U.S. patent application number 14/414388 was filed with the patent office on 2015-07-23 for method for inducing immune stimulation using non-proliferative allogeneic leukocytes.
The applicant listed for this patent is CANADIAN BLOOD SERVICES. Invention is credited to Mark D. Scott, Wendy M. Toyofuku, Duncheng Wang.
Application Number | 20150202229 14/414388 |
Document ID | / |
Family ID | 49914159 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202229 |
Kind Code |
A1 |
Scott; Mark D. ; et
al. |
July 23, 2015 |
Method for Inducing Immune Stimulation Using Non-Proliferative
Allogeneic Leukocytes
Abstract
This invention relates to cellular-based therapies for
decreasing the level of regulatory T cells (Treg) and/or increasing
the level of pro-inflammatory T cells (Th17) to favor immune
stimulation. To provide these therapeutic effects, a
non-proliferative allogeneic leukocyte population is contacted with
another leukocyte population capable of proliferating. The
leukocyte populations are contacted so as to allow pro-inflammatory
allo-recognition. Cellular-based preparations and processes for
achieving cellular therapy are also provided.
Inventors: |
Scott; Mark D.; (Surrey,
CA) ; Wang; Duncheng; (Greenville, NC) ;
Toyofuku; Wendy M.; (Surrey, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CANADIAN BLOOD SERVICES |
Ottawa |
|
CA |
|
|
Family ID: |
49914159 |
Appl. No.: |
14/414388 |
Filed: |
July 12, 2013 |
PCT Filed: |
July 12, 2013 |
PCT NO: |
PCT/CA2013/050546 |
371 Date: |
January 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61670636 |
Jul 12, 2012 |
|
|
|
61670694 |
Jul 12, 2012 |
|
|
|
Current U.S.
Class: |
424/184.1 ;
424/278.1 |
Current CPC
Class: |
A61K 2039/515 20130101;
A61K 2035/122 20130101; A61P 37/02 20180101; A61K 35/17 20130101;
C12N 5/0637 20130101; C12N 2500/50 20130101; C12N 2501/65 20130101;
C12N 15/113 20130101; A61K 39/0008 20130101; C12N 2501/00 20130101;
A61K 35/00 20130101; A61K 47/60 20170801; A61P 35/02 20180101; C12N
2310/141 20130101; A61K 35/14 20130101; A61K 47/6901 20170801; A61K
2035/124 20130101; A61K 39/001 20130101; A61K 9/0019 20130101; A61K
47/69 20170801; C12N 2502/1114 20130101; A61K 2039/577 20130101;
C12N 5/0638 20130101 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 9/00 20060101 A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2012 |
CA |
2782942 |
Claims
1. A method of decreasing a ratio of the level of regulatory T
(Treg) cells to the level of pro-inflammatory T cells in a subject
in need thereof, said method comprising administering to the
subject a therapeutic amount of: (i) a first cellular preparation
comprising a first leukocyte, wherein the first leukocyte is
allogeneic to the subject and is non-proliferative; (ii) a cultured
cellular preparation comprising a leukocyte from the subject and
obtained by culturing the leukocyte from the subject with the first
leukocyte; and/or (iii) a supernatant of a cell culture of a second
leukocyte and a third leukocyte, wherein the second leukocyte is
allogeneic to the third leukocyte and wherein at least one of the
second or third leukocyte is non-proliferative; thereby decreasing
the ratio of the level of Treg cells to the level of
pro-inflammatory T cells in the subject.
2. The method of claim 1, wherein the first leukocyte, the second
leukocyte and/or the third leukocyte is irradiated.
3-5. (canceled)
6. The method of claim 1, wherein the first leukocyte, the
leukocyte from the subject, the second leukocyte and/or the third
leukocyte is a T cell.
7. The method of claim 6, wherein the T cell is a CD4-positive T
cell or a CD8-positive T cell.
8. (canceled)
9. The method of claim 1, wherein the leukocyte from the subject is
expanded in vitro prior to administration to the subject.
10. The method of claim 1, wherein the first leukocyte is removed
from the cultured cellular preparation prior to administration to
the subject.
11. The method of claim 1, wherein the second leukocyte or the
third leukocyte is from the subject.
12. The method of claim 1, wherein the decreased ratio between the
level of Treg cells and the level of pro-inflammatory T cells is
for treating, preventing and/or alleviating the symptoms associated
with a condition caused or exacerbated by a reduced immune response
in the subject.
13. The method of claim 12, wherein the condition is a
proliferation-associated disorder or an infection.
14. The method of claim 13, wherein the proliferation-associated
disorder is cancer.
15. (canceled)
16. The method of claim 13, wherein the infection is a parasitic
infection.
17-32. (canceled)
33. A process for increasing and/or providing the ability of a
cellular-based preparation to decrease a ratio of regulatory T
(Treg) cells to pro-inflammatory T cells in a subject, said process
comprising: (i) at least one of: preventing a first leukocyte to
from proliferating to provide a first modified leukocyte, wherein
the first leukocyte is allogeneic to the subject; culturing the
first modified leukocyte with a leukocyte from the subject to
obtain a cultured cellular preparation; or preventing one of a
second leukocyte or a third leukocyte from proliferating, culturing
the second leukocyte with the third leukocyte, isolating the cell
culture supernatant to obtain a cell culture supernatant, wherein
the second leukocyte is allogeneic to the third leukocyte; and (ii)
formulating the first modified leukocyte, the cultured cellular
preparation and/or the cell culture supernatant for administration
to the subject.
34. The process of claim 33, wherein step (ii) further comprises
formulating the first modified leukocyte, the cultured cellular
preparation and/or the cell culture supernatant for intravenous
administration to the subject.
35. The process of claim 33, further comprising irradiating the
first leukocyte, the second leukocyte and/or the third leukocyte
for preventing proliferation.
36. The process of claim 33, wherein the second leukocyte or the
third leukocyte is from the subject.
37-39. (canceled)
40. The process of claim 33, wherein the first leukocyte, the
leukocyte from the subject, the second leukocyte and/or the third
leukocyte is a T cell.
41. The process of claim 40, wherein the T cell is a CD4-positive T
cell or a CD8-positive cell.
42. (canceled)
43. The process of claim 33, further comprising expanding the
leukocyte from the subject in vitro prior to step (ii).
44. The process of claim 33, further comprising removing the first
leukocyte from the cultured cellular preparation prior to step
(ii).
45. The process of claim 33, wherein step (ii) further comprises
formulating the first modified leukocyte, the cultured cellular
preparation and/or the cell culture supernatant in a vaccine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from CA patent application
2782942, U.S. provisional patent application 61/670636 and U.S.
provisional patent application 61/670694 all filed on Jul. 12,
2012. Their content is incorporated herewith in their entirety.
TECHNOLOGICAL FIELD
[0002] This invention relates to the use of cellular-based
preparations using non-proliferative allogeneic leukocytes to
decrease the level of regulatory T (Treg) cells and/or decrease the
level of pro-inflammatory T cells for inducing of an immune
stimulation or a pro-inflammatory state in the treated subject.
These cellular-based preparations are useful for the treatment of
various conditions associated with decreased or inappropriate
immune responses, such as proliferation-associated diseases and
infections.
BACKGROUND
[0003] Failure of an animal's immune system to recognize and
destroy abnormal cells arising from normal progenitor cells can
result in uncontrolled growth and formation of tissue masses that
may cause significant morbidity and mortality in the absence of
ineffective therapeutic interventions. This is commonly
exemplified, but not limited to, cancer cells caused by spontaneous
genetic mutation and deletions or exposure to environmental agents
leading to similar genetic and cellular changes. Currently, most
therapeutic drugs consist of chemical cytotoxic agents targeting
proliferating cells, of which the cancer cells are preferentially
affected due to their higher mitotic rate, but with limited direct
specificity towards the cancer cells.
[0004] New cellular based approaches attempt to overcome the lack
of specificity of cytotoxic drugs by inducing (in vivo or ex vivo)
a target cell antigen-specific response by clonal expansion of a
subset of reactive leukocytes from the affected individual or
animal. This can be done by isolating either the "target cell"
(e.g. cancer cell) itself or by molecular mimicking of a target
cell antigen. However, this approach is expensive and may or may
not effectively stimulate the desired pro-inflammatory state in
vivo. Moreover, in some cases, infective agents (e.g. viruses and
parasites) may be established and persist in a subject due to a
failure of the subjects immune system to effectively respond to the
infective agent/organism via a pro-inflammatory mechanism. Indeed,
in many cases the infective organism may actively exert an anergic
effect yielding a decreased ratio of Treg to pro-inflammatory
cells.
[0005] It would be highly desirable to be provided with a
cellular-based preparation capable of inducing a state of immune
stimulation by decreasing the ratio of the level of regulatory T
cells (such as Treg) to pro-inflammatory T cells (such as Th1 and
Th17). The cellular-based preparation could induce an immune
stimulation either by decreasing Treg levels, increase
pro-inflammatory T cell levels or both. These preparations could be
useful for treating, preventing and/or alleviating the symptoms
associated to a condition associated with a low or inappropriate
immune response (e.g. anergy or tolerance for example), such as a
proliferation-associated disorder (cancer for example) or an
infection (a parasitic infection for example).
BRIEF SUMMARY
[0006] One aim of the present invention is to provide
cellular-based preparations capable of inducing a state of immune
stimulation by decreasing the ratio of the level of regulatory T
cells (such as Treg) to the level of pro-inflammatory T cells (such
as Th1 and Th17). The cellular-based preparations could induce
immune stimulation either by decreasing Treg levels, increasing
pro-inflammatory T cell levels or both. These cellular-based
preparations are useful for treating, preventing and/or alleviating
the symptoms associated to a condition caused/exacerbated by a low
or inappropriate immune response. The cellular-based preparations
and therapies presented herewith are derived from the contact of at
least two distinct leukocyte populations which are considered
allogeneic with respect to one another and wherein one of the
leukocyte population is non-proliferative. The two leukocyte
populations are contacted under conditions so as to allow
pro-inflammatory allo-recognition and ultimately immune
stimulation. The two leukocyte populations can be contacted in
vitro, ex vivo or in vivo to induce immune stimulation and/or a
pro-inflammatory state.
[0007] In accordance with the present invention, there is provided
a method of decreasing a ratio of the level of regulatory T (Treg)
cells to the level of pro-inflammatory T cells in a subject in need
thereof. Broadly, the method comprises administering: a cellular
preparation comprising a first leukocyte being allogeneic to the
subject as well as being non-proliferative; a cultured cellular
preparation comprising a leukocyte from the subject which has been
obtained by culturing it with the first leukocyte and/or a
supernatant of a cell culture of a second leukocyte and a third
leukocyte wherein the second leukocyte is allogeneic to the third
leukocyte and one of the second or the third leukocyte is
non-proliferative. The method is designed to provide a decrease in
the ratio of the level of Treg cells to the level of
pro-inflammatory T cells in the treated subject. In an embodiment,
the leukocyte is irradiated to prevent it from proliferating. In
another embodiment, the leukocyte is modified with a biocompatible
polymer. In such embodiment, it is contemplated that the
cytoplasmic membrane of the leukocyte has a membrane-associated
protein covalently bound to the biocompatible polymer. In some
embodiment, the biocompatible polymer is a polyethylene glycol
(PEG) or 2-alkyloxazoline (POZ). In yet another embodiment, the
leukocyte described herein is a T cell (such as, for example, a
CD4-positive or a CD8-positive T cell). In another embodiment, in
the cultured cellular preparation, the leukocyte from the subject
is expanded in vitro (or ex vivo) prior to administration to the
subject. In yet another embodiment, in the cultured cellular
preparation, the first leukocyte is removed prior to administration
to the subject. In an embodiment of the cell culture supernatant,
the second leukocyte or the third leukocyte is from the subject. In
still another embodiment, the decreased ratio between the level of
Treg cells and the level of pro-inflammatory T cells is for
treating, preventing and/or alleviating the symptoms associated to
a condition caused/exacerbated by a reduced immune response (e.g.
for example, a state of anergy or tolerance, a
proliferation-associated disorder such as cancer or an infection
such as a parasitic infection).
[0008] In accordance with the present invention, there is provided
a cellular-based preparation for decreasing a ratio of regulatory T
(Treg) cells to pro-inflammatory T cells in a subject. The
cellular-based preparation comprises a first cellular preparation
comprising a first leukocyte being allogeneic to the subject as
well as being non-proliferative; a cultured cellular preparation
comprising a leukocyte from the subject which has been obtained by
culturing it with the first allogeneic and non-proliferative
leukocyte and/or a supernatant of a cell culture of a second
leukocyte and a third leukocyte, wherein the second leukocyte is
allogeneic to the third leukocyte and one of the second or the
third leukocyte is non-proliferative. The cellular-based
preparation can be admixed with an appropriate excipient prior to
administration to the subject. Embodiments with respect to the type
of non-proliferative cells, the type of polymer modifications that
can be made to the leukocyte, the first leukocyte, the leukocyte
from the subject, the second leukocyte, the third leukocyte as well
as the various uses of the preparations have been described above
and do apply herein.
[0009] In accordance with the present invention, there is provided
the use of the cellular-based preparation described herein for
decreasing a ratio of regulatory T (Treg) cells to pro-inflammatory
T cells in a subject. There is provided the use of the
cellular-based preparation described herein for the preparation of
a medicament for decreasing a ratio of regulatory T (Treg) cells to
pro-inflammatory T cells in a subject. The cellular-based
preparation comprises a first cellular preparation comprising a
first leukocyte being allogeneic to the subject as well as being
non-proliferative; a cultured cellular preparation comprising a
leukocyte from the subject which has been obtained by culturing it
with the first leukocyte and/or a supernatant of a cell culture of
a second leukocyte and a third leukocyte wherein the second
leukocyte is allogeneic to the third leukocyte and one of the
second or the third leukocyte is non-proliferative. The
cellular-based preparation can be admixed with an appropriate
excipient prior to administration to the subject. Embodiments with
respect to the type of non-proliferative cells, the type of polymer
modifications that can be made to the leukocyte, the first
leukocyte, the leukocyte from the subject, the second leukocyte,
the third leukocyte as well as the various uses of the preparations
have been described above and do apply herein.
[0010] In accordance with the present invention, there is provided
a process for increasing and/or for providing the ability of a
cellular-based preparation to decrease a ratio of regulatory T
(Treg) cells to pro-inflammatory T cells in a subject. Broadly, the
process comprises (i) at least one of 1/ preventing a first
leukocyte from proliferating to obtain a first modified leukocyte
(wherein the first leukocyte is allogeneic to the subject), 2/
culturing the first modified leukocyte with a leukocyte from the
subject to obtain a cultured cellular preparation and/or 3/
preventing one of a second leukocyte or a third leukocyte from
proliferating, culturing the second leukocyte with the third
leukocyte (wherein the second leukocyte is allogeneic to the third
leukocyte), isolating the cell culture supernatant to obtain a cell
culture supernatant; and (ii) formulating the first modified
leukocyte, the cell cultured cellular preparation or the cell
culture supernatant for administration to the subject (such as, for
example, intravenous administration). The formulating step can also
encompass formulating the first modified leukocyte, the cell
cultured cellular preparation or the cell culture supernatant in a
vaccine. In an embodiment, the method can comprise modifying the
first leukocyte, the leukocyte from the subject, the second
leukocyte and/or the third leukocyte with a biocompatible polymer.
For example, the method can comprise covalently binding the
biocompatible polymer to a membrane-associated protein of the
cytoplasmic membrane of the first leukocyte, the leukocyte from the
subject, the second leukocyte and/or the third leukocyte. In a
further embodiment, the biocompatible polymer is a polyethylene
glycol (PEG) or 2-alkyloxazoline (POZ). Embodiments with respect to
type of non-proliferative cells, the first leukocyte, the leukocyte
from the subject, the second leukocyte, the third leukocyte as well
as the various uses of the preparations have been described above
and do apply herein.
[0011] Throughout this text, various terms are used according to
their plain definition in the art. However, for purposes of
clarity, some specific terms are defined below.
[0012] Allogeneic cell. A cell is considered "allogeneic" with
respect to another cell if both cells are derived from the same
animal species but presents sequence variation in at least one
genetic locus. A cell is considered "allogeneic" with respect to a
subject if the cell is derived from the same animal species as the
subject but presents sequence variation in at least one genetic
locus when compared to the subject's respective genetic locus.
Allogeneic cells induce an immune reaction (such as a cell-based
immune reaction, a rejection for example) when they are introduced
into an immunocompetent host. In an embodiment, a first cell is
considered allogeneic with respect to a second cell if the first
cell is HLA-disparate (or HLA-mismatched) with the second cell.
[0013] Allo-recognition. As it is known in the art, the term
"allo-recognition" (also spelled allorecognition) refers to an
immune response to foreign antigens (also referred to as
alloantigens) from members of the same species and is caused by the
difference between products of highly polymorphic genes. Among the
most highly polymorphic genes are those encoding the MHC complex
which are most highly expressed on leukocytes though other
polymorphic proteins may similarly result in immune recognition.
These polymorphic products are typically recognized by T cells and
other mononuclear leukocytes. In the context of the present
invention, the term "pro-inflammatory allo-recognition" refers to
an immune response associated with the expansion of
pro-inflammatory T cells and/or the differentiation of naive T
cells into pro-inflammatory T cells. Pro-inflammatory
allo-recognition in vivo mediates cell or tissue injury and/or
death and loss of cell or tissue function. Still in the context of
the present invention, the term "pro-tolerogenic allo-recognition"
refers to an immune response associated with the expansion of Treg
cells and/or the differentiation of naive T cells into Treg cells.
A pro-tolerogenic allo-recognition is usually considered weaker
than a pro-inflammatory allo-recognition. Further, an in vivo
pro-tolerogenic allo-recognition does not lead to significant cell
or tissue injury and/or death nor loss of cell or tissue
function.
[0014] Anergy and Tolerance. In the present context, the term
"anergy" refers to a non-specific state of immune unresponsiveness
to an antigen to which the host was previously sensitized to or
unsensitized to. It can be characterized by a decrease or even an
absence of lymphokine secretion by viable T cells when the T cell
receptor is engaged by an antigen. In the present context, the term
"tolerance" (also referred to as a pro-tolerogenic state) refers to
an acquired specific failure of the immunological mechanism to
respond to a given antigen, induced by exposure to the antigen.
Tolerance refers to a specific nonreactivity of the immune system
to a particular antigen, which is capable, under other conditions,
of inducing an immune response. However, in the present context,
the terms "anergy" and "tolerance" are used interchangeably since
the compositions and methods presented herewith can be used to
achieve both anergy and tolerance.
[0015] Autologous cell. A cell is considered "autologous" with
respect to another cell if both cells are derived from the same
individual or from genetically identical twins. A cell is
considered "autologous" to a subject, if the cell is derived from
the subject or a genetically identical twin. Autologous cells do
not induce an immune reaction (such as a rejection) when they are
introduced into an immuno-competent host.
[0016] Conditions associated with a reduced (low or inappropriate)
immune response. In the context of the present invention, the
subjects afflicted by these conditions have increased ratio of Treg
to pro-inflammatory T cells when compare to the ratio observed in
sex- and age-matched healthy subjects. In some embodiments, the
immune system of subjects afflicted by a condition associated with
a low, repressed or inappropriate immune response is in a state of
anergy. The immune system of some of the subjects afflicted by
these conditions fails to produce target specific pro-inflammatory
cell (T and B lymphocytes) capable of recognizing and destroying
abnormal cells (e.g., cancer cells or infected cells).
Alternatively, the immune system of some of the subjects afflicted
by these conditions exhibit elevated levels of regulatory T and B
cells that inhibit normal pro-inflammatory T and B cells from
exerting their function (i.e. inducing a partial or complete immune
suppression) thereby preventing destruction of an abnormal cell of
cell aggregates. One of these conditions is a
proliferation-associated disorder (such as, for example, cancer).
Another of these conditions is an infection (such as for example a
parasitic infection).
[0017] Proliferation-associated disorders. These disorders (also
referred to as hyperproliferative disorders) form a class of
diseases where cells proliferate more rapidly, and usually not in
an ordered fashion, than corresponding healthy cells. The
proliferation of cells cause an hyperproliferative state that may
lead to biological dysfunctions, such as the formation of tumors
(malignant or benign). One of the proliferation-associated disorder
is cancer. Also known medically as a malignant neoplasm, cancer is
a term for a large group of different diseases, all involving
unregulated cell growth. In cancer, cells divide and grow
uncontrollably, forming malignant tumors, and invade nearby parts
of the body. The cancer may also spread to more distant parts of
the body through the lymphatic system or bloodstream. In an
embodiment, the cancer is a carcinoma (e.g. a cancer of the
epithelial cells). Other types of cancer include, but are not
limited to sarcoma, lymphoma, leukemia, germ cell tumor and
blastoma.
[0018] Immune stimulation. In the present context, the term "immune
stimulation" or "pro-inflammatory state" refers to a state of
immune responsiveness to an antigen independent of the host
previously sensitization to the antigen. It can be characterized by
an increase or a modulation in the level of lymphokine secretion by
viable T cells when the T cell receptor is engaged by an antigen.
In the present context, the term "stimulation" refers to an
acquired specific activation of the immunological mechanism to
respond to a given antigen, induced by exposure to the antigen. In
the context of the present invention, the immune stimulation is
considered therapeutic and specifically excludes inflammatory
diseases, conditions and/or disorders.
[0019] Immunogenic cell. A first cell is considered immunogenic
with respect to a second cell when it is able to induce an immune
response in the latter cell. In some embodiment, the immune
response is in vitro (e.g. a mixed lymphocyte reaction) or can be
observed in vivo (e.g. in a subject having the second cell and
having received the first cell). The second cell can be located in
an immunocompetent subject. Preferably, the immune response is a
cell-based immune response in which cellular mediator can be
produced. In the context of this invention, the immunogenic cells
are immune cells, such as white blood cells or leukocytes.
[0020] Immunogenic cell culture conditions. A cell culture is
considered to be conducted in immunogenic conditions when it allows
the establishment of a pro-inflammatory immune response between two
distinct and unmodified leukocytes (and, in an embodiment,
allo-recognition). Preferably, the pro-inflammatory immune response
is a cell-based immune response in which cellular mediator can be
produced. For example, the cell culture conditions can be those of
a mixed lymphocyte reaction (primary or secondary).
[0021] Infection. As used in the context of the present invention,
the term "infection" or "infective disease" is a condition caused
by the presence and proliferation of an infectious agent which
induces a state of low or repressed immune response (e.g. anergy).
In some embodiments, the infection is caused by a parasite and in
such instances, it is referred to as a "parasitic" infection. There
are mainly three classes of parasites which can cause infections,
at least in humans, protozoa (causing protozoan infection),
helminths (causing an helminthiasis) and ectoparasites. As it is
known in the art, parasites have the intrinsic ability, upon
infecting their host, to upregulate or enhance Treg's levels and/or
activity and thereby induce a state of immune tolerance. This is
exemplified by filarial nematodes in which the nematode secretes
substances that cause an increase in the host's Treg lymphocytes
levels. The increase in Tregs actively down-regulate the Th1 and
Th2 responses necessary for eradication of the parasite.
Administration of an agent that can reverse the parasite's induced
Treg increase would enhance the ability of the subjects immune
system to eradicate the parasitic infection.
[0022] Leukocyte. As used herein, a leukocyte (also spelled
leucocyte) is defined as a blood cell lacking hemoglobin and having
a nucleus. Leukocytes are produced and derived from hematopoietic
stem cells. Leukocytes are also referred to as white blood cells.
Leukocytes include granulocytes (also known as polymorphonuclear
leucocytes), e.g. neutrophils, basophils and eosoniphils.
Leukocytes also include agranulocytes (or mononuclear leucocytes),
e.g. lymphocytes, monocytes and macrophages. Some of the
lymphocytes, referred to as T cells (or T-cell), bear on their
surface a T-cell receptor. T cell are broadly divided into cells
expressing CD4 on their surface (also referred to as CD4-positive
cells) and cells expressing CD8 on their surface (also referred to
as CD8-positive cells). Some of the lymphocytes, referred to as B
cells (or B-cells), bear on their surface a B-cell receptor.
[0023] Non-proliferative leukocyte. As used herein, the term
"non-proliferative leukocyte" refers to a leukocyte which has been
modified to no longer being capable of cellular proliferative (e.g.
performing at least one complete division cycle). In some
embodiments, this modification may be temporary and the
non-proliferative properties of a leukocyte may be limited in time.
For example, when a leukocyte is modified from a contact with a
pharmacological agent capable of limiting its proliferation, the
removal of the pharmacological agent from the cell culture can
allow the leukocyte to regain its proliferative properties. In
other embodiments, the modification is permanent and the modified
leukocyte cannot regain its proliferative properties. For example,
when a leukocyte is irradiated, it is not possible for it to regain
its proliferative properties. In the context of the present
application, the expressions "non-proliferative leukocyte" or
"leukocyte limited from proliferating" are used
interchangeably.
[0024] Peripheral blood mononuclear cells (PBMC). This term refers
to the cell population recuperated/derived from the peripheral
blood of a subject (usually a mammal such as a human). PBMC usually
contains T cells, B cells and antigen presenting cells.
[0025] Pharmaceutically effective amount or therapeutically
effective amount. These expressions refer to an amount (dose) of a
cellular preparation effective in mediating a therapeutic benefit
to a patient (for example prevention, treatment and/or alleviation
of symptoms of an immune-associated disorder or infection in which
the ratio of Tregs to pro-inflammatory T cells is high when
compared to sex- and aged-matched healthy subjects). It is also to
be understood herein that a "pharmaceutically effective amount" may
be interpreted as an amount giving a desired therapeutic effect,
either taken in one dose or in any dosage or route, taken alone or
in combination with other therapeutic agents.
[0026] Prevention, treatment and alleviation of symptoms. These
expressions refer to the ability of a method or cellular
preparation to limit the development, progression and/or
symptomology of a immune-associated disorder associated to
conditions caused/exacerbated by a low or inappropriate immune
response (also known as a state of anergy or tolerance). The
subjects being afflicted with these conditions/disorders s ratio of
Tregs to pro-inflammatory T cells which is considered high when
compared to sex- and aged-matched healthy subjects. Broadly, the
prevention, treatment and/or alleviation of symptoms encompasses
decreasing the levels of Treg cells and/or increasing the levels of
pro-inflammatory T cells. A method or cellular-based preparation is
considered effective or successful for treating and/or alleviating
the symptoms associated with the disorder when a reduction in the
pro-tolerogenic state (when compared to an untreated and afflicted
individual) in the treated individual (previously known to be
afflicted with the disorder) is observed. A method or
cellular-based preparation is considered effective or successful
for preventing the disorder when a reduction in the pro-tolerogenic
state (when compared to an untreated and afflicted individual) in
the treated individual is observed upon an immunological challenge
(such as, for example, an antigenic challenge). In instances where
the conditions to be treated is cancer, exemplary symptoms which
can be alleviated with the cellular-based preparations described
herewith include, but are not limited to, number and/or size of
metastasic tumors, presence and/spread of metastatic tumors and/or
size of primary tumor. In instances where the conditions to be
treated is an infection, exemplary symptoms which can be alleviated
with the cellular-based preparations described herewith include,
but are not limited to, infectious agent's burden, infectious
agent's presence and fever.
[0027] Pro-inflammatory T cells. In the present context,
pro-inflammatory T cells are a population of T cells capable of
mediating an inflammatory reaction. Pro-inflammatory T cells
generally include T helper 1 (Th1 or Type 1) and T helper 17 (Th17)
subsets of T cells. Th1 cells partner mainly with macrophage and
can produce interferon-.gamma., tumor necrosis factor-.beta., IL-2
and IL-10. Th1 cells promote the cellular immune response by
maximizing the killing efficacy of the macrophages and the
proliferation of cytotoxic CD8+ T cells. Th1 cells can also promote
the production of opsonizing antibodies. T helper 17 cells (Th17)
are a subset of T helper cells capable of producing interleukin 17
(IL-17) and are thought to play a key role in autoimmune diseases
and in microbial infections. Th17 cells primarily produce two main
members of the IL-17 family, IL-17A and IL-17F, which are involved
in the recruitment, activation and migration of neutrophils. Th17
cells also secrete IL-21 and IL-22.
[0028] Regulatory T cells. Regulatory T cells are also referred to
as Treg and were formerly known as suppressor T cell. Regulatory T
cells are a component of the immune system that suppress immune
responses of other cells. Regulatory T cells usually express CD3,
CD4, CD8, CD25, and Foxp3. Additional regulatory T cell populations
include Tr1, Th3, CD8.sup.+CD28.sup.-, CD69.sup.+, and Qa-1
restricted T cells. Regulatory T cells actively suppress activation
of the immune system and prevent pathological self-reactivity, i.e.
autoimmune disease. The critical role regulatory T cells play
within the immune system is evidenced by the severe autoimmune
syndrome that results from a genetic deficiency in regulatory T
cells. The immunosuppressive cytokines TGF-.beta. and Interleukin
10 (IL-10) have also been implicated in regulatory T cell function.
Similar to other T cells, a subset of regulatory T cells can
develop in the thymus and this subset is usually referred to as
natural Treg (or nTreg). Another type of regulatory T cell (induced
Treg or iTreg) can develop in the periphery from naive CD4.sup.+ T
cells. The large majority of Foxp3-expressing regulatory T cells
are found within the major histocompatibility complex (MHC) class
II restricted CD4-expressing (CD4.sup.+) helper T cell population
and express high levels of the interleukin-2 receptor alpha chain
(CD25). In addition to the Foxp3-expressing CD4.sup.+CD25.sup.+,
there also appears to be a minor population of MHC class I
restricted CD8.sup.+ Foxp3-expressing regulatory T cells. Unlike
conventional T cells, regulatory T cells do not produce IL-2 and
are therefore anergic at baseline. An alternative way of
identifying regulatory T cells is to determine the DNA methylation
pattern of a portion of the foxp3 gene (TSDR,
Treg-specific-demthylated region) which is found demethylated in
Tregs.
[0029] Splenocytes. This term refers to the cell population
obtained from the spleen of a subject (usually a mammal such as a
rodent). Splenocytes usually comprise T cell, B cell as well as
antigen presenting cells.
[0030] Syngeneic cell. A cell is considered "syngeneic" with
respect to a subject (or a cell derived therefrom) if it is
sufficiently identical to the subject so as to prevent an immune
rejection upon transplantation. Syngeneic cells are derived from
the same animal species.
[0031] Viable. In the present context, the term "viable" refers to
the ability of a cell to complete at least one cell cycle and,
ultimately proliferate. A viable cell is thus capable of
proliferating. By opposition, the term "non-viable" or
"non-proliferative" both refer to a cell which is no longer capable
of completing at least one cell cycle. By comparison, the term
"cycle arrest" refers to a cell which has been treated to halt its
cell cycle progression (usually with a pharmacological agent) but
which is still capable of re-entering the cell cycle (usually when
the pharmacological agent is removed).
[0032] Xenogeneic cell. A cell is considered "xenogeneic" with
respect to a subject (or a cell derived from the subject) when it
is derived from a different animal species than the subject. A
xenogeneic cell is expected to be rejected when transplanted in an
immunocompetent host.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Having thus generally described the nature of the invention,
reference will now be made to the accompanying drawings, showing by
way of illustration, a preferred embodiment thereof.
[0034] FIG. 1 shows diagrammatically the conditioned media
protocol. A primary (1.degree.) two-way mixed lymphocyte reaction
(MLR) was initiated using two HLA-disparate populations consisting
of unmodified or polymer-grafted (1 mM SVAmPEG; 5 kDa) PBMC. Within
the mPEG-MLR, only one donor population was PEGylated. At 72 h, the
conditioned media from the wells were collected. Secondary
(2.degree.) mixed lymphocyte reactions using control and PEGylated
PBMC from the same donors were initiated. A mitogen (PHA)
stimulation control was added to assure that the media collected
would support proliferation. 1.degree. MLR Conditioned media:
1=Resting unmodified PBMC; 2=Resting mPEG-PBMC; 3=Control MLR; and
4=mPEG-MLR. 1.degree. MLR/2.degree. MLR Cell Types or Stimulation:
A=Resting PBMC; B=Resting mPEG PBMC; C=MLR; D=mPEG MLR; P=PHA
stimulation.
[0035] FIG. 2 shows 1.degree. mixed lymphocyte reaction (MLR)
results. Primary(1.degree.) media cytokine levels at 72 h. IL-2
(A), IFN-.gamma. (B) IL-17A (C), TNF-.alpha. (D) and IL-6 (E),
levels are significantly increased in the control two-way MLR
utilizing unmodified PBMC populations from HLA disparate
individuals. The cytokine profile (ng/mL) was analyzed using the BD
cytometric bead array. Values shown are the mean.+-.SD of a minimum
of four independent experiments. Percent non-viable cells within
the control and PEGylated (SVAmPEG; 5 kDa) resting PBMC was
assessed by propidium iodine exclusion (F).
[0036] FIG. 3 shows 2.degree. mixed lymphocyte reaction (MLR)
results. Shown is the proliferation index (percent PBMC
proliferation) of the secondary MLR (.quadrature. resting PBMC,
control MLR, .smallcircle. mPEG MLR, .box-solid. PHA stimulation)
that were conducted in the indicated (x-axis) conditioned media. As
shown, relative to all other conditioned media, the media from the
1.degree. plate control MLR demonstrated a significant (p<0.01)
pro-proliferative effect in the 2.degree. MLR. This effect was
noted on even resting PBMC and PHA-stimulated cells. No significant
differences were noted between fresh media in a parallel secondary
plate and the resting PBMC conditioned media. Shown are the
individual results of four independent experiments and the mean
(line). PEGylated cells were modified with 1 mM SVAmPEG (5
kDa).
[0037] FIG. 4 illustrates the effects of the various conditioned
media on the levels of Treg and Th17. Use of non-modified human
lymphocytes (control MLR) resulted in a significant in vitro
immunomodulatory effects as noted by changes in the percentage of
Treg (A) and Th17 (B) T cell populations. Results are provided for
Treg levels (upper panel and columns), for Th17 levels (lower panel
and columns) as well as percent PBMC proliferation (line) for
2.degree. plates (dark gray column.fwdarw.resting PBMC; light grey
column.fwdarw.control MLR; white column.fwdarw.mPEG MLR; hatched
column.fwdarw.PHA stimulation) having received 1.degree.
conditioned media (defined in x-axis). As shown, the 1.degree.
media from the control MLR enhanced Th17 cell production and
greatly inhibited Treg levels. The relative ratio of Th17:Treg was
highly correlated with lymphocyte proliferation as denoted by the
right y-axis and the embedded line graph. An increased level of
Th17 cells was associated with the 1.degree. media from the control
MLR and PHA stimulation. PEGylated cells were modified with 1 mM
SVAmPEG (5 kDa). Percent PBMC proliferation is provided in the
right y-axis and by line on both panels.
[0038] FIG. 5 illustrates Treg levels in the spleen (A), in the
brachial lymph nodes (B) or in the blood (C) in function of time
(hours post injection) following administration of donor
splenocytes or control (.DELTA. naive; .tangle-solidup. soluble
mPEG; .quadrature. syngeneic cells; .box-solid. mPEG syngeneic
cells; allogeneic cells; .smallcircle. mPEG allogeneic cells). A
significant decrease in Tregs (relative to naive mice) is noted in
mice transfused with unmodified allogeneic splenocytes. In
comparing the absolute difference between the control PEGylated
splenocytes (dotted area or .DELTA.d) the differential impact of
donor cell PEGylation can be fully appreciated. Importantly, as
noted at 120 h, transfusion of soluble mPEG, syngeneic cells or
mPEG-syngeneic cells had no significant effect on the Treg
lymphocyte population. The range observed in naive mice is denoted
by the grey bars. PEGylated murine splenocytes were modified with 1
mM SVAmPEG (20 kDa).
[0039] FIG. 6 illustrates Th17 levels in the spleen (A), in the
brachial lymph nodes (B) or in the blood (C) in function of time
(hours post injection) following administration of donor
splenocytes or control (.DELTA. naive; .tangle-solidup. soluble
mPEG; .quadrature. syngeneic cells; .box-solid. mPEG syngeneic
cells; allogeneic cells; .smallcircle. mPEG allogeneic cells).
PEGylation of allogeneic donor murine splenocytes resulted in a
significant in vivo immunomodulatory effect as evidenced by
baseline levels of Th17 lymphocytes. As shown, unmodified
allogeneic splenocytes resulted in a dramatic increase (p<0.001
at all time points>24 h) in Th17 lymphocytes. In comparing the
absolute difference between the control and PEGylated splenocytes
(dotted area or .DELTA.d) the differential impact of donor cell
PEGylation can be fully appreciated. Importantly, as noted at 120
h, transfusion of soluble mPEG, syngeneic cells or mPEG-syngeneic
cells had no significant effect on the Th17 lymphocyte population.
The range observed in naive mice is denoted by the grey bars.
PEGylated murine splenocytes were modified with 1 mM SVAmPEG (20
kDa).
[0040] FIG. 7 shows the ratio of Treg/Th17 levels five days
following administration of donor splenocytes. Administration of
unmodified allogeneic donor murine splenocytes resulted in a
significant in vivo immunomodulatory effect. The panels in (A) show
the ratio of Treg/Th17 levels in spleen (A1), "brachial" lymph node
(A2), and peripheral blood (A3). *=p<0.01 relative to control
animals; #=p<0.01 relative to unmodified allogeneic cells. The
graph in (B) compares the ratio when non-modified allogeneic cells
(right side) or PEGylated allogeneic cells (left side) are
administered demonstrating the reduction in the Treg:Th17 ratio
induced by the unmodified allogeneic cells. * denotes statistical
significance (p<0.001).
[0041] FIG. 8 shows the long-term immunomodulatory effects of donor
cells. The immunomodulatory effects of the unmodifed splenocytes is
long lived (>30 days) and is systemic in nature. The systemic
nature would be of importance in limiting metastases or in
rejection of parasitic infections. Results are shown for percentage
of Tregs (upper panels) and Th17 cells (lower panels) in the spleen
(right panels), brachial lymph nodes (middle panels) and peripheral
blood (left panels) for mice transfused with allogeneic splenocytes
(.box-solid.) and mPEG allogeneic splenocytes (.quadrature.).
Thirty days post-transfusion with allogeneic splenocytes
(.smallcircle.), mice still demonstrated significantly lowered Treg
levels demonstrating persistence of the immunomodulation. When mice
previously challenged with allogeneic splenocytes were rechallenged
30 days later with unmodified allogeneic splenocytes ( ) no
increase in Treg or decrease in Th17 cells were observed
demonstrating immune stimulation. Shaded area on the graph indicate
Treg and Th17 levels in naive mice. PEGylated murine splenocytes
were modified with 1 mM SVAmPEG (20 kDa).
[0042] FIG. 9 shows that immunomodulation is not
haplotype-specific. Initial one-way MLR (.smallcircle.) was
conducted and consisted of C57Bl/6 (H-2b) splenocytes challenged
with unmodified or PEGylated irradiated Balb/c (H2-d) splenocytes.
Following 48 h of challenge, duplicate samples were challenged with
unmodified-non-irradiated C3H (H-2k) splenocytes (two-way MLR or ).
Results are shown as .sup.3H-thymidine incorporation in function of
polymer (mPEG 5 kDa) grafting concentration (in mM). The addition
of the fresh responder cells from a third, H2-disparate mouse
strain (C3H), at 48 h did not reverse the attenuation of
proliferation in responder cells co-incubated with irradiated,
cmPEG-modified Babl/c splenocytes. In contrast, the proliferation
in the control (0 mM) MLR was significantly (p<0.001) enhanced
by the addition of the C3H splenocytes (.DELTA.C3H). The data shown
represented the co-culturing of 5.12.times.10.sup.6 C57Bl/6
splenocytes with 5.12.times.10.sup.6 irradiated, mPEG-derivitized
Balb/c splenocytes. After 48 h of incubation, fresh C3H responder
cells were added to duplicate wells. The results were expressed as
the average mean.+-.standard deviation of triplicate samples from a
representative experiment. PEGylated murine splenocytes were
modified with the indicated concentrations (mM) of activated mPEG
(5 kDa). For comparative purposes, the anti-proliferative
dose-response effect of cyclosporine A (CSA; which induces a
pharmacologically-induced anergy) in a one-way murine MLR under the
same experimental condition is shown in the insert.
[0043] FIG. 10 provides an hypothetical representation of
cellular-mediated immune modulation. (A) Current immunomodulation
therapy almost exclusively targets the recipient's immune system
and does not address the inherent antigenicity and immunogenicity
of allogeneic tissues. Response to non-self is in large part
mediated by cell-cell interactions between Antigen Presenting Cells
(APC; e.g., dendritic cells) and naive T lymphocytes (Thp). This
cell-cell interaction is characterized by adhesion, allorecognition
and co-stimulation events. Consequent to allorecognition,
cytokine/chemokine burst occurs followed by proliferation of
pro-inflammatory T cells (e.g., CTL, Th17, Th1 populations),
immunoglobulin production and decreased evidence of regulatory T
cells (Treg). (B) In contrast, polymer modification of donor PBMC
results in loss of appropriate cell-cell interaction leading to
loss of the cytokine burst, decreased/absent proliferation,
evidence of apoptosis of alloresponsive T cells and increased
levels of Regulatory T (Treg) cells that, in aggregate, provides a
tolerogenic/anergic state both in vitro and in vivo. Shown with the
schematic is a DNA laddering gel of an unmodified MLR (A) and a
PEGylated MLR (B) showing enhanced apoptosis consequent to
PEGylation. Size of T cell population denotes increase or decrease
in number. Size of B cell indicates antibody response.
[0044] FIG. 11 illustrates significant changes in the levels of
Th17 and Treg lymphocytes are noted in the spleen (upper panels),
brachial lymph node (middle panels) and pancreatic lymph nodes
(lower panels) upon conversion of NOD mice from non-diabetic (left
panels) to diabetic (right panels). These changes are characterized
by dramatically increased Th17 (in the spleen, from 0.03 to 3.84%;
in the brachial lymph node from 0.01% to 0.67%; in the pancreatic
lymph node from 0.05% to 1.05%) and significantly decreased Treg
(in the spleen, from 16.5% to 2.0%; in the brachial lymph node from
11.8% to 1.8% and in the pancreatic lymph node, from 12.7% to 4.1%)
lymphocytes. Tregs: *, p<0.001 from non-diabetic NOD mice. Th17:
**p<0.001 from non-diabetic NOD mice.
[0045] FIG. 12 illustrates cellular proliferation in a 2-way MLR of
PEGylated or POZylated cells at day 10. Results are shown for the
mPEG-MLR (.box-solid.) and POZ-MRL (.quadrature.) as a percentage
of proliferation (with respect to the proliferation of the control
MLR; i.e., 0 mM) as a function of grafting density.
[0046] FIG. 13 illustrates the immunomodulatory effects of
allogeneic and mPEG-allogeneic splenocytes upon injection in mice.
Carrier (PBS), allogeneic splenocytes (SPL) or mPEG allogeneic
splenocytes (mPEG-SPL) were injected in mice. (A) In vivo apoptosis
is provided as percentage of apoptotic cells (e.g., Annexin
V-positive cells) in in the spleen (grey bars) or the lymph node
(white bars) in function of type of injection (PBS=control,
SPL=unmodified allogeneic splenocytes, mPEG-SPL=mPEG allogeneic
splenocytes). (B) Percentage of CD4-positive cells having a
depolarized mitochondria in the spleen (grey bars) or the lymph
node (white bars) in function of type of injection (PBS=control,
SPL=unmodified allogeneic splenocytes, mPEG-SPL=mPEG allogeneic
splenocytes). (C) Percentage of intracellular IL-10-positive and
CD4-positive cells in the spleen (grey bars) or the lymph node
(white bars) in function of type of injection (PBS=control,
SPL=unmodified allogeneic splenocytes, mPEG-SPL=mPEG allogeneic
splenocytes). (D) 5-day weight gain (g) in mouse in function of
type of injection (PBS=control, SPL=unmodifiedallogeneic
splenocytes, mPEG-SPL=mPEG allogeneic splenocytes). In (D), the SPL
treated mice showed a loss of weight relative to PBS of mPEG-SPL
treated mice (0.64 g; approximately a 4% decrease in relative body
weight). *=p<0.01 relative to PBS treated animal; #=p<0.01
relative to unmodified splenocytes.
[0047] FIG. 14 illustrates the effects of allogeneic splenocytes
numbers and mPEG-grafting density on T cell differentiation in
vivo. As shown, the dose of allogeneic cells can be adjusted to
achieve variable changes in the relative abundance of Treg and Th17
cells; hence a variable change in the Treg:Th17 ratio. Percentage
of CD4-positive Tregs (white bars, percentage indicated on left
y-axis) and Th17 cells (grey bars, percentage indicated on right
y-axis) measured in resting Balb/c mice, mice having received
unmodified allogeneic (e.g. C57BL/6) splenocytes (5, 20 or
40.times.10.sup.6 cells) or mice having received mPEG-modified (at
a density of 0.5 mM, 1 mM or 4 mM) allogeneic (e.g. C57BL/6)
splenocytes (5, 20 or 40.times.10.sup.6 cells). *=p<0.01
relative to naive animal; #=p<0.01 relative to animal
administered unmodified splenocytes.
[0048] FIG. 15 illustrates the effects of allogeneic splenocytes on
CD279 expression of CD4-positive cells in vivo. Saline, syngeneic
splenocytes (syngeneic), allogeneic splenocytes (allogeneic) or
mPEG-allogeneic splenocytes (mPEG-Allo) have been injected
intravenously once (at day 0) or trice (at days 0, 2 and 5) in
recipient mice. CD4-positive cells have been harvested 5
(.smallcircle.) or 10 ( ) days after the last injection. The
percentage of CD4-positive and CD279-positive cells is shown in
function of type of injection (saline, syngeneic splenocytes,
allogeneic splenocytes or mPEG-allogeneic splenocytes) and number
of injections (once=1, trice=3). (A) Results are shown for
CD4-positive spleen cells. (B) Results are shown for CD4-positive
lymph node cells. *=p<0.01 relative to naive (shaded area)
animal; #=p<0.01 relative to animal administered unmodified
allogneic splenocytes.
[0049] FIG. 16 illustrates the effects of allogeneic splenocytes on
the percentage of Natural Killer (NK) cells in vivo. Saline,
syngeneic splenocytes (syngeneic), allogeneic splenocytes
(allogeneic) or mPEG-allogeneic splenocytes (mPEG-Allo) have been
injected intravenously once (at day 0) or trice (at days 0, 2 and
5) in recipient mice. NK cells have been harvested 10 days after
the last injection. The percentage of NK cells is shown in function
of type of injection (saline, syngeneic splenocytes, allogeneic
splenocytes or mPEG-allogeneic splenocytes), number of injections
(once=1, trice=3) and location of the NK cells ( =spleen,
.smallcircle.=brachial lymph node). Shaded area refers to the
percentage of NK levels in non-treated animals. *=p<0.01
relative to naive (shaded bar) animal; #=p<0.01 relative to
animal administered unmodified allogneic splenocytes.
[0050] FIG. 17 illustrates the effects of allogeneic splenocytes on
the thymus in vivo. Saline, allogeneic splenocytes (Allo) or
mPEG-allogeneic splenocytes (mPEG-Allo) have been injected
intravenously once in recipient mice. Thymic cells have been
harvested 5 days after the injection. (A) The percentage of
CFSE-positive donor cells (with respect to the total CD4-positive
cells) is shown in function of type of injection (saline,
allogeneic splenocytes or mPEG-allogeneic splenocytes). White bar
in mPEG-Allo sample represents the number of donor Tregs injected.
(a) denotes CFSE positive donor cells showing that no thymic
microchimerisim is achieved in vivo (i.e., donor cells do not
migrate to, or survive in, the recipient thymus). (b) denotes the
proliferative expansion of the donor Treg yielding thymic
microchimerism. *p<0.01 relative to saline treated animal.
#p<0.01 relative to allogeneic treated animal. (B) The
percentage of Treg cells or CD25-positive cells (with respect to
the total CD4-positive cells) is shown in function of type of
injection (saline, allogeneic splenocytes or mPEG-allogeneic
splenocytes). *p<0.01 relative to saline treated animal.
#p<0.01 relative to allogeneic treated animal. (a) denotes
decrease in Treg in allogeneic treated animals. (b) denotes
increase in Tregs in mPEG-allogeneic treated animals over that of
naive animals. (c) denotes the proliferative expansion of the donor
Treg yielding thymic microchimerism. *p<0.01 relative to saline
treated animal. #p<0.01 relative to allogeneic treated animal.
(C) The percentage of Treg cells (white bars, percentage indicated
in left y-axis, with respect to the total CD4-positive cells) and
Th17 cells (grey bars, percentage indicated in right y-axis, in
view of the total CD4-positive cells) is shown in function of type
of injection (saline (naive), allogeneic splenocytes (Allo),
gamma-irradiated allogneneic splenocytes (Ir-Allo), mPEG-allogeneic
(mPEG-Allo) or gamma-irradiated allogeneic splenocytes (Ir
mPEG-Allo)). Gamma-irradiated donor cells are incapable of
proliferation and are non-viable demonstrating that they can also
be used to alter the immune response. Changes in T cell subsets in
thymus are recipient-derived (e.g., CFSE-Negative, data not
shown).
[0051] FIG. 18 illustrates that conditioned murine plasma modulates
the Treg and Th17 differentiation levels in vivo. Conditioned
murine plasma (obtained from donor mice 5 days post leukocyte
transfer) was administered once or thrice to mice and Treg/Th17
levels were measured in the spleen and the lymph nodes. (A) Results
are shown as the percentage of Treg cells (in function of CD4.sup.+
cells) (white bars, left y axis) and as the percentage of Th17
cells (in function of CD4.sup.+ cells) (grey bars, right y axis) in
the spleen of animals treated once (1) or thrice (3) with a control
(Saline), a negative control conditioned plasma from animals having
received saline (Plasma (Saline)), a conditioned plasma from
animals having received unmodified allogeneic splenocytes (Plasma
(Allo)) or a condition plasma from animals having received
polymer-modified allogeneic splenocytes (Plasma (mPEG-Allo)). (B)
Results are shown as the percentage of Treg cells (in function of
CD4.sup.+ cells) (white bars, left y axis) and as the percentage of
Th17 cells (in function of CD4.sup.+ cells) (grey bars, right y
axis) in the brachial lymph nodes of animals treated once (1) or
thrice (3) with a control (Saline), a negative control conditioned
plasma from animals having received saline (Plasma (Saline)), a
conditioned plasma from animals having received unmodified
allogeneic splenocytes (Plasma (Allo)) or a conditioned plasma from
animals having received polymer-modified allogeneic splenocytes
(Plasma (mPEG-Allo)). *=p<0.01 relative to saline control
animal; #=p<0.01 relative to animal administered with the
unmodified allogeneic splenocytes (Plasma (Allo)).
[0052] FIG. 19 illustrates that conditioned murine plasma induces
long-term changes in cytokines expression levels in vivo.
Conditioned murine plasma (obtained from donor mice 5 days post
leukocyte transfer) was administered once or thrice to mice and
intracellular cytokine positive cells were measured in the spleen
and the lymph nodes. Results are shown as the percentage of
intracellular cytokine positive cells (in function of CD4.sup.+
cells) in the spleen of animals treated once (1) or thrice (3) with
a negative conditioned plasma from animals having received saline
(light grey bars), a conditioned plasma from animals having
received unmodified syngeneic splenocytes (dark gray bars), a
conditioned plasma from animals having received unmodified
allogeneic splenocytes (hatched bars) and a conditioned plasma from
animals having received polymer-modified allogeneic splenocytes
(white bars). Results are shown for IL-10, IL-2, TNF-.alpha.,
IFN-.gamma. and IL-4 either 30 or 60 days following the last
administration of the conditioned serum or control. Similar results
have been obtained with the leukocytes obtained from the brachial
lymph nodes of these treated animals (data not shown).
[0053] FIG. 20 illustrates that conditioned murine plasma modulates
multiple Treg subsets in vivo. Conditioned murine plasma (obtained
from donor mice 5 days post allogeneic leukocyte transfer) was
administered mice and multiple Treg subset levels were measured in
the spleen and the lymph nodes. Results are shown as the percentage
of Treg subset (in function of CD4.sup.+ cells) in the spleen and
brachial lymph node of animals administered with a control
(Saline), a negative control conditioned plasma from animals having
received saline (Plasma (Saline)), a conditioned plasma from
animals having received unmodified allogeneic splenocytes (Plasma
(Allo)) or a condition plasma from animals having received
polymer-modified allogeneic splenocytes (Plasma (mPEG-Allo)).
Results are shown for Foxp3.sup.+ cells (white bars in the spleen,
light gray bars in the lymph node), CD25.sup.+ cells (hatches bars
in the spleen, dark grey bars in the lymph node) and CD69.sup.+
cells (horizontal hatched bars in the spleen, diagonal hacthed bars
in the lymph node).
[0054] FIG. 21 illustrates that conditioned murine plasma prepared
from mice injected with saline, allogeneic or mPEG allogeneic cells
modulates Treg and Th17 differentiation levels in vivo. Conditioned
murine plasma (obtained from donor mice 5 days post leukocyte
transfer) was administered to mice and Treg/Th17 levels were
measured in the spleen, the lymph nodes and the blood five days
after treatment. Results are shown for naive animals (white bars)
and animals receiving conditioned plasma prepared from animals
having received saline (Plasma (Saline); light grey bars), animals
having received unmodified allogeneic splenocytes (Plasma (Allo);
dark grey bars) or polymer-modified allogeneic splenocytes (Plasma
(mPeg-All); hatched bars). Results are shown as the percentage of
Treg cells (in function of CD4.sup.+ cells) in the spleen (A), the
lymph node (B) or the blood (C). Results are also shown as the
percentage of Th17 cells (in function of CD4.sup.+ cells) in the
spleen (D), the lymph node (E) or the blood (F). *=p<0.01
relative to saline control animal; #=p<0.01 relative to animal
administered with Plasma(Allo)-conditioned plasma.
DETAILED DESCRIPTION
[0055] In accordance with the present invention, there is provided
cellular-based preparations for decreasing the level of regulatory
T cells and/or increasing the level of pro-inflammatory T cells for
inducing immune stimulation and/or a pro-inflammatory state in a
subject in need thereof. The cellular-based preparations and
therapies presented herewith concern the contact of at least two
distinct leukocyte populations which are considered allogeneic with
respect to one another and wherein at least one of the leukocyte
population is considered non-proliferative. The contact between the
two leukocyte populations occurs under conditions to allow
pro-inflammatory allo-recognition but limit or prevent
pro-tolerogenic recognition. The cellular-based preparations can be
a first leukocyte (which is allogeneic to the treated subject)
which has been prevented from proliferating. The cellular-based
preparation can also be a cultured cellular preparation obtained by
culturing the first leukocyte with a leukocyte from the subject (or
syngeneic to the subject). Alternatively, the cellular-based
preparation can be a cell culture supernatant (or a sample thereof)
obtained by isolating the cell culture supernatant of a co-culture
a second and a third leukocytes, wherein the second leukocyte is
allogeneic to the third leukocyte and one of the second or third
leukocyte is considered non-proliferative.
[0056] As it will be shown below, the modification of allogeneic
leukocyte to prevent them from proliferating provides a significant
opportunity to modulate the responsiveness (i.e., immunoquiescent
versus pro-inflammatory) of the recipient's immune system. Of
importance, the allogeneic leukocyte, besides being prevented from
proliferating, does not need to be further manipulated to mediate
its therapeutic effect. However, in some embodiments, the surface
of the allogeneic leukocyte can be further modified (for example
cross-linked and/modified with a polymer) to increase its
antigenicity. The allogeneic leukocyte can be expanded in vitro
prior to a co-culture step or its administration to the subject in
need thereof.
[0057] As shown herein, the contact between two leukocyte
populations (wherein one population has been refrained from
proliferating) which are considered allogeneic to one another
induces an immune stimulation (e.g. a pro-inflammatory state). More
specifically, the contact between two leukocyte populations
decreases the level of Treg cells and especially the levels of
CD69.sup.+ Treg cells. In addition, the contact between two
leukocyte populations potentiates natural killer (NK) cells. Taken
together, this indicates that the contact between the two leukocyte
populations can induce therapeutic effects in subjects experiencing
a low or inappropriate immune response (for example having elevated
levels of Treg cells (especially CD69.sup.+ Treg cells) and/or
having a low level or inactive NK cells) by favoring immune
stimulation via the induction of a pro-inflammatory state in
subjects experiencing anergy.
[0058] As it is known in the art, the administration of a
population of viable allogeneic leukocyte preparation can induce
the onset of graft-vs.-host disease. As shown herein, the
administration of non-viable/non-proliferative allogeneic
leukocytes (or products derived therefrom) can induce an immune
stimulation (in vivo as well as in vitro) and can be used to shift
the recipient's immune system from a pro-tolerogenic state to a
pro-inflammatory state while preventing graft-vs.-host disease.
These cellular preparations provide therapeutic tools for the
treatment of conditions associated with a pro-tolerogenic state
(e.g. anergy), such as proliferation-associated disorders as well
as infections. These cellular preparations provide tools for
shifting the immune system in a non-specific manner and to bolster
the immune system.
[0059] Methods for Modulating the Treg/Pro-inflammatory T Cells
Ratio
[0060] The present invention provides methods and cellular
preparations for decreasing the ratio of the level of regulatory T
cells with respect to the level of pro-inflammatory T cells. In the
present invention, the ratio can be decreased either by lowering
the level of regulatory T cells in the subject or increasing the
level of pro-inflammatory T cells in the subject. Alternatively,
the ratio can be decreased by lowering the level of regulatory T
cells in the subject and increasing the level of pro-inflammatory T
cells in the subject. When the Treg/pro-inflammatory T cells ratio
is decreased in a subject, it is considered that a state of immune
stimulation is induced or present in the subject. The induction of
a state of immune stimulation in subjects experiencing an
abnormally decreased immune state can be therapeutically beneficial
for limiting the symptoms or pathology associated with the
abnormally low immune reaction or an acquired state of anergy. In
some embodiments, it is not necessary to induce a state of complete
immune stimulation, a partial induction of immune stimulation can
be beneficial to prevent, treat and/or alleviate the symptoms of a
disorder associated with a pro-tolerogenic state (such as, for
example, a proliferation-associated disorder or an infection).
[0061] In order to decrease the Treg/pro-inflammatory T cells
ratio, an allogeneic cellular preparation can be administered to
the subject in a therapeutically effective amount. In such
instance, the cellular preparation can comprise a first leukocyte
that has been modified to be considered to be non-proliferative.
Prior to its modification, the first leukocyte is considered
immunogenic (e.g. allogeneic for example) with respect to the
subject because it is able to induce an immune response (e.g. a
cell-mediated immune response) when administered to the subject. As
indicated above, it is possible to determine if two cells are
considered immunogenic with respect to one another by conducting
conventional in vitro assays, such as a mixed lymphocyte reaction.
It is also expected that MHC-disparate cells would be considered
immunogenic with respect to one another. In an embodiment, the
first leukocyte can be xenogeneic to the subject. However, the
first leukocyte cannot be autologous or syngeneic to the subject.
Importantly, the first leukocyte, prior to its modification, is
also considered viable and capable of cellular proliferation. The
first leukocyte can even be optionally expanded in vitro
(preferably under conditions favoring the expansion of
pro-inflammatory T cells or the differentiation of naive T cells in
pro-inflammatory T cells), however, in such embodiment, the first
leukocyte is modified to become non-proliferative prior to its
administration to the subject. In an embodiment, the first
allogeneic leukocyte can be modified to bear on its surface a
polymer. However, the polymer, when present, must be selected or
grafted at a density so as to allow the pro-inflammatory
allo-recognition of the first leukocyte by the recipient. When the
first leukocyte is modified to bear on its surface a polymer, it
can be modified to be non-proliferative either prior to or after
the polymer modification.
[0062] Alternatively, in order to decrease the
Treg/pro-inflammatory T cells ratio, a cultured cellular
preparation can be administered to the subject in a therapeutically
effective amount. In order to do so, the first leukocyte is placed
in contact in vitro with a leukocyte from the subject or a
leukocyte syngeneic to the subject. One of the two leukocyte
population is prevented from proliferating. In some embodiments,
the first leukocyte is refrained from proliferating while the
leukocyte from the subject (or syngeneic to the subject) is viable
and capable of proliferation. The two cell populations are cultured
under immunogenic conditions to provide a cultured cellular
preparation in which a pro-inflammatory allo-recognition occurs. In
an embodiment, the two cells populations are cultured under
conditions favoring the expansion (e.g. proliferation) and/or
differentiation (e.g. naive to pro-inflammatory T cells) of the
cultured cells (preferably the leukocytes from the subject). This
expansion/proliferation can occur before, during or after the
co-culture of the two leukocyte populations. In some embodiments,
it is preferable to remove the first leukocyte from the cultured
cellular preparation prior to the administration of the cultured
cellular preparation to the subject. Methods of separating the two
cellular populations are known to those skilled in the art and
include, without limitation, cell sorting and magnetic beads. In an
embodiment, the first allogeneic leukocyte and/or the leukocyte
from the subject can be modified to bear on its surface a polymer.
However, the polymer, when present, must be selected or grafted at
a density so as to allow the pro-inflammatory allo-recognition of
the first leukocyte by the leukocyte from the subject (or syngeneic
to the subject). When the leukocyte is modified to bear on its
surface a polymer, it can be modified to be non-proliferative
either prior to or after the polymer modification.
[0063] An alternative way of decreasing the Treg/pro-inflammatory T
cell ratio concerns the administration of the supernatant of a cell
culture of a second leukocyte and a third leukocyte. In such
embodiment, one of the second or third leukocyte is limited from
proliferating when both leukocyte populations are cultured together
under immunogenic conditions. In some embodiments, the cell culture
supernatant can comprise leukocytes or leukocyte fractions (for
example a part of the cytoplasmic membrane) and/or even cellular
products present in the cell culture. In such embodiment, the
second leukocyte is considered immunogenic (e.g. allogeneic) with
respect to the third leukocyte because when the second leukocyte is
placed into contact with the third leukocyte, an immune response
(e.g. a cell-mediated immune response) occurs (provided that the
cell culture is performed under immunogenic conditions). It is
possible to determine if two cells are considered immunogenic with
respect to one another by conducting conventional in vitro assays,
such as the mixed lymphocyte reaction. It is also expected that
MHC-disparate cells would be considered immunogenic with respect to
one another. In another embodiment, the second leukocyte cell can
be xenogeneic to the third leukocyte However, the second leukocyte
cannot be autologous or syngeneic to the third leukocyte. In the
methods and cellular compositions described herein, it is possible
that one of the second or third leukocyte be syngeneic or derived
from the subject which will be treated with the cell culture
supernatant. In addition, in other embodiments, both the second
and/or third leukocytes can be considered allogeneic or xenogeneic
to the subject which will be treated. In some embodiment, the
leukocytes are being cultured in conditions favoring in vitro
expansion and/or differentiation of naive T cells to
pro-inflammatory cells of the leukocyte population that is not
refrained from proliferating. Such expansion/differentiation can
occur prior to, during or after the co-culture of the two leukocyte
populations. Importantly, the cell culture supernatant, apart from
being optionally filtered to remove cells and cellular debris, is
not submitted to further extraction/size fractionation or specific
enrichment of one of its components prior to its administration to
the subject. The cell culture supernatant thus comprises the
conditioned media from the cell culture (e.g. cellular by-products
such as cytokines for example). In an embodiment, the second
leukocyte and/or the third leukocyte can be modified to bear on its
surface a polymer. However, the polymer, when present, must be
selected or grafted at a density so as to allow the
pro-inflammatory allo-recognition of the second leukocyte by the
third leukocyte. When the leukocyte is modified to bear on its
surface a polymer, it can be modified to be non-proliferative
either prior to or after the polymer modification.
[0064] An alternative way of decreasing the Treg/pro-inflammatory T
cell ratio in a subject to be treated, is to administer the
conditioned blood (or fraction thereof such as plasma or serum) of
a test subject that has been administered with a first
non-proliferative allogeneic leukocyte. The animal is transfused
with in conditions so as to allow a pro-inflammatory
allo-recognition but to prevent the onset of GVHD. In some
embodiments, this conditioned blood can comprise the first
leukocyte or a derivative thereform (a part of the cytoplamsic
membrane from the first leukocyte for example). The first leukocyte
is considered immunogenic (e.g. allogeneic) with respect to the
test subject because when the first leukocyte is transfused into
the animal, an immune response (e.g. a cell-mediated immune
response, preferably a pro-inflammatory allo-recognition) occurs.
In another embodiment, the first leukocyte can be xenogeneic with
respect to the animal. However, the first leukocyte cannot be
autologous or syngeneic to the animal. In some embodiments, the
first leukocyte can be allogeneic or xenogeneic to the subject
which will be treated with the conditioned blood. In alternative
embodiment, the first leukocyte can be syngeneic or derived from
the subject which will be treated with the conditioned blood. In an
embodiment, the first allogeneic leukocyte can be modified to bear
on its surface a polymer. However, the polymer, when present, must
be selected or grafted at a density so as to allow the
pro-inflammatory allo-recognition of the first leukocyte by the
recipient. When the first leukocyte is modified to bear on its
surface a polymer, it can be modified to be non-proliferative
either prior to or after the polymer modification.
[0065] In the context of the present invention, some of the
leukocytes used in the cellular preparations are both modified for
bearing a low-immunogenic biocompatible polymer and being modified
to no longer be capable of proliferation. The order in which the
leukocytes are modified (modification with polymer and modification
to prevent proliferation) is not important. Leukocytes can be first
modified to bear the polymer and then modified to refrain from
proliferating. Alternatively, the leukocytes can be first modified
to refrain from proliferating and then modified to bear the
polymer.
[0066] The leukocytes described herein can be derived from any
animals, but are preferably derived from mammals (such as, for
example, humans and mice).
[0067] In the methods and cellular preparations provided herewith,
the surface of the leukocyte can be modified with a low-immunogenic
biocompatible polymer. The polymer must be grafted at
concentrations or polymer size that will allow pro-inflammatory
allo-recognition while preventing or limiting pro-tolerogenic
allo-recognition. For some specific applications, it may be
preferable to modify the surface of the leukocyte with a single
type of low-immunogenic biocompatible polymer. However, for other
applications, it is possible to modify the surface of the leukocyte
with at least two different types of low-immunogenic biocompatible
polymers.
[0068] In order to achieve these modifications, the low-immunogenic
biocompatible polymer can be covalently bound to the cytoplasmic
membrane of the leukocyte and, in a further embodiment, a
membrane-associated protein of the surface of the leukocyte or
inserted, via a lipophilic tail, in the cytoplasmic membrane of the
leukocyte. When the polymer is bound to a membrane-bound protein,
the membrane-associated protein must have at least a portion which
is accessible on the external surface of the cytoplasmic membrane
of the leukocyte for being covalently attached to the polymer. For
example, the membrane-associated protein can be partially embedded
in the cytoplasmic membrane or can be associated with the external
surface of the membrane without being embedded in the cytoplasmic
membrane. The low-immunogenic biocompatible polymer can be
covalently bound to a plurality of membrane-associated proteins. In
an alternative or complementary embodiment, the low-immunogenic
biocompatible polymer can be inserted in the cytoplasmic membrane
by using a lipid-modified polymer.
[0069] In some embodiment, the low-immunogenic biocompatible
polymer can be polyethylene glycol (methoxy polyethylene glycol for
example). The polyethylene glycol can be directly and covalently
bound to a membrane-associated protein or, alternatively, a linker
attaching the low-immunogenic biocompatiable polymer can be used
for attaching the polymer to the protein. Exemplary linkers are
provided in U.S. Pat. No. 8,007,784 (incorporated herewith in its
entirety). In alternative embodiments, the low-immunogenic polymer
can be POZ or HPG.
[0070] In the methods and cellular preparations provided herewith,
the leukocytes can be mature leukocytes or be provided in the form
of stem cells. For example, leukocytes can be obtained from
isolating peripheral blood mononuclear cells (PBMC) from the
subject. Optionally, the PBMCs can be differentiated in vitro into
DC or DC-like cells. Alternatively, the leukocytes can be obtained
from the spleen (e.g. splenocytes). Leukocytes usually include T
cells, B cells and antigen presenting cells. In the methods and
cellular preparations provided herewith, the leukocytes are not
erythrocytes. However, traces of erythrocytes in the leukocytic
preparations are tolerated (for example, less than about 10%, less
than about 5% or less than about 1% of the total number of cells in
the preparation).
[0071] Even though it is not necessary to further purify the
leukocytes to conduct the method or obtain the cellular
preparations, it is possible to use a pure cell population or a
relatively homogenous population of cells as leukocytes. This pure
cell population and relative homogenous population of cells can,
for example, essentially consist essentially of a single cell type
of T cells, B cells, antigen presenting cells (APC) or stem cells.
Alternatively, the population of cells can consist essentially of
more than one cell type. The population of cells can be obtained
through conventional methods (for example cell sorting or magnetic
beads). In an embodiment, when the population of cells consist of a
single cell type (for example, T cells), the percentage of the cell
type with respect to the total population of cells is at least 90%,
at least 95% or at least 99%. The relatively homogenous population
of cells are expected to contain some contaminating cells, for
example less than 10%, less than 5% or less than 1% of the total
population of cells.
[0072] The cell culture supernatant used in the method or in the
cultured cellular preparation can be obtained by co-culturing a
second leukocyte population with a third leukocyte population. It
is also possible to co-culture a second leukocyte homogenous cell
population (such as, for example, a T pure cell population or a
substantially pure T cell population) with a third leukocyte
preparation. It is also contemplated to culture a second leukocyte
population with a third leukocyte population (such as, for example,
a pure T cell population or a substantially pure T cell
population).
[0073] In the methods and preparations presented herewith, it is
required to inhibit/limit the proliferation of one of the two
leukocyte populations. For example, a leukocyte can be
treated/modified prior to cell culture or its administration into
the subject in order to inhibit/limit the cell from proliferating
in the subject. For example, the cell can be irradiated (e.g.
.gamma.-irradiation) prior to its introduction in the subject or
its introduction into a culture system. Upon irradiation, the
leukocyte is not considered viable (e.g. capable of proliferation).
In an embodiment, polymer grafting can be used to affect the
leukocyte viability and utlimately refrain the leukocyte from
proliferating. In a further embodiment of irreversible
non-proliferation, a cell can be treated with a fixation agent
(e.g. glutaraldehyde). Alternatively, leukocyte can be treated with
a pharmacological agent which halts cell cycle progression. Upon
the administration of such pharmacological agent, the leukocyte is
considered viable since it can resume cellular proliferation when
the agent is removed from the cell-containing medium. When the
first leukocyte is administered to the subject in need thereof, it
is preferable that the leukocyte is modified in order to
permanently being refrained from proliferating.
[0074] When the cell culture supernatant is used in the method or
in the cellular preparations, it is required to inhibit/limit the
proliferation of one of the two or the two leukocyte populations.
As indicated above, the inhibition of cellular proliferation can be
achieved by various means, including irradiation and the use of a
polymer, a fixation agent or a pharmacological agent. In this
particular embodiment, it is important that only one of the two
cell populations be inhibited/limited from proliferating and that
the other cell population be able to proliferate.
[0075] The conditioned blood that can be used in the method can be
obtained by administering (preferably transfusing or intravenously
administering) to a test subject (such as a rodent), a first
leukocyte which has been modified so as to limit, preferably
permanently, its ability to proliferate. It is also possible to
transfuse a first leukocytic homogenous cell population (such as,
for example, a T pure cell population or a substantially pure T
cell population) to the test subject. The blood (or a fraction
thereof) is recuperated from the test subject after a time
sufficient to induce in the subject a state of immune stimulation
or a pro-inflammatory state. In order to obtain a blood fraction
(such as serum or plasma) from the animal, it is possible to submit
the blood of the animal to a centrifugation step and, optionally,
eliminate red blood cells via cellular lysis.
[0076] As shown herein, the administration of the cellular
preparations induce a state of immune stimulation in the treated
subject. In some embodiments, the state of stimulation can persist
long after the administration of the cellular preparation or the
cell culture supernatant (as shown below, at least 270 days in
mice). In an optional embodiment, the state of stimulation does not
revert back to a pro-tolerogenic state. Consequently, the methods
and cellular preparations described herein are useful for the
treatment, prevention and/or alleviation of symptoms associated
with conditions caused/exacerbated by a low or inappropriate immune
response.
[0077] A state of immune stimulation can be considered
therapeutically beneficial in subjects experiencing (or at risk of
experiencing) a repressed immune response (anergy or tolerance),
such as for example those observed upon the induction and
maintenance of an proliferation-associated disorder (such as
cancer). Some of these conditions are associated with either a high
level of Tregs and/or a low level of pro-inflammatory T cells (such
as Th17 and/or Th1) when compared to sex- and aged-matched healthy
subjects. Because it is shown herein that the cellular-based
preparations are beneficial for decreasing the ratio
Tregs/pro-inflammatory T cells, it is expected that administration
of the cellular-based preparations to afflicted subjects will
treat, prevent and/or alleviate symptoms associated with the
proliferation-associated disorder.
[0078] A state of immune stimulation can also be considered
therapeutically beneficial in subjects at risk of developing an
abnormally repressed immune response, a state or anergy or a
pro-tolerogenic state. Such abnormally repressed immune responses
can be observed in subjects being afflicted by or susceptible to be
afflicted by a proliferation-associated disorder such as cancer. In
this embodiment, the methods and cellular preparations can be
applied to prevent or limit the onset or maintenance of a repressed
immune response. The cellular-based preparation can be
co-administered with the other therapeutics currently used to
managed the proliferation-associated disorder. The cellular-based
preparation can be administered to any subjects in need thereof,
including humans and animals.
[0079] Such abnormally repressed immune responses can be also
observed in subjects being infected, especially by a parasite or a
virus. In these conditions, the methods and cellular preparations
can be applied to prevent or limit the onset or maintenance of a
repressed immune response. The cellular-based preparation can be
co-administered with the other therapeutics currently used to
managed the infection.
[0080] The cellular-based preparation can be administered to any
subjects in need thereof, including humans and animals.
[0081] In an embodiment, the state of abnormal repression of the
immune system is not caused by an infection of the immune cells
themselves (e.g. EBV or HIV for example). However, in other
embodiment, in instances where an infection of the immune cells is
afflicting the subject, it is possible to use cellular preparations
described to treat or alleviate the symptoms of the viral
infection. For example, a leukocyte from the subject (preferably a
cytotoxic T cell which is specific to the infectious agent) can be
co-cultured, under immunogenic conditions, with a first allogeneic
and non-proliferative leukocyte. After the co-culture, the cultured
leukocyte can be reintroduced in the infected subject to treat
and/or alleviate the symptoms associated to the infection (a viral
infection, for example, an EBV or HIV infection).
[0082] In the methods and cellular preparations described herein,
it is contemplated that the cellular-based preparations be
optionally administered with other therapeutic agents known to be
useful for the treatment, prevention and/or alleviation of symptoms
of conditions associated to a condition caused/exacerbated by a low
or inappropriate immune response, such as, for example, IL-2, IL-4,
TNF-.alpha. and/or INF-.gamma..
[0083] Processes for Obtaining Cellular Preparations
[0084] The cellular-based preparations described herein are
obtained by contacting two distinct and allogeneic leukocyte
populations. One of the two leukocyte population is
non-proliferative or modified to become non-proliferative. In this
first step, it is important that this modification is made without
interfering substantially with the intrinsic ability of the first
leukocyte to induce a pro-inflammatory allo-recognition by the
leukocyte (either in vivo or in vitro). In order to determine if
pro-inflammatory allo-recognition occurs (or alternatively is
substantially reduced), various techniques are known to those
skilled in the art and include, but are not limited to, a standard
mixed lymphocyte reaction (MLR), high molecular weight mitogen
stimulation (e.g. PHA stimulation) as well as flow cytometry (Chen
and Scott, 2006, Wang et al. 2011).
[0085] In order to prevent a leukocyte from proliferating, the cell
can be irradiated (e.g. .gamma.-irradiation) prior to its
introduction in the subject or its introduction into a culture
system. Upon irradiation, the leukocyte is not considered viable
(e.g. capable of proliferation). In a further embodiment, the
surface of the leukocyte can be modified by a polymer to alter or
limit its viability. In another embodiment, the leukocyte can be
treated with a fixation agent to prevent it from proliferating.
Alternatively, leukocyte can be treated with a pharmacological
agent which halts cell cycle progression. Upon the administration
of such pharmacological agent, the leukocyte is considered viable
since it can resume cellular proliferation when the agent is
removed from the cell-containing medium.
[0086] To provide the cellular preparations described herewith, the
leukocytes used can be mature leukocytes or be provided in the form
of stem cells. For example, leukocytes can be obtained from
isolating peripheral blood mononuclear cells (PBMC) from the
subject. Optionally, the PBMCs can be differentiated in vitro into
dendritic (DC) or DC-like cells. Alternatively, the leukocytes can
be obtained from the spleen (e.g. splenocytes). Leukocytes usually
include T cells, B cells and antigen presenting cells. For
providing the cellular preparations, the leukocytes are not
erythrocytes. However, traces of erythrocytes in the leukocyte
population used are tolerated (for example, less than about 10%,
less than about 5% or less than about 1% of the total number of
cells in the preparation).
[0087] Even though it is not necessary to further purify the
leukocytes to provide the cellular preparations, it is possible to
use a pure cell population or a relatively homogenous population of
cells as leukocytes. This "pure" cell population and "relative
homogenous population" of cells can, for example, essentially
consist essentially of a single cell type of T cells, B cells,
antigen presenting cells (APC) or stem cells. Alternatively, the
population of cells can consist essentially of more than one cell
type. The population of cells can be obtained through conventional
methods (for example cell sorting or magnetic beads). In an
embodiment, when the population of cells consist of a single cell
type (for example, T cells), the percentage of the cell type with
respect to the total population of cells is at least 90%, at least
95% or at least 99%. The relatively homogenous population of cells
are expected to contain some contaminating cells, for example less
than 10%, less than 5% or less than 1% of the total population of
cells.
[0088] The leukocytes can be obtained from any animals, but are
preferably derived from mammals (such as, for example, humans and
mice). In an embodiment, the leukocytes can be obtained from a
subject intended to be treated with the cellular preparations.
[0089] In the embodiment where an allogeneic leukocyte population
is administered to the treated subject (optionally to recuperate
the conditioned blood), it is contemplated that it can be
expanded/differentiated (e.g. from naive to pro-inflammatory) prior
to the administration.
[0090] In embodiments where two leukocyte populations are
co-cultured in vitro, the step of preventing the leukocyte from
proliferating occurs prior to the co-culture. However, it is
contemplated, in this embodiment, that the leukocyte population
which is going to be prevented from proliferating can be
expanded/differentiated (e.g. from naive to pro-inflammatory) prior
to the co-culture. The co-culture of the two leukocyte populations
is performed in immunogenic conditions so as to allow a
pro-inflammatory allo-recognition in the leukocyte population which
has not been modified (e.g. which can exhibit cellular
proliferation). Since a physical contact between the two leukocyte
populations is important for allowing pro-inflammatory
allo-recognition, it is important that the two leukocyte population
be cultured into conditions allowing for such physical contact (for
example in a culture vessel which does allow physical contact
between the two leukocyte populations).
[0091] When a co-culture system is used, it is possible to culture
a first leukocytic population (such as, for example a PBMC or
splenocyte) with a leukocytic population from a subject (such as,
for example a PBMC or splenocyte). It is also possible to culture a
first leukocytic relatively homogenous cell population (such as,
for example, a T cell population) with a leukocytic population from
a subject (such as, for example a PBMC or splenocyte). It is also
contemplated to culture a first leukocytic population (such as, for
example a PBMC or splenocyte) with a leukocytic relatively
homogenous population of cells from the subject (such as, for
example, a T cell population). It is further completed to culture a
first leukocytic relatively homogenous cell population (such as,
for example, a T cell population) with a leukocytic relatively
homogenous population of cells from the subject (such as, for
example, a T cell population).
[0092] Usually, the cultured cellular preparation (between the
first leukocyte and the leukocyte from the subject or syngeneic to
the subject) is obtained at least 24 hours after the initial
contact between the first leukocyte and the leukocyte from the
subject. In some embodiments, the cultured cellular preparation is
obtained at least 48 hours or at least 72 hours after the initial
contact between the first leukocyte and the leukocyte from the
subject. When the incubation takes place in a 24-well plate, the
concentration of each leukocyte population can be at least
1.times.10.sup.6 cells.
[0093] In yet a further optional embodiment, the modified second
leukocyte can be placed in a cell culture with the a third
leukocyte and the supernatant of this cell culture can be
administered to the subject in need thereof. The supernatant can be
modified (e.g. filtered) to remove the second and/or third
leukocyte and the cellular debris associated thereto. However, no
specific size fractionation nor enrichment of a specific fraction
of the supernatant is applied to the cell culture supernatant prior
to administering it to the subject. The second and third leukocytes
are cultured in the same medium (or in the same culture system),
one of the two cell populations is inhibited/limited from
proliferating (as long as the other cell populations remains
capable of proliferating). In an embodiment, the modified second
leukocyte can first be expanded/differentiated and then
inhibited/limited from proliferating prior to its co-culture with
the third leukocyte. Alternatively, the third leukocyte can be
expanded/differentiated prior to its co-culture with the modified
second leukocyte or afterwards. As indicated above, in the cell
culture system, the second leukocyte is allogeneic to the third
leukocyte. In some embodiments, the second leukocyte can be
allogeneic to the subject and to third leukocyte. Alternatively,
the second leukocyte can be xenogeneic to the subject and/or to the
third leukocyte. Optionally, one of the second or third leukocyte
can be syngeneic or derived from the subject.
[0094] When a co-culture system is used, it is possible to culture
a second leukocytic population (such as, for example a PBMC or
splenocyte) with a third leukocytic population (such as, for
example a PBMC or splenocyte). It is also possible to culture a
second leukocytic relatively homogenous cell population (such as,
for example, a T cell population) with a third leukocytic
population (such as, for example a PBMC or splenocyte). It is also
contemplated to culture a second leukocytic population (such as,
for example a PBMC or splenocyte) with a third leukocytic
relatively homogenous population of cells (such as, for example, a
T cell population). It is further completed to culture a second
leukocytic relatively homogenous cell population (such as, for
example, a T cell population) with a third leukocytic relatively
homogenous population of cells (such as, for example, a T cell
population).
[0095] Usually, the cultured cellular preparation is obtained at
least 24 hours after the initial contact between the second
leukocyte and the third leukocyte. In some embodiment, the cultured
cellular preparation is obtained at least 48 hours or at least 72
hours after the initial contact between the second leukocyte and
the third leukocyte. When the incubation takes place in a 24-well
plate, the concentration of each leukocyte population can be at
least 1.times.10.sup.6 cells.
[0096] In other embodiments, a conditioned blood can be used. The
conditioned blood used can be obtained by administering a first
leukocyte, a first leukocyte population or a first leukocytic
relatively homogeneous population (e.g. all modified to be
refrained or inhibited from proliferating) to the test subject
(usually an animal, such as a mouse). The blood (or a fraction
thereof) is recuperated from the subject after a time sufficient to
induce in the test subject a state of immune stimulation. It is
important that the first leukocyte be administered to an immune
competent test subject and that the blood or blood fraction be
obtained at a later a time sufficient to provide a conditioned
blood. The test subject is a subject being immune competent and
having a Treg/pro-inflammatory T cell ratio which is substantially
similar to age- and sex-matched healthy subjects. As used herein,
the conditioned blood refers to physical components present in the
blood and obtained by administering the first leukocyte to the
immune competent test subject and having the pro-inflammatory
properties described herein. It is recognized by those skilled in
the art that the conditioned blood may be obtained more rapidly by
increasing the amount of leukocytes being administered or
administering more than once (for example one, twice or thrice) the
modified leukocyte. Usually, the conditioned blood is obtained at
least one day after the administration of the first leukocyte. In
some embodiment, the conditioned blood is obtained at least 2, 3,
4, 5, 6, 7 or 8 days after the administration of the first
leukocyte. In an embodiment, the conditioned blood can be obtained
by administering at least 5.times.10.sup.6 leukocytes to the test
subject (e.g. a mice) and recuperating the plasma five days later.
In some embodiment, the conditioned blood can be obtained by
administering at least 20.times.10.sup.6 polymer-modified
leukocytes. Methods for obtaining the blood or its fractions (such
as serum or plasma) are known to those in the art and usually
involve centrifugation and cell lysis.
[0097] As indicated herein, it is possible to modify the surface of
the leukocyte with a biocompatible polymer. It is important that
the polymer used exhibits biocompatibility once introduced into a
cell culture system or administered to the test subject. Such
biocompatible polymer include, but are not limited to polyethylene
glycol (particularly methoxypoly(ethylene glycol)), POZ and
hyperbranched polyglycerol (HPG). In some embodiments, it is
preferable to use a single type of polymer to modify the surface of
leukocytes. In other embodiments, it is possible to use at least
two distinct types of polymers to modify the surface of the
leukocyte.
[0098] In an embodiment, the biocompatible polymer can be
covalently associated with the membrane-associated protein(s) of
the leukocyte by creating a reactive site on the polymer (for
example by deprotecting a chemical group) and contacting the
polymer with the leukocyte. For example, for covalently binding a
methoxypoly(ethylene glycol) to the surface of a leukocyte, it is
possible to incubate a methoxypoly(-ethylene glycol) succinimidyl
valerate (reactive polymer) in the presence of the leukocyte. The
contact between the reactive polymer and the leukocyte is performed
under conditions sufficient for providing a grafting density which
will prevent/limit pro-tolergenic allo-recognition and allow
pro-inflammatory allo-recognition. In an embodiment, the polymer is
grafted to a viable leukocyte and under conditions which will
retain the viability of the leukocyte. A linker, positioned between
the surface of the leukocyte and the polymer, can optionally be
used. Examples of such polymers and linkers are described in U.S.
Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In another
embodiment, the biocompatible polymer can be integrated within the
lipid bilayer of the cytoplasmic membrane of the leukocyte by using
a lipid-modified polymer.
[0099] As indicated above, it is important that the biocompatible
polymer be grafted at a density sufficient for preventing/limiting
pro-tolerogenic allo-recognition and allow pro-inflammatory
allo-recognition. In an embodiment, the polymer is polyethylene
glycol (e.g. linear) and has an average molecular weight between 2
and 40 KDa as well as any combinations thereof. In a further
embodiment, the average molecular weight of the PEG to be grafted
is at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 kDa. In
another embodiment, the average molecular weight of the PEG to be
granted is no more than 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2
kDa. In another embodiment, the grafting concentration of the
polymer (per 20.times.10.sup.6 cells) is no more than 2.4, 2.0,
1.0, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or 0.005 mM. In still
another embodiment, the grafting concentration of the polymer (per
20.times.10.sup.6 cells) is equal to or lower than 0.005, 0.01,
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.4 mM. In embodiments
where the polymer is grafter to affect the viability of the
leukocyte (for example by creating cellular instability, cellular
fragmentation or vesiculization, the concentration of the polymer
(per 20.times.10.sup.6 cells) is equal to or higher than 10 mM. In
order to determine if pro-inflammatory allo-recognition occurs (or
is prevented), various techniques are known to those skilled in the
art and include, but are not limited to, a standard mixed
lymphocyte reaction (MLR), high molecular weight mitogen
stimulation (e.g. PHA stimulation) as well as flow cytometry (Chen
and Scott, 2006). In order to determine if a weak pro-tolerogenic
allo-recognition occurs (or is prevented), various techniques are
known to those skilled in the art and include, but are not limited
to, the assessment of the level of expansion and differentiation of
Treg cells and or prevention of Th17 expansion/differentiation.
[0100] Once the cellular preparations have been obtained, they can
be formulated for administration to the subject. The formulation
step can comprise admixing the cellular preparations (at a
therapeutically effective dose) with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, and/or
carriers. The formulations are preferably in a liquid injectable
form and can include diluents of various buffer content (e.g.,
Tris-HCl, acetate, phosphate), pH and ionic strength, additives
such as albumin or gelatin to prevent absorption to surfaces. The
formulations can comprise pharmaceutically acceptable solubilizing
agents (e.g., glycerol, polyethylene glycerol), anti-oxidants
(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
thimerosal, benzyl alcohol, parabens), bulking substances or
tonicity modifiers (e.g., lactose, mannitol).
[0101] The present invention will be more readily understood by
referring to the following examples which are given to illustrate
the invention rather than to limit its scope.
EXAMPLE I
Material and Methods
[0102] Human PBMC and dendritic cell preparation. Human whole blood
was collected in heparinized vacutainer blood collection tubes (BD,
Franklin Lakes, N.J.) from healthy volunteer donors following
informed consent. PBMC were isolated from diluted whole blood using
FicollePaque PREMIUM.TM. (GE Healthcare Bio-Sciences Corp,
Piscataway, N.J.) as per the product instructions. The PBMC layer
was washed twice with 1.times. Hank's Balanced Salt Solution (HBSS;
without CaCl.sub.2 and MgSO.sub.4; Invitrogen by Life Technologies,
Carlsbad, Calif.) and resuspended in the appropriate media as
needed for mixed lymphocyte reactions and flow cytometric analysis
of Treg and Th17 phenotypes. Dendritic cells (DC) were prepared
from isolated PBMC as described by O'Neill and Bhardwaj (O'Neill et
al., 2005). Briefly, freshly isolated PBMC were overlaid on Petri
dishes for 3 h in AIM V serum free culture medium (Invitrogen,
Carlsbad, Calif.). Non-adherent cells were gently washed off the
plate. The adherent cells (monocyte rich cells) were treated with
IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&D Systems,
Minneapolis, Minn.) in AIM V medium. Cells were again treated with
IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifuged
and resuspended in fresh media supplemented with DC maturation
factors (TNF-.alpha., IL-1.beta., IL-6; R&D Systems,
Minneapolis, Minn.) and prostaglandin E2 (Sigma Aldrich, St. Louis,
Mo.). The mature DC-like cells were harvested on day 7 and CD80,
CD83, CD86 and HLA-DR expressions were determined to confirm DC
maturation via flow cytometry (FACSCalibur.TM. Flow Cytometer, BD
Biosciences, San Jose, Calif.).
[0103] Murine splenocyte and tissue harvesting. All murine studies
were done in accordance with the Canadian Council of Animal Care
and the University of British Columbia Animal Care Committee
guidelines and were conducted within the Centre for Disease
Modeling at the University of British Columbia. Murine donor cells
used for the in vivo donation and in vitro studies were euthanized
by CO.sub.2. Three allogeneic strains of mice were utilized for
syngeneic and allogeneic in vitro and in vivo challenge: Balb/c,
H-2.sup.d; C57Bl/6, H-2.sup.b; and C3H, H-2.sup.k. Murine spleens,
brachial lymph nodes, and peripheral blood were collected at the
indicated days. Mouse spleens and brachial lymph nodes were
dissected and placed into cold phosphate buffered saline (PBS; 1.9
mM NaH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, and 154 mM NaCl, pH
7.3) containing 0.2% bovine serum albumin (BSA; Sigma Aldrich, St.
Louis, Mo.) and kept on ice until ready to process. Whole blood was
collected in heparinized tubes via cardiac puncture. Murine donor
splenocytes were prepared from freshly harvested syngeneic or
allogeneic spleens via homogenization into a cell suspension in PBS
(0.2% BSA) using the frosted end of two microscope slides. The
resultant cell suspension was spun down at 500.times.g. The
splenocyte pellet was resuspended in 1 mL of 1.times. BD Pharm
LYSE.TM. lysing buffer (BD Biosciences, San Diego, Calif.) and
incubated for 1 min at room temperature. Lymph node cells were
harvested via tissue homogenization as described above, washed
twice and resuspended in PBS (0.2% BSA) for flow cytometric
analysis of Th17, Treg and murine haplotype. Recipient peripheral
blood lymphocytes were prepared via lysis of the red cells (BD
Pharm Lyse lysing buffer; BD Biosciences, San Diego, Calif.) at
1.times. concentration, followed by washing (1.times.) and
resuspension in PBS (0.2% BSA) for flow analysis of Th17, Treg and
murine haplotype.
[0104] mPEG modification (PEGylation) of PBMCs and splenocytes.
Human PBMC and murine splenocytes were derivatized using
methoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA;
Laysan Bio Inc. Arab, Ala.) with a molecular weight of 5 or 20 kDa
as previously described (Scott et al., 1997; Murad et al, 1999A;
Chen et al., 2003; Chen et al., 2006). Grafting concentrations
ranged from 0 to 5.0 mM per 4.times.10.sup.6 cells/mL. Cells were
incubated with the activated mPEG for 60 min at room temperature in
isotonic alkaline phosphate buffer (50 mM K.sub.2HPO.sub.4 and 105
mM NaCl; pH 8.0), then washed twice with 25 mM HEPES/RPMI 1640
containing 0.01% human albumin. Human PBMC were resuspended in AIM
V media at a final cell density of 2.0.times.10.sup.6 cells/mL for
use in the MLR. Murine splenocytes used for in vivo studies were
resuspended in sterile saline at a final cell density of
2.0.times.10.sup.8 cells/ml for i.v. injection. To determine if the
simple presence of the mPEG polymer itself altered the immune
response either in vitro and in vivo, additional studies were done
with unactivated polymer incapable of covalent grafting to the cell
surface. For these studies, allogeneic human (in vitro studies) or
syngeneic and allogeneic murine splenocytes (in vivo studies) were
treated with non-covalently bound mPEG (soluble mPEG) under the
same reaction conditions described for the covalent grafting
studies. For clarity, "soluble mPEG" refers to cells treated with
non-covalently grafted polymer while "mPEG-modified" refers to
treatment with activated polymer resulting in the covalent grafting
of the mPEG to the cell membrane.
[0105] In vitro and in vivo cell proliferation. Cell proliferation
(both in vitro and in vivo) was assessed via flow cytometry using
the CELLTRACE.TM. CFSE (Carboxyfluorescein diacetate, succinimidyl
ester) Cell Proliferation Kit (Invitrogen by Life Technologies e
Molecular probes, Carlsbad, Calif.). Human and murine cells
labeling was done according to the product insert at a final
concentration of 2.5 mM CFSE per 2.times.10.sup.6 cells total.
Donor and recipient cell proliferation was differentially
determined by haplotype analysis. In some experiments, cell
proliferation was measured by .sup.3H-thymidine incorporation. In
these experiments, donor splenocytes (5.12.times.10.sup.6 cells per
well) were co-incubated in triplicate in 96-well plates at
37.degree. C., 5% CO.sub.2 for 3 days. On day 3, all wells were
pulsed with .sup.3H-thymidine and incubated for 24 h at 37.degree.
C., 5% CO.sub.2. Cellular DNA was collected on filter mats using a
Skatron cell harvester (Suffolk, U.K.) and cellular proliferation
was measured by .sup.3H-thymidine incorporation.
[0106] Mixed lymphocyte reaction (MLR)--control and conditioned
media. The effects of polymer grafting (5 kDa SVAmPEG) on
allorecognition in vitro were assessed using two-way MLR (Murad et
al, 1999A; Chen et al., 2003; Chen et al., 2006). PBMC from two
MHC-disparate human donors were label with CFSE as described. Each
MLR reaction well contained a total of 1.times.10.sup.6 cells
(single donor for resting or mitogen stimulation or equal numbers
for disparate donors for MLR). Cells were plated in multiwell
flat-bottom 24-well tissue culture plates (BD Biosciences,
Discovery Labware, Bedford, Mass.). PBMC proliferation, cytokine
secretion, as well as Treg and Th17 phenotyping was done at days 10
and 14. For flow cytometric analysis, the harvested cells were
resuspended in PBS (0.1% BSA). While time course studies (Days 4,
7, 10 and 14) were done, the presented studies show days 10 and 14.
These extended studies are, in fact, the most stringent test of the
immunomodulatory effects of the grafted polymer as membrane
remodeling over this time could have resulted in a latter onset of
proliferation. To investigate in vitro whether polymer grafting to
allogeneic PBMC gave rise to tolerance or anergy, secondary
(2.degree.) MLR studies were conducted using conditioned media.
Conditioned media from a primary (1.degree.) 2 way-MLR was
collected at 72 h for conducting a secondary (2.degree.) MLR as
schematically shown in FIG. 1. Conditioned media from the 1.degree.
MLR included: A1) resting unmodified PBMC; B2) resting PEGylated
PBMC; C3) two-way MLR; and D4) two-way mPEG-MLR. The 2.degree. MLR
utilized freshly prepared MHC-disparate donors (either the same as
or different from) the initial plate and plated as described above.
As shown in FIG. 1, the 2.degree. MLR samples included: A) resting
PBMC; B) two-way MLR; P) mitogen stimulation; D) two-way mPEG-MLR.
For these studies, PBMC were derivatized using 1 mM 5 kDa SVAmPEG.
Mitogen stimulation (PHA-P; Sigma-Aldrich, St. Louis, Mo.) of donor
PBMC in the secondary plates was done to assess the proliferation
potential and viability of cells incubated in the conditioned
media. Human PBMC were challenged with 2 mg/ml per 1.times.10.sup.6
cells of PHA-P. All plates were incubated at 37.degree. C. (5%
CO.sub.2). Following 13 days of incubation (37.degree. C., 5%
CO.sub.2), the cell culture supernatants were collected and cells
were harvested from the 2.degree. MLR plates. Cell proliferation
was measured via CSFE-dilution of CD3.sup.+CD4.sup.+ lymphocytes by
flow cytometry.
[0107] Immunophenotyping by flow cytometry. The T lymphocytes
populations (double positive for CD3.sup.+ and CD4.sup.+) in both
the in vitro and in vivo studies were measured by flow cytometry
using fluorescently labeled CD3 and CD4 monoclonal antibodies (BD
Pharmingen, San Diego, Calif.). Human and mouse Regulatory T
lymphocytes (Treg) were CD3.sup.+/CD4.sup.+ and FoxP3.sup.+
(transcription factor) while inflammatory Th17 lymphocytes cells
were CD3.sup.+/CD4.sup.+ and IL-17.sup.+ (cytokine) as measured per
the BD Treg/Th17 Phenotyping Kit (BD Pharmingen, San Diego,
Calif.). After the staining, the cells (1.times.10.sup.6 cells
total) were washed and resuspended in PBS (0.1% BSA) prior to flow
acquisition. Isotype controls were also used to determine
background fluorescence. All samples were acquired using the
FACSCalibur.TM. flow cytometer (BD Biosciences, San Jose, Calif.)
and CellQuest Pro.TM. software for both acquisition and
analysis.
[0108] Cytokine quantitation. Cell culture supernatants were
collected from the 1.degree. two-way MLR plate and stored at
-80.degree. C. prior to analysis. Conditioned media aliquots from a
minimum of four independent experiments were used for
quantification of supernatant cytokine levels using the BD
Cytometric Bead Array (CBA) system (BD Biosciences, San Diego,
Calif.) for flow cytometry. The CBA system is a multiplexed bead
based immunoassay used to quantitate multiple cytokine levels in a
single sample simultaneously by fluorescence-based emission and
flow cytometry. Cytokine measured included: IFN.gamma.,
TNF-.alpha., IL-10, IL-5, IL-4, and IL-2 using the BD Human Th1/Th2
Cytokine Kit I.TM.. The IL-6 and IL-17A levels were measured using
the BD CBA Human Soluble Protein Flex Set.TM.. Both assays were
performed following the manufacturer's product instruction manual.
Briefly, cell culture supernatants of resting unmodified PBMC,
unmodified MLR, PEGylated (5 kDa SVAmPEG; one donor) resting PBMC,
PEGylated MLR, and mitogen (PHA) stimulated PBMC were incubated at
room temperature in the dark with a mixture of each cytokine
antibody-conjugated capture bead and the PE-conjugated detection
antibody. Following the incubation, the samples were washed and
acquired using a FACSCalibur.TM. flow cytometer and analyzed using
Cell-Quest Pro.TM. software. Cytokine protein levels were
determined using the BD Cytometric Bead Array.TM. and FCAP
Array.TM. analysis software (BD Biosicences, San Diego, Calif. and
Soft Flow Inc, St. Louis Park, Minn.).
[0109] In vivo murine studies. To investigate whether mPEG grafting
to leukocytes would have systemic in vivo effects, a murine
adoptive transfer system was employed using three genetically
different strains: Balb/c, H-2.sup.d; C57Bl/6, H-2.sup.b; and C3H,
H-2.sup.k (Chen et al., 2003; Chen et al., 2006). All mice (donors
and recipients) were 9-11 weeks old. Donor splenocytes were
prepared and CSFE labeled as described. control and mPEG-grafted (1
mM, 20 kDa SVAmPEG) syngeneic or allogeneic cells
(20.times.10.sup.6 splenocytes) were transfused intravenously
(i.v.) via the tail vein into recipient animals. BALB/c and C57BL/6
mice injected with sterile saline served as control animals.
Animals were euthanized by CO.sub.2 at predetermined intervals at
which time blood, brachial lymph nodes and spleen were collected
and processed for Th17/Treg phenotyping analysis and splenocyte
proliferation studies by flow cytometry. Donor cell engraftment and
proliferation were assessed via flow cytometry using murine
haplotype (H-2K.sup.b vs. H-2K.sup.d) analysis. To determine the
persistence of the immunomodulation, mice were re-challenged
(2.degree. challenge) 30 days after the initial transfer of
allogeneic or mPEGallogeneic splenocytes with unmodified allogeneic
cells. At 5 days post 2.degree. challenge, Treg and Th17
phenotyping of murine splenocytes isolated from the spleen, lymph
node and peripheral blood was again assessed via flow
cytometry.
[0110] Statistical analysis. Data analysis was conducted using
SPSS.TM. (v12) statistical software (Statistical Products and
Services Solutions, Chicago, Ill., USA). For significance, a
minimum p value of <0.05 was used. For comparison of three or
more means, a one-way analysis of variance (ANOVA) was performed.
When significant differences were found, a post-hoc Tukey test was
used for pair-wise comparison of means. When only two means were
compared, student-t tests were performed.
EXAMPLE II
In Vitro and In Vivo Immunomodulation
[0111] The material and methods used in this example are provided
in Example I.
[0112] To determine the effects of polymer-grafting on the immune
response, initial in vitro experiments examined the cytokine burst
characterizing control and polymer modified MLR. The
polymer-mediated immunocamouflage of human PBMC resulted in
significant changes in the cytokine profile of the conditioned
media obtained from the 1.degree. MLR plate (FIGS. 1 and 2). As
shown in FIG. 2, control MLRs yielded elevated concentrations of
IL-2, IFN-.gamma., IL-17A, TNF-.alpha. and IL-6 relative to resting
unmodified or PEGylated PBMC. Conversely, the MLR media contained
reduced concentrations of IL-10, a cytokine favoring immune
suppression (1.33.+-.0.73 for the control MLR versus 2.01.+-.1.26
and 8.90.+-.2.10 ng/ml for resting PBMC and mPEG-MLR,
respectively).
[0113] The conditioned media produced from the initial 72 h MLR
exerted a significant effect on the 2.degree. MLR as demonstrated
in FIG. 3. While the 1.degree. media from resting PBMC showed no
significant effect on the 2.degree. MLR, the media from the
1.degree. Control MLR demonstrated a significant (p<0.01)
pro-proliferative effect in the 2.degree. MLR. As shown, the mean
proliferation index of the 2.degree. MLR increased from
26.05.+-.12.47 to 44.72.+-.17.13 in the presence of conditioned
media from the 1.degree. Control MLR. The pro-inflammatory effect
of the 1.degree. MLR media was noted on even the resting PBMC and
PHA-stimulated cells. In contrast, the 1.degree. conditioned media
from the mPEG-MLR demonstrated a significant (p<0.001)
anti-proliferative effect in not only the 2.degree. MLR but also
the PHA-stimulated cells. The differential proliferation response
between the control and mPEG-MLR conditions for matching
experiments is noted by the lines connecting paired
experiments.
[0114] Furthermore, as shown in FIG. 4, the proliferation index was
positively correlated with an increased population of Th17 T cells
and inversely correlated with Treg lymphocytes numbers. As
demonstrated, the 1.degree. conditioned media from the control MLR
yielded elevated levels of Th17 cells and decreased levels of Treg
lymphocytes. In comparison, the 1.degree. media from the mPEG-MLR
resulted in significantly elevated (p<0.001) levels of Treg
cells and a virtually non-existent population of Th17 lymphocytes.
The source of the conditioned media also impacted the efficacy of
PHA stimulation. As shown, conditioned media from the control MLR
significantly enhanced proliferation relative to media from resting
PBMC (p<0.01) and resting mPEG-PBMC (p<0.001). In contrast,
conditioned media from the mPEG-MLR significantly inhibited mitogen
proliferation.
[0115] Hence, the in vitro experiments demonstrated that allogeneic
PBMC results in a pro-inflammatory effect governed in part by
changes in the Th17 and Treg populations and subsequent ratio of
these cell populations. Moreover, these conditioned media
experiments demonstrated that this immunomodulatory effect arises
from soluble factors that might be able to induce a systemic effect
in vivo. To determine if similar effects would be observed in vivo,
a murine splenocyte adoptive transfer model was utilized. As
demonstrated in FIG. 5, unmodified and PEGylated allogeneic donor
splenocytes resulted in a significant in vivo immunomodulatory
effect giving rise to altered ratios of Treg to pro-inflammatory
lymphocytes within the spleen, brachial lymph node, and peripheral
blood. As noted, in all three tissues, a significant (p<0.001 at
120 h) time-dependent decrease in Treg lymphocytes over that
observed in naive mice was noted in mice receiving allogeneic donor
cells. In stark contrast, a significant (p<0.001) increase in
Tregs (.gtoreq.48 h post-injection relative to naive mice) is noted
in mice transfused with mPEG-modified allogeneic splenocytes. The
absolute difference between the unmodified (control) and PEGylated
splenocytes, shown by the stippled area, demonstrates the true
magnitude of the differential impact of unmodified versus
mPEG-modified allogeneic cells. As noted at 120 h, transfusion of
soluble mPEG, syngeneic cells or mPEG-syngeneic cells had no
significant effect on the Treg or Th17 lymphocyte populations.
[0116] As foreshadowed by our in vitro human PBMC findings (Example
II), murine Th17 lymphocyte levels were differentially influenced
by the administration of unmodified or mPEG-modified allogeneic
donor cells (FIG. 6). Importantly, unmodified allogeneic murine
donor cells resulted in a significant (p<0.001), time-dependent,
increase in the Th17 cell population in the spleen, brachial lymph
node and peripheral blood relative to naive mice, mice transfused
with syngeneic splenocytes or mice transfused with mPEG-modified
allogeneic cells. The absolute difference between the unmodified
and mPEG-modified donor cells is denoted by the stippled area. As
with the Treg population, transfusion of soluble mPEG, syngeneic
cells or mPEG-syngeneic cells had no significant effect on the Th17
lymphocyte population at 120 h.
[0117] As also shown on FIG. 7, normal mice have significantly
higher levels of Tregs (Spleen .about.10% total CD4+ T cells)
relative to Th17 T Cells (Spleen .about.0.05% total CD4+ T cells).
Further, treatment with unmodified allogeneic cells results in
production of Th17 cells and loss of Tregs resulting in a
significant (p<0.001) decrease in the Treg:Th17 ratio thus
inducing a systemic pro-inflammatory state as evidenced by the
changes in the spleen, brachial lymph node and circulating blood of
the mouse. In contrast, polymer modified allogeneic cells maintain
(even increase) Tregs and prevents Th17 production.
[0118] As might be anticipated, the allogeneic splenocyte mediated
increase in Th17 cells in the peripheral blood samples occurred
later in the studied time course (96 h) compared to either of the
lymphatic tissues (spleen and lymph nodes; 48 h). This clearly
suggests that T cell proliferation initially occurred within the
lymphatic tissues and secondarily migrated into the peripheral
blood. A similar time dependency was noted with the Treg
proliferation induced by the mPEG-modified splenocyte populations.
Proliferation initially occurred within lymphatic tissue within
.about.48 h and only appeared within the peripheral blood after
.about.96 h.
[0119] Of importance was the observation that the immunomodulatory
effects of the allogeneic splenocytes were long lived. As shown in
FIG. 8, 30 days post transfusion with allogeneic splenocytes Th17
cell remain elevated in the spleen, lymph node and blood while the
Treg levels remain significantly decreased relative to naive mice
yield a significantly decreased Treg:Th17/Th1 ratio. In contrast to
mice treated with allogeneic splenocytes, a secondary adoptive
transfer of unmodified allogeneic splenocytes (30 days post
1.degree. challenge; measured at 120 h) to mice previously treated
with PEGylated allogeneic showed no significant decrease in Treg
cells, or increase in Th17 cells, relative to the day 30 levels.
This was in direct contrast to that observed in naive mice (FIG. 5)
injected with unmodified allogeneic cells that demonstrated a
dramatic decrease in Treg lymphocytes.
[0120] To determine if the observed in vivo murine findings gave
rise to a tolerance to a specific H-2 haplotype or a more
generalized anergy to allogeneic tissues, in vitro two-way murine
MLR studies of three allogeneic splenocyte populations (Balb/c,
H-2.sup.d; C57Bl/6, H-2.sup.b; and C3H, H-2.sup.k) were done. As
demonstrated in FIG. 9, unmodified (viable or non-viable)
allogeneic cells give rise to a pro-inflammatory, pro-proliferative
state. This effect is reduced or lost upon the covalent grafting of
low immunogenic polymers as demonstrated by use of unmodified and
polymer-grafted H-2 disparate splenocytes. As shown, PEGylation of
stimulator (i.e., irradiated and incapable of proliferation)
splenocytes very effectively attenuated allorecognition and
proliferation of the responder cell population within a one-way
MLR. Moreover, for comparative purposes, the anti-proliferative
dose-response effect of cyclosporine A (CSA; which induces a
pharmacologically-induced anergy) in a one-way murine MLR under the
same experimental condition is shown. Interestingly, the type of
unmodified or polymer-modified cell is important. Human lymphocytes
and murine splenocytes express high levels of "self-antigens"
(Human Leukocyte Antigens (HLA) and mouse H-2 proteins). If cells
devoid of these highly immunogenic antigens are used in the murine
model, no changes in either Tregs or Th17 cells are observed. In
mice injected with unmodified allogeneic erythrocytes, Treg levels
within the spleen, lymph node and peripheral blood were
(respectively): 91.7%, 95.0% and 107.0% of control mouse values.
Similarly unchanged, Th17 levels were (respectively): 71.2%, 112.1%
and 79.2% of control mouse values. Thus, allogeneic murine RBC do
not elicit any significant changes in the systemic levels of either
Treg or Th17 lymphocytes. This finding was observed despite some
antigenic differences between the RBC in H-2 disparate mice. In
support of the low immunogenicity of these genetically different
RBC, allogeneic RBC exhibit normal in vivo circulation nor do they
elicit a significant immune response. Hence, polymer coupled to a
low-immunogenicity allogeneic cell can not induce the
immunomodulation noted with the highly immunogenic splenocytes.
[0121] As demonstrated herein, allogeneic lymphocytes (human PBMC
or murine splenocytes) relative to mPEG-modified allogeneic cells
dramatically increase allorecognition and pro-inflammatory effects
at both the local (cell:cell; MLR) and systemic (in vivo murine
models) levels. Importantly, as demonstrated in our in vivo
studies, it is not the donor cells that differentiate into Th17 (or
other pro-inflammatory subpopulations) or Treg cells, rather it is
the recipients immune system that responds to the unmodified or
PEGylated splenocytes and upregulates production of either Th17
(upon challenge with unmodified splenocytes) or Treg (upon
challenge with mPEG-splenocytes) populations. This was noted by
both the absence of CFSE-staining (only donor cells were stained)
and H-2 phenotyping of the Th17 and Treg cell populations.
[0122] The observed proinflammatory state induced by unmododified
lymphocyte preparations is surprisingly long lasting in vivo. As
noted in FIG. 8, the elevated levels of Th17 and decreased levels
of Treg lymphocytes noted at day 5 persist to day 30. Moreover, the
presence of these Treg (as well as other probable immunological
events) prevents a pro-inflammatory response to unmodified
allogeneic splenocytes administered at day 25. Indeed, no increase
in Th17 lymphocytes is noted in the immunomodulated mice. Moreover,
for the systemic pro-inflammatory response to occur, a highly
immunogenic cell type (e.g., lymphocyte and/or antigen presenting
cells) must be employed as less immunogenic cells, such as H-2
disparate erythrocytes, do not alter the immune (Treg/Th17)
response. While allogeneic murine erythrocytes do express antigenic
differences at the membrane, these cells are only weakly
immunogenic eliciting weak IgG responses and typically remain in
the vascular circulation with a near normal half-life. Moreover,
the induction of both local and systemic pro-inflammatory state can
be modified or lost upon the covalent grafting of a polymer to the
cell. However, soluble mPEG.+-.allogeneic cells has no effect on
the population dynamics of either Treg or Th17 lymphocytes in vitro
or in vivo.
[0123] The balance between Treg and Th17 cells has been identified
as a key factor that orchestrates the tolerance/inflammation level
of human immune system. Regulatory T cells provide suppressor
effect and maintain tolerance, while Th17 cells mediate and are
indicative of a pro-inflammatory state. Hence, modulation of this
balance (either increasing or decreasing the Treg:Th17 ratio) may
be clinically useful. Recent findings have shown that cyclosporine,
a clinically used immunosuppressive agent, has substantial effects
on the Treg/Th17 cell response; though this may be mediated by Th17
cytotoxicity as Treg cells cultured in the presence of rapamycin,
but not cyclosporine A, are found to suppress ongoing alloimmune
responses. Additionally, mycophenolic acid, another
immunosuppressive agent, was found to shift the lymphocyte
polarization by inhibiting IL-17 expression in activated PBMC in
vitro. Of clinical importance, all of these pharmacologic agents
exert significant systemic toxicity and their ongoing use requires
substantial monitoring.
[0124] While induction of tolerance or anergy in transfusion and
transplantation medicine by the polymer-mediated immunocamouflage
of allogeneic leukocytes may provide a less toxic approach than
current conventional pharmacologic agents, other situations exist
in which enhancing the pro-inflammatory state of a subject would be
beneficial. Indeed, increased Treg levels (or Treg:Th17/Th1 ratio)
may prevent desired immunological responses to cancer cells,
parasitic infections, or viral infection. While an abundant number
of approaches are being investigated to prevent and/or regulate the
consequences of allorecognition exist (as exemplified by phenotype
matching (ranging from blood group to HLA matching) and the use of
immunosuppressive agents; FIG. 10A) few pharmacological approaches
exist for effectively stimulating the immune system. Most commonly
used to achieve this goal are cytokine administration. While
extensive tissue matching (e.g., blood groups, HLA) can
dramatically enhance transfusion or transplantation success, the
necessity of tissue matching dramatically reduces the potential
pool of donor tissues. Even in a tissue as plentiful as blood,
extensive non-ABO matching for chronically transfused patients
(e.g., sickle cell disease), while considered desirable, is costly
and often difficult to achieve due to the scarcity of appropriately
matched donor cells. This difficulty is greatly exaggerated with
less common tissues and organs (e.g., islets and kidneys).
[0125] Thus, while pharmacological interventions have been employed
to enhance the probability of successful donor tissue engraftment
(FIG. 10A), engraftment may also be accomplished by induction of
tolerance or anergy. As shown in FIG. 10B, polymer grafting "of",
or grafting "to", allogeneic donor tissue may be used to enhance or
replace pharmacologic agents by induction tolerance or anergy as
evidenced by altered Treg:Th1/Th17 ratios. Tolerance and/or anergy
is induced by the prechallenge of a potential tissue recipient with
PEGylated donor specific (or simply allogeneic; see FIG. 9) PBMC
several (.about.5) days prior to tissue transplantation could be
used to induce a tolerogenic state within the recipient as shown in
FIG. 10B. Elevated levels of Tregs and the down-regulation of Th17
cells would diminish the risks of both hyper-acute and acute
rejection of the donor tissue. There are several substantial
advantages for this approach. Primary amongst these are the easy
collection of donor specific (or simply allogeneic) PBMC, the ease
of PEGylation of the PBMC as well as the ease of administration to
the transplant recipient. While a potential risk of lymphocyte
transfusions is transfusion associated graft versus host disease
(TA-GVHD) in immunosuppressed patients, it was previously
demonstrated that PEGylation effectively blocks TA-GVHD in a murine
model (Chen et al., 2003; Chen et al., 2006). Moreover, this
process could be used in conjunction with irradiated PBMC thus
obviating any risk of TAGVHD. Irradiated cells retain their
allo-stimulatory effects and PEGylation similarly inhibits this
allorecognition and proliferation.
[0126] Conversely to the induction of tolerance, the administration
of immunogenic allogeneic or xenogeneic cells (e.g., leukocytes)
that retain all or part (e.g. a partial modification via low levels
of grafted polymer) of their inherent immunogenicity can be used to
stimulate the immune system as evidenced by a reduced Treg:Th1/Th17
ratio. This approach can be used to counter a pre-existing state of
anergy or tolerance arising either inherently or due to an
infective agent (e.g. parasite). As shown by administration of
allogeneic, non-viable or viable, leukocytes, or preparations
thereof, a proinflammatory state can be induced. Said state will
provide an enhanced systemic pro-inflammatory response allowing for
overcoming an immunosuppressive state and a more effective response
abnormal cells or cell aggregates (e.g. cancer cells) can be
achieved as well as an enhanced response to infective agents (e.g.
nematodes) that induce an immunosuppressive state.
[0127] In summary, administration of viable or non-viable
allogeneic donor lymphocytes can be used to induce or enhance a
pro-inflammatory state in subjects exhibiting a spontaneous or
induced immunosuppressive state. The enhanced proinflammatory state
exists at the cell:cell level and also gives rise to systemic
immunomodulation. The systemic immunomodulation is evidenced by a
significant up-regulation of pro-inflammatory Th17 cells and/or a
significant down-regulation of Treg cells. This immunomodulation is
persistent (.about.30 days). The clinical use of unmodified or
partially modified (e.g., PEG or other covalently grafted polymers)
allogeneic leukocytes may be useful in inducing a pro inflammatory
state and enhancing the destruction of abnormal cells or cell
aggregates (e.g. cancer cells and cancer tumors) and/or enhancing
the immunological response to infective agents (e.g., parasitic
nematodes).
EXAMPLE III
In Vivo Immunomodulation in Nod Mice
[0128] Some of the material and methods referred to in this example
are provided in Example I.
[0129] In the NOD mice, autoimmune destruction of the pancreatic
islets occurs within approximately 16 weeks and was confirmed with
elevated blood glucose measures. The lymphocytes from pre-diabetic
and diabetic animals has been obtained from the spleen, the
brachial lymph node and the pancreatic lymph node. These
lymphocytes have been submitted to flow cytometry using anti-IL-17A
(PE) and anti-FoxP3 (Alexa 697) antibodies. As shown in FIG. 11,
significant changes in the levels of Th17 and Treg lymphocytes are
noted in the spleen, brachial lymph node and pancreatic lymph nodes
upon conversion of NOD mice from non-diabetic to diabetic state.
These changes are characterized by dramatically increased Th17 (top
numbers in each panels) and significantly decreased Treg (lower
numbers in each panels) lymphocytes. Tregs: *, p<0.001 from
non-diabetic NOD mice. Th17: **p<0.001 from non-diabetic NOD
mice.
[0130] The NOD mice (8 to 10 week-old) have been treated with
allogeneic leukocytes (as described in Example I) and
mPEG-allogeneic leukocyte (as described in Example I) and were
compared to untreated control mice (naive or NOD in Table 1). Th17
levels have been measured in various tissues (as described in
Example I). Peripheral blood samples of the groups were pooled for
analysis, all other samples were measured individually. Five male
NOD mice per group were used. The results are shown in Table 1
provided below. As noted in Table 1, the level of Th17
(pro-inflammatory) cells can be increased by treatment with
unmodified allogeneic cells. This pro-inflammatory state results in
increased tissue destruction. While the use of unmodified or
partially modified allogeneic cells would not be used
therapeutically in this disease model, the finding provide evidence
of an increased pro-inflammatory state and increased killing of
specific cells (i.e., pancreatic islets of Langerhans) types.
TABLE-US-00001 TABLE 1 Treatment of NOD mice with unmodified or
mPEG-modified allogeneic cells. Unmodified cells results in a
potent inflammatory state as shown by increased Th17 cells. In
contrast, administration of mPEG-allogeneic cells does not induce
inflammation. Th17 mPEG Tissue NOD Allogeneic Allogeneic Blood 0.38
0.67 0.17* Spleen 0.10 .+-. 0.01 2.32 .+-. 0.38 0.11 .+-. 0.01*
Brachial L. Node 0.08 .+-. 0.01 1.25 .+-. 0.35 0.06 .+-. 0.01*
Pancreatic L. Node 0.05 .+-. 0.01 0.27 .+-. 0.08 0.07 .+-. 0.01* *p
< 0.001 relative to unmodified allogeneic cell treated.
EXAMPLE IV
Poz Polymer for Inducing Pro-Inflammatory State
[0131] Some of the material and methods referred to in this example
are provided in Example I.
[0132] Human PBMC and dendritic cell preparation. Human whole blood
was collected in heparinized vacutainer blood collection tubes (BD,
Franklin Lakes, N.J.) from healthy volunteer donors following
informed consent. PBMC were isolated from diluted whole blood using
FicollePaque PREMIUM.TM. (GE Healthcare Bio-Sciences Corp,
Piscataway, N.J.) as per the product instructions. The PBMC layer
was washed twice with 1.times. Hank's Balanced Salt Solution (HBSS;
without CaCl.sub.2 and MgSO.sub.4; Invitrogen by Life Technologies,
Carlsbad, Calif.) and resuspended in the appropriate media as
needed for mixed lymphocyte reactions and flow cytometric analysis
of Treg and Th17 phenotypes. Dendritic cells (DC) were prepared
from isolated PBMC as described by O'Neill and Bhardwaj (O'Neill et
al., 2005). Briefly, freshly isolated PBMC were overlaid on Petri
dishes for 3 h of in AIM V serum free culture medium (Invitrogen,
Carlsbad, Calif.). Non-adherent cells were gently washed off the
plate. The adherent cells (monocyte rich cells) were treated with
IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&D Systems,
Minneapolis, Minn.) in AIM V medium. Cells were again treated with
IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifuged
and resuspended in fresh media supplemented with DC maturation
factors (TNF-a, IL-1b, IL-6; R&D Systems, Minneapolis, Minn.)
and prostaglandin E2 (Sigma-Aldrich, St. Louis, Mo.). The mature
DC-like cells were harvested on day 7 and CD80, CD83, CD86 and
HLA-DR expressions were determined to confirm DC maturation via
flow cytometry (FACSCalibur.TM. Flow Cytometer, BD Biosciences, San
Jose, Calif.).
[0133] mPEG modification (PEGylation) of PBMCs and splenocytes.
Human PBMC and murine splenocytes were derivitized using
methoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA;
Laysan Bio Inc. Arab, Ala.) with a molecular weight of 20 kDa as
described in Example I. Grafting concentrations ranged from 0 to
3.0 mM per 4.times.10.sup.6 cells/mL.
[0134] POZ modification (POZylation) of PBMCs and splenocytes.
N-hydoxysuccinimidyl ester of polyethyloxazoline propionic acid
(SPA-PEOZ; Serina Therapeutics, Huntsville, Ala.) with a molecular
weight of 20 kDa were grafted on the cells as described in Example
I. Grafting concentrations ranged from 0 to 3.0 mM per
4.times.10.sup.6 cells/mL.
[0135] In vitro and in vivo cell proliferation. Cell proliferation
(both in vitro and in vivo) was assessed via flow cytometry using
the CellTrace.TM. CFSE (Carboxyfluorescein diacetate, succinimidyl
ester) Cell Proliferation Kit (Invitrogen by Life Technologies e
Molecular probes, Carlsbad, Calif.) as described in Example I.
[0136] Mixed lymphocyte reaction (MLR)--control and conditioned
media. The effects of polymer grafting (20 kDa SVAmPEG or 20 kDa
POZ) on allorecognition in vitro were assessed using two-way MLR
(Murad et al, 1999A; Chen et al., 2003; Chen et al., 2006) as
described in Example I.
[0137] A 2-way MLR was conducted using either PEGylated or
POZylated human cells. As shown on FIG. 12, the grafting of
equimolar concentrations of wither 20 kDa mPEG or PEOZ (POZ) on a
human mixed lymphocyte reaction (MLR) had similar effects on
cellular proliferation.
V--In Vivo Modulation of Treg:Th17 Ratio by Polymer-Modified
Lymphocytes
[0138] Some of the material and methods referred to in this example
are provided in Example I.
[0139] Non-modified allogeneic splenocytes (20.times.10.sup.6) and
mPEG-modified allogeneic splenocytes (20.times.10.sup.6) have been
intravenously administered to mouse (naive 8-week old Balb/c mouse;
10 mice per treatment condition). After 5 days, the spleen and the
lymph nodes were harvested and the CD4-positive cells they
contained were further analyzed by flow cytometry. As shown in
FIGS. 13A (annexin V staining) and 13B (mitochondrial
depolarization), the administration of unmodified and mPEG-modified
allogeneic splenocytes, when compared to the administration of
phosphate buffered saline (PBS), increased the number of apoptotic
CD4-positive cells. As shown in FIG. 13C, the administration of
mPEG-modified allogeneic splenocytes increased the intracellular
expression of IL-10 in CD4-positive cells. In contrast, unmodified
splenocytes significantly reduced the expression of IL-10 positive
cells when compared to mPEG-modified allogeneic splenocytes.
Further, the administration of non-modified allogeneic splenocytes
caused a mean decrease in mouse weight whereas the administration
of mPEG-modified allogeneic splenocytes caused a mean increase in
mouse weight (FIG. 13D). Weight loss, or failure to gain weight, is
indicative of a pro-inflammatory state.
[0140] Non-modified allogeneic splenocytes (either 5, 20 or
40.times.10.sup.6 C57BL/6 cells) and mPEG-modified allogeneic
splenocytes (either 5, 20 or 40.times.10.sup.6 C57BL/6 cells
grafted at a density of 0.5 mM, 1 mM or 4 mM) have been
intravenously administered to mouse (5 Balb/c mice/treatment
condition). After 5 days, the spleen and the lymph nodes were
harvested and the CD4-positive cells they contained were further
analyzed by flow cytometry. As shown in FIG. 14, the administration
of non-modified allogeneic splenocytes decreased the percentage of
Treg cells and increased the percentage of Th17 cells. As also
shown in FIG. 14, the administration of mPEG-modified allogeneic
splenocytes increased the percentage of Treg cells and decreased
the percentage of Th17 cells. Surprisingly, the increase in Treg
cell counts observed after the administration of mPEG-modified
allogeneic splenocytes occurred without an increase in spleen
weight while the increase in Th17 cell counts observed after the
administration of the non-modified allogeneic splenocytes
correlated with an increase in spleen weight (a mean 1.5.times.
increase, data not shown).
[0141] Saline, syngeneic splenocytes, non-modified allogeneic
splenocytes (20.times.10.sup.6 C57BL/6 cells) and mPEG-modified
allogeneic splenocytes (20.times.10.sup.6 C57BL/6 cells grafted at
a density of 1 mM PEG) have been intravenously administered to
mouse either once (at day 0, e.g. condition 1) or thrice (at days
0, 2 and 4, e.g. condition 3) (20.times.10.sup.6 C57BL/6 cells
grafted at a density of 1 mM PEG). After 5 or 10 days, the spleen
and lymph nodes were harvested and the CD4-positive cells they
contained were further analyzed by flow cytometry with an
anti-CD279 antibody. As shown in FIGS. 15A and B, the
administration of non-modified allogeneic splenocytes decreased the
number of CD279-positive cells (with respect to the total number of
CD4-positive cells), in the spleen and in the lymph nodes, when
compared to mock-treated or syngeneic-treated animals. As also
shown in FIGS. 15A and B, the administration of mPEG-modified
allogeneic splenocytes increased the number of CD279-positive cells
(with respect to the total number of CD4-positive cells), in the
spleen and in the lymph nodes, when compared to mock-treated or
syngeneic-treated animals. Ten days after the administration of
un-modified allogeneic splenocytes a significant increase in the
percentage of NK cells was observed in both the spleen and the
brachial lymph node was noted (FIG. 16). NK cells are implicated in
the destruction of tumor cells. In contrast, the administration of
PEG-modified allogeneic splenocytes was also shown to decrease the
percentage of NK cells in both the spleen and the brachial lymph
node (FIG. 16) relative to naive mice and mice treated with
non-modified allogenic cells. Further, as shown in Table 2 below,
the administration of non-modified allogeneic splenocyte increased
the NK Cell alloresponse and baseline levels in recipient mice (as
measured by flow cytometry using a NK1.1 antibody while mPEG
modified cells reduced NK1.1 levels to below that seen in naive
mice.
TABLE-US-00002 TABLE 2 Percentage of NK1.1-positive cells in mice
having received saline, syngeneic splenocytes, non-modified
allogeneic splenocytes and mPEG-modified allogeneic splenocytes.
Cells were harvested 10 days after the last injection Type of cells
administered Number of Percentage of NK1.1- (20 .times. 10.sup.6
cells) doses positive cells None (saline) 1 1.12 None (saline) 3
0.97 Syngeneic 1 0.94 Syngeneic 3 0.91 Non-modified allogeneic 1
2.26 Non-modified allogeneic 3 2.30 mPEG-modified 1 0.29 allogeneic
mPEG-modified 3 0.21 allogeneic
[0142] The thymus of these animals has also been harvested and the
thymic cells characterized. As shown in FIG. 17A, the
administration of mPEG-modified allogeneic splenocytes increased
microchimerism in the thymus of recipient animals as shown by the
number of CFSE labeled allogeneic donor cells in the thymus. In
contrast non-modified cells did not. Under normal conditions only 6
to 10% of the injected donor CD4-positive splenocytes are Treg
(17A; open bar segment). Moreover as shown in FIG. 17B, while the
administration of mPEG-modified splenocytes increased the total
percentage in thymic Treg cells (donor, open bar; recipient grey
bar) in the recipient, non-modified allogeneic cells decreased Treg
levels in the recipient's thymus. Further, the administration of
non-modified allogeneic splenocytes increased the percentage of
thymic pro-inflammatory Th17 cells, while the administration of the
mPEG-modified allogeneic splenocytes decreased the percentage of
thymic Th17cells (FIG. 17C).
VI--In Vivo Modulation of Treg:Th17 Ratio by Conditioned Media
Obtained via Polymer-Modified Lymphocytes
[0143] Some of the material and methods referred to in this example
are provided in Example I.
[0144] Conditioned serum. Conditioned serum was obtained (by
bleeding the animal and separating the cellular components of blood
from the serum via centrifugation) five days after mice (Balb/c;
N=5) received saline, unmodified syngeneic splenocytes (Balb/c),
unmodified allogeneic splenocytes (20.times.10.sup.6 C57BL/6 cells)
or mPEG-modified allogeneic splenocytes (20.times.10.sup.6 C57BL/6
cells grafted at a density of 1 mM PEG). The serum from naive
animals was also obtained as a control. The conditioned or naive
serum (100 .mu.l) was then administered (i.v. tail vein injection)
once (at day 0) or thrice (at days 0, 2 and 4) to recipient mice
(Balb/c; N=5). Five days after the last administration, a blood
sample, the spleen and the brachial lymph nodes were obtained from
the treated animals and the leukocytes they contained were
analyzed.
[0145] As shown on FIG. 18, the administration of the conditioned
serum from animals having received unmodified allogeneic
splenocytes caused in vivo a reduction in the levels of Tregs,
while increasing the levels of Th17 cells in both the spleen and
the lymph nodes. As also shown on FIG. 18, the administration of
the conditioned serum from animals having received polymer modified
allogeneic splenocytes caused in vivo an increase in the levels of
Tregs as well as a decrease in the levels of Th17 cells, both in
the spleen and the lymph node.
[0146] This modulation in Treg/Th17 ratio was also shown to be
associated in the long term modification of the expression of
pro-/anti-inflammatory cytokine positive CD4+ lymphocytes. As shown
on FIG. 19, the administration of the conditioned serum from
animals having received unmodified allogeneic splenocytes caused in
vivo an increase in the expression of pro-inflammatory cytokines
(IL-2, TNF-.alpha., IFN-.gamma. and IL-4) positive lymphocytes
while the administration of the conditioned serum from animals
having received polymer modified allogeneic splenocytes caused in
vivo an increase in the expression of anti-inflammatory cytokines
(IL-10) in CD4.sup.+ lymphocytes. These results were observed for
at least 30 days and 60 days after the last administration. Similar
observations have been observed 270 days after the last
administration (data not shown).
[0147] The administration of the conditioned medium also caused a
shift in the Treg subsets. As shown on FIG. 20, the administration
of the conditioned serum from animals having received unmodified
allogeneic splenocytes caused in vivo decrease in all Treg subsets
(Foxp3.sup.+, CD25.sup.+ and CD69.sup.+) in the spleen and the
lymph nodes. The administration of the conditioned serum from
animals having received polymer modified allogeneic splenocytes
caused in vivo an increase all Treg subsets with the largest change
noted in CD69.sup.+ cells. Non-modified cells had a roughly
equivalent inhibitory effect on all Treg subsets tested for further
demonstrating a potent proinflammatory response.
[0148] As shown on FIG. 21, the administration of the conditioned
serum from animals having received unmodified allogeneic
splenocytes caused in vivo a reduction in the levels of Tregs,
while increasing the levels of Th17 cells in the spleen, the lymph
nodes and the blood. As also shown on FIG. 21, the administration
of the conditioned serum from animals having received polymer
modified allogeneic splenocytes caused in vivo an increase in the
levels of Tregs as well as a decrease in the levels of Th17 cells,
in the spleen, the lymph node and the blood.
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[0155] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
* * * * *