U.S. patent application number 17/062877 was filed with the patent office on 2021-04-22 for methods and materials for the generation of regulatory t cells.
The applicant listed for this patent is LIFE TECHNLOGIES AS. Invention is credited to Tanja AARVAK, Anne BRUNSVIG, Gunnar KVALHEIM, Walter Gabriell Borelli PIEDRAS, Anne-Marie RASMUSSEN.
Application Number | 20210115401 17/062877 |
Document ID | / |
Family ID | 1000005315817 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210115401 |
Kind Code |
A1 |
AARVAK; Tanja ; et
al. |
April 22, 2021 |
Methods and Materials for the Generation of Regulatory T Cells
Abstract
Methods are disclosed for the generation of immunosuppressive
regulatory T cells. The methods can include contacting a population
of CD4+CD25- T cells with a T cell receptor (TCR)/CD3 activator, a
TCR co-stimulator activator, and rapamycin. Kits for the generation
of immunosuppressive regulatory T cells, methods of use, and cell
populations are also disclosed.
Inventors: |
AARVAK; Tanja; (Oslo,
NO) ; RASMUSSEN; Anne-Marie; (Oslo, NO) ;
KVALHEIM; Gunnar; (Oslo, NO) ; PIEDRAS; Walter
Gabriell Borelli; (Montevideo, UY) ; BRUNSVIG;
Anne; (Stabekk, NO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNLOGIES AS |
Oslo |
|
NO |
|
|
Family ID: |
1000005315817 |
Appl. No.: |
17/062877 |
Filed: |
October 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16057274 |
Aug 7, 2018 |
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17062877 |
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14806403 |
Jul 22, 2015 |
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16057274 |
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13689530 |
Nov 29, 2012 |
9119807 |
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14806403 |
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12604263 |
Oct 22, 2009 |
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13689530 |
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12279713 |
Oct 30, 2009 |
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PCT/IB2007/050508 |
Feb 15, 2007 |
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12604263 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/12 20130101;
C12N 2501/2302 20130101; C12N 2501/04 20130101; C12N 2501/2304
20130101; A61K 35/17 20130101; C12N 5/0637 20130101; C12N 2501/999
20130101; C12N 2501/998 20130101; C12N 5/0636 20130101; A61K
2035/122 20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; A61K 31/12 20060101 A61K031/12; A61K 35/17 20060101
A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 15, 2006 |
GB |
603081.1 |
Claims
1. A method of generating immunosuppressive regulatory T cells from
a sample, the method comprising: providing an initial sample
containing a population of CD4+CD25- T cells; and contacting the
population with a T cell receptor (TCR)/CD3 activator, a TCR
co-stimulator activator, and rapamycin to generate a final sample
comprising immunosuppressive regulatory T cells.
2. The method of claim 1, further comprising purifying the
population prior to the contacting step.
3. The method of claim 2, wherein the purifying step comprises
isolating CD4+ T cells.
4. The method of claim 1, wherein the generated immunosuppressive
regulatory T cells are CD4+CD25+.
5. The method of claim 1, wherein the generated immunosuppressive
regulatory T cells are CD4+CD25+FOXP3-.
6.-8. (canceled)
9. The method of claim 1, wherein the final sample comprises at
least about 50% CD4+CD25+ cells.
10. The method of claim 1, wherein the population is contacted with
the rapamycin before the T cell receptor (TCR)/CD3 activator and
TCR co-stimulator activator.
11.-16. (canceled)
17. The method of claim 1, wherein the T cell receptor (TCR)/CD3
activator is an antibody or a ligand for TCR/CD3.
18.-19. (canceled)
20. The method of claim 1, the TCR co-stimulator activator is an
antibody.
21.-36. (canceled)
37. A kit for the generation of immunosuppressive regulatory T
cells, the kit comprising: a T cell receptor (TCR)/CD3 activator, a
TCR co-stimulator activator, and rapamycin.
38. The kit of claim 37, wherein: the T cell receptor (TCR)/CD3
activator is immobilized on a solid phase; and the TCR
co-stimulator activator is immobilized on a solid phase.
39. (canceled)
40. A composition comprising a population of immunosuppressive
regulatory T cells, wherein at least 80% of the immunosuppressive
regulatory T cells are CD4+FoxP3- T cells.
41. The composition of claim 40, wherein the population of
immunosuppressive regulatory T cells contains CD4+CD25+ T
cells.
42. The composition of claim 40, wherein the population of
immunosuppressive regulatory T cells contains at least 50%
CD4+CD25+ T cells.
43. The composition of claim 40, wherein the population of
immunosuppressive regulatory T cells contains CD4+CD25- T
cells.
44. The composition of claim 40, wherein the population of
immunosuppressive regulatory T cells is present in a culture
medium.
45. A method of treating a mammal, the method comprising
administering the composition of claim 40 to the mammal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 16/057,274, filed on Aug. 7, 2018, which is a
divisional application of U.S. application Ser. No. 14/806,403,
filed on Jul. 22, 2015, now abandoned, which is a continuation
application of U.S. application Ser. No. 13/689,530, filed Nov. 29,
2012 and issued as U.S. Pat. No. 9,119,807 on Sep. 1, 2015, which
is a continuation of U.S. application Ser. No. 12/604,263, filed
Oct. 22, 2009, now abandoned, which is a continuation application
of U.S. application Ser. No. 12/279,713, filed Oct. 30, 2009, now
abandoned, which is a 371 U.S. national application of
international application No. PCT/IB2007/050508, filed Feb. 15,
2007, which claims priority to G.B. patent application Serial No.
603081.1, filed Feb. 15, 2006, each of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods and materials for the
generation of regulatory T cells.
SUMMARY OF THE INVENTION
[0003] The proliferation of T cells can be stimulated by contacting
the cells with a T cell receptor/CD3 activator, a TCR
co-stimulatory activator, and rapamycin. Methods for generating
regulatory T cells from CD4+CD25+FOXP3- T cells and the use of
these methods in the generation of T cell populations are
disclosed. These cells can be used in a variety of applications
including immunotherapy.
DESCRIPTION OF THE FIGURES
[0004] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these figures in combination with the
detailed description of specific embodiments presented herein.
[0005] FIG. 1 shows the extent of proliferation of various ratios
of T cells cultured with or without rapamycin in a standard
proliferation assay. CD25- T cells (T cells cultured without
Rapamycin) were analyzed for proliferative capacity in a standard
proliferation assay using .sup.3H Thymidine incorporation after 4
days stimulation. CD25+ T cells (T cells cultured with Rapamycin)
were added to the CD25- T cells in a ratio from 1:1 to 1:32. The
proliferative capacity of CD25- T cells alone was defined as 100%
proliferation. The x-axis is the ratio of CD25+ cells to CD25-
cells. The y-axis is percent proliferation.
[0006] FIGS. 2A and 2B show flow cytometry analysis of CD4 and CD25
expression on CD4+ T cells expanded (FIG. 2A) with rapamycin or
(FIG. 2B) without rapamycin.
DETAILED DESCRIPTION OF THE INVENTION
[0007] While compositions and methods are described in terms of
"comprising" various components or steps (interpreted as meaning
"including, but not limited to"), the compositions and methods can
also "consist essentially of" or "consist of" the various
components and steps, such terminology should be interpreted as
defining essentially closed-member groups.
[0008] Aspects of the present invention relate to methods for
generating CD4+CD25+FOXP3- regulatory T cells and the use of these
methods in the generation of T cell populations which have
applications in for example immunotherapy.
[0009] Naturally occurring regulatory T (Treg) cells suppress
immune responses and play an important role in immunotherapy
against autoimmune diseases and provide transplantation tolerance.
Various populations of Treg cells have been described and include
naturally occurring CD4+CD25+FOXP3+ cells and induced Tr1 and Th3
cells that secrete IL-10 and TGF.beta. respectively. The natural
occurring CD4+CD25+FOXP3+ Treg cells represents 5-10% of the CD4+ T
cells in the peripheral blood and are in a hypoproliferative state
which has hampered detailed characterization and the potential use
of these cells in immune therapy. In vivo uses therefore have
relied on expansion protocols to generate sufficient numbers of
Treg cells for in vivo use. The clinical use of Treg cells is
limited by the lack of appropriate isolation and expansions
protocols to generate sufficient numbers for in vivo infusion.
[0010] The present invention addresses this need by providing a
method of generating a population of immunosuppressive Treg cells
from the abundant CD4+CD25- T cell population. This provides Treg
cells in sufficient numbers for in vivo infusions. The protocol can
be used both for generating Treg cells for research purposes and
for clinical use by infusion in patients.
[0011] The invention thus provides a method in which Treg cells
expressing CD4, CD25 but not FOXP3 are generated. In one
embodiment, the protocol uses a solid support carrying CD3/CD28
antibodies, Rapamycin and optionally cytokines, such as IL-4 and/or
IL-2 to activate and expand CD4+ T cells isolated either from
peripheral blood or leukopheresis products.
[0012] The use of anti-CD3/CD28, for example, provides the
activation signal for the T cell population. T cells require at
least two signals for activation. Signal one is antigen specific
and is elicited by peptide/major histocompatibility complex (MHC)
complexes presented by antigen-presenting cells (APC) and received
through the T-cell receptor (TCR)/CD3 complex. Signal two (which is
antigen non-specific) is also delivered by antigen presenting cells
and one of the candidate molecules for its receptor is the T cell
antigen CD28. It is thought that when both the TCR/CD3 and CD28 T
cell receptors are occupied by appropriate ligands, T cells are
stimulated to proliferate and produce IL-2 (a cytokine essential
for T cell proliferation), whereas occupation of the T cell
receptor alone favors T cell anergy or apoptosis.
[0013] In vitro it has been shown that T cell growth and cytokine
production can be stimulated by culturing T cells with anti-CD3
antibodies which have been immobilized to a solid phase (for
example beads or tissue culture plates) and adding soluble CD28
antibodies (Sommer et al., 1993. Eur. J. Immunol. 23:2498-2502,
Sunder-Plassmann et al., 1996. Blood 87:5179-5184). More recently
it has been shown that co-immobilising both CD3 and CD28 antibodies
to the same solid phase or to different solid phases can also
induce T cell proliferation (Levine et al., 1997. J. of Immunol.
159: 5921-30; Li et al., 1999. Science 283:848-851).
[0014] Rapamycin is an immunosuppressive agent used to prevent
allograft rejection. Recently, the cellular target for Rapamycin in
vitro has been discovered, and shown to selectively expand
naturally occurring CD4+CD25+FOXP3+.times. regulatory T cells. The
present inventors have developed a new protocol using Rapamycin to
generate new Treg cells from CD4+CD25- T cells. In contrast to
naturally occurring CD4+CD25+ Treg cells that express FOXP3, the
Treg cells generated in accordance with the invention do not
express FOXP3 but still show very strong suppressive
capacities.
[0015] Treg cells generated according to the invention have many
potential uses, including experimental and therapeutic uses. In
particular it is envisaged that such T cell populations will be
extremely useful in suppressing undesirable or inappropriate immune
responses. In such methods a small number of T cells are removed
from a patient and then manipulated and expanded ex vivo before
reinfusing them into the patient. Examples of diseases which may be
treated in this way are autoimmune diseases and conditions in which
suppressed immune activity is desirable, e.g. for
allo-transplantation tolerance. A therapeutic method could comprise
providing a mammal, obtaining CD4+CD25- T cells from the mammal;
expanding/activating the T cells ex vivo in accordance with the
methods of the invention as described above; and administering the
expanded/activated T cells to the mammal to be treated. The first
mammal and the mammal to be treated can be the same or different.
The mammal can generally be any mammal, such as cats, dogs,
rabbits, horses, pigs, cows, goats, sheep, monkeys, or humans. The
first mammal ("donor") can be syngeneic, allogeneic, or xenogeneic.
Therapy could be administered to mammals having aberrant immune
response (such as autoimmune diseases including, for example
diabetes, multiple sclerosis, myasthenia gravia, neuritis, lupus,
rheumatoid arthritis, psoriasis, and inflammatory bowel disease),
tissue transplantation, or fertility treatments.
[0016] The main technical hurdles involved in such therapies
include the purification of the cells of interest from the patient
and the expansion and/or the manipulation of the cells in vitro.
Such therapies generally require a large number of cells and thus
it can be seen that it is vital to optimize the methods of inducing
in vitro T cell proliferation in order to maximize the number of T
cells produced and minimize the time required to produce the T
cells in sufficient numbers.
[0017] Thus viewed in a first aspect the present invention provides
a method for stimulating proliferation of CD4+CD25- T cells
comprising contacting the cells with a T cell receptor (TCR)/CD3
activator, a TCR co-stimulator activator and rapamycin. The cells
can be cultured under conditions and for a time suitable to achieve
levels of proliferation as described hereinafter. This method
generates a CD4+CD25+ T cell population. T cells which are FOXP3-
may be used and the generated T cell population in that case remain
FOXP3-.
[0018] An alternative embodiment of the invention provides a method
of preparing a T cell population of CD4+CD25+ T cells, preferably
CD4+CD25+FOXP3- T cells, comprising contacting CD4+CD25- T cells
(preferably CD4+CD25-FOXP3- cells) with a TCR/CD3 activator, a TCR
co-stimulatory activator and rapamycin to stimulate proliferation
of the CD4+CD25- T cells and optionally isolating the T cell
population. The CD4+CD25+(preferably CD4+CD25+FOXP3-) T cell
population thus formed provides a further aspect of the invention.
Preferably, the T cell population thus formed is immunosuppressive.
Thus, the cells are able to suppress proliferation of syngeneic T
cells in vitro, e.g. as described in the Examples herein. At a 1:1
ratio of the test T cells, with, for example, T cells prior to
stimulation, immunosuppressive cells preferably achieve at least
70, 80 or 90% suppression of proliferation, i.e. reduction in cell
numbers relative to control over a suitable culture period, e.g.
between 2 and 14 days, for example 4 days. Preferably, the
CD4+CD25- T cells used in the stimulation methods and the CD4+CD25+
T cells generated according to the invention as described herein
are FOXP3-.
[0019] The TCR/CD3 activator can be an antibody or ligand for
TCR/CD3, for example a CD3 antibody. The TCR co-stimulatory
activator can be an antibody or ligand for CD28, CD137 (4-1BB),
GITR, B7-1/2, CD5, ICOS, OX40 or CD40. Preferably the TCR/CD3
activator is a CD3 antibody and the TCR co-stimulatory activator is
a CD28 antibody.
[0020] Preferably the activators, e.g. antibodies, are immobilized
to a solid phase. The activators, e.g. antibodies may be provided
at variable concentration on the solid support, such as at a ratio
of about 1:10 to about 10:1 of CD28 antibody to TCR/CD3 antibody.
Optionally more than one of the TCR/CD3 activators and/or more than
one of the TCR co-stimulatory activators may be used in methods of
the invention.
[0021] "Stimulating proliferation" as used herein refers to any
event which results in a detectable increase or expansion in the
number of T cells present when compared with the number present in
the absence of such stimulation. The daughter cells which are
generated by the proliferation may have a different phenotype as a
result of stimulation, in particular, stimulation results in CD25
expression thus resulting in the generation of CD4+CD25+ cells from
CD4+CD25- cells.
[0022] Such an increase in number may be relatively small, but is
preferably a significant increase such as for example an increase
in cell number of at least about 2 fold, preferably at least about
5 fold, about 20 fold or about 50 fold and more preferably at least
about 100 fold, about 500 fold, or greater than about 1000 fold.
Such increases in number may be measured at any appropriate time
point in the cell expansion protocol, such as for example at day 4
up to day 12 of the cell expansion protocol.
[0023] T cells which are considered positive for a specific
antigen, e.g. CD3, carry detectable levels of the antigen, e.g. as
determined by cell sorting (e.g. flow cytometry) or the use of a
solid support to which an appropriate binding partner, e.g.
antibody is bound.
[0024] As discussed above stimulation of T cells with a TCR/CD3
activator and a TCR co-stimulatory activator, e.g. anti-CD3 and
anti-CD28 antibodies also provides the two signals required for T
cell activation. Thus, viewed in a yet further alternative way, the
present invention provides a method of activating CD4+CD25- T cells
comprising contacting the cells with a TCR/CD3 activator and a TCR
co-stimulatory activator (e.g. CD28 antibodies and T cell receptor
(TCR)/CD3 antibodies) and rapamycin.
[0025] Thus, throughout the discussion of the present invention,
reference to methods of stimulating proliferation and use of T
cells so stimulated should also be read to include methods of
stimulating T cell activation and the use of such activated T
cells.
[0026] Any CD4+CD25- T cell population may be expanded/activated
using the present method. For example the T cell population may
comprise CD4+CD25-CD8+ T cells. In a preferred aspect, CD4+CD25- T
cells are separated from the source starting material prior to
stimulation and expansion. Thus, in a preferred aspect the present
invention provides a method for stimulating proliferation of
CD4+CD25- T cells comprising at least the steps of: (i) isolating
CD4+CD25- T cells from a sample, and (ii) contacting the cells with
a TCR/CD3 activator and a TCR co-stimulatory activator (e.g. CD28
antibodies and T cell receptor (TCR)/CD3 antibodies) and
rapamycin.
[0027] An additional embodiment of the invention provides a method
of preparing a T cell population of CD4+CD25+(preferably
CD4+CD25+FOXP3-) T cells, comprising (i) isolating CD4+CD25- T
cells from a sample, and (ii) contacting the cells with a TCR/CD3
activator and a TCR costimulatory activator (e.g. CD28 antibodies
and T cell receptor (TCR)/CD3 antibodies) and rapamycin to
stimulate proliferation of the CD4+CD25- T cells and optionally
isolating the T cell population.
[0028] The CD4+CD25- cells which are used in methods of the
invention may comprise the entire CD4+CD25- T cell population or a
portion of that population. For example, a sub-population may be
used in the method, e.g. CD4+CD25-FOXP3-, which sub-population may
be used in its entirety or a portion of that sub-population may be
used. Thus the isolated CD4+CD25- cells subjected to the
stimulation method of the invention may contain at least about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
or about 90% of the CD4+CD25- cells (or a sub-population thereof)
in the sample from which the CD4+CD25- cells were isolated.
Preferably however, in order to generate sufficient levels of Treg
for in vivo uses, essentially the entire CD4+CD25- population from
the starting sample is used in methods of the invention.
[0029] The stimulation method may be performed in the presence of
other cells, e.g. other T cells, such as CD4+CD25+ cells which
themselves will proliferate during the method of the invention.
Thus in the above described protocol, the step of isolating
CD4+CD25- T cells may comprise isolating a significant portion
(i.e. at least 20, 30, 40, 50, 60, 70, 80 or 90%) or all CD4+
cells, i.e. which include both CD25- and CD25+, from the sample.
Thus in a preferred embodiment of the invention, the isolation step
comprises the isolation of CD4+ cells. Furthermore, other cells may
also be present such that the e.g. CD4+, CD4+CD25- and/or
CD4+CD25-FOXP3- cells form only a portion of the cells used in the
stimulatory method, before or after the isolation step. Thus in the
stimulatory step the CD4+CD25- may be present as a portion of the
cells subject to stimulation, i.e. an enriched preparation may be
used, such as comprising at least about 50, about 60, about 70,
about 80 or about 90% of the total cells subjected to
stimulation.
[0030] Preferably however, at least some CD4+CD25+ cells are
absent, e.g. at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, or about 90% of the
cells which appeared in the starting material are absent.
Alternatively expressed the cells for stimulation/expansion are
preferably substantially all CD4+CD25- cells, such as at least
about 80%, about 90%, about 95% or about 98% CD4+CD25- T cells are
used in the method. Especially preferably, at least some CD4+FOXP3+
cells are absent in the cells used for expansion, e.g. at least 10,
20, 30, 40, 50, 60, 70, 80 or 90% of the cells which appeared in
the starting material of the sample are absent. Alternatively
expressed the cells for stimulation/expansion are preferably
substantially all CD4+FOXP3- (preferably CD4+CD25-FOXP3-) T cells,
e.g. at least 80, 90, 95 or 98% CD4+FOXP3- or CD4+CD25-FOXP3- T
cells are used in the method.
[0031] Sources of T cells and methods of isolating particular T
cell populations (e.g. CD4+ cells) which can be expanded by
stimulation according to the methods of the present invention are
well known and described in the literature. Thus for example T
cells may conveniently be isolated from the blood e.g. from a
peripheral blood mononuclear cell (PBMC) population isolated from
blood, or from other blood-derived preparations such as
leukopheresis products or from bone marrow, lymph, thymus, spleen
or umbilical cord. Examples of T cell populations which can be
selected and expanded according to the methods of the present
invention include those which are derived by negative selection
from PBMC where B cells and monocytes are depleted, (optionally
negative selection of CD25- and/or FOXP3- cells), positive
selection of the CD4+ T cells using beads coupled with Abs against
CD4 (or flow cytometry) and positive selection of T cells using
sheep red blood cells. T cell populations may be derived from any
appropriate source, including human or animal sources.
[0032] Immobilization of the activators (e.g. CD28 and CD3/TCR
antibodies) to a solid phase according to the present invention
means that the activators are immobilized to (or on) the same or
different solid supports. Preferably, the antibodies are
co-immobilized to (or on) the same surface.
[0033] "Immobilized" or "Immobilization" as used herein refers to
any means by which activators, e.g. antibodies can be bound,
attached or otherwise fixed to a solid phase. Such immobilization
may be direct (i.e. the activators themselves are attached to the
solid phase) or indirect (i.e. the activators are attached via an
intermediate entity) and may occur by way of any chemical or
non-chemical attachment method. Such methods include immobilization
of activators by adsorption and/or by covalent attachment or via
affinity between molecules (e g immobilization on an avidin-coated
surface of biotinylated antibodies, or the immobilization of an
anti CD3 or anti CD28 via a secondary antibody or antibodies which
are themselves attached to the solid phase). The immobilization
must be of sufficient strength that the activators are not removed
under the conditions used to prepare the solid phase with the
activators attached (for example the immobilized activators must
withstand the washing conditions etc. associated with the
preparation of the solid phase) and are also not removed under the
conditions used to stimulate T cell proliferation.
[0034] The step of "contacting" the population of T cells with the
activators, e g immobilized to the solid phase, may be carried out
in any convenient or desired way. When the method is performed
without immobilized activators, the activators are added into the
solution containing the cells to be expanded. When one or more of
the activators is carried on a solid support, e.g. if the solid
phase was a tissue culture plate or flask or some other flat
surface, the population of T cells, conveniently in an aqueous
medium such as for example an appropriate cell culture medium, may
simply be added to the solid phase under appropriate conditions.
Alternatively, if the solid phase is particulate (for example
beads) the solid phase itself may be added to the T cell population
under appropriate conditions. In any event, in the contacting step,
the cells are contacted with activators which are already
immobilized i.e. the activators are bound to the solid support
before they bind to the cells, i.e. before the contacting step.
[0035] The activators are provided in a molar equivalent or molar
excess to the cells to be stimulated. Thus, for example in the case
of anti-CD3/anti-CD28 solid supports, e.g. beads, the bead to T
cell ratio may range from 1:10 to 1:1, e.g. 3:1. Beads for this
purposes are well known in the art (e.g. from Dynal Biotech
ASA).
[0036] Appropriate conditions will be such conditions which are
suitable for T cell growth and should be chosen depending on for
example the T cell population concerned and the particular cell
culture medium used. Such media and conditions are well known in
the art. Typical conditions may be maintenance of cells in a
humidified atmosphere containing 5 or 10% CO.sub.2 at 37.degree. C.
or under low O.sub.2 concentration (e.g. 2%). Appropriate culture
medium is selected depending on the T cell population being
expanded. For example for T cell populations the culture medium X
vivo 15 supplemented with 5% human AB serum is appropriate or RPMI
1640 with FCS, CellGro DC, or other media suited for cell culture
may be used.
[0037] Rapamycin is contacted with the cells prior to,
simultaneously with, and/or subsequent to contact of the cells with
the activators. Rapamycin is preferably present throughout the
proliferation/expansion step in the method according to the
invention. The rapamycin may be added in one or more steps. Thus
for example, as described in the examples herein, isolated CD4+
cells may be stimulated with activators (CD3 antibodies and CD28
antibodies) and rapamycin at the same time. In this method,
subsequent growth and passaging is performed in the presence of
rapamycin, but not the activators.
[0038] Rapamycin may be used at a concentration of from about 0.01
.mu.M to about 10 .mu.M, such as about 0.5 .mu.M to about 2 .mu.M,
or about 1 .mu.M. Rapamycin is a protein kinase inhibitor with a
molecular weight of 914.2, also referred to as Sirolimus, Rapamune,
AY-22989, RAPA and NSC-226080, available from Sigma, Calbio Chem,
LC Labs etc. Rapamycin is available from a variety of commercial
sources, such as A.G. Scientific, Inc. (San Diego, Calif.,
USA).
[0039] Other cytokines and/or growth factors may be added to the
cultures as appropriate. Such cytokines and/or growth factors are
added at appropriate concentrations and time points. For example
IL-2 and/or IL-4 may be added to enhance the proliferation of the T
cells and other cytokines may be added to induce particular
differentiation patterns if required (e.g. TGF-.beta. and IL10).
For example IL-4 has been shown to trigger differentiation of T
cell populations into the Th2 subpopulation and IFN-(to trigger
differentiation into the Th1 subpopulation (Sunder-Plassmann,
supra). Thus in preferred embodiments of the invention, a cytokine,
e.g. IL-2 is preferably added, e.g. at a final concentration of
10-2,000 U/ml, e.g. at 20 U/ml during stimulation and at 1,000 U/ml
periodically during the initial culture period and 20 U/ml in the
period prior to harvest. IL-4 is preferably added, e.g. at a final
concentration of 1,000-5,000 U/ml, e.g. at 1,000 U/ml during
stimulation periodically during the initial culture period and in
the period prior to harvest. Appropriate cytokines and growth
factors and their effects on T cells are well known and described
in the art. Once the T cell population has been brought into
contact with the activators and rapamycin under appropriate
conditions for growth of the T cells, growth is allowed to progress
for a time period selected according to the final number of T cells
required and the rate of expansion of the cells. Passaging of the
cells may be undertaken during this period. Such a time period is
normally between 3 and 10 days but can be as long as 14 to 20 days
or even longer providing the viability and continued proliferation
of the T cells is maintained.
[0040] If necessary the T cell population which is being
expanded/activated according to the methods of the present
invention may be re-stimulated by contacting the cells with further
activators in a similar way to the initial stimulation. In general,
re-stimulation is only necessary or desired if the T cells are to
be cultured for a long period of time, e.g. more than 20 days.
[0041] Treg cells having immunosuppressive properties can be
assessed by analysis of their phenotype (CD4+CD25+FOXP3-) and their
ability to suppress proliferation of syngeneic T cells in vitro,
e.g. as described in the Examples herein.
[0042] Once a T cell population has been expanded to a required
level as described above, the expanded population can then be
separated from the solid phase in an appropriate way. For example,
if the activators are attached to a solid phase and that solid
phase is a tissue culture well, plate or bottle, the T cell
population can be obtained by removal of the culture medium which
contains the T cells. If the solid phase comprises for example
magnetic beads, a magnetic field is used to attract the beads to
the side of the vessel and the culture medium containing the T
cells can then be poured off. Other particulate solid supports e.g.
non-magnetic beads may be centrifuged or filtered away from the
cells. Although the majority of T cells will be located in the
culture medium, some T cells are likely to be attached to the solid
phase after expansion. If desired such T cells can be detached by
for example resuspension using a pipette or other suitable means.
Such a resuspension will normally be carried out before the T cells
are separated from the solid phase to improve the yield. Once
separated from the solid phase the T cell population can then be
further treated and/or manipulated in any desired way or used
directly for suitable applications such as for example in vitro
experiments and research, non-therapeutic applications, therapeutic
applications etc. as discussed below.
[0043] When soluble activators are employed, these may be removed
by competition with appropriate ligands, e.g. CD3 or CD28, but more
preferably the T cells are collected from the culture medium and
used for applications as described herein without further
refinement. Conveniently, for large scale applications, appropriate
isolation and preparation platforms may be used for selection of
the T cell populations for stimulation and/or for the expansion
protocol and/or isolation of the generated T cell population. In
this regard special mention may be made of Dynal's ClinExVivo.TM.
platforms in which closed sterile disposable bags may be used for
any magnetic cell separation steps which are performed, e.g. for
cell isolation prior to, or after, expansion.
[0044] Thus in a preferred aspect the present invention provides a
method for stimulating proliferation of CD4+CD25- T cells
comprising at least the steps of: (i) isolating CD4+CD25- T cells
from a sample, preferably by a method comprising at least the steps
of contacting the sample with a solid support carrying CD4
antibodies and isolating the T cells which bind to the solid
support, or using negative isolation of CD4+ T cells by removing
all unwanted cells with a mix of mAbs followed by magnetic beads to
capture cells (ii) contacting the cells with a) a solid support
carrying CD28 antibodies and TCR/CD3 antibodies and b) rapamycin,
(iii) incubating the cells under conditions to allow proliferation,
preferably in the presence of IL-4 and/or IL-2 and (iv) isolating
the T cells after the incubation.
[0045] When the activator used in accordance with the invention is
an antibody reactive with TCR co-stimulatory activators: CD28,
CD137 (4-1 BB), GITR, B7-1/2, C D5, ICOS, OX40, CD40 or CD137 or
TCR/CD3 activators: CD3 or TCR may be used. More than one antibody
from each group may be used. Preferably a TCR or CD3 antibody is
used in conjunction with a CD28 antibody. Thus, using CD28 and
TCR/CD3 antibodies as an example, a "CD28 antibody" and a "TCR/CD3
antibody" according to the present invention are antibodies capable
of binding to (or immunoreactive with) CD28 or TCR/CD3
respectively. Preferred are antibodies capable of binding
specifically to CD28 or TCR/CD3 in a manner which distinguishes
from the binding to other "non-target" molecules, i.e. preferred
antibodies are those which exhibit detectable binding affinity for
CD28 or CD3/TCR but whose binding to other molecules, for example
other cell surface molecules is negligible, insignificant or
non-detectable. In preferred aspects of the invention, the method
of the invention may involve the use of antibodies to (i) CD3 and
CD28, (ii) CD3 and CD137, or (iii) CD3 and CD28 and CD137.
[0046] A number of CD28, CD137 (4-1BB), GITR, B7-1/2, CD5, ICOS,
OX40, CD40, CD3 or TCR antibodies (or anti-CD28 etc.) are known in
the literature or commercially available. Any such antibody, or its
fragments or derivatives may be used (so long as they retain
binding activity).
[0047] Antibodies for use in methods of the present invention may
be of any species, class or subtype providing that such antibodies
can react with the target of interest, e.g. CD3, the TCR, or CD28
as appropriate.
[0048] Thus "antibodies" for use in the present invention
include:
[0049] (a) any of the various classes or sub-classes of
immunoglobulin, e.g. IgG, IgA, IgM, IgD or IgE derived from any
animal e g any of the animals conventionally used, e.g. sheep,
rabbits, goats, or mice, or egg yolk
[0050] (b) monoclonal or polyclonal antibodies
[0051] (c) intact antibodies or fragments of antibodies, monoclonal
or polyclonal, the fragments being those which contain the binding
region of the antibody, e.g. fragments devoid of the Fc portion
(e.g. Fab, Fab', F(ab')2, scFv), the so called "half molecule"
fragments obtained by reductive cleavage of the disulphide bonds
connecting the heavy chain components in the intact antibody. Fv
may be defined as a fragment containing the variable region of the
light chain and the variable region of the heavy chain expressed as
two chains.
[0052] (d) antibodies produced or modified by recombinant DNA or
other synthetic techniques, including monoclonal antibodies,
fragments of antibodies, "humanised antibodies", chimeric
antibodies, or synthetically made or altered antibody-like
structures. Also included are functional derivatives or
"equivalents" of antibodies e.g. single chain antibodies,
CDR-grafted antibodies etc. A single chain antibody (SCA) may be
defined as a genetically engineered molecule containing the
variable region of the light chain, the variable region of the
heavy chain, linked by a suitable polypeptide linker as a fused
single chain molecule.
[0053] Methods of preparation of antibody fragments and synthetic
and derivatized antibodies are well known in the art and widely
described in the literature and will not be described herein.
[0054] In a preferred embodiment of the invention CD3 and CD28
antibodies are used and more preferably these antibodies are
monoclonal antibodies. Preferred CD28 antibodies are L293 (Becton
Dickinson), Mab 9.3 (an IgG2A antibody, Dr. Ledbetter, Bristol
Myers Squibb Corporation, Seattle, Wash.), Mab Kolt-2 (IgG1), 15E8
(IgG1), 248.23.2 (IgM), YTH913.12 (Monosan), CD28.2 (Coulter),
EX5.3D10 (IgG2A), and B-T3 clone (Diaclone, France). Preferred CD3
antibodies are Spv.T3b, OKT-3 (Ortho Pharmaceutical), MEM-57 and
WT32 (Monosan), SK7 (Becton Dickinson), UCHTI (Coulter), HIT3a
(Pharmigan), and clone BC3 (Fred Hutchinson Cancer Research Centre,
Seattle). Preferred TCR antibodies are the clone BL-A
(Monosan).
[0055] Solid phases for use in the present invention may be any
solid surfaces to which activators, e.g. antibodies or antibody
fragments can be immobilized either directly (i.e. the antibodies
themselves are attached to the solid phase) or indirectly (i.e. the
antibodies are attached via an intermediate entity, for example a
secondary antibody, which is itself attached to the solid phase).
For example, such solid phases may comprise glass, silica, latex,
polymeric materials, plastic, tissue culture plastic, dextran,
cellulose and PEG, iron and other metals. Such solid supports may
take the form of any of the well known supports or matrices which
are currently used for immobilization or separation, for example
particles, beads, bottles, tubes, strips plates or wells, sheets,
fibres, capillaries, needles, combs, pipette tips, microarrays,
chips, filters, membranes, and so on.
[0056] Preferably the solid phase is a particulate material.
Conveniently, a particulate solid support used according to the
invention will comprise spherical beads. The size of the beads or
indeed any other particulate form of support is not critical, but
they may for example be of the order of diameter of at least 1 and
preferably at least 2 .mu.m, and have a maximum diameter of
preferably not more than 10 .mu.m and more preferably not more than
6 .mu.m. For example, preferred beads for use in the present
methods have a diameter of 4.5 .mu.m though smaller particles such
as those with a diameter of 1 .mu.m or 2.8 .mu.m may be used. Small
particles, for example beads of diameter less than 1 .mu.m, may be
cross-linked to provide larger complexes.
[0057] Monodisperse particles, that is those which are
substantially uniform in size (e.g. size having a diameter standard
deviation of less than 5%) have the advantage that they provide
very uniform reproducibility of reaction. Monodisperse polymer
particles produced by the technique described in U.S. Pat. No.
4,336,173 are especially suitable.
[0058] Non-magnetic polymer beads suitable for use in the method of
the invention are available from Dynal Biotech ASA (Oslo, Norway)
as well as from Qiagen, Amersham Pharmacia Biotech, Serotec,
Seradyne, Merck, Nippon Paint, Chemagen, Promega, Prolabo,
Polysciences, Agowa and Bangs Laboratories.
[0059] However, to aid manipulation and separation, magnetic or
magnetizable beads are preferred. The term "magnetic" as used
herein means that the support is capable of having a magnetic
moment imparted to it when placed in a magnetic field, and thus is
displaceable under the action of that field. In other words, a
support comprising magnetic particles may readily be removed by
magnetic aggregation, which provides a quick, simple and efficient
way of separating the particles, and is a far less rigorous method
than traditional techniques such as centrifugation which generate
shear forces which may disrupt cells.
[0060] Thus, the magnetic particles may be removed onto a suitable
surface by application of a magnetic field e.g. using a permanent
magnet. It is usually sufficient to apply a magnet to the side of
the vessel containing the sample mixture to aggregate the particles
to the wall of the vessel.
[0061] Especially preferred particles are superparamagnetic
particles as magnetic aggregation and clumping of the particles
during reaction can be avoided. Such particles are described, for
example, by Sintef in EP-A-106873. The well-known magnetic
particles sold by Dynal Biotech ASA (Oslo, Norway) under the trade
mark DYNABEADS.RTM., are particularly suited for use in the present
invention. Particularly preferred beads for use in the present
invention are Dynabeads M-450.
[0062] Functionalized coated particles for use in the present
invention may be prepared by modification of the beads according to
U.S. Pat. Nos. 4,336,173, 4,459,378 and 4,654,267 or by other
procedures known in the art. Thus, beads, or other supports, may be
prepared having different types of functionalized surface, for
example positively or negatively charged, hydrophilic or
hydrophobic. Hydrophobic surfaces (for example hydrophobic resins)
are particularly preferred for use in the attachment of antibodies,
as, by way of a hydrophobic interaction, the antibodies will be
adsorbed to the surface of the beads. This adsorption is optionally
followed by chemical linkage, for example covalent linkage of the
antibodies to the surface of the beads. Methods for forming such
chemical or other linkages are well known and documented in the
art.
[0063] Based on the varying physical and/or chemical properties of
the activators, e.g. antibodies used, such as hydrophobicity and
isoelectric point, binding profiles will vary slightly for
different activators. This is dealt with by adapting the reaction
conditions in order to obtain the required levels of activators,
e.g. by modifying the ratio of activators immobilized on the solid
surface, when a solid support is used. An indication of the ratios
of activators which become bound to a solid phase under certain
reaction conditions can be assessed by a person skilled in the art
for example by immobilizing anti-CD3 and anti-CD28 antibodies of
different sub-classes onto the solid phase under certain conditions
and then using individual reagents which react specifically with
antibodies of the particular sub-classes in question to determine
in turn the amount and hence the ratio of the different antibodies
bound.
[0064] Magnetic or magnetizable beads are particularly preferred
because of the ease of manipulation. In addition, as described
above, preferably the beads have a hydrophobic surface. Thus, to
prepare beads for use in the methods of the present invention the
magnetic and hydrophobic beads are incubated with an appropriate
mixture of the activators, e.g. CD3/(or TCR) and CD28 antibodies
under appropriate reaction conditions to facilitate adsorption and
optionally chemical linkage of activators to the surface of the
beads.
[0065] The activators, in appropriate ratios, are generally mixed
together before they are put into contact with the beads.
"Appropriate reaction conditions" as discussed above will vary
according to the particular activators used but exemplary
conditions may comprise for example an incubation of the beads and
activators, e.g. antibodies in a phosphate buffer (for example 0.5
M phosphate) of pH 7.4 and a particle concentration of
4.times.10.sup.8 beads/ml. Human serum albumin or other serum
albumin such as bovine serum albumin may optionally be present
(e.g. at a concentration of about 0.05% w/v) to stabilize the
activators and block any remaining hydrophobic patches on the
surface of the beads.
[0066] Once the appropriate reaction mixture has been set up the
reaction is allowed to progress for an appropriate time and under
appropriate conditions to facilitate the activator absorption to
the surface in the required ratio. Again the particular conditions
can be varied depending on the components of the reaction mixture
but exemplary conditions include incubation for 16-24 hours at
37.degree. C. with slow tilt and rotation.
[0067] Whatever the conditions chosen, once the immobilization
reaction is complete the beads with activators immobilized to their
surfaces in the appropriate ratio can be removed from the remaining
aqueous medium by placing a magnet at the side of the reaction
vessel and discarding the supernatant. The beads will generally be
washed to remove any excess non bound antibodies and are then ready
for use. Such beads, once prepared, can be used immediately or can
be stored for future use.
[0068] A yet further aspect of the invention provides the use of
the methods of the invention in the expansion/activation of T cell
populations for use in in vitro experiments and research. A yet
further aspect of the invention provides the use of the methods of
the invention in the expansion/activation of T cell populations for
the generation of T cell populations for immune suppression
therapy. In such a therapy a T cell sample is taken from a patient
and manipulated ex vivo as described hereinbefore to isolate and
expand an appropriate T cell population before reinfusing the T
cell population to a patient to suppress the patient's immune
response.
[0069] Thus, a yet further aspect of the invention provides a
method of treatment of a mammal with a condition or disease
typified by an aberrant immune response or in which immune
suppression would be advantageous, comprising administering to the
mammal T cells prepared in accordance with the method of the
invention. Thus in a particular embodiment, the method
comprises:
[0070] a) obtaining CD4+CD25- T cells from a first mammal;
[0071] b) expanding/activating the T cells ex vivo in accordance
with the methods of the invention as described above; and
[0072] c) administering the expanded/activated T cells to the
mammal to be treated.
[0073] This method thus provides a method of achieving an
immunosuppressive effect in a mammal, i e a method of preventing an
immune response. An effective amount of the T cells is administered
to achieve the desired therapeutic effect, e.g. by intravenous
injection in a pharmaceutically acceptable diluent.
[0074] The mammal to be treated may be the same as the first mammal
from which the T cells are sourced or may be a different mammal,
i.e. a recipient of T cells from a donor. When a donor is used, the
donor is preferably syngeneic, but may also be allogeneic or even
xenogeneic provided the cells are subject compatible.
[0075] The condition or disease typified by an aberrant immune
response may be an autoimmune disease, for example diabetes,
multiple sclerosis, myasthenia gravia, neuritis, lupus, rheumatoid
arthritis, psoriasis or inflammatory bowel disease. Conditions in
which immune suppression would be advantageous include conditions
in which a normal or an activated immune response is
disadvantageous to the mammal, e g allo-transplantation of e.g.
body fluids or parts, to avoid rejection, or in fertility
treatments in which inappropriate immune responses have been
implicated in failure to conceive and miscarriage. The use of such
cells before, during, or after transplantation avoids extensive
chronic graft versus host disease which may occur in patients being
treated (e.g. cancer patients). The cells may be expanded
immediately after harvest or stored (e.g. by freezing) prior to
expansion or after expansion and prior to their therapeutic use.
The therapies may be conducted in conjunction with known
immunosuppressive therapies.
[0076] As used herein, "treating" refers to the reduction,
alleviation or elimination, preferably to normal levels, of one or
more of the symptoms of the disease or condition which is being
treated, e.g. alleviation of immune dysfunction or avoidance of
transplant rejection, relative to the symptoms prior to
treatment.
[0077] Preferably the mammal is a domestic or livestock animal
(e.g. cats, dogs, rabbits or horses, pigs, cows, goats, sheep) or a
primate, e.g. a human.
[0078] Alternatively viewed, this aspect of the invention also
provides the use of a T cell population obtained by the stimulation
method of the present invention in the preparation of a therapeutic
composition for achieving an immunosuppressive effect in a mammal,
e.g. for treating a mammal with a condition or disease typified by
an aberrant immune response or in which immune suppression would be
advantageous.
[0079] Alternatively, aspects of the invention provide the use of a
TCR/CD3 activator, a TCR co-stimulatory activator, and/or rapamycin
in the preparation of a medicament comprising a T cell population
obtained by the stimulation method of the present invention for
achieving an immunosuppressive effect in a mammal, e g for treating
a mammal with a condition or disease typified by an aberrant immune
response or in which immune suppression would be advantageous.
[0080] Methods of obtaining a T cell sample from a patient (or an
animal source if appropriate) and isolating CD4+ cells for
expansion/activation are well known and documented in the art. For
example, peripheral blood mononuclear cells can be obtained from
buffy coats of normal blood donors by density gradient
centrifugation and leukopheresis. Adherent mononuclear cells can
then be separated from non-adherent mononuclear cells by adherence
to a solid surface such as for example beads or tissue culture
plastic under appropriate conditions (e.g. 30 minutes to 2 hours at
37.degree. C.). Enriched T cells can be used directly for
subsequent procedures or T cells can then be separated from other
non adherent mononuclear cells by a technique known as "rosetting".
Negative selection of cells can also be used. Isolation of
sub-populations of T cells can then be carried out with the aid of
appropriate antibodies to cell surface antigens displayed on the
surface of particular populations of T cells. Such techniques are
sometimes referred to as positive or negative selection.
[0081] Once an appropriate T cell population or sub population has
been isolated from a patient or animal, genetic or any other
appropriate modification or manipulation may optionally be carried
out before the resulting T cell population is expanded using the
methods and supports of the invention. The manipulation may for
example take the form of re-stimulation of the T cells with
anti-CD3 and anti-CD28 antibodies to re-activate them.
[0082] Genetic or other manipulation of the T cells is an optional
step and is not necessary for some therapies.
[0083] A yet further aspect of the invention provides a kit for
expanding/activating CD4+CD25- T cells comprising a T cell receptor
(TCR)/CD3 activator, a TCR co-stimulator activator and rapamycin.
Preferably the activators are CD28 antibodies and CD3/TCR
antibodies, which are preferably immobilized on a solid
support.
[0084] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor(s) to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1: General Expansion Protocol for Generation of
CD4+CD25+FOXP3- Regulatory T Cells
[0085] 1) Isolate CD4+ T cells from peripheral blood or
leukopheresis products by the use of Dynal.RTM. CD4 Positive
isolation kit, Dynal.RTM. CD4 Negative Isolation kit,
Dynabeads.RTM. MyPure.TM. CD4 T cell kit, or CD4 Microbeads
MACS.RTM..
[0086] 2) Stimulate 1.5.times.10.sup.6 CD4+ T cells with
anti-CD3/CD28 Dynabeads (3:1 bead: T cell ratio) in X-vivo medium
with 1000 U/ml IL-4 (optional), 20 U/ml IL-2 and 1 .mu.M final
concentration of Rapamycin. Total medium volume should be 1.5 ml
per 1.5.times.10.sup.6 CD4+ T cells in a 24 well plate. The
protocol can be scaled up.
[0087] 3) On day 2, add a 10.times. concentrate that contains
10,000 U/ml IL-4 (optional) and 10,000 U/ml IL-2. Add this in a
volume that is 10% of the initial culture volume.
[0088] 4) On day 4, add the cytokine concentrate similar to day
2.
[0089] 5) On day 6, add the 10.times. cytokine concentrate again
and take an aliquot to count the cells. This is the first time that
the culture should be disturbed. Based on the count adjust the cell
concentration to 1.times.10.sup.6 CD4 cell/ml. Split wells and add
culture medium containing Rapamycin, 1000 U/ml IL-4 (optional), 20
U/ml IL-2 to maintain cell concentration at 0.5.times.10.sup.6
cells/ml.
[0090] 6) From day 7 to day 12 count the cells daily. Split wells
and add culture medium containing Rapamycin, 1000 U/ml IL-4
(optional), 20 U/ml IL-2 to maintain the cell concentration at
0.5.times.10.sup.6 cells/ml. On day 12 the beads are removed using
a magnet.
[0091] 7) On day 14-15 the cells can be analyzed for surface marker
expression and functional properties.
Example 2: Analysis of Properties of Expanded T Cells
[0092] CD4+ T cells were isolated from peripheral blood by negative
and/or positive isolation as described in Example 1. After
expansion with IL-2, +/-IL-4, CD3/CD28 Dynabeads.RTM., with or
without Rapamycin the expanded cells were analyzed for various
surface markers (CD4, CD25, CD62L, CRTh2, CCR4, CXCR3, CCR7) and
for intracellular FOXP3 expression and cytokine secretion.
Suppressive capacity was analyzed in standard functional assays as
described below.
[0093] The above mentioned surface markers are as follows:
[0094] CD62L: Lymph node homing adhesion molecule, expressed on
naive T cells and naturally occurring CD4+CD25+ Treg cells.
[0095] CRTh2: A marker expressed on all Th2 cells.
[0096] CCR4: Chemokine receptor expressed on Treg cells and Th2
cells.
[0097] CXCR3: Chemokine receptor expressed on inflammatory Th1
cells.
[0098] CCR7:Lymph node homing chemokine receptor expressed on naive
T cells, central memory T cells and naturally occurring CD4+CD25+
Treg cells.)
[0099] CD4+ T cells expanded without Rapamycin show low expression
of CD25, CD62L and CCR7, and higher expression of CXCR3. The
cytokine profile shows high levels of both Th1 and Th2 cytokines.
In contrast, the CD4+ T cells expanded with Rapamycin have a
phenotype of Treg cells with high CD25, low CXCR3 and low secretion
of cytokines. Approximately 70% of the expanded cells express high
levels of CD25. None of the expanded T cells with or without
Rapamycin have an increased expression of FOXP3. 10% of freshly
isolated CD4+ T cells expressed FOXP3 and after expansion still
only 10% of the cells expressed FOXP3.
[0100] Standard proliferation assays were performed using 75,000
CD25- T cells supplemented with CD25+ T cells to provide the ratios
indicated in FIG. 1 and thymidine incorporation was analyzed after
a period of 4 days. T cells expanded with Rapamycin (CD25+) were
found to be capable of suppressing proliferation of non Rapamycin
expanded T cells (CD25-). In the presence of CD25+ T cells, the
CD25- T cells capacity to proliferate was inhibited by 90%. (FIG.
1).
[0101] FIG. 2 illustrates the expression of CD4 and CD25 in CD4+ T
cells expanded with or without rapamycin. Rapamycin expanded cells
express 80% CD4+CD25+ T cells wherein in the absence of rapamycin
only 20% of the T cells were CD4+CD25+.
[0102] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the scope and concept of the invention.
* * * * *