSF, GMCSF, granulocyte colony-stimulating factor (GCSF),
FLT3-ligand, and other natural and artificial cytokines and
hematopoietic growth factors. Previously, however, it was not known
whether the APCs induced by such treatments were tolerance-inducing
or non-tolerance-inducing or a mixture of both. The present
invention provides the discovery that by measuring IDO expression
following isolation and in vitro treatment of the desired APC
population, the effectiveness of such in vivo treatments can be
evaluated and improved upon. In addition, as described herein, the
present invention provides methods to quantify IDO expression, both
on a cell-by-cell basis and as a biological assay for bulk
populations. Thus, in an embodiment, tolerogenic APCs or their
precursors in peripheral blood from a donor may be increased by
treatment with selected cytokines prior to isolation for in vitro
culture and delivery to a recipient for the purpose of inducing
transplantation tolerance.
[0066] Thus, in one aspect, the present invention comprises a
method for increasing the number of tolerance-inducing
antigen-presenting cells (APCs) in a subject comprising treating
the subject to increase the production of APCs or their precursors
(APC progenitors) expressing levels of indoleamine 2,3-dioxygenase
(IDO) enzyme activity sufficient to suppress proliferation of T
cells (IDO.sup.+ APCs).
[0067] In another aspect, the present invention comprises a method
for enhancing tolerance in a subject comprising the steps of:
[0068] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject;
[0069] (b) treating the cells to select for APCs expressing levels
of indoleamine 2,3-dioxygenase (IDO) enzyme activity sufficient to
suppress proliferation of T cells (IDO.sup.+ APCs); and
[0070] (c) administering the treated cells of step (b) to the
original subject or to a second subject in an amount effective to
generate a tolerance-promoting response in said recipient
subject.
[0071] Preferably, the tolerance-promoting response reduces T cell
activation in the recipient subject. Also preferably, the
tolerance-promoting response prolongs the survival of transplanted
cells or tissues in the recipient subject. Also preferably, the
tolerance-promoting response reduces the symptoms of an autoimmune
disease in the recipient subject.
[0072] In an embodiment, the subject from which the APCs or APC
progenitors are isolated comprises a tissue donor to the recipient
subject. In another embodiment, the subject from which the APCs or
APC progenitors are isolated comprises a mammal with an autoimmune
disorder.
[0073] The method may include exposing the APCs or APC progenitors
to at least one source of antigen after isolation from the first
subject and treatment to select for IDO.sup.+ APCs. Preferably, the
antigen comprises a synthetic or natural polypeptide. Also
preferably, the antigen comprises at least one antigen expressed by
a donor tissue graft. Alternatively, the antigen may comprise at
least one antigen to which the recipient subject has an autoimmune
disorder. In another embodiment, the method may comprise
transfecting or genetically engineering the APCs selected as
IDO.sup.+ to express at least one antigenic polypeptide.
[0074] The present invention also provides compositions for
enhancing T cell tolerance. Such tolerizing APCs may be used to
promote acceptance of graft or transplant tissue from a donor
subject in a recipient or to treat a patient with autoimmune
disease. In this aspect, the present invention comprises isolated
antigen-presenting cells (APCs) selected as comprising APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity sufficient to suppress proliferation of T cells (IDO.sup.+
APCs). In another aspect, the present invention comprises an
isolated antigen-presenting cell selected as comprising expression
of indoleamine 2,3-dioxygenase (IDO) enzyme activity at a level
sufficient to suppress proliferation of T cells. In yet another
aspect, the present invention comprises antigen-presenting cells
comprising expression of indoleamine 2,3-dioxygenase (IDO) enzyme
activity at a level sufficient to suppress proliferation of T cells
(IDO.sup.+ APCs) made by the methods of the invention.
[0075] Preferably, the isolated IDO.sup.+ APCs comprise at least
90% of the APC population expressing IDO at levels of at least
2-fold over background. More preferably, the isolated IDO.sup.+
APCs comprise at least 95% of the APC population expressing 1D0 at
levels of at least 2-fold over background. Also preferably, the
isolated IDO.sup.+ APCs comprise suppressor activity comprising an
at least a 2-fold increase in T cell proliferation in the presence
of an DO inhibitor as compared to in the absence of an IDO
inhibitor. In an embodiment, the isolated 1D0.sup.+ APCs express at
least one antigenic polypeptide.
[0076] In an embodiment, the isolated cells comprise at least one
cell surface marker that identifies the cells as expressing levels
of indoleamine 2,3-dioxygenase (IDO) enzyme activity sufficient to
suppress T cell proliferation (IDO.sup.+ APCs). Preferably, the
marker comprises CD123, CD11c and CCR6.
[0077] In an embodiment, the composition of the present invention
includes a pharmaceutically acceptable carrier. Also preferably,
the composition of the present invention includes one or more
immunosuppressive pharmaceuticals in a unit dosage form.
[0078] In another aspect, the present invention comprises the
generation of immunostimulatory cells. Such cells may be used to
stimulate the immune response, as for example, to cancer-related
antigens or viral-related antigens. Thus, in this aspect, the
present invention comprises a method of making antigen-presenting
cells (APCs) for enhancing T cell dependent immunologic activation
in a subject comprising the steps of:
[0079] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a subject; and
[0080] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs).
[0081] Preferably, the IDO.sup.LO APCs comprise a population of
APCs having less than 10% of the population expressing IDO at a
level of greater than 2-fold over background. More preferably, the
IDO.sup.LO APCs comprise a population of APCs having less than 5%
of the population expressing IDO at a level of greater than 2-fold
over background. Alternatively, the IDO.sup.LO APCs may be
quantitated using a T cell proliferation assay such as a mixed
leukocyte reaction or similar methods, wherein IDO.sup.LO APCs
comprise an absence of suppressor activity comprising a less than a
1.5-fold increase in T cell proliferation in the presence of an IDO
inhibitor as compared to in the absence of an IDO inhibitor.
Preferably, the IDO inhibitor comprises 1-methyl-(D,L)-tryptophan,
.beta.-(3-benzofuranyl)-(D,L)-alanine,
.beta.-(3-benzo(b)thienyl)-(D,L)-alanine, or
6-nitro-(D,L)-tryptophan. Also preferably, the IDO inhibitor
comprises 1-methyl-(D)-tryptophan or 6-nitro-(D)-tryptophan.
[0082] Preferably, the isolated APCs or APC progenitors comprise
mature blood-derived dendritic cells, mature tissue dendritic
cells, monocyte-derived macrophages, non-dendritic APCs, B cells,
plasma cells, or any mixture thereof. Also preferably, the isolated
APCs or APC progenitors comprise a cell type bearing markers of
antigen presentation and costimulatory function. Also preferably,
the APCs or APC progenitors are isolated from peripheral blood,
bone marrow, lymph nodes or a solid organ from a human or other
mammal.
[0083] The treatment to select for IDO.sup.LO APCs may comprise
predetermined culture conditions or physical selection. In an
embodiment, treatment of APCs and progenitor APCs to select for
IDO.sup.LO APCs comprises culturing the cells in the presence of
serum-free medium. In other embodiments, treatment of APCs and
progenitor APCs to select for IDO.sup.LO APCs comprises culturing
the cells in the presence of MCSF, or GMCSF, or interferon-a, or
combinations thereof. In an embodiment, treatment of APCs and
progenitor APCs to select for IDO.sup.LO APCs comprises culturing
the cells with an agent to cause maturation of those APCs that
express low levels of IDO (mow APCs). Preferably, the maturation
agents comprise TNF.alpha., CD40-ligand (CD40L), activating
anti-CD40 antibodies, cells engineered to express cell-surface
CD40-ligand, proinflammatory bacterial or pathogen products, or any
combination thereof. Alternatively (or additionally), the APCs may
genetically engineered to express CD40-ligand. The treatment may
also comprise culturing the cells in the presence of neutralizing
antibodies for IL10 and/or TGF.beta.. Thus, for selection of
IDO.sup.LO APCs, these agents may be combined singly, or added
together with other agents used for the maturation of DCs
(Jonuleit, H., et al., Eur. J. Immunol. 27: 315-3142 (1997); Reddy,
A., et al., Blood 90: 3640-3646 (1997).
[0084] Alternatively, step (b) may comprise genetically modifying
the APCs or APC progenitors such that the final preparation
comprises APCs expressing levels of indoleamine 2,3-dioxygenase
(IDO) enzyme activity not sufficient to suppress T cell
proliferation (IDO.sup.LO APCs).
[0085] In an embodiment, the method utilizes cell surface proteins
or other markers for selection of IDO.sup.LO cells from other APCs.
Thus, in an embodiment, the method includes measuring expression of
at least one cell surface marker that identifies the APCs as
expressing levels of IDO not sufficient to suppress T cell
proliferation (EDO.sup.Lo) or as expressing levels of IDO
(IDO.sup.+). Preferably, the cell surface marker is used to
separate APCs that express low levels of IDO (IDO.sup.LO APCs) from
APCs that express high levels of IDO sufficient to suppress T cell
proliferation (IDO.sup.+ APCs). The markers used for differential
selection of IDO.sup.LO cells from IDO.sup.+ cells include, but are
not limited to, CD123, CD11c, CCR6, CD14 or any combination
thereof. Alternatively, the method may include differential
adhesion to a substrate to separate APCs that express levels of IDO
not sufficient to suppress T cell proliferation (IDO.sup.LO APCs)
from APCs that express levels of IDO sufficient to suppress T cell
proliferation (IDO.sup.+ APCs).
[0086] One object of the present invention is to develop
immunogenic APCs that present a specific subset of antigens of
interest. For example, APCs depleted of tolerance-inducing cells
(i.e. IDO.sup.LO APCs) may be used to present antigens expressed by
a tumor or a pathogen to a subject to increase the immune response
to such antigens. Thus, in an embodiment the method includes
exposing the treated APC preparation to at least one source of
antigen after isolation and selection of IDO.sup.LO APCs. In an
embodiment, the antigen comprises a at least one synthetic or
natural polypeptide. Preferably, the antigen is expressed by a
tumor. Also preferably, the antigen is expressed by a pathogen.
[0087] Alternatively, the method may comprise transfecting or
genetically engineering the IDO.sup.LO APCs to express at least one
antigenic polypeptide.
[0088] In another aspect, the present invention comprises a method
for increasing the number of non-suppressive antigen-presenting
cells (APCs) in a subject comprising treating the subject to
increase the population of APCs or their precursors (APC
progenitors) expressing levels of indoleamine 2,3-dioxygenase (IDO)
enzyme activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs).
[0089] In another aspect, the present invention comprises a method
for increasing the protective immune response in a subject
comprising the steps of:
[0090] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject;
[0091] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs); and
[0092] (c) administering the treated cells of step (b) back into
the subject in an amount effective to generate a protective immune
response in the subject.
[0093] Preferably, a protective immune response comprises a
reduction in proliferation of tumor cells or a reduction in the
clinical progression of a malignancy. Also preferably, a protective
response is associated with a reduced pathogen load or increased
resistance to at least one pathogen.
[0094] In an embodiment, the method is used to increase the immune
response to a specific subset of antigens. Thus, in an embodiment,
the method includes exposing the treated cells of step (b) to at
least one source of antigen after isolation from the first subject.
In an embodiment, the antigen comprises an natural or synthetic
polypeptide. Preferably, the antigen is expressed by a tumor. Also
preferably, the antigen is expressed by a pathogen. In another
embodiment, the method comprises transfecting or genetically
engineering the treated cells of step (b) to express at least one
antigenic polypeptide.
[0095] The present invention also provides composition for
increasing T cell activation. Such compositions may be used to
increase the T cell response to antigens in a subject. In this
aspect, the present invention comprises isolated antigen-presenting
cells (APCs) selected as comprising APCs expressing levels of
indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient to
cause suppression of T cell proliferation (IDO.sup.LO APCs). In
another aspect, present invention comprises an isolated
antigen-presenting cell (APC) selected as comprising levels of
indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient to
cause suppression of T cell proliferation. In yet another aspect,
the present invention comprises antigen-presenting cells (APC)
comprising levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs) made by the methods of the
invention.
[0096] Preferably, the IDO.sup.LO cells comprise a population of
APCs having less than 10% of the population expressing IDO at a
level of greater than 2-fold over background. More preferably, the
IDO.sup.LO cells comprise a population of APCs having less than 5%
of the population expressing IDO at a level of greater than 2-fold
over background. Also preferably, the IDO.sup.LO APCs comprise an
absence of suppressor activity comprising less than a 1.5-fold
increase in T cell proliferation in the presence of an DO inhibitor
as compared to in the absence of an IDO inhibitor.
[0097] In an embodiment, the IDO.sup.LO APCs comprise at least one
cell surface marker that identifies the cells as expressing levels
of indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient
to cause suppression of T cell proliferation (DO.sup.W).
Preferably, the marker comprises CD14. Also preferably, the marker
causes preferential adhesion of the cells to plastic. Preferably,
the composition comprises a pharmaceutically acceptable
carrier.
APCs as IDO.sup.+ and IDO.sup.LO Populations
[0098] The present invention relies on the discovery that APCs
expressing high levels of the intracellular enzyme indoleamine
2,3-dioxygenase (IDO) are capable of suppressing T cell responses
in vitro and in vivo. Thus, the present invention is based on the
discovery that the tryptophan-degrading enzyme indoleamine
2,3-dioxygenase (IDO) is an intrinsic attribute of APCs that
determines whether or not the APC is immunosuppressive or
immunostimulatory.
[0099] Immunologic tolerance is operationally defined as the
absence of an immunologic rejection response toward specific
tissues or antigens. Conceptually, there are two types of
tolerance: pre-existing tolerance to self, and acquired tolerance
to new antigens. For example, immunocompetent mice become allergic
(non-reactive) even to non-self antigens when these are introduced
on tumors (Staveley-O'Carroll, K., et al., Proc. Natl. Acad. Sci.
USA, 95: 1178-1183 (1998)). This anergy is apparently caused, not
by the tumor cells themselves, but by cross-presentation of tumor
antigens by tolerogenic bone marrow-derived APCs (Sotomayor, E. M.,
et al., Blood, 98: 1070-1077 (2001)).
[0100] Tolerogenic APCs are potent regulators of the immune
response because they can create networks of immunoregulatory
(suppressor) T cells. These regulatory T cell networks are
apparently involved in both maintaining normal tolerance to self,
and also in mediating a state of acquired unresponsiveness to
non-self antigens (e.g. Sakaguchi, S., Cell, 101: 455-459 (2000);
H. Waldmann and S. Cobbold, Immunity, 14: 399-406 (2001); Shevach,
E. M., J. Exp. Med., 193: F41-F46 (2001)). For example, it has been
shown that tumor-specific regulatory T cells exist, and that
blocking or depleting these cells facilitates the ability to break
tolerance to tumor antigens (Sutmuller, R. P. M., et al., J. Exp.
Med., 194: 823-832 (2001); van Elsas, A., et al., J. Exp. Med.,
190: 355-366 (1999); van Elsas, A., et al., J. Exp. Med., 194:
481-490 (2001)). Once established, this type of unresponsiveness is
self-perpetuating, transferable, and can even "spread" to encompass
new antigens encountered in the same context as those to which the
network is already tolerant (S. Cobbold and H. Waldmann, Curr.
Opin. Immunol., 10: 518-524 (1998)). When present, regulatory T
cells tend to be dominant, enforcing functional tolerance
throughout the entire immune system even in the face of other,
non-tolerant T cells (Honey, K., et al., Immunol. Res., 20: 1-14
(1999)). It is known that certain types of human APCs are able to
promote such regulatory T cells (Jonuleit, H., Trends in Immunol.
22: 394-400 (2001); Dhodapkar, M. V., et al., J. Exp. Med., 193:
233-238 (2001)). However the mechanism by which this occurs is
unknown. Clearly, the ability to create such potent regulatory T
cells is highly desirable in settings such as organ transplantation
or autoimmunity. Conversely, it is undesirable (but often occurs)
to inadvertently create such cells when immunizing against antigens
(e.g. from pathogens or tumors).
[0101] The enzyme indoleamine 2,3-dioxygenase (IDO) is an
intracellular heme-containing enzyme that catalyzes the initial
rate-limiting step in tryptophan degradation along the kynurenine
pathway (M. W. Taylor and G. Feng, FASEB J., 5, 2516-2522 (1991)).
It has been proposed that IDO suppresses T cell proliferation by
degrading tryptophan in the local environment (Munn, D. H., et al.,
J. Exp. Med., 189: 1363-1372 10 (1999)). Two types of human APCs,
(1) monocyte-derived macrophages (Munn, D. H., et al., J. Exp.
Med., 189: 1363-1372 (1999)), and (2) monocyte-derived dendritic
cells (Hwu, P., et al., J. Immunol. 164: 3596-3599 (2000)), which
suppress T cell activation in vitro have been shown to express the
tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (DO). In
mice, IDO has been implicated in the tolerance displayed by the
maternal immune system toward the immunologically disparate fetus
(Mellor, A. L., et al., Nat. Immunol. 2: 64-68 (2001); Munn, D. H.,
et al., Science, 281: 1191-1193 (1998)), as well as in acquired
tolerance toward antigens presented by murine CD8a.sup.+ dendritic
cells (Grohmann, U. et al., J. Immunol., 167: 708-714 (2001)).
Also, IDO is required for the induction of spontaneous tolerance by
liver allografts (Miki, T., et al., Transplantation Proceedings 33:
129-130 (2000)), a process which is thought to be mediated by graft
associated DCs (Thompson, A. W. and Lu., L., Immunol. Today 20:
2731 (1999)). A direct mechanistic link between DO gene expression
and suppression of antigen-specific T cell responses in vivo has
been shown in a mouse model by the inventors (Mellor, A. L., et
al., I Immunol. 168: 3771-3776 (2002)), wherein transfection of the
mouse IDO gene into murine cell lines causes: (1) suppression of T
cell responses to antigens presented by the IDO-expressing cell
lines; and (2) abrogation of the ability of the cells to prime an
allogenic T cell response in vivo to antigens.
[0102] There are several ways to measure IDO expression. As defined
herein, cells comprising high levels of IDO activity comprise: (1)
a level of IDO activity sufficient to suppress T cell proliferation
either in vitro or in vivo; (2) a level of IDO protein or RNA
significantly above the background level of the assay; or (3) at
least 90% of APCs in the preparation expressing IDO as enumerated
on a cell-by-cell basis. For example, high level IDO expression
(IDO.sup.+) is defined by flow cytometry quantitatively on a cell
by cell basis as expression of antigenic IDO protein at a level of
at least 2-fold above background, more preferably, at a level of at
least 5-fold above background, and even more preferably, at a level
of at least 10-fold over background. In this assay, background may
be defined as neutralization of an anti-IDO antibody using standard
techniques such as binding with an excess of an immunizing peptide
(polyclonal antibody assay) or binding of an isotype-matched
control (monoclonal antibody assay). Thus, in an embodiment of the
present invention, tolerance-inducing IDO+ APCs comprise at least
10 90% of the APC population expressing IDO at levels of at least
2-fold over background, and more preferably, at least 95% of the
APC population expressing IDO at levels of at least 2-fold over
background.
[0103] IDO protein and RNA levels can also be measured by other
techniques including western blot, immunohistochemistry, northern
blot, reverse-transcriptase polymerase chain reaction (RT-PCR), or
in situ hybridization. Preferably, using the techniques of
immunohistochemistry or in situ hybridization, IDO expression is be
measured on a cell-by-cell basis. Cells expressing IDO are defined
relative to the appropriate negative control for the particular
assay as understood by one skilled in the art. Preferably, the
IDO-expressing APCs comprise at least 90% of the APC population in
such an assay, and 20 more preferably, at least 95% of the APC
population. IDO can also be measured by western blot, northern
blot, RT-PCR, and other assays that measure DO in a bulk
population. High level IDO expression (IDO.sup.+) for a bulk
population is defined as DO-specific signal of at least 2-fold over
the negative control for the particular assay; as understood by one
skilled in the art, or preferably, at a level of at least 5-fold
over background, and more preferably, at a level of at least
10-fold over background.
[0104] Low levels of IDO expression (IDO.sup.LO) may also be
defined by flow cytometry or other assays quantitatively on a
cell-by-cell basis with reference to the percentage of cells
expressing IDO. Thus, in an embodiment, IDO.sup.LO cells comprise
APCs wherein a minority of APCs in the preparation expressing IDO
protein at a level of at least 2-fold over background. In an
IDO.sup.LO preparation of APCs, preferably less than 10% of the
APCs express IDO protein at a level of at least 2-fold over
background, more preferably less than 5% of the APCs express IDO
protein at a level of at least 2-fold over background.
Alternatively, IDO is measured by immunohistochemistry, in situ
hybridization or other techniques that measure IDO on a
cell-by-cell basis, and an IDO.sup.LO preparation is defined as
comprising less than 20% IDO-expressing cells, or more preferably
less than 10% IDO-expressing cells, and even more preferably, less
than 5% IDO-expressing cells. Alternatively, DO expression is
measured in a bulk population, such that IDO-specific signal is
less than 2-fold over the negative control for the particular
assay.
[0105] Alternatively, an assay to measure biological activity such
as a T cell proliferation assay is used to quantify DO activity. A
T cell proliferation assay includes, but is not limited to, a mixed
leukocyte reaction (MLR) assay, or stimulation of T cells with
antigen or mitogen.
[0106] Thus, in an embodiment, high level DO expression (IDO.sup.+)
is defined as a greater than 2-fold increase in T-cell
proliferation when an inhibitor of IDO is added to MLRs containing
the preparation of interest. This assay provides a physiological
basis to quantify the amount of T-cell proliferation that has been
suppressed by IDO (i.e. the MLR without the IDO inhibitor compared
to the MLR with the IDO inhibitor). Preferably, the MLR contains
the APC preparation to be administered plus allogeneic or
xenogeneic T cells. Alternatively, the T cell proliferation assay
may contain the APC preparation to be administered plus autologous
T cells and an antigen or mitogen to serve as the stimulus for T
cell proliferation. High level IDO expression (IDO'') is defined as
a greater than 2-fold increase in T cell proliferation when an
inhibitor of IDO is added to co-cultures containing the preparation
of interest.
[0107] T cell proliferation assays may also be used to quantify low
DO activity. Thus, in an embodiment, low IDO activity (IDO.sup.LO)
is defined by an allogenic MLR or autologous antigen or
mitogen-stimulation assay as less than 1.5 fold increase in T cell
proliferation when an inhibitor of IDO is added to co-cultures
containing the APC preparation of interest.
[0108] As defined herein, an inhibitor of IDO is an agent capable
of preventing tryptophan degradation and/or kynurenine production
by IDO enzyme in a cell free system, or by cells expressing IDO.
For example, the inhibitor of IDO is an agent capable of preventing
tryptophan degradation and/or kynurenine production by isolated
human monocyte-derived macrophages activated by interferon-.gamma.
(Munn, D. H., et al., 0.1 Exp. Med., 189: 1363-1372 (1999)).
Preferably, the inhibitor of IDO is an analogue of tryptophan. More
preferably, the inhibitor of IDO is the (D) isomer analogue of
tryptophan rather than the (L) analogue, as in some cases only the
(D) isomer reveals true suppression of T-cell activation by IDO.
Thus in an embodiment, the inhibitor of IDO comprises
1-methyl-(D,L)-tryptophan, .beta.-(3-benzofuranyl)-DL-alanine (the
oxygen analog of tryptophan) (1-MT),
.beta.[3-benzo(b)thienyl]-(D,L)-alanine (the sulfur analog of
tryptophan) (S. G. Cady and M. Sono, Arch. Biochem. Biophys. 291,
326 (1991)), or 6-nitro-(D,L)-tryptophan. More preferably, the
inhibitor of IDO comprises 1-methyl-(D)-tryptophan or
6-nitro-(D)-tryptophan.
[0109] Thus, the present invention describes a method of making
antigen-presenting cells (APCs) for enhancing T-cell tolerance
comprising the steps of:
[0110] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject; and
[0111] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity sufficient to suppress proliferation of T cells (IDO.sup.+
APCs).
[0112] The present invention also describes a method of making
antigen-presenting cells (APCs) for enhancing T-cell activation in
an individual comprising the steps of:
[0113] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a subject; and
[0114] (b) treating the isolated cells to select for APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity not sufficient to cause suppression of T cell
proliferation (IDO.sup.LO APCs).
[0115] As defined herein, isolated APCs or progenitor APCs comprise
populations of cells which are either able to express high levels
of IDO (IDO.sup.+ APCs) or which constitutively express low levels
of IDO (IDO.sup.LO APCs) as measured by protein levels (flow
cytometry), mRNA levels, or T cell proliferation assays. The
isolated APCs or APC progenitors may comprise mature blood-derived
dendritic cells, mature tissue dendritic cells, monocyte-derived
macrophages, non-dendritic APCs, B cells, plasma cells, or any
mixture thereof. In an embodiment, the isolated APCs or APC
progenitors comprise a cell type bearing markers of antigen
presentation and costimulatory function.
[0116] As defined herein, non-dendritic APCs comprise cells
isolated directly from peripheral blood, bone marrow, or solid
organ or tissue, or derived by in vitro culture of cells from
peripheral blood, bone marrow, or solid organ or tissue, which
cells do not express CD83, but which do express high levels of
MI-IC class II antigen as well as at least one marker of APC
function. Such markers of APC function include, but are not limited
to, CD80, CD86, and B7-H1 (Dong et al., Nature Med., 5: 1365-1369
(1999)). Such non-dendritic APCs may express high constitutive or
inducible levels of IDO (IDO.sup.+), low levels of IDO (DO.sup.LO),
or may comprise a mixture of IDO.sup.+ and IDO.sup.LO cells.
Non-dendritic APCs include, but are not limited to, endothelial
cells, tissue macrophages, and other cells expressing constitutive
or inducible MHC II.
[0117] Non-dendritic APC include cultured blood-derived
non-dendritic APCs. As defined herein, cultured blood-derived
non-dendritic APCs comprise isolated peripheral blood mononuclear
cells or a fraction thereof which following culture in vitro, do
not express CD83 but do express high levels of MHC class II
antigens as well as one or more markers of APC co-stimulatory
function, such as, but not limited to, CD80, CD86 or B7-H1 (Dong et
al., Nature Med., 5: 1365-1369 (1999)), either constitutively or
following exposure to maturation agents. Blood-derived
non-dendritic APCs may be cultured in a medium with or without
cytokines including, but not limited to, MCSF, GMCSF, IL4, IL3, IL
10, and TNF.alpha.. For example, in an embodiment, monocyte derived
macrophages cultured in MCSF express high levels of IDO (IDO.sup.+)
(Munn, D. H., et al., J. Exp. Med., 189: 1363-1372 (1999)). In
another embodiment, CD14+/CD83-cells following culture in GMCSF+IL4
(which differentially adhere to plastic culture dishes) show no IDO
mediated suppression) (IDO.sup.LO).
[0118] As defined herein, dendritic cells (DCs) comprise cells
isolated directly from peripheral blood, bone marrow, organs or
tissues, or derived by culture of cells isolated form peripheral,
bone marrow, organs, tissues, or isolated CD34.sup.1 stem cells
collected from peripheral blood or bone marrow which cells express
CD83 constitutively or following culture and maturation. DCs may be
cultured in medium with or without cytokines, including, but not
limited to GMCSF, 1L4, IL3, and IL10.
[0119] Thus, as defined herein, immature dendritic cells (DCs)
comprise DCs which express low levels of MHC class II antigens. As
defined herein, low levels of MHC class II antigens are levels less
than 2-fold greater than the negative control used in the assay to
measure MHC class II antigen expression. Low levels of MHC class II
may also be determined by comparison to mature DCs, and preferably
comprise less than half the level of expression of MHC class II
antigens found on mature DCs. MHC class II antigens may be measured
by flow cytometry or other methods known in the art.
[0120] As defined herein, mature dendritic cells (DCs) comprise DCs
which constitutively express high levels of MHC class II, or which
have been treated with agents to cause maturation. As defined
herein, high levels of MHC class II antigens are levels at least
2-fold greater than the negative control used in the assay to
measure MHC class H antigen expression. Maturation can also be
defined by comparison with the same population of DCs prior to
treatment with agents to induce maturation. Defined in this way,
maturation comprises at least a 2-fold upregulation of MHC class II
antigen. Agents causing maturation comprise TNF.alpha., CD40-ligand
(CD4OL), activating anti-CD40 antibodies, cells engineered to
express cell surface CD40-ligand, or bacterial or pathogen
products.
[0121] As defined herein, B cells comprise bells isolated from
blood, bone marrow, lymph nodes or other tissue which express one
or more markers of B cell differentiation such as, but not limited
to, CD19, CD20, CD21, or surface immunoglobulin, wherein B cell
markers may be measured by flow cytometry or other methods known in
the art.
[0122] As defined herein, plasma cells comprise cells isolated from
blood, lymph node or other tissue which express CD38 and
cytoplasmic immunoglobulin as measured by flow cytometry or other
methods known in the art.
[0123] As defined herein, selection of APCs which comprise
IDO.sup.+ APCs or IDO.sup.LO APCs may comprise selective culturing
of the cells, including a predetermined regimen of cytokines and/or
maturation agents. For example, a cytokine cocktail such as those
known in the art (Jonuleit, H., et al., Eur. J. Immunol., 27:
3135-3142 (1997)) may be employed. Thus, for selection of
IDO.sup.LO APCs or IDO.sup.LO APCs, cytokines may be combined
singly, or added together with other agents used for the maturation
of DCs (Jonuleit, H., et al., Eur. J. Immunol. 27: 315-3142 (1997);
Reddy, A., et al., Blood 90: 3640-3646 (1997)). Selection also
comprises physical selection techniques such as selecting
immunosorting of either IDO.sup.+ or IDO.sup.LO cells. This is
possible in that certain cell-surface antigens are associated with
the IDO.sup.+ and IDO.sup.LO phenotypes in APCs. In another
embodiment, sorting comprises differential adherence of either
IDO.sup.LO or IDO.sup.+ cells to a substrate, presumably due to the
expression of a specific cell surface marker that increases
adherence.
[0124] T cell responses comprise allogeneic, xenogeneic,
mitogen-driven, or antigen-driven responses. As defined herein,
allogeneic T cells comprise T cells from a different individual of
the same species, wherein such T cells proliferate in response to
the presence of antigenic differences between the individuals.
Xenogeneic T cells comprise T cells from an individual of a
different species, wherein such T cells proliferate in response to
the presence of antigenic differences between the species. As an
example, T cells from a human recipient are xenogeneic to a porcine
tissue donor.
[0125] As defined herein, CD40-ligand (CD4OL) comprises isolated
polypeptides, multimers of such peptides, or other compositions
that bind to the extracellular binding region of a CD40 receptor of
human, porcine or other origin (e.g. U.S. Pat. No. 6,290,972,
incorporated by reference in its entirety herein).
[0126] As defined herein, proinflammatory bacterial and pathogen
products comprise materials isolated from bacteria or other
pathogens, or synthetic compositions derived from compounds
produced by such organisms, including, but not limited to,
lipopolysaccharide, CpG DNA, or monophosphoryl lipid A, which have
as their defining property that they cause up-regulation of WIHC
class II molecules and/or costimulatory molecules (CD80 or CD86) on
immature dendritic cells or non-dendritic APCs.
[0127] Thus, the present invention relies on the discovery that
APCs may express high levels of IDO (IDO.sup.+/POS) or low levels
of IDO (IDO.sup.LO/NEG) depending upon their hematopoietic lineage
or state of maturation including the effect of conditioning and/or
licensing signals encountered during development. As an example,
FIG. 1 summarizes a proposed 3-step model for the regulation of IDO
in the specific case of monocyte-derived dendritic cell DC
differentiation. In step 1, monocytes begin to differentiate along
the DC lineage. Step 2 occurs during later DC differentiation and
maturation, when there is a cryptic commitment of each individual
DC to subsequently become either IDO.sup.+/POS or IDO.sup.LO/NEG.
In an embodiment, those DCs that are negative for the cell surface
marker CD123 commit to becoming IDO-negative (IDO.sup.LO/NEG),
suggesting that there is some degree of inherent heterogeneity or
"pre-commitment" within in the circulating monocyte pool. In
contrast, cells that are CD123 positive (CD123.sup.+)still have the
option to become either functionally IDO.sup.+/POS or
IDO.sup.LO/NEG, based on the conditions present during maturation.
Thus, the CD123.sup.+ cells will commit to the IDO.sup.LO/NEG
(non-suppressor) phenotype if step 2 is driven solely by
pro-inflammatory factors (e.g., CD40L, TNF.alpha.). If
counter-regulatory cytokines such IL10 or TGF.beta. are present
during maturation, then the CD123.sup.+ cells will commit to the
IDO.sup.+IPOS (suppressor) phenotype.
[0128] Although the cells are committed at step 2, the functional
IDO.sup.+/POS phenotype is not manifest until the DCs are
activated, as for example, by the cytokine interferon-.gamma. and
possibly additional signals would come from the T cell during
antigen presentation (step 3). Thus, although the same signal is
delivered to both "non-suppressor" and "suppressor" DCs, the
response of the DC to this signal, either IDO-mediated suppression
of T-cell activation (IDO.sup.+/POS), or downregulation of IDO
(IDO.sup.LO/NEG) such that the DC able to promote T-cell
activation, depends on its history in step 2.
[0129] This model is consistent with existing models under which
DCs undergo a "licensing" or "conditioning" process (corresponding
to Step 2), either through direct cell-cell interaction with a
helper T cell (Cella, M., et al., J. Exp. Med., 184: 747-752
(1996); Ridge, J. P., et al., Nature 393: 474-478 (1998);
Schoenberger, S. P., et al., Nature 393: 480-483 (1998); Bennett,
S. R., et al, Nature 393: 478-480 (1998)) or via signals from the
local cytokine milieu (Gallucci, S., et al., Nat. Med. 5: 1249-1255
(1999); Kourilsky, P,. et al., Trends in Immunol., 22: 502-509
(2001)). One of the previously undescribed aspects of DC maturation
shown by the model in FIG. 1 is that DCs can be "licensed" to
suppress, and that ability of DCs to become suppressive may be
regulated in vitro by culture conditions. Additionally, the model
teaches that suppressive and non-suppressive DC populations can be
distinguished by IDO expression (and cell surface markers
associated with IDO.sup.+/POS and/or IDO.sup.LO/NEG phenotypes). In
vivo, the cytokines driving commitment to the suppressor phenotype
(e.g., IL10, TGF.beta. may be provided by interaction with
regulatory T cells (H. Waldmann and S. Cobbold, Immunity 14:
399-406 (2001); Maloy, K. G., et al., Nature Immunol., 2: 816-822
(2001)) or may be present in a generalized tolerogenic milieu
(Kourilsky, P, et al., Trends in Immunol., 22: 502-509 (2001);
Fiocchi, Clin. Invest., 108: 523-526 (2001); Chen, W. et al.,
Immunity 22:14:715-725 (2001); Jonuleit, H. et al., Trends in
Immunol. 22: 394-400 (2001)). In vitro, the regulatory cytokines
may be supplied as recombinant cytokines during maturation.
[0130] Thus, the present invention teaches that this developmental
scheme can be modeled in vitro to provide IDO.sup.+ and IDO.sup.LO
APCs. In humans, DC maturation has been associated with improved
antigen-presenting function (Dhodapkar, M. V., et al., J. Clin.
Invest. 105: R9-R14 (2000)) which as often been assumed to
correspond to a loss of tolerogenic activity (Dhodapkar, M. V., et
al., J. Exper. Med., 193: 233-238 (2001); Roncarolo, M. G. et al.,
J. Exp. Med. 193: F5-F9 (2001)). However, maturation may not be
associated with a loss of tolerogenic activity. Instead, tolerance
may be related to an additional signal, as yet undescribed, and
which is distinct from other antigen presentation and
co-stimulatory factors (Albert, M. L., et al., Nature Immunol., 2:
1010 (2001); Shortman, K., et al., Nature Immunol., 2: 988-989
(2001); T. Blankenstein and T. Shuler, Trends Immunol., 23: 171-173
(2002)). The present invention teaches that this additional signal
is the expression of IDO, such that mature DCs that express IDO
will be tolerogenic and mature DCs that do not express IDO will be
immunogenic.
[0131] Thus, the inventors believe that immature DCs are generally
tolerogenic because in immature DCs, the activating population is
ineffective, and therefore the tolerogenic population, although not
optimized, is unopposed. The significant drawbacks to using
immature DCs for therapy are that: (1) such cells constitute an
uncharacterized mixture of cells, and in many applications even a
minor contaminating admixture of the undesired type of APC (i.e.
immunogenic instead of tolerogenic) may render the APC population
unusable or even harmful for the desired application; (2) immature
DCs are inherently unstable and may mature (thus, providing an
undesired and potentially activating population of cells); and (3)
immature DCs are inherently poor antigen-presenting cells due to
their immature status, so the tolerogenic subset does not function
as effectively as would a pure population of mature tolerogenic
DCs.
[0132] The present invention therefore provides a method to produce
relatively pure populations of suppressive and nonsuppressive APCs.
Most DCs and other APC populations contain a mixture of suppressive
and nonsuppressive populations. The present invention teaches that
the conditions under which the APCs are derived can markedly affect
the ratio of tolerogenic APCs as compared to immunogenic APCs.
Previously, the existence of different tolerogenic and immunogenic
subsets in humans could not be shown, nor was it possible to
isolate specific tolerogenic (or immunogenic) subsets of APCs.
Thus, the ability to use APCs for clinical applications was
severely compromised as the presence of immunosuppressive APCs in a
preparation of cells being used to enhance the T cell response
(e.g. an anti-tumor vaccine) would result in antagonism of the
desired effect. Similarly, the presence of immunogenic APCs in a
preparation of cells being used to suppress the T cell response
(e.g. transplant therapy) would be counter-productive.
[0133] Referring now to FIG. 2, APCs may be treated by culturing
under conditions to favor production of (i.e. to select for) APCs
that express high levels of IDO (IDO.sup.+ APCs). In an embodiment,
the isolated cells are cultured in medium which is essentially free
of serum. Alternatively (or additionally), the cells may be
cultured in the presence of macrophage colony stimulating factor
(MCSF) or granulocyte-macrophage colony stimulating factor (GMCSF).
When GMCSF is used, the concentration may range from 10 ng/ml to
1000 ng/ml, or more preferably from 50 ng/ml to 500 ng/ml.
Alternatively (or additionally), the cells may be cultured in the
presence of cytokines such as, but not limited to, TGF.beta., IL
10, IL 4, IL3, or any combinations thereof.
[0134] In an embodiment, the cells are also be treated with an
agent to cause maturation of those APCs that express high levels of
IDO. Preferably, the maturation agents comprise TNF.alpha., IL10,
TGF.beta., CD40-ligand, activating anti-CD40 antibodies, cells
engineered to express cell surface CD40-ligand, proinflammatory
bacterial or pathogen products, or any combination thereof. Thus,
these agents may be combined singly, or added together with other
agents used for the maturation of DCs (Jonuleit, H., et al., Eur.
J. Immunol., 27: 3135-3142 (1997); Reddy, et al., Blood 90:
3640-3646 (1997)).
[0135] Following culture and maturation steps, further purification
can be accomplished by differential adherence to a selected
substrate chosen and optimized to yield preferential enrichment of
the desired IDO.sup.+ population, by methods described herein.
Alternatively (or additionally), the purity of the IDO+ population
is increased by immunosorting based on cell-surface antigens that
associate with IDO.sup.+ APCs. For example, in an embodiment, CD123
and CCR6 are associated with IDO.sup.+ APCs and CD14 is associated
with IDO.sup.LO APCs. Thus, the present invention contemplates that
IDO.sup.+ APCs isolated directly from tissues may only require
maturation and immunosorting to comprise a pure population.
Alternatively, when using CD34.sup.+ APC progenitors, culture may
be required for differentiation, and the cytokines chosen for use
during culture are selected based on their ability to increase the
IDO.sup.+ population.
[0136] FIG. 2 also shows that APCs may be treated by culturing
under conditions to favor production of APCs that express low
levels of IDO (IDO.sup.LO APCs). For example, cells may be cultured
in the presence of GMCSF+IL4 and then matured in the presence of
TNF-.alpha. and CD40 ligand. In an embodiment, APCs are cultured in
the presence of neutralizing antibodies to IL10 and TGF.beta.. The
cells may also be treated with an agent to cause maturation of
those APCs that express low levels of [DO. Preferably, the
maturation agents comprise TNF.alpha., CD40-ligand, activating
anti-CD40 antibodies, cells engineered to express cell-surface
CD40-ligand, proinflammatory bacterial or pathogen products, or any
combination thereof. Thus, these agents may be combined singly, or
added together with other agents used for the maturation of DCs
(Jonuleit, H., et al., Eur. J. Immunol., 27: 3135-3142 (1997);
Reddy, et al., Blood 90: 3640-3646 (1997)).
[0137] As described above, and referring now to FIG. 3, the present
invention further includes the step of measuring expression of at
least one cell surface antigenic marker that identifies the cells
as expressing high levels of IDO (IDO.sup.+ APCs) or not expressing
high levels of IDO (IDO.sup.LO APCs). Preferably, the absence or
presence of the cell surface marker associated with high IDO is
used to select for, and isolate, APCs that express high levels of
IDO (.IDO.sup.+ APCs) from APCs that do not express high levels of
IDO (IDO.sup.LO APCs). For example, markers associated with high
levels of IDO in APCs comprise CD123 and CCR6. A less preferred
marker is CD11c. In an embodiment, the presence of a cell-surface
marker associated with low levels of IDO expression (IDO.sup.LO) is
used to deplete the preparation of non-tolerogenic cells.
Preferably, a marker associated with low levels of IDO in APCs is
CD14. For example, in an embodiment, monocytes may be treated with
a cytokine cocktail to induce differentiation and expression of
IDO. The cells which express high levels of IDO, and are
tolerance-inducing, are then separated from those cells which do
not express IDO using at least one cell surface marker which shows
a correspondence with IDO expression.
[0138] In another embodiment, the expression of uncharacterized
cell surface proteins is used to facilitate separation of IDO.sup.+
cells from IDO.sup.LO APCs. For example, in an embodiment, cells
cultured in serum-free medium display enhanced adherence of the
IDO.sup.LO APCs to the plastic culture dish. Thus, by selecting
those cells which do not adhere to the plastic dish, a population
of IDO.sup.+ cells is selected.
[0139] For example, and referring now to FIG. 4, DCs (immature)
cultured in serum-free medium and enriched by non-adherence of
IDO.sup.F cells to plastic culture wells (i.e. the IDO.sup.LO cells
adhere) show moderate suppression of allogeneic T cell
proliferation when still immature (FIG. 4A). Suppression may be
measured as the finding more APCs (i.e. a low T cell:APC ratio)
results in less T cell proliferation (FIG. 4A). Preferably, the
inhibition is substantially due to IDO expression, as evidenced by
reversal of the inhibition by 1-methyl-(D)-tryptophan (1-MT), an
inhibitor of IDO. In contrast, mature DCs exhibit a much higher
level of suppression, with suppression occurring even at T cell:APC
ratios of 100:1 (FIGS. 4B and 4C). Addition of 1-MT (to inhibit IDO
mediated suppression) causes a significant increase in T cell
proliferation, to levels greater than immature DCs. Thus, contrary
to current models, using the methods of the present invention,
maturation actually enhances the suppression of T cells, and the
enhanced suppression is due to IDO.
[0140] Referring now to FIG. 5, cultured blood-derived APCs derived
in bovine serum based medium (and not fractionated by differential
adherence) produce a preparation comprising a mixture of IDO.sup.+
and IDO.sup.LO cells. In an embodiment, a population of immature
DCs which express the cell surface marker CD123 (CD123+)
constitutively express immunoreactive IDO protein (FIGS. 5A and C
for myeloid DCs derived in GMCSF+IL4; FIG. 5B for macrophages
derived in MCSF, respectively). Maturation for 2 days with TNFct,
or with CD4OL, or with a published cocktail of cytokines (Jonuleit
H., et al., Eur. J. Immunol., 27: 3135-3142 (1997), or
monocyte-condition medium (Reddy at al., Blood 90: 3640-3646
(1997)) does not affect IDO expression in the subset of CD123+
cells (not shown). In an embodiment, CD123 positive (CD 123.sup.+)
cells expressing high levels of IDO (IDO.sup.+) also express high
levels of the cytokine receptor CCR6. (FIG. 5C). In contrast, cells
selected as adhering to the culture dishes comprise primarily
IDO.sup.LO non-dendritic APCs. Preferably, expression of IDO
protein correlates with the ability of the cells to stimulate T
cell proliferation as measured by tritiated thymidine incorporation
into T cell DNA (FIG. 5E).
[0141] Association of Cell Surface Markers with IDO Expression
[0142] Because DO is an intracellular enzyme, expression of the
enzyme is not easy to detect in the intact (i.e. living) cell.
Thus, the present invention utilizes the discovery that specific
cell surface markers are associated with expression of IDO in
antigen-presenting cells (FIG. 3).
[0143] For markers associated with cells having high levels of IDO
expression (IDO.sup.+), the marker preferably comprises is a cell
surface protein (antigen) for which greater than 75% of the cells
express high levels of IDO by flow cytometry or suppression of T
cell proliferation as measured using T cell proliferation assays,
and more preferably, for which greater than 90% of the cells
express high levels of IDO by flow cytometry or suppression of T
cell proliferation as measured using T cell proliferation assays,
and even more preferably, for which greater than 95% of the cells
express high levels of IDO by flow cytometry or suppression of T
cell proliferation as measured using T cell proliferation
assays.
[0144] In an embodiment, the cell surface marker associated with
IDO expression is CD123. CD123 (the IL3-receptor a chain) is
expressed on the small population of lymphoid-lineage
"plasmacytoid" dendritic cells in peripheral blood (Liu, Y. J.,
Cell, 106: 259-262 (2001)), but it is also expressed at lower
levels on a poorly-defined subset of myeloid-lineage dendritic
cells in vivo (Olweus, J., et al., Proc. Natl. Acad. Sci., USA, 94:
12551-12556 (1997); Summers, K. L., et al., Am. J. Pathol., 159:
285-295 (2001)).
[0145] Referring now to FIG. 5, preferably there is a 1:1
correspondence between APCs expressing IDO (IDO.sup.+) and at least
one cell surface marker. For example, in an embodiment,
monocyte-derived DCs cultured for 7 days in GMCSF+IL4 (FIG. 5A) or
macrophage-derived DCs cultured in MCSF (FIG. 5B) display a
discrete subset of cells that express high levels of IDO
(IDO.sup.+), and express the cell surface marker CD123 (FIGS. 5A
and B).
[0146] In addition, other cell surface markers may be used to
identify IDO.sup.+ cells. Thus, in an embodiment, a majority of
LDO.sup.+ APCs express the myeloid-lineage marker CD11 (FIGS. 5A
and B).
[0147] Preferably, another marker highly associated with IDO
expression is the chemokine receptor CCR6. CCR6 is the receptor for
the chemokine mip-3.alpha., a chemotactic factor for immature
dendritic cells (Yang, D., et al., J. Immunol., 163: 17371741
(1999)). Different subsets of dendritic cells express distinct
patterns of chemokine receptors (Sozzani, S., et al., J. Leukocyte
Biol. 66: 1-9 (1999)). CCR6 is expressed on CD34.sup.+-derived
dendritic cells at immature stages of differentiation, and on
immature monocyte-derived dendritic cells cultured with
transforming growth factor (TGF)-13, but is lost under some
conditions when dendritic cells mature (Yang, D., et al., J.
Immunol. 163: 1737-1741 (1999)). The present invention shows that
under conditions favoring high expression of IDO, over 90% of APCs
which express IDO also express CCR6 (FIG. 5C). Thus,
IDO-expressing, tolerance-inducing APCs may comprise the cell
surface markers CD123, CCR6, and in some cases, CD11c.
[0148] The specific pattern of markers that identifies the IDO (or
IDO.sup.LO) population varies depending on the conditions used to
isolate and culture the APCs. For example, CD11e is expressed at
low levels in IDO.sup.LO cells cultured in bovine calf serum based
medium (high seeding density and no differential adherence
selection; FIG. 5A) but is expressed at higher levels for the
IDO.sup.LO culture in serum-free medium (low seeding density and a
final fractionation by differential adherence of non-dendritic APCs
to the culture dish).
[0149] In an embodiment, enrichment using the cell surface marker
alters the composition of the preparation such that it displays a
higher level of IDO activity as measured by suppression of a T cell
proliferation assay (e.g. an allogenic MLR). For example, and
referring now to FIG. 6, CD123 enriched (CD123.sup.+) APCs are
markedly less efficient at stimulating T-cell proliferation than
either the original unfractionated mixture, or the CD123 depleted
subset (CD123.sup.LO) that remains after sorting. The lack of
T-cell activation is due to IDO expression, as shown by the ability
of the IDO inhibitor, 1-methyl-(D,L)-tryptophan (1-MT), to prevent
suppression. Thus, addition of 1-MT allowed the APCs to stimulate
T-cell proliferation at near control levels, indicating that IDO is
involved in the suppression. Enrichment may be accomplished by
positive selection using magnetic beads comprising antibodies to
the marker of interest, adhesion, flow cytometric sorting or other
selections techniques known in the art.
[0150] Alternatively, when a pure population of IDO.sup.+ APCs are
desired, cell sorting techniques may be used to generate a
population of APCs depleted of IDO.sup.LO cells using a cell
surface antigen that correlates with low levels of IDO expression.
Preferably, the cell surface antigen is a marker for which greater
than 75% of the antigen-bearing cells do not express high levels of
IDO by flow cytometry or suppression of T cell proliferation
assays, more preferably, greater than 90% of the antigen-bearing
cells do not express high levels of IDO by flow cytometry or
suppression of T cell proliferation assays, and even more
preferably, greater than 95% of the antigen-bearing cells do not
express high levels of IDO by flow cytometry or suppression of T
cell proliferation assays.
[0151] In an embodiment, the marker associated with IDO.sup.LO
cells comprises CD14. CD14 (the endotoxin-binding protein receptor)
is a well-accepted marker for cells of the monocyte-macrophage
lineage (Szabolcs, P., et al., Blood 87: 4520-30 (1996)).
Monocyte-derived dendritic cells down-regulate CD14 to undetectable
(background) levels when they differentiate along the dendritic
cell lineage (Pickl, W. F., et al., J. Immunol. 157: 3850-3859
(1996)). Mature myeloid dendritic cells do not express CD14 (K.
Shortman and Y.-J. Liu, Nature Reviews; Immunology 2: 151-161
(2002)). Thus, in a culture comprising both mature DCs and a second
population of non-dendritic APCs expressing CD14, the expression of
CD14 can be used to distinguish between the two populations.
[0152] Thus, in yet an embodiment, a population of cells comprising
low levels of IDO expression (IDO.sup.LO) is generated by depleting
the APCs of IDO.sup.+ APCs (e.g. by selection with CD123 or other
markers associated with high IDO) or by positive selection for
markers associated with low IDO expression, such as CD14.
[0153] In yet another embodiment, the expression of uncharacterized
cell surface proteins is used to facilitate separation of IDO.sup.+
cells from IDO.sup.LO cells. For example, cells cultured in
serum-free medium display enhanced adherence of the IDO.sup.LO APCs
to the plastic culture dish. Thus, by selecting those cells which
do not adhere to the plastic dish, a population of IDO.sup.+ cells
is selected. Examples of substrates which may be used for selection
of cells by adherence include, but is not limited to, plastic (for
example, plastic culture flasks or petri dishes), plastic treated
by chemical or other means to facilitate adherence of tissue
culture cells (tissue culture plastic), gas-permeable collection
bags used in isolation and storage of blood products, filters,
protein coatings, protein-lipid films and the like.
[0154] For example, FIG. 5D shows adherent cells taken from culture
of monocytes in serum-free medium supplemented with GMCSF+IL4 and
matured with a cocktail of TNF.alpha., IL.beta., 1L6 and PGE2 as
previously described (Jonuleit, H. et al., Eur. J. Mumma, 27:
3135-3142 (1997)). In this type of culture, the adherent cells are
normally discarded because they are not dendritic cells (Jonuleit,
H., et al., Eur. J. Immunol., 27: 3135-3142 (1997); Reddy, A., et
al., Blood 90: 3640-3546 (1997)). These cells are in fact quite
valuable, as they represent a substantially purified preparation of
IDO.sup.LO cells. These cells are not EDO.sup.+, but they express
markers of APC function (MHC class 11, CD80, and CD86) at levels
similar to non-adherent (fD0.sup.+) cells from the same cultures.
Greater than 95% of the IDO.sup.LO adherent cells express CD14,
whereas less than 10% of the IDO.sup.LO adherent cells express CD
123 or CCR6.
[0155] IDO.sup.+ APCs as Transplant Therapeutics
[0156] Cells which are tolerance-inducing APCs (IDO.sup.+ APCs),
may be used to promote tolerance in a subject. Thus, the invention
comprises a method of preparing cells comprising tolerance-inducing
APCs and the use of the cells to enhance immunological tolerance in
an individual.
[0157] Thus, in one aspect, the present invention comprises a
method to generate APCs for enhancing T cell tolerance towards
cells, tissues and specific antigens in an individual comprising
administration of a cell preparation in which the
antigen-presenting cells (APCs) express high levels of IDO. In this
aspect, the present invention relies on the discovery that APCs
expressing high levels of IDO (IDO.sup.+) are associated with
reduced ability to activate T-cells (FIG. 6). In this way,
IDO.sup.+ APCs are used to increase the likelihood of acceptance of
a graft or transplant from a first donor mammal to a second
recipient mammal.
[0158] Thus, in one aspect, the present invention comprises a
method for enhancing tolerance in a subject comprising the steps
of:
[0159] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a first subject;
[0160] (b) treating the cells to select for APCs that expressing
levels of indoleamine 2,3-dioxygenase (IDO) enzyme activity
sufficient to suppress proliferation of T cells (IDO.sup.+ APCs);
and
[0161] (c) administering the treated cells back to the original
subject or to a second subject in an amount effective to generate a
tolerance-promoting response in the recipient subject.
[0162] In an embodiment, a tolerance-promoting response reduces
T-cell activation in the recipient subject. In an embodiment, the
tolerance-promoting response prolongs the survival of transplanted
cells or tissues in the recipient subject. In yet another
embodiment, the treated cells are administered back to the original
subject, and the tolerance-promoting response reduces the symptoms
of an autoimmune disease in the subject.
[0163] As described herein, the present invention also comprises
compositions for enhancing T cell tolerance. For example, such
compositions may be used to promote acceptance of a tissue
transplant or graft. Thus, the present invention also comprises a
composition for enhancing T cell tolerance comprising
antigen-presenting cells (APCs) selected as comprising APCs
expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme
activity sufficient to suppress proliferation of T cells (IDO+
APCs). In one aspect, the compositions are be made by the methods
of the present invention.
[0164] In an embodiment, `DO.sup.+ APCs of the methods and
compositions of the present invention are evaluated by measurement
of the number of cells expressing IDO. Preferably, the
tolerance-inducing IDO APCs comprise a cell population wherein at
least 90% APCs expressing IDO at levels at least 2-fold greater
than background, and more preferably at least 95% of the APCs
express IDO at levels at least 2-fold greater than background,
where background comprises a negative control for IDO.
[0165] Alternatively or additionally, IDO activity is quantified
using a biological assay. In an embodiment, the tolerance-inducing
IDO APCs comprise suppression of T cell proliferation comprising at
least a 2-fold increase in T cell proliferation in the presence of
an IDO inhibitor as compared to T cell proliferation in the absence
of an IDO inhibitor. Preferably, the inhibitors comprise
1-methyl-(D,L)-tryptophan, .beta.-(3-benzofuranyl)-(D,L)-alanine,
.beta.[3-benzo(b)thienyl]-(D,L)-alanine, or
6-nitro-(D,L)-tryptophan. More preferably, the inhibitors comprises
1-methyl-(D)-tryptophan or 6-nitro-(D)-tryptophan.
[0166] Preferably, the isolated APCs of the methods and
compositions of the present invention comprise non-dendritic APCs,
mature blood-derived dendritic cells, mature tissue dendritic
cells, monocyte-derived macrophages, non-dendritic cells, B cells,
plasma cells, or any mixture thereof. Also preferably, the APCs
comprise markers of antigen presentation and co-stimulatory
function. Preferably, the APCs are isolated from peripheral blood,
bone marrow, lymph nodes or a solid organ from a mammal. More
preferably, the subject is human.
[0167] One object of the present invention is to develop
tolerance-promoting APCs that present a specific subset of antigens
of interest. For example, tolerance-promoting APCs that present
antigens from a donor may be administered to a transplant recipient
to promote acceptance of a graft or transplant. Thus, in an
embodiment, the subject from which the APCs or APC progenitors of
the methods and compositions of the present invention are isolated
comprises a tissue donor to a second subject. In another
embodiment, the APCs or APC progenitors are isolated from a subject
with an autoimmune disorder for subsequent preparation of IDO.sup.+
APCs for use in treating the disorder.
[0168] Alternatively, the treated APCs of the methods and
compositions of the present invention are exposed to at least one
source of antigen after isolation from a first subject and
selection as IDO.sup.+ APCs. Preferably, the source of antigen
comprises antigens expressed by a donor tissue graft. Also
preferably, the source of antigen comprises protein or other
material to which a patient has an autoimmune disorder (see e.g.
Yoon, J.-W., et al., Science 284: 1183-1187 (1999) for examples of
such proteins). Thus, in an embodiment, the subject from which the
APCs or progenitor APCs are isolated comprises a patient with an
autoimmune disorder. In an embodiment, the antigen comprises a
purified, or a synthetic or recombinant polypeptide representing a
specific antigen to which it is desired that tolerance be induced,
or a short synthetic polypeptide fragment derived from the amino
acid sequence of such an antigen. In an embodiment, the isolated
APCs are transfected or genetically engineered to express at least
one antigenic polypeptide (see e.g. Nair, S R., et al., Int. J.
Cancer, 82: 121-124 (1999); Heiser, A., et al., J. Clin. Invest.,
109: 409-417 (2002)).
[0169] In an embodiment, selection of IDO.sup.+ cells of the
methods and compositions of the present invention is facilitated
using a cell surface marker that identifies the cells as expressing
levels of DO sufficient to suppress T cell proliferation (IDO.sup.+
APCs) or expressing levels of IDO not sufficient to suppress T cell
proliferation (IDO.sup.LO APCs) as described herein. The markers
used may include, but are not limited to, CD123, CCR6, CD11c, CD14,
or any combination thereof. Alternatively, the method may include
differential adhesion to a substrate to separate APCs that express
low levels of IDO (IDO.sup.LO APCs)) from APCs that express high
levels of IDO (IDO.sup.+ APCs).
[0170] In an embodiment, the composition used to enhance T cell
tolerance may include a pharmaceutically acceptable carrier.
Alternatively, the composition may include one or more
immunosuppressive pharmaceuticals in a unit dosage form.
[0171] As defined herein, a graft is a tissue specimen for
transplantation from one mammal into another mammal. A host mammal
is defined as the recipient of a graft specimen from a donor
mammal, wherein the donor mammal and the host mammal are distinct
entities. The grafts may either be homografts (a graft transplanted
between mammals of the same species) or xenografts (a graft
transplanted between mammals of the different species).
[0172] Isolation of mammalian cells for use as APCs of the present
invention may be accomplished in accordance with the methods
described in the Examples below. In addition, U.S. Pat. Nos.
5,849,589, 6,008,004, 6,194,204, and 6,274,378 describe isolation
of mixed populations (i.e. not selected as tolerance-inducing)
dendritic cells for use with the selection methods described herein
and are hereby incorporated by reference in their entirety. Thus,
U.S. Pat. No. 5,849,589 describes a method to induce
differentiation of a monocyte into a dendritic cell describes a DC
precursor found among bone marrow CD34.sup.+ cells; U.S. Pat. No.
6,194,204 describes a serial separation technique for isolating DC
from peripheral blood; and U.S. Pat. No. 6,274,378 describes a
method to increase the yield of DCs by culturing in GMCSF and IL-4.
Those skilled in the art would be able to implement modifications
to the disclosed methods of isolating cells for propagation as APCs
without the exercise of undo experimentation.
[0173] U.S. Pat. Nos. 5,871,728 and 6,224,859, the disclosure of
which is incorporated by reference in full herein, describe the
isolation of dendritic cells from a donor for use in a transplant
regimen. In contrast to the present invention, however, the
dendritic cells in U.S. Pat. Nos. 5,871,728 and 6,224,859 131 are
cultured under conditions to remain in an immature state and
therefore do not present DCs which are optimized to promote a
tolerogenic response, nor are they purified to enrich for the
tolerogenic population and/or the immunogenic population.
[0174] In an embodiment, autologous tolerogenic APCs are pulsed
with an autoantigen (i.e. a protein or other material to which a
patient has developed an autoimmune immunologic response as part of
an autoimmune disorder) and administered back to a patient with an
autoimmune disease. In another embodiment, tolerogenic APCs are
isolated from the donor of a bone marrow transplant or
hematopoietic stem-cell transplant and administered to a second
subject who is receiving the transplant so as to prevent
graft-versus-host disease in the recipient subject. Alternatively
administration of tolerogenic APCs may be used to treat established
graft-versus-host disease in a transplant recipient. In both cases,
the goal is to initiate the development of a regulatory T cell
response to a target antigen or set of antigens which will reduce
the autoimmune or graft-versus-host T cell responses (H. Waldmann
and S. Cobbold, Immunity 14: 399-406 (2001)).
[0175] Tolerogenic APCs may be administered prior to
transplantation. In an embodiment, prophylactic administration may
be commenced as early as 1 month prior to transplantation, and more
preferably at about 1 week prior to transplantation. Alternatively,
or additionally, administration may be at the time of
transplantation and for several months following the
transplantation, and at least several weeks following the
transplantation, or even more preferably, several days following
transplantation.
[0176] Immunosuppressive agents may also be included as part of the
transplant regimen. Immunosuppressive reagents are typically
administered at the time of transplantation and at least daily for
some period afterwards. The amount of immunosuppressive agent is
preferably adjusted based upon the success of the cell-driven
response (i.e. the response as a result of administration of
IDO.sup.+ APCs.)
[0177] Pharmaceutical formulations can be prepared by procedures
known in the art. For example, the compounds can be formulated with
common excipients, diluents (such as phosphate buffered saline),
tissue-culture medium, or carriers (such as autologous plasma or
human serum albumin) and administered as a suspension.
[0178] The invention contemplates methods of administration which
are well known in the art. Administration of the APCs of the
present invention may include, but is not limited to, intravenous,
subcutaneous, and intra-tumoral modes of administration.
Additionally, the compounds are well suited to formulation as
sustained release dosage forms of at least part of the preparation
(e.g. immunosuppressive agents). Preferably, the cells are
administered in a dose ranging from 5.times.10.sup.5 to
5.times.10.sup.10 cells per dose. More preferably, the cells are
administered in a dose ranging from 5.times.10.sup.6 to
5.times.10.sup.9 cells per dose. Even more preferably, the cells
are administered in a dose ranging from 5.times.10.sup.7 to
1.times.10.sup.8 cells per dose.
[0179] IDO.sup.LO APCs as Immunogenic Vaccines
[0180] The present invention also provides a means to isolate cells
enriched for immunostimulatory APCs that show enhanced T-cell
activation. For example, cells depleted of tolerance-inducing APCs
can be used as anti-cancer vaccines or anti-viral vaccines to
increase the host response to cancer or viral antigens,
respectively.
[0181] In this aspect, the present invention comprises a method for
increasing the protective immune response to at least one specified
antigen in a subject comprising the steps of:
[0182] (a) isolating antigen-presenting cells (APCs) or their
precursors (APC progenitors) from a subject;
[0183] (b) treating the cells to select for APCs expressing levels
of indoleamine 2,3-dioxygenase (IDO) enzyme activity not sufficient
to cause suppression of T cell proliferation (IDO.sup.LO APCs);
and
[0184] (c) administering the treated cells back to the subject in
an amount effective to generate a protective immune response in
said subject.
[0185] In an embodiment, a protective immune response comprises a
reduction in size of a tumor or a reduction in the clinical
progression of a malignancy. In another embodiment, a protective
immune response is associated with increased resistance to at least
one pathogen. For example, such increased resistance to a pathogen
may comprise an increased resistance to infection, a reduced
pathogen load, or increased production of pathogen specific
antibodies or T cells.
[0186] The present invention also comprises compositions for
increasing T cell activation. Such compositions are useful for
generating a protective immune response. Thus, in another aspect
the present invention comprises isolated antigen-presenting cells
selected as comprising APCs expressing low levels of indoleamine
2,3-dioxygenase (IDO) enzyme activity) (IDO.sup.LO), wherein the
IDO.sup.LO cells comprise a population of APCs expressing IDO at a
level not sufficient to cause suppression of T cell proliferation
(IDO.sup.LO APCs). The IDO.sup.LO APCs may be prepared by the
methods described in the present invention or other methods known
in the art.
[0187] Preferably, IDO.sup.LO cells of the methods and compositions
of the present invention comprise a population of APCs having less
than 10% of the population expressing IDO at a level of greater
than 2-fold over background. More preferably, IDO.sup.LO cells of
the methods and compositions of the present invention comprise a
population of APCs having less than 5% of the population expressing
IDO at a level of greater than 2-fold over background. Also
preferably, IDO.sup.LO APCs comprise non-suppressor activity
quantified by a mixed leukocyte reaction as less than 1.5 fold
increase in T cell proliferation in the absence of an IDO inhibitor
as compared to T cell proliferation in the presence of an EDO
inhibitor. Preferably, the IDO inhibitors comprise
1-methyl-(D,L)-tryptophan, .beta.-(3-benzofuranyl)-(D,L)-alanine,
.beta.[3-benzo(b)thienyl]-(D,L)-alanine, or
6-nitro-(DL)-tryptophan. Also preferably, the IDO inhibitors
comprise 1-methyl-(D)-tryptophan or 6-nitro-(D)-tryptophan.
[0188] Preferably, the isolated APCs or progenitor APCs comprise
mature blood-derived dendritic cells, mature tissue dendritic
cells, monocyte-derived macrophages, nondendritic APCs, B cells,
plasma cells, or any mixture thereof. Also preferably, the isolated
APCs progenitor APCs comprise a cell type bearing markers of
antigen presentation and costimulatory function. Also, in preferred
embodiments, the APCs progenitor APCs are isolated from peripheral
blood, bone marrow, lymph nodes or a solid organ from a human or
other mammal.
[0189] In an embodiment the treated APCs of the methods and
compositions of the present invention are exposed to at least one
source of antigen after isolation from a subject and selection as
IDO.sup.LO APCs. Preferably, the antigen is a polypeptide expressed
by a tumor. Alternatively, the antigen may be expressed by a
pathogen. Preferably, the antigen comprises an unpurified tumor or
viral preparation. In another embodiment, the antigen is a
synthetic or recombinant protein representing a known antigen to
which it is desired to induce an immune response, or a short
synthetic polypeptide derived from the amino acid sequence of such
a protein. In another embodiment, the APCs selected as IDO.sup.LO
are transfected or genetically engineered to express at least one
such antigenic protein or polypeptide.
[0190] In an embodiment, selection of IDO.sup.LO cells of the
methods and compositions of the present invention is facilitated
using a cell surface marker that identifies the cells as expressing
high levels of 1DO (IDO.sup.+ APCs) or not expressing high levels
of IDO (IDO.sup.LO APCs) as described herein. The markers used may
include, but are not limited to CD123, CD11c, CCR6, CD14, or any
combination thereof. Alternatively, the method may include
differential adhesion to a substrate to separate APCs that express
low levels of IDO (IDO.sup.LO APCs) from APCs that express high
levels of IDO (IDO.sup.+ APCs).
[0191] As described herein, the APCs selected as IDO.sup.LO APCs of
the methods and compositions of the present invention may be
exposed to at least source of antigen after isolation from said
first subject. Preferably, the antigen is a polypeptide expressed
by a tumor. Alternatively, the antigen may be expressed by a
pathogen. For example, U.S. Pat. Nos. 6,228,640, 6,210,662,
6,080,409, 5,994,126, 5,851,756, and 5,582,831 describe the
manipulation of isolated dendritic cells to produce
immunostimulatory vaccines specific to certain antigens. The
disclosures of U.S. Pat. Nos. 6,228,640, 6,210,662, 6,080,409,
5,994,126, 5,851,756, and 5,582,831 are hereby incorporated in full
by reference.
[0192] Alternatively, the isolated APCs are transfected or
genetically engineered to express at least one antigenic
polypeptide (see e.g. Nair, S. K., et al., Int. J Cancer, 82:
121-124 (1999); Heiser, A., et al., J. Clin. Invest., 109: 409-417
(2002)
[0193] Antigens may also be physically introduced into cells. For
example, U.S. Pat. No. 6,228,640 BI describes pulsing DCs with
tumor RNA or expression products to prepare APCs comprising
expression of specific antigens. Also, U.S. Pat. Nos. 6,210,662 and
6,080,409 describe methods and compositions generated by activating
APCs by contact with a polypeptide complex constructed by joining
together a dendritic cell-binding protein (GMCSF) and a polypeptide
antigen. U.S. Pat. Nos. 5,994,126 and 5,851,756 describes a method
for producing mature DCs pulsed with antigen, including
particulates where the antigenic material is expressed on the
surface of the cells as immunogens for vaccines. U.S. Pat. No.
5,582,831 describes formulation of tumor vaccines by exposing tumor
cells to a cross-linking agent to generate antigenic protein
complexes.
[0194] In an embodiment, the isolated APCs of the methods and
compositions of the present invention further comprise at least one
cell surface antigenic marker that identifies the cells as
expressing low levels of IDO (IDO.sup.LO APCs) or expressing high
levels of IDO (IDO' APCs). The marker may include, but is not
limited to CD123, CD11c, CCR6, or any combination thereof.
Alternatively, the method may include differential adhesion to a
substrate to separate APCs that express low levels of IDO
(IDO.sup.LO APCs) from APCs that express high levels of IDO
(IDO.sup.+ APCs).
[0195] The compositions comprising IDO.sup.LO APCs may also include
a pharmaceutically acceptable carrier, where pharmaceutically
acceptable carriers include, but are not limited to the carriers
described herein.
[0196] Methods and Kits to Measure the Amount of Immunosuppressive
Cells in a Mixed Population of APCs
[0197] The present invention also describes methods to quantitate
the levels of immunosuppressive APCs in a population of APCs. For
most applications it would be preferable, if not absolutely
required, to determine the nature of the cell population being
used. For example, when utilizing a preparation of cells for
inducing tolerance in a host, a level of contaminating IDO.sup.LO
cells of less than 10%, and more preferably less than 5%, is
desired. Conversely, when utilizing a preparation of cells for
increasing the immune response in a host, the level of
contaminating IDO.sup.+ cells should be determined.
[0198] Thus, in one aspect, the present invention comprises a
method to determine the number of tolerance-inducing
antigen-presenting cells (APCs) in a cell population comprising
measuring the number of cells expressing levels of indoleamine
2,3-dioxygenase (IDO) enzyme sufficient to suppress proliferation
of T cells (IDO.sup.+ APCs) in the cell population. In an
embodiment, IDO is quantified on a cell-by-cell basis. In an
embodiment, IDO is quantified in a bulk population of APCs.
[0199] In another aspect, the present invention comprises a kit for
determining the number of tolerance-inducing antigen-presenting
cells (IDO.sup.+ APCs) in a cell population comprising reagents to
measure levels of indoleamine 2,3-dioxygenase (IDO) enzyme in the
APCs, wherein the reagents are packaged in at least one individual
container.
[0200] The immunosuppressive IDO.sup.+ APCs may also be quantified
using a biological assay. Thus, in another aspect, the present
invention comprises a method to quantify the ability of a
population of APCs to suppress T cell proliferation comprising
measuring the ability of the cell population to increase in T cell
proliferation in the presence of an IDO inhibitor as compared to in
the absence of an IDO inhibitor. The present invention also
comprises a kit for determining the ability of a population of APCs
to suppress T cell proliferation comprising an IDO inhibitor
packaged in at least one individual container. In an embodiment,
the kit includes individual assay vessels which provide a
predetermined cell density. Preferably, the assay vessels comprise
round-bottomed or V-shaped wells.
[0201] IDO.sup.+ APCs and Mip-3a as Markers of Tumors
[0202] Because tolerance-inducing APCs reduce the host's ability to
reject foreign antigens which are present on tumor cells, the
presence of tolerance-inducing APCs in a tumor is associated with a
less favorable prognosis than in cases where tolerance-inducing
APCs are not present. Thus, the present invention also describes
assessing the relative risk of tumor progression by assaying tissue
from a tumor or tumor draining lymph node for antigen-presenting
cells of myeloid-lineage which have high levels of expression of
the intracellular enzyme indoleamine 2,3-dioxygenase (IDO).
[0203] Thus, in another aspect, the present invention comprises a
method for assessing the relative risk of tumor progression in a
subject comprising the steps of:
[0204] (a) assaying a sample of tissue from a tumor or tumor
draining lymph node from a subject for expression of the enzyme
indoleamine 2,3-dioxygenase (DO); and
[0205] (b) correlating the risk of tumor progression to DO
expression in the tissue sample, wherein IDO expression is
positively correlated with an increase in the risk of tumor
progression.
[0206] In an embodiment, the method further includes identification
of cell surface markers associated with high DO expression.
Preferably, the cell surface markers comprise CD123, CD11c or
CCR6.
[0207] The present invention also comprises kits for assessing the
relative risk of tumor progression in a subject. For example, in
one aspect, the present invention comprises a kit for assessing the
relative risk of tumor progression in a subject comprising reagents
for detection of the enzyme indoleamine 2,3-dioxygenase (IDO) in a
sample of tissue from a tumor or tumor draining lymph node from a
subject, wherein the reagents are packaged in at least one
individual container. Preferably, the kit further comprises
reagents for detection of cell surface or immunohistochemical
markers associated with high DO expression by APCs. More
preferably, the cell surface markers detected using the kit
comprise CD123, CD11c or CCR6.
[0208] The present invention also relies on the discovery that
tumor cells that recruit tolerance-inducing APCs to the tumor
exhibit increased tolerance. CCR6, a marker highly associated with
IDO expression (IDO), is a receptor for the chemokine mip-3.alpha.,
a chemotactic factor for immature dendritic cells (D. Yang, O. M.
Howard, Q. Chen, J. J. Oppenheim, J. Immunol. 163: 1737-1741
(1999)). Elevated mip-3.alpha. expression has been seen in certain
tumors (Bell, D., et al., J. Exp. Med., 190: 1417-1426 (1999)).
Thus, tolerance-inducing APCs that express receptors for
chemoattractant factors secreted by the tumors play a role in the
development of tumor-induced tolerance.
[0209] Thus, in another aspect, the present invention also
comprises a method for assessing the risk of tumor progression in a
subject comprising the steps of:
[0210] (a) assaying a sample of tissue from a tumor or tumor
draining lymph nodes from a subject for mip-3.alpha. expression;
and
[0211] (b) correlating the risk of tumor progression to
mip-3.alpha. expression in the tissue sample, wherein mip-3.alpha.
expression is positively correlated with an increase in the risk of
tumor progression.
[0212] In another aspect, the present invention comprises a kit for
assessing the relative 5 risk of tumor progression in a subject
comprising reagents for detection of relative levels of expression
of mip-3.alpha. in a sample of tissue from a tumor or tumor
draining lymph node from a subject, wherein the reagents are
packaged in at least one individual container.
[0213] For example, malignant melanoma is a tumor with well-defined
T cell antigens but which `nevertheless is not eliminated by the
immune system. In tumor specimens comprising both primary and
metastatic lesions, a majority show infiltration of IDO.sup.+ cells
(FIG. 8B). In addition, recruitment of IDO.sup.+ dendritic cells is
also seen in carcinoma of the breast, lung, colon and pancreas.
Accumulation of these cells occurs primarily around the margins of
the tumor and infiltrating along the fibrous stoma, or along the
vessels in perivascular cuffs and are not a normal constituent of
skin or connective tissue.
[0214] Tumor-draining lymph nodes may be a critical site for
initiation of anti-tumor immune responses (Ochsenbein, A. F., et
al., Nature 411: 1058-1064 (2001)). In an analysis of over 300
tumor-draining lymph nodes from 26 patients with malignant
melanoma, markedly abnormal accumulation of IDO.sup.+ cells is seen
(FIG. 8C-E). The IDO.sup.+ cells are found to extensively
infiltrate the lymphoid regions of the lymph nodes, largely
concentrating in the interfollicular and T cell zones. There is
also frequent accumulation around blood vessels (FIG. 8D) and
accumulation at the interface between lymphoid tissue and tumor
metastases or medullary sinuses (FIG. 8E). Normal lymphoid tissue
(tonsillectomy specimens with minimal hypertrophy, or lymph node
dissections from patients with early-stage node-negative breast
cancer) show only scattered IDO.sup.+ cells (FIG. 8F), and do not
display the extensive focal collections and confluent areas of
IDO.sup.+ cells seen in tumor-draining nodes. Also, many primary
and metastatic tumors contain individual tumor cells (FIG. 8O) or
entire localized regions within the tumor that express mip-3.alpha.
by immunohistochemistry. Quantitative analysis of mip-3.alpha. mRNA
by real-time PCR confirms mip3a expression in samples of malignant
melanoma (M), renal carcinoma (R), and non-small cell lung cancer
(L) (FIG. 9).
[0215] Thus, the present invention identifies IDO.sup.+ myeloid
dendritic cells as a novel immunoregulatory subset of human APCs.
The IDO.sup.+ dendritic cell population appears to be distinct from
the previously described "plasmacytoid" or pre-DC2 dendritic cells
(Liu, Y. J., Cell 106, 259-262 (2001)). Thus, pre-DC2 express CD123
do not express CD11c, whereas CD11c is found on essentially all
IDO.sup.+ cells in vitro. In addition, while it is possible that
that pre-DC2 cells may express IDO in some situations, the majority
of IDO-expressing cells appear to be myeloid-derived.
[0216] The present invention differs from previous application of
tolerogenic DCs in that: (1) mature DCs which are more effective
than immature DCs at suppressing T cells are used; (2) a relatively
pure preparation of tolerogenic cells (i.e. expressing IDO) is
used, rather than a preparation contaminated by non-tolerogenic
(IDO.sup.LO) cells; and (3) APCs of a type other than DCs may be
employed, where the non-dendritic APCs are selected based on
expression of IDO. Thus, in an embodiment, the isolated tolerogenic
APCs comprise mature dendritic cells cultured under conditions
optimized to yield a preparation of IDO.sup.+ cells or mature cells
enriched for IDO.sup.+ cells by selection of IDO.sup.+ cells from a
mixed population of IDO.sup.+/IDO.sup.LO cells. Selection of
IDO.sup.+ cells may take advantage of differential expression of
cell surface antigens by either the IDO.sup.+ or the IDO.sup.LO
cells. Thus, cells may be separated by immunosorting, differential
adherence or other methods known in the art.
[0217] The present invention differs from previous application of
immunizing (non-tolerogenic) DCs in that: (a) a relatively pure
preparation of non-tolerogenic cells (i.e. not expressing
immunosuppressive levels of IDO) is used, rather than a preparation
contaminated by tolerogenic (IDO.sup.+) cells; and (2) APCs of a
type other than DCs may be employed, where the non-dendritic APCs'
are selected based on low or negative expression of IDO. Thus, in
an embodiment, the isolated non-tolerogenic APCs comprise mature
dendritic cells cultured under conditions optimized to yield a
preparation of IDO.sup.LO cells, or mature cells enriched for
IDO.sup.LO cells by selection of IDO.sup.LO cells from a mixed
population of IDO.sup.+/IDO.sup.1.degree. cells. Selection of
IDO.sup.LO cells may take advantage of differential expression of
cell surface antigens by either IDO.sup.LO or IDO.sup.+ cells.
Thus, cells may be separated by immunosorting, differential
adherence or other methods known in the art. In another embodiment,
the isolated non-tolerogenic APCs comprise mature non-dendritic
APCs cultured under conditions optimized to yield a preparation of
IDO.sup.LO cells, with or without further enrichment by selection
of IDO.sup.LO cells from a mixed population of IDO+/IDO.sup.LO
cells.
[0218] The present invention teaches that IDO-expressing APCs cells
are tolerogenic, and are found in large numbers in tumors and
draining lymph nodes. One mechanism contributing to the
accumulation of these cells may be tumor-derived mip-3.alpha..
Mip-3.alpha. is the only known ligand for CCR6, and CCR6 appears to
selectively associate with the EDO.sup.+ dendritic cell phenotype
in vitro. The ability to isolate these IDO.sup.+ and IDO.sup.LO
APCs cells in vitro provides a means to use specific subsets of the
IDO expressing monocytes as therapeutics to either increase or
decrease immunologic tolerance.
EXAMPLES
Example 1
Cell Culture
[0219] Human monocytes and lymphocytes were isolated as separate
fractions by leukocytapheresis and counterflow elutriation (D. H.
Munn et al., J. Exp. Med. 189, 1363-1372 (1999)). Monocytes
(typically >95% purity) were cultured in 100 mm tissue culture
petri dishes in RPMI-1640 medium with 10% newborn calf serum
(Hyclone) and including penicillin/streptomycin and glutamine.
Cultures received either MCSF (200 U/ml, Genetics Institute) on day
0, or GMCSF (50 ng/ml, R&D Systems)+IL4 (50 ng/ml, R&D
Systems) on days 0, 2 and 4. For experiments where CCR6 expression
was of interest, cultures received a single dose of GMCSF+IL4 (100
ng/ml each) on day 0, with no further supplementation. Loosely
adherent dendritic cells (GMCSF+IL4) were harvested by gentle
aspiration; adherent macrophages (MCSF) and non-dendritic APCs
(GMCSF+IL4) were harvested with EDTA. Other cultures were conducted
in serum-free medium (X-vivo 15; BioWhitaker, Walkersville, Md.)
plus cytokines.
Example 2
Production of Antibodies
[0220] All antibodies were obtained commercially except for
polyclonal antiserum against human IDO which was manufactured as a
work for hire by ZCB Inc., Hopkinton, Mass. All commercial
antibodies and reagents were from BD Biosciences-Pharmingen (San
Jose, Calif.) unless specified otherwise. For detection of cell
surface antigens, DCs were triple-stained with anti-CD123-biotin
(clone 7G3; it was found that clone 9F5 gave suboptimal results
with dendritic cells) followed by streptavidin-perCP, plus
anti-CD11 callophycocyanin (clone S-HCL-3) or anti-CCR6-fluorescein
(clone 53103.111, R&D systems, Minneapolis, Minn.). CCR6
results were also confirmed using a second anti-CCR6 antibody
(clone 11A9; Pharmingen). For detection of IDO, cells were fixed
and permeabalized (Cytofix/Cytoperm), and then stained with rabbit
anti-IDO antibody prepared against the peptide followed by
polyerythrin-labeled anti-rabbit secondary antibody (Jackson
Immunoresearch, West Grove Pa.) cross-adsorbed against mouse, human
and bovine IgG, for multiple labeling). Dendritic cells were gated
on forward and side scatter to exclude contaminating lymphocytes
and debris.
[0221] For preparation of rabbit anti-IDO antibody, the peptide
DLIESGQLRERVEKLNMLC (SEQ ID NO: 1) was prepared based on the
GenBank sequence of human IDO (M34455) and conjugated to keyhole
limpet cyanogen. Rabbits were immunized with conjugated peptide in
Freund's adjuvant (all immunization, antibody preparation and
affinity purification steps were performed as a work for hire (QCB,
Inc., Hopkinton, Mass.). This peptide gave the best results out of
several different sequences screened for their ability to detect
human IDO in formalin-fixed paraffin-embedded tissue and by flow
cytometry. Validation studies showed that this antibody
immununoprecipitated the expected 45 kD band from cell lysates,
correlated with IDO mRNA and functional enzymatic activity in
vitro, identified an interferon-.gamma. inducible antigen in two
known-positive cell lines (THP-1 and HeLa), and detected an antigen
by immunohistochemistry which was specifically localized to cells
with known expression of IDO (the syncytiotrophoblast cells of
human placenta; Y. Kudo and C. A. Boyd, Biochetn. Biophys. Acta
1500, 119-124 (2000)). Results were consistent from animal to
animal, and from lot to lot of antibody.
Example 3
Regulation of IDO Expression During DC Maturation
[0222] In humans DCs, maturation has been associated with loss of
tolerogenic activity (Dhodapkar, M. V., et al., J. Exp. Med., 193:
233-238 (2001)). The experiments described in FIG. 4 addressed the
issue of whether DC maturation down-regulates IDO mediated
suppressor activity. Monocyte-derived DCs were cultured for 7 days
in X-vivo medium with GMCSF+IL4 (non-adherent cell population,
>95% IDO.sup.+, >95% CD123.sup.+). During the final two days,
the cells were either (A) left as immature DCs (no additions); (B)
matured using a cytokine cocktail comprising TNF.alpha., IL113, IL6
and PGE2 (Jonuleit, H. et al., Eur. J. Immunol., 27: 3135-3142
(1997)); or (C) matured using monocyte-conditioned medium (Reddy,
A., et al., Blood 90: 3640-3546 (1997)). Each group was harvested
and added to 5.times.10.sup.5 allogeneic T cells in V-bottom 96
well microliter wells in 200 ul medium (10% fetal calf serum in
RPMI)). Differing numbers of DCs were added to a fixed number of T
cells to produce the T cell to APC ratios shown (thus, the greater
number of APCs are on the left of the axis, the lesser numbers on
the right). Replicate groups of wells received either 200 .mu.M
1-methyl-(D)-tyrptophan (1-MT) (.quadrature.), or saline control
(.DELTA.), to disclose IDO-mediated suppression (defined as the
amount of proliferation restored at each T cell:APC ratio by adding
1-MT and shown for one point as an arrow in panel B). After 5 days,
T cell proliferation was measured as the incorporation of tritiated
thymidine (Munn, D. H. et al., J. Exp. Med., 189: 1363-1372
(1999).
[0223] FIG. 4A shows that higher numbers of immature dendritic
cells (lower APC: T cell ratios) were associated with increased IDO
mediated suppression (shown as the reduced T cell proliferation at
the lower T cell:APC cell ratios, and as enhancement of
proliferation when 1-methyl-(D)-tryptophan was added). In FIGS. 4B
and 4C, the DCs were matured. It can be seen that the mature DCs
show greater IDO mediated suppression (with suppression occurring
at T cell:APC cell ratios as low as 100:1). Although the mature DCs
were highly suppressive due to the presence of IDO, the mature
forms actually function better as antigen-presenting cells compared
to the immature form, as revealed by the higher T cell
proliferation achieved when suppression was prevented by 1-MT (i.e.
the difference between .quadrature. and .DELTA. for each
experiment). Thus, mature DCs derived under conditions optimized in
accordance with the invention were both more suppressive and more
effective as APCs than immature DCs. Both of these attributes are
desirable for induction of tolerance.
Example 4
Co-Expression of IDO with Cell Surface Markers CD123, CC11c and
CCR6 in Myeloid APCs
[0224] Expression of IDO in immature monocyte-derived (myeloid)
dendritic cells (Dhodapkar, M. V., et al., J. Exp. Med. 193:
233-238 (2001)) and in immunosuppressive monocyte-derived
macrophages (Munn, D. H., et al., J. Exp. Med. 189: 1363-1372
(1999)) was analyzed. FIG. 5 shows the expression of IDO and CCR6
by myeloid antigen-presenting cells which express the cell surface
antigen CD123 (CD123.sup.+). Human monocytes were cultured as
described above (Example 1) for 7 days with GMCSF+IL4 to produce
myeloid dendritic cells (FIGS. 5A and 5C), or for 7 days in MCSF to
produce macrophages (FIG. 5B) (Munn, D. H., et al., J. Exp. Med.
189: 1363-1372 (1999)). Prior to analysis, cells were treated with
interferon-.gamma. (INF.gamma.) for 18 hrs to induce maximal
expression of IDO. Harvested cells were triple-stained for CD123,
CD11c and IDO. For FIG. 5D, cells were cultured as in Example 1
except in a commercial, FDA-approved serum-free medium formulation
(X-vivo 15; BioWhitaker, Waldersville, Md.).
[0225] As shown in FIGS. 5A and B, both preparations contained a
discrete subset of cells that expressed IDO following interferon-7
treatment. Characterization of these IDO cells showed that they all
expressed the myeloid-lineage marker CD11c, and CD123, wherein
>90% of the IDO.sup.+ expressed the myeloid-lineage marker CD11c
and >99% of the IDO.sup.+ cells expressed CD123. To test whether
these were truly DCs, additional phenotyping was performed. Cells
were matured with TNF.alpha. during the last 2 days of culture, in
order to upregulate maturation and costimulatory markers, and
non-adherent cells were harvested. Following TNFu, all non-adherent
cells displayed a veiled/dendritic morphology. Three-color
phenotyping showed that the CD123.sup.+/IDO.sup.+ subset of cells
were uniformly CD14.sup.- and CD83.sup.+, consistent with their
identity as dendritic cells; uniformly CD11b.sup.+ and BDCA-2.sup.-
(Dzionek, A., et al., J. Immunol, 165: 6037-6046 (2000)) consistent
with their myeloid origin, and distinguishing them from
plasmacytoid DCs (Grouard, G., et al., J. Exp. Med., 185: 1101-1111
(1997)); and 100% positive for CD80, CD86 and MHC class II
(HLA-DR). Under these conditions (bovine serum-based medium) CD11c
expression was high on the CD123.sup.+ subset, and was lower and
variable on the CD123.sup.LO subset.
[0226] In addition, when monocytes were cultured under conditions
that favored expression of CCR6 (serum-free medium, single-dose
GMCSF+IL4), the CD123.sup.+/IDO.sup.+ cells were almost all
(>99%) CCR6.sup.+ (FIG. 5C). For experiments where CCR6
expression was of interest, cultures received a single dose of
GMCSF+IL4 (100 ng/ml each) on day 0, with no further
supplementation. Moreover, within the myeloid dendritic cell
population, IDO and CCR6 expression were coincident. T and B cells,
which also express CCR6, were excluded from analysis by forward and
side scatter properties during flow cytometric analysis. The
cell-surface CCR6 on these cells was functional: when immature
dendritic cells containing a mixture of CCR6-positive and -negative
cells were placed in chemotaxis chambers, the CCR6-positive cells
selectively migrated in response to a mip3oc gradient (data not
shown).
[0227] Expression of IDO is not found in all types of dendritic
cells. Analysis of plasmacytoid dendritic cells, defined as the
population of peripheral blood mononuclear cells expressing CD123
but negative for lineage-specific markers (Lin-1 marker cocktail,
BD-Pharmingen), revealed no detectable expression of IDO following
activation for 6 hrs or 24 hrs with interferon-.gamma., in the
presence of IL3 to support viability (data not shown). Moreover,
when the adherent cells (comprising the non-dendritic APC
population) from cultures of peripheral blood mononuclear cells in
GMCSF+IL4 were examined, they were found to express very low levels
of IDO and little CD 123 (FIG. 5D). Additional phenotyping of the
non-dendritic APCs showed that they were uniformly CD14-positive
and CD83-negative (thus, distinguishing them unambiguously from
mature dendritic cells, but were >95% positive for CD80 and CD86
(thus, identifying them as mature antigen-presenting cells), and
expressed high levels of the MHC class II antigen HLADR (further
distinguishing them from immature dendritic cells, and identifying
them as mature APCs). Consistent with the observed absence of IDO
expression, these non-dendritic APCs showed excellent APC function
without any detectable IDO-mediated suppression (i.e. no increase
in proliferation in the presence of 1-methyl-(D)-typtophan (1-MT)
(FIG. 5E), where stippled bars are standard MLR and striped bars
are the MLR with 1-MT. The T cell:APC ratio in FIG. 5E was the same
(20:1) for both DCs and non-dendritic APCs and both populations
were isolated from the same culture of mononuclear cells in GMCSF
IL4 and tested against the same population of T cells in parallel
MLRs.
Example 5
Suppression of Allogeneic T Cell Proliferation by Dendritic Cells
Expressing IDO
[0228] The experiments shown in the previous example demonstrated
that distinct IDO.sup.+ and IDO.sup.LO subsets can exist in the
same preparation of dendritic cells. This example shows that IDO
expressing dendritic cells from such a mixture suppress allogeneic
T cell proliferation (FIG. 6).
[0229] Myeloid dendritic cells (derived in bovine serum-based
medium as in FIG. 5A) were activated for 24 hrs with TNF.alpha. (10
ng/ml, BD), labeled with anti-CD123 antibody, then enriched by
sorting with goat anti-mouse secondary antibody conjugated to
magnetic beads (Miltenyi Biotec). Since expression of cell-surface
CD123 correlated closely with possession of inducible IDO,
immunomagnetic sorting based on CD123 was used to enrich for the
IDO.sup.+ subset. Cells selected as CD123.sup.+ cells (85-90%
purity) by immunomagnetic sorting were then tested as stimulators
in an allogeneic MLR. Dot-plots show analysis before ("Pre-sort")
and after ("CD123 enrichment (FIG. 6A).
[0230] The CD123.sup.+-enriched cells were used as APCs in an
allogeneic MLR. Dendritic cells were mixed with purified allogeneic
lymphocytes (<1% monocytes, 80-85% T cells, with the balance
being B and natural killer (NK) cells) at a 1:10 ratio in V-bottom
culture wells. After 5 days, proliferation was measured by 4 hr
thymidine incorporation assay. Controls shown include the
unfractionated population ("Pre-sort") and the cells remaining
after positive selection for CD123 ("Depleted"). Typically <10%
of the "Depleted" cell population was CD123.sup.+. Solid bars show
conventional MLR; open bars show MLR in the presence of 200 uM
1-methyl-(D,L)-tryptophan (1-MT) (Sigma-Aldrich, St. Louis, Mo.),
an inhibitor of IDO. In a similar set of experiments, 3 different
pairs of donors, each allogeneic to the other, and each pair
pre-tested to produce an active MLR were used without 1-MT (solid
bars) or with 1-MT (open bars) (FIG. 6C).
[0231] As shown in FIGS. 6B and C, the CD123-enriched (CD123.sup.F)
IDO cells were markedly less efficient at stimulating T cell
proliferation than either the original unfractionated mixture, or
than the CD123-depleted LDO.sup.LO subset that remained after
sorting. To test the hypothesis that this lack of proliferation was
due to active suppression by IDO, cultures were treated with
1-methyl-(D,L)-tryptophan (1-MT), a pharmacologic inhibitor of IDO.
In the presence of 1-methyl-(D,L)-tryptophan, the CD123.sup.+
dendritic cells stimulated proliferation at or near control levels
(FIGS. 6B and C), demonstrating that IDO causes suppression.
Example 6
Sorting on the Basis of Cell Surface CD123+ Results in Enrichment
of the IDO+ Population
[0232] This example shows that sorting dendritic cells to select
for CD123.sup.+ cells results in a population of cells which
exhibits high levels of IDO expression. In this experiment,
monocyte derived dendritic cells (DCs) were labeled with anti-CD123
antibody and selected using immunomagnetic sorting. Immediately
after sorting, cells were dual-stained for CD123 (surface) and IDO
(intracellular). As seen in FIG. 7, the positively selected cells
were approximately 90% CD123.sup.+. In addition, all (>99%) of
the cells showed high levels of IDO as detected by staining. In
contrast, the residual cells following CD123 depletion were mostly
CD123 negative, and expressed low, or undetectable levels of IDO.
Thus, it was found that the CD123 depleted population had 10-100
fold lower levels of IDO than the CD123.sup.+ population (FIG.
7).
Example 7
Detection of IDO-Expressing CD123+ Dendritic Cells in Human Tumors
and Draining Lymph Nodes
[0233] This example shows that CD123.sup.+ dendritic are associated
with human tumors and draining lymph nodes. Samples of tumor and
tumor-draining lymph nodes were chosen from patients with malignant
melanoma, a tumor with well-defined T cell antigens but which
nevertheless is not eliminated by the immune system. Recruitment of
1D0.sup.+ dendritic cells was also seen in carcinoma of the breast,
lung, colon and pancreas, tumors which account for almost half of
all cancer deaths in the United States.
[0234] Archival pathology specimens were stained for expression of
DO and other antigens by immunohistochemistry. Paraffin sections (5
um) were deparaffinized, treated for 8 min with proteinase K (Dako,
Carpinteria, Calif.), and stained with rabbit anti-human IDO
antibody (5 .mu.g/ml in Tris buffered saline with 0.05% Tween-20
and 10% goat serum). Detection was via secondary antibody
conjugated to alkaline phosphatase (LSAB-rabbit kit, Dako) with
Fast Red chromogen, or horseradish peroxidase (LSAB2, Dako) and
diaminobenzidine. Negative controls consisted of the anti-IDO
antibody neutralized with a 100-fold molar excess of the immunizing
peptide. Mip-3.alpha. (goat polyclonal, R&D Systems) was used
following antigen retrieval with citrate (Target, Dako). For
dual-staining, the first antibody was applied following appropriate
antigen retrieval and detected with peroxidase/diaminobenzidine.
Stained slides were then subjected to additional antigen retrieval
if required and stained for the second antigen by alkaline
phosphatase/Fast Red. Secondary antibodies were cross-adsorbed
against mouse, human and bovine IgG for multiple labeling.
[0235] In all of these studies, the IDO.sup.+ cells observed
appeared to be of the same cell type, displaying a characteristic
morphology resembling plasmaeytoid DCs (Cella, M., et al., Nature
Medicine 5: 919-923 (1999)); Grouard, G., et al., I Exp. Med., 185:
1101-1111 (1997); Facchetti, F., et al., J. Pathol., 158: 57-65
(1989)). They were neither histiocytic (macrophage-like) nor
classically dendritic in appearance, and did not mark with HamS6 (a
macrophage marker) or S100 (a marker of classical dendritic cells)
(data not shown). Shown in FIG. 8A is a known positive control for
detection of IDO (brown, diaminobenzidine chromogen) in
synrytiotrophoblast cells of term human placenta (Kudo, Y., et al.,
Biochem. Biophys. Acta 1500: 119-124 (2000)). The inset shows the
same tissue, but with anti-IDO antibody neutralized by an excess of
the immunizing peptide. (Bar=100 um, inset at half-scale).
[0236] For normal lymphoid tissue controls, non-inflamed tonsil
(from routine tonsillectomy, pathologic diagnosis of "hypertrophy")
and lymph nodes from patients with node-negative breast cancer who
never developed metastases or recurred in 5 years Following
resection were used. Although not technically "normal," these
specimens were the least inflamed lymphoid tissue removed in
routine clinical practice. Over 20 of these specimens have been
examined, and they consistently show only rare, scattered IDO.sup.+
cells, usually localized to germinal centers (FIG. 8F).
[0237] For tumor-draining lymph nodes from regional lymph node
dissections in patients with a variety of solid tumors (breast,
colon, lung, and pancreatic carcinoma, and malignant melanoma) were
used. Most of these nodes were not mapped by lymphoscintigraphy, so
not all would actually drain the tumor, but many would. In all 5
types of tumor examined, approximately one-third to one-half of
patients had one or more lymph nodes showing markedly abnormal
collections of IDO+ cells (FIG. 8C). In these nodes, often massive
infiltrates of IDO+ cells were localized to the perifollicular and
interfollicular areas, often adjacent to the medullary sinuses, or
collected in dense perivascular cuffs around high endothelial
venules (FIG. 8D). In 328 lymph nodes from 26 patients with
melanoma, abnormal infiltration of IDO+ cells was found in 14/26
patients. Where micro-metastases to lymph nodes were present, IDO+
cells often surrounded the margins of the tumor collections (FIG.
8E).
[0238] Thus, FIG. 8C shows a draining lymph node of a malignant
melanoma showing accumulation of IDO-expressing cells (red) in the
lymphoid and perivascular regions of the node, but sparing the
macrophage-rich sinuses (asterisk). (Bar=100 um). FIG. 8D shows a
higher magnification of panel C, showing a characteristic
collection of IDO-expressing cells around a high-endothelial venule
(V). (Bar=50 um). FIG. 8E shows a low-power view of a draining
lymph node containing heavily pigmented metastatic melanoma cells
(endogenous melanin, black; darkest signal), with confluent
infiltration of IDO-expressing cells (red; next darkest signal)
around the tumor deposits.
[0239] For solid tumors, 14 malignant melanoma tumors were examined
with 8/14 found to display collections of IDO.sup.+ cells at the
site of the primary tumor. Usually these were in the connective
tissue immediately surrounding the tumor (FIG. 8B, arrows) rather
than in the tumor parenchyma itself. Similar infiltrates of
IDO.sup.+ cells have been seen in breast, lung, and pancreatic
tumors.
[0240] For inflamed lymphoid tissue tonsils known to be infected
(either by clinical diagnosis or by histopathologic diagnosis) and
lymph node biopsies bearing the histopathologic diagnosis of
"reactive lymph node" were examined. Many of these specimens showed
focal or regional collections of IDO' cells. In tonsils these
collections frequently occurred in a subepithelial location beneath
the mucosa and along the crypts (not shown).
[0241] Finally, gut-associated lymphoid tissue from the (human)
small intestine was examined since IDO DCs derived in vitro
expressed CCR6, and mice with a targeted disruption of CCR6
(Varona, R., et al., J. Clin. Invest., 107: R37-45 (2001)) fail to
recruit a population of myeloid DCs into the lymphoid tissue of the
gut. FIG. 6G shows prominent collections of IDO.sup.+ cells in the
lamina propria overlying lymphoid aggregates in the gut,
congregating near cells expressing mip-3.alpha. (the ligand for
CCR6 (Sozzani, S. et al., J. Leukocyte Biol. 66: 1-9 (1999);
Zlotnik, A., et al., Immunity 12: 121-127 (2000)).
[0242] Thus, it was found that cells expressing IDO (and CCR6)
co-localized with cells expressing mip-3.alpha.. Sections of normal
human small intestine were used as a positive control for
mip-3.alpha. expression, since marine studies have shown that
mip-3.alpha. is highly expressed in the subepithelial tissues
overlying mucosal lymphoid aggregates of the small intestine (A.
Iwasaki and B. L. Kelsall, J. Exp. Med. 191: 1381-1394 (2000)). As
shown in FIG. 8G, the corresponding region in humans contained
focal collections of cells expressing mip-3.alpha., along with
extensive co-localization of IDO-expressing dendritic cells to the
same areas. Thus, FIGS. 8G and H shows co-localization of cells
expressing IDO (brown; darkest cytoplasmic signal) and mip-3.alpha.
(red; next darkest signal) in the lamina propria of the small
intestine, particularly in the subepithelial areas overlying
mucosal lymphoid aggregates (LA). FIG. 8H shows a higher
magnification of the region in panel G indicated by the arrow.
Bar=50 um.
[0243] Examination of mip-3.alpha. expression in malignancies
showed that many primary and metastatic tumors contained individual
tumor cells (FIG. 8I) or entire localized regions within the tumor
that expressed mip-3.alpha. by immunohistochemistry. Although both
mip-3.alpha. and IDO expressing cells are found in the tumor, they
did not appear to be located in identical cells. Thus, FIG. 8I
shows expression of mip-3.alpha. (red) (arrow, lower right) by
tumor cells in a lesion of malignant melanoma metastatic to lymph
node. The mip-3.alpha..sup.+ cells are scattered throughout the
tumor (T), while the IDO.sup.+ cells are congregated at the margins
of the metastasis but confined to the residual lymph node tissue
(LN). FIG. 8J shows a higher magnification of the region in panel M
indicated by the arrow, showing mip-3.alpha. expression in tumor
cells where the bar=50 um.
[0244] In addition, the morphology of these cells showed that they
were tumor cells, not stroma or other host-derived cells.
Quantitative analysis of mip-3.alpha. mRNA by real-time PCR
confirmed expression in 8/18 samples of malignant melanoma (see
Example 8). To ensure that this was not an idiosyncratic property
of melanomas, additional RNA samples were analyzed from tumors of
unrelated histology and cell of origin (renal cell carcinoma and
non-small cell lung cancer). This confirmed that a variety of tumor
types express mip-3.alpha. (D. Bell et Exp. Med. 190, 1417-1426
(1999)).
Example 8
Quantification of Mip-3.alpha. Expression in Human Tumors
[0245] It was found that human tumors express mip-3.alpha.. RNA was
isolated from melanomas (M, n=18), renal cell carcinomas (R, n=19)
or non-small cell lung cancers (L, n=9) and analyzed for expression
of mip-3.alpha. by quantitative RT-PCR (FIG. 9). The RNA was
reverse-transcribed using random hexamer priming and analyzed using
the LightCycler real-time PCR system (Roche,. Indianapolis, Ind.)
and FastStart DNA Amplification Kit (SYBR Green 1, Roche). The
primers used were: GAPDH (GenBank GI:7669491, sense basepairs (bp)
87-104, antisense by 289-307) and mip-3.alpha. (GenBank GI:4759075,
sense by 103-121, antisense bp410-428). Standard curves were
prepared from U937 cells induced with phorbol myristate acetate for
24 hrs, and were linear (r=-0.99) in the range of 100 pg to 100 ng
total RNA.
[0246] It was found that there was an increase in mip-3.alpha. mRNA
in all three tumor types assayed (FIG. 9). To permit comparison
between different samples the data are presented as an index,
calculated as the ratio of mip-3.alpha. to the GAPDH housekeeping
gene in each sample, normalized to the value of the control cell
line (resting U937 cells). The data shown thus represent fold
increase of mip-3.alpha. expression over that for GAPDH.
[0247] The invention has been described in detail with particular
reference to preferred embodiments thereof, but it will be
understood that variations and modifications can be effected within
the spirit and scope of the invention. References cited herein are
incorporated in their entirety by reference unless otherwise noted.
Sequence CWU 1
1
1119PRTHomo sapiens 1Asp Leu Ile Glu Ser Gly Gln Leu Arg Glu Arg
Val Glu Lys Leu Asn1 5 10 15Met Leu Cys
* * * * *