U.S. patent application number 10/661804 was filed with the patent office on 2005-05-12 for cd4+cd25+ regulatory t cells from human blood.
Invention is credited to Dieckmann, Detlef, Schuler, Gerold.
Application Number | 20050101012 10/661804 |
Document ID | / |
Family ID | 34553512 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050101012 |
Kind Code |
A1 |
Schuler, Gerold ; et
al. |
May 12, 2005 |
CD4+CD25+ regulatory T cells from human blood
Abstract
The present invention provides suppressive and/or regulative
human CD4.sup.+CD25.sup.+ T cells, a method for expanding same, and
the use of the suppressive and/or regulative human
CD4.sup.+CD25.sup.+ T cells and the expanded T cells as regulatory
agent.
Inventors: |
Schuler, Gerold; (Spardorf,
DE) ; Dieckmann, Detlef; (Uttenreuth, DE) |
Correspondence
Address: |
JENKINS & WILSON, PA
3100 TOWER BLVD
SUITE 1400
DURHAM
NC
27707
US
|
Family ID: |
34553512 |
Appl. No.: |
10/661804 |
Filed: |
September 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10661804 |
Sep 12, 2003 |
|
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PCT/EP02/02671 |
Mar 12, 2002 |
|
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Current U.S.
Class: |
435/372 ;
424/144.1 |
Current CPC
Class: |
C12N 5/0636 20130101;
C12N 2501/51 20130101; C12N 2501/23 20130101; C12N 2501/515
20130101; G01N 33/505 20130101; A61P 37/06 20180101; C12N 2500/14
20130101; C12N 2501/01 20130101; A61P 31/00 20180101; A61K 35/17
20130101; G01N 33/5002 20130101 |
Class at
Publication: |
435/372 ;
424/144.1 |
International
Class: |
A61K 039/395; C12N
005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2001 |
DE |
01106033.2 |
Claims
1. Suppressive and/or regulative human CD4.sup.+CD25.sup.+ T
cells.
2. The T cells according to claim 1 which are isolatable from human
peripheral blood, preferably by suitable monoclonal antibodies and
using magnetic separation or immunoadsorption methods.
3. The cells according to claim 1 which are CTLA-4.sup.+ and
possess regulatory properties.
4. A method for expanding CD4.sup.+CD25.sup.+ T cells as defined in
claim 1, which method comprises stimulating the cells with a T cell
stimulating agent or with antigen-presenting cells ex vivo and in
vivo.
5. The method of claim 4, wherein the T cell stimulating agent is a
composition comprising: (a) anti-CD3 and/or anti-CD28
ligands/monoclonal antibodies, including superagonistic antibodies;
(b) a ligand/antibody to T cell receptors on the surface of
CD4.sup.+CD25.sup.+ T cells or to T cell receptor components; (c)
MHC-peptide complexes binding to the T cell receptors expressed on
the surface of regulatory T cells; or (d) a phorbolester and a
calcium ionophor.
6. The method of claim 4 wherein the antigen-presenting cells are
selected from autologous, non-autologous, artificial
antigen-presenting cells, etc., preferably the antigen presenting
cells are dendritic cells.
7. The method according to claim 4, wherein the T cell stimulating
agent and antigen-presenting cells are used together with IL2
and/or IL-5, IL-7 and/or IL-9, IFN-.alpha. and/or IL-10.
8. Expanded human CD4.sup.+CD25.sup.+ T cells obtainable by the
method according to claim 4.
9. The expanded human CD4.sup.+CD25.sup.+ T cells of claim 8 which
are fixated CD4.sup.+CD25.sup.+cells, preferably obtainable by ex
vivo treatment with paraformaldehyde.
10. A pharmaceutical composition comprising the human
CD4.sup.+CD25.sup.+ T cells according to claim 1.
11. Use of CD4.sup.+CD25.sup.+ T cells according to claim 1 for
preparing a regulatory medicament.
12. A method to identify, monitor and/or remove CD4.sup.+CD25.sup.+
T cells from human blood and other tissues ex vivo or in vivo,
which method comprises: (a) utilizing agents/ligands specifically
binding to the CD4, and/or CD25, and/or CTL-A4 (CD154) entities on
the T cells, preferably anti-CD4 and/or anti-CD25, and/or
anti-CTL-A4 antibodies; (b) utilizing immunoadsorption methods;
and/or (c) utilizing a stimulating agent or antigen presenting
cells as defined in claim 4.
13. Use of a T cell stimulating agent or antigen presenting cells
as defined in claim 5 for preparing an agent to induce regulatory
CD4.sup.+CD25.sup.+ T cells in vivo, preferably for preparing an
agent for treating autoimmune diseases in a patient.
14. Use of CD4.sup.+CD25.sup.+ T cells according to claim 1: (a) in
assays that will allow to identify other growth and/or functionally
modifying (inhibitory/enhancing)/apoptotic or anti-apoptotic
factors; (b) for identifying molecules expressed by the
CD4.sup.+CD25.sup.+ T cells including identification of novel
molecules on said cells, or if presenting molecules which are
deemed pharmaceutically active; or (c) for identifying precursor
cells or progeny of the regulatory CD4.sup.+CD25.sup.+ T cells.
15. Use of the enriched CD4.sup.+CD25.sup.+ T cells of claim 1 for
preparing an agent for adoptive transfer therapy, an agent for
treating diseases with enhanced immunity including but not limited
to autoimmune diseases, or an agent for preventing/treating
transplantation reactions such as graft versus host disease, graft
rejections, etc.
16. A method for adoptive transfer therapy which comprises
injecting/infusing back into the patients enriched/expanded
autologous or non-autologous regulatory CD4.sup.+CD25.sup.+ T cells
according to claim 1 to prevent or treat any immune reactions that
are too strong and/or pathogenic, or to prevent/treat
transplantation reactions such as graft versus host disease and
graft rejections.
17. A method for preparing CD4.sup.+CD25.sup.+ T cells with a
particular desired antigen-specific T cell receptor which
comprises: (a) activating/stimulating/expanding the
CD4.sup.+CD25.sup.+ T cells according to claim 1 with antigen
presenting cells, preferably immature or mature dendritic cells
(DC), presenting said antigen in vitro or in vivo; or (b) utilizing
a ligand/antibody to a particular T cell receptor expressed on
(subsets of) CD4.sup.+CD25.sup.+ regulatory T cells, or a
MHC-peptide Complex binding to a particular T cell receptor on
(subsets of) CD4.sup.+CD25.sup.+ T cells.
18. The method of claim 17, wherein the antigen-presenting cells
are pulsed/loaded/fed with tissue or any defined or undefined
antigens, wherein (a) the defined antigens preferably are
autoantigens (including, but not limited to, desmoglein 3 in the
case of pemphigus vulgaris, melanA or tyrosinase in case of
vitiligo; thyreoglobulin in case of thyreoiditis), foreign antigens
(including pathogen-derived antigens such as Hepatitis C), or
alloantigens/transplantation antigens; and (b) the undefined
antigens preferably are tissue or cell-derived antigens (including,
but not limited to, antigens which are in the form of necrotic or
apoptotic cells or tissue derived RNA or hybrids between cells of
interest and dendritic cells/antigen presenting cells, other forms
of delivery of undefined antigens into dendritic cells or other
antigen presenting cells) or pathogen-derived antigens.
19. CD4.sup.+CD25.sup.+ T cells having a particular desired
antigen-specific T cell receptor and being obtainable by the method
of claim 17, or by transfection of a T cell receptor of desired
antigen specificity into ex vivo isolated or expanded T cells.
20. Pharmaceutical composition comprising the T cells of claim 19,
preferably said pharmaceutical composition being suitable to treat
diseases with enhanced immunity including, but not limited to,
autoimmune diseases, graft versus host disease and graft
rejections.
21. Use of agents specifically binding to the CD4 and/or CD25
and/or CTL-A4 (CD154) entities on the T cells, including but not
limited to ligands/antibodies, such as anti-CD25 and/or anti-CTL-A4
mAb, or antibodies or MHC-peptide Complexes or other ligands
binding to T cell receptors on (subsets of) CD4.sup.+CD25.sup.+ T
cells for preparing a medicament for removal or functional
impairment of CD4.sup.+CD25.sup.+ T cells in vivo in order to
enhance immune responses, including dampen regulation by
CD4.sup.+CD25.sup.+ T cells in vivo, for example, to enhance tumor
immunity.
22. Use of the expanded T cells of claim 8 for preparing an agent
for adoptive transfer therapy, an agent for treating diseases with
enhanced immunity including but not limited to autoimmune diseases,
or an agent for preventing/treating transplantation reactions such
as graft versus host disease, graft rejections, etc.
23. Use of the fixated T cells of claim 9 for preparing an agent
for adoptive transfer therapy, an agent for treating diseases with
enhanced immunity including but not limited to autoimmune diseases,
or an agent for preventing/treating transplantation reactions such
as graft versus host disease, graft rejections, etc.
Description
[0001] The present invention provides suppressive and/or regulative
human CD4.sup.+CD25.sup.+ T cells, a method for expanding same, and
the use of the suppressive and/or regulative human
CD4.sup.+CD25.sup.+ T cells and of the corresponding expanded T
cells as regulatory agent.
BACKGROUND OF THE INVENTION
[0002] Immunological self-tolerance is critical for the prevention
of autoimmunity and maintenance of immune homeostasis. The ability
of the immune system to discriminate between self and non-self is
controlled by mechanisms of central and peripheral tolerance.
Central tolerance involves deletion of self-reactive T lymphocytes
in the thymus at an early stage of development (Rocha, B. and von
Boehmer, H., Science 251:1225-1228 (1991); Kisielow, P. et al.,
Nature 333:742-746 (1988)). Several mechanisms of peripheral
tolerance have been described, including T cell anergy and
ignorance (Rocha, B. and von Boehmer, H., Science 251:1225-1228
(1991); Kisielow, P. et al., Nature, 333:742-746 (1988); Schwartz,
R. H., Science 248:1349-1356 (1990); Miller, J. F. A. P. and Heath,
W. R., Immunol. Rev. 133:131-150 (1993)). Studies ongoing for more
than a decade in rodents have provided firm evidence for the
existence of a unique CD4.sup.+CD25.sup.+ population of
"professional" regulatory/suppressor T cells that actively and
dominantly prevent both the activation as well as the effector
function of autoreactive T cells that have escaped other mechanisms
of tolerance (Sakaguchi, S. et al., J. Immunol. 155:1151-1164
(1995); Takahashi, T. et al., Int. Immunol. 10:1969-1980 (1998);
Itoh, M. et al., J. Immunol. 162:5317-5326 (1999)). The elimination
or inactivation of these cells resulted in severe autoimmune
disease, and was also found to enhance immune responses to
alloantigens and even tumors (Sakaguchi, S. et al., J. Immunol.
155:1151-1164 (1995); Itoh, M. et al., J. Immunol. 162:5317-5326
(1999); Shimizu, J. et al., J. Immunol. 163:5211-5218 (1999)).
Recent studies revealed that the CD4.sup.+CD25.sup.+ regulatory T
cells constitute a rather homogenous population (Thornton, A. M.,
Shevach, E. M., J. Immunol. 164:183-190 (2000)), are derived from
the thymus (Itoh, M. et al., J. Immunol. 162:5317-5326 (1999)), are
naturally non-proliferative (i.e. anergic) to stimulation via the
TCR, but require activation via their TCR to become suppressive and
to inhibit the proliferation of CD4.sup.+ or CD8.sup.+ T cells.
Once activated, however, their regulatory/suppressor function was
completely antigen-nonspecific, cytokine-independent yet cell
contact dependent (Thornton, A. M., Shevach, E. M., J. Immunol.
164:183-190 (2000)). The exact mechanisms of suppression, notably
the cell surface and/or short-range soluble molecules involved in
the T-T interaction, have yet to be characterized. New in vitro
data suggest that the CD4.sup.+CD25.sup.+ T cells inhibit the
proliferation of responders by inhibiting their IL-2 production
(Thornton, A. M., Shevach, E. M., J. Exp. Med. 188:287-296 (1998)).
Recent in vivo studies suggest that the function of
CD4.sup.+CD25.sup.+ T cells is crucially dependent on signaling via
the CTLA-4/CD152 molecule which was found to be constitutively
expressed on CD4.sup.+CD25.sup.+ T cells (Read, S. et al., J. Exp.
Med. 192:295-302 (2000); Salomon, B. et al., Immunity 12:431-440
(2000); Takahashi, T. T. et al., J. Exp. Med. 192:303-310
(2000)).
[0003] Although it has been evident for years that in rodents the
CD4.sup.+CD25.sup.+ T cell population constitutes a unique lineage
of "professional" regulatory/suppressor T cells which are crucial
for the prevention of spontaneous autoimmune disease (Sakaguchi, S.
et al., J. Immunol. 155:1151-1164 (1995)), it is unknown to date,
whether CD4.sup.+4T cells exhibiting similar functional properties
are naturally occurring in man. The removal and/or functional
impairment of these cells in vivo in mice e.g. by anti-CD25 and/or
anti-CTLA-A4 mAb treatment of animals induces various spontaneous
autoimmune diseases AND rejection of tumors. The mechanism is that
removal/impairment of these cells removes their negative control of
autoreactive T cells so that these T cells become active. If one
gives back the CD4.sup.+CD25.sup.+ T cells by adoptive transfer
into these animals regluation is restored and autoimmunity/tumor
rejection is stopped.
[0004] As set forth above, it is totally unknown to date whether
CD4.sup.+ T cells exhibiting similar functional properties are
naturally present in man. The preparation of human T cells with
regulatory properties, which are, however, no CD4.sup.+CD25.sup.+ T
cells is known in the art. E. g. Jonuleit, H. et al., J. Exp. Med
192:1213-1222 (2000) describe the induction of regulatory T cells
from human nave T cells by repetitive stimulation with immature
dendritic cells. Most of this work was done with T cells from cord
blood which is the richest source of truly nave T cells. It is to
be noted that CD4 CD25.sup.+ T cells are constitutively detectable
in the human blood from early time points on. The subject of
Jonuleit et al. is not a naturally occurring population. De Jong,
P. et al., Int. Immunol. 6:631-638 (1994) describe the effect of
TGF-.beta.1 on nave and memory CD4.sup.+ T cells. A differential
effect is shown, with stimulatory effect on primarily activated
CD45 RA.sup.+ CD4.sup.+ T cells. Proliferation of CD45 RO.sup.+
CD4.sup.+ T cells or secondary stimulated CD45 RO.sup.+ cells is
suppressed. In the case of CD45 RA.sup.+T cells TGF-.beta. leads to
an increased mean of fluorescens of CD25. The effects described
here solely relate to proliferation of T cells. A regulatory
capacity of nave TGF-.beta. treated T cells is not shown.
[0005] Surprisingly it was now found that CD4.sup.+CD25.sup.+,
primarly CD45RO.sup.+ T cells (mean 6% of CD4.sup.+ T cells),
hereinafter shortly referred to as "CD4.sup.+CD25.sup.+ T cells",
are present in human blood, in particular the peripheral blood of
adult healthy volunteers. In the past T cells exhibiting the
phenotype (i.e., suppressive/regulatory CD4.sup.+CD25.sup.+ T
cells) have been known for years but they were misinterpreted to be
conventional memory cells (Kanegane, H. et al., Int. Immunol.,
3:1349-1356 (1991); Taka, K. et al., Immunol. 72:15-19 (1990);
Jackson, A. L. et al., Clin. Immunol. Innunopathol. 54:126-133
(1990)). In contrast to previous reports the human
CD4.sup.+CD25.sup.+ T cells are not conventional memory cells but
rather regulatory cells exhibiting functional properties identical
to their rodent counterparts. CTLA-4 (CD152), for example, which is
essential for the in vivo suppressive activity of
CD4.sup.+CD25.sup.+ T cells, was constitutively expressed, and
remained strongly upregulated after stimulation. The cells were
non-proliferative to stimulation via their TCR, but the anergic
state was partially reversed by IL-2 and IL-15. Upon stimulation
with allogeneic (but not syngeneic) mature dendritic cells or
plate-bound anti CD3+ anti-CD28 the CD4.sup.+CD25.sup.+ T cells
released IL-10, and in coculture experiments suppressed the
activation and proliferation of CD4.sup.+ and CD8.sup.+ T cells.
Suppression proved IL-10 independent, yet contact dependent as in
the mouse. The identification of regulatory CD4.sup.+CD25.sup.+ T
cells has important implications for the study of tolerance in man,
notably in the context of autoimmunity, transplantation, and
cancer.
SUMMARY OF THE INVENTION
[0006] The present invention provides
[0007] (1) suppressive and/or regulative human CD4.sup.+CD25.sup.+
T cells;
[0008] (2) a method for expanding CD4.sup.+CD25.sup.+ T cells as
defined in (1) above, which method comprises stimulating the cells
with a T cell stimulating agent or with antigen-presenting cells ex
vivo and in vivo;
[0009] (3) expanded human CD4.sup.+ CD25.sup.+ T cells obtainable
by the method as defined in (2) above, preferably by the ex vivo
method defined in (2) above;
[0010] (4) a pharmaceutical composition comprising the human
CD4.sup.+ CD25.sup.+ T cells as defined in (1) or (3) above;
[0011] (5) use of CD4.sup.+ CD25.sup.+ T cells as defined in (1) or
(3) above for preparing a regulatory medicament;
[0012] (6) a method to identify, monitor and/or remove
CD4.sup.+CD25.sup.+ T cells from human blood and other tissues ex
vivo or in vivo, which method comprises
[0013] (i) utilizing agents/ligands specifically binding to the
CD4, and/or CD25, and/or CTL-A4 (CD154) entities on the T cells,
preferably anti-CD4 and/or anti-CD25, and/or anti-CTL-A4
antibodies, and/or
[0014] (ii) utilizing immunoadsorption methods; and/or
[0015] (iii) utilizing a stimulating agent or antigen presenting
cells as defined in (2) above;
[0016] (7) use of a T cell stimulating agent or antigen presenting
cells as defined in (2) above for preparing an agent to induce
regulatory CD4.sup.+CD25.sup.+ T cells in vivo, preferably for
preparing an agent for treating autoimmune diseases in a
patient;
[0017] (8) use of CD4.sup.+CD25.sup.+ T cells as defined in (1) or
(3) above
[0018] (a) in assays that will allow to identify other growth
and/or functionally modifying (inhibitory/enhancing)/apoptotic or
anti-apoptotic factors
[0019] (b) for identifying molecules expressed by the
CD4.sup.+CD25.sup.+ T cells including identification of novel
molecules on said cells, or if presenting molecules which are
deemed pharmaceutically active, or
[0020] (c) for identifying precursor cells or progeny of the
regulatory CD4.sup.+CD25.sup.+ T cells;
[0021] (9) use of the enriched CD4.sup.+CD25.sup.+ T cells of (1)
above or the expanded T cells of (3) above for preparing an agent
for adoptive transfer therapy, an agent for treating diseases with
enhanced immunity including but not limited to autoimmune diseases,
or an agent for preventing/treating transplantation reactions such
as graft versus host disease and graft rejections;
[0022] (10) method for adoptive transfer therapy which comprises
injecting/infusing back into the patients enriched/expanded
autologous or non-autologous regulatory CD4.sup.+CD25.sup.+ T cells
as defined in (1) or (3) above to prevent or treat any immune
reactions that are too strong and/or pathogenic, or to
prevent/treat transplantation reactions such as graft versus host
disease and graft rejections;
[0023] (11) a method for preparing CD4.sup.+CD25.sup.+ T cells with
a particular desired antigen-specific T cell receptor which
comprises (i) activating/stimulating/expanding the
CD4.sup.+CD25.sup.+ T cells according to (1) above with antigen
presenting cells, preferably immature or mature dendritic cells
(DC), presenting said antigen in vitro or in vivo; or
[0024] (ii) utilizing a ligand/antibody to a particular T cell
receptor expressed on (subsets of) CD4.sup.+CD25.sup.+ regulatory T
cells, or a MHC-peptide complex binding to a particular T cell
receptor on (subsets of) CD4.sup.+CD25.sup.+ T cells;
[0025] (12) CD4.sup.+CD25.sup.+ T cells obtainable by the method as
defined in (11) above, or by transfection of a T cell receptor of
desired antigen specificity into ex vivo isolated or expanded T
cells;
[0026] (13) pharmaceutical composition comprising the T cells of
(12) above, preferably said pharmaceutical composition being
suitable to treat diseases with enhanced immunity including, but
not limited to, autoimmune diseases, graft versus host disease and
graft rejections; and
[0027] (14) use of agents specifically binding to the CD4 and/or
CD25 and/or CTL-A4 (CD154) entities on the T cells, including but
not limited to ligands/antibodies, such as anti-CD25 and/or
anti-CTL-A4 mAb, or antibodies or MHC-peptide complexes or other
ligands binding to T cell receptors on (subsets of)
CD4.sup.+CD25.sup.+ T cells for preparing a medicament for removal
or functional impairment of CD4.sup.+CD25.sup.+ T cells in vivo in
order to enhance immune responses, including dampen regulation by
CD4.sup.+CD25.sup.+ T cells in vivo, for example, to enhance tumor
immunity.
SHORT DESCRIPTION OF THE FIGURES
[0028] FIG. 1 shows that CD4.sup.+CD25.sup.+ T cells exhibit
distinct phenotypical differences as compared to
CD4.sup.+CD25.sup.- T cells.
[0029] FIG. 2 shows that CD4.sup.+CD25.sup.+ T cells are
nonproliferative/anergic to both allogeneic and polyclonal
stimulation, which is partially reversed by the addition of IL-2
and/or IL-15, but not by neutralizing anti IL-10 antibodies.
[0030] FIG. 3 shows that CD4.sup.+CD25.sup.+ T cells, if stimulated
via the TCR, suppress the activation of CD4.sup.+ and CD8.sup.+ T
cells in a cell contact- and dose-dependent manner.
[0031] FIG. 4 shows that the cytokine profiles of
CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells are
different.
[0032] FIG. 5 shows that activated and fixated CD4.sup.+CD25.sup.+
cells still exhibit regulatory capacity.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The embodiments (1) to (14) of the invention are hereinafter
described in more detail:
[0034] (A) The phenotypical characterization of the human
CD4.sup.+CD25.sup.+ T cells now allows the identification,
monitoring (e.g. by FACS), isolation and removal of these cells
from human blood and other tissues ex vivo (hereinafter
occasionally referred to as "in vitro") or in vivo. This isolation
or removal can be achieved by contacting the human blood or other
tissues with suitable agents ex vivo. Suitable agents are in
particular anti-CD4, anti-CD25, anti-CTL-A4 (CD154) antibodies,
etc.
[0035] (B) The CD4.sup.+CD25.sup.+ T cells can be expanded ex vivo
by stimulation of the cells by treatment with suitable T cell
stimulating agents or with antigen presenting cells (hereinafter
shortly referred to as "APCs"). Suitable T cell stimulating agents
suitable for the methods of (2) and (6) above include, but are not
limited to, compositions comprising
[0036] (a) anti-CD3 and/or anti-CD28 ligands/monoclonal antibodies
(mAb) including superagonistic antibodies; (b) a ligand/antibody to
T cell receptors on the surface of CD4.sup.+CD25.sup.+ T cells or
to T cell receptor components; or
[0037] (c) MHC-peptide complexes binding to the T cell receptors
expressed on the surface of regulatory T cells; or
[0038] (d) PMA (as well as other phorbolester)+ionomycin (or other
calcium ionophores).
[0039] "Ligand" or "ligands" in accordance with the present
invention relates to all kinds of compounds capable of binding to
specific molecules (including polynucleotides and polypeptides such
as cell receptors, CD25, CTL-A4, etc.). A preferred ligand is a
monoclonal antibody or a fragment thereof. The terms "ligand" and
"antibody" are used interchangeable throughout the application
text.
[0040] Suitable APCs include autologous or non-autologous or
artificial antigen-presenting cells (e.g. dendritic cells (for the
preparation of dendritic cells see e.g. WO 93/20185, WO 97/29182
and EP-A-0 922 758), etc.). Said T cell stimulating agents and
antigen-presenting cells can be used together with IL-2 or IL-15 or
a combination thereof. Instead of IL-2/IL-15, other cytokines that
use the gamma chain common to several cytokine receptors can also
be used (including, but not restricted to, IL-7 and IL-9).
Furthermore, IFN alpha and IL-10, which are known to promote the
generation of other regulatory cells (Groux, H. et al., Nature
389:737-742 (1997); Roncarolo, M. G., J. Exp. Med. 193:F5-F9
(2001)) also promote the generation/expansion of these cells.
Finally, other methods for polyclonal or oligoclonal (e.g. by
superantigens) T cell stimulation are also applicable (anti-CD3
and/or anti-CD28 lingands/antigens including superagonistic
antibodies; PMA+ ionomycin; etc.). These stimulating agents or
antigen presenting cells may be used for the expansion in amounts
well-known for a person skilled in the art.
[0041] (C) The identification of the CD4.sup.+CD25.sup.+ T cells
also allows to monitor and expand CD4.sup.+CD25.sup.+ T cells in
vivo (e.g. by adminstering the T cells stimulating agents defined
above, such as IL-2+IL-15 or IL-10+IFN.alpha. to patients, with or
without the dendritic cells/antigen presenting cells) to induce
regulatory T cells e.g. to fight autoimmune diseases.
[0042] (D) The CD4.sup.+CD25.sup.+ T cells or expanded
CD4.sup.+CD25.sup.+ T cells of (1) or (3) above can be used in
assays that will allow to identify other growth and/or functionally
modifying (inhibitory/enhancing)/(apoptotic or anti-apoptotic)
factors that can be used in vitro or in vivo (e.g. to stimulate or
to remove or functionally disturb the CD4.sup.+CD25.sup.+ T cells).
These T cells are also suitable for identifying molecules expressed
by the CD4.sup.+CD25.sup.+ T cells by methods such as but not
restricted to mAb generation, proteomics, DNA chip analysis,
subtractive hybridization (in particular (a) identification of
"novel", i.e., hitherto unknown, molecules, or (b) if these
molecules are suitable pharmaceutical target structures, to
therewith develop novel stimulating, inhibiting or
apoptosis-inducing medicaments, for "switching on" or "switching
off" of CD4.sup.+CD25.sup.+ T cells in vitro and in vivo) and for
identifying precursor cells or progeny of the regulatory
CD4.sup.+CD25.sup.+ T cells.
[0043] (E) The CD4.sup.+CD25.sup.+ T cells or expanded
CD4.sup.+CD25.sup.+ T cells of (1) or (3) above can be used in
adoptive transfer therapy, i.e. inject/infuse back into the
patients enriched/expanded autologous or non-autologous regulatory
T cells to prevent or treat any immune reactions that are too
strong and/or pathogenic, i.e. to treat autoimmunity in its
broadest sense (including but not limited to classical autoimmune
diseases such as lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, pemphigus vulgaris or thyreoiditis as well as
diseases that have at least some autoimmune aspect in their
pathogenesis such as vitiligo, atopic dermatitis, psoriasis
vulgaris, etc.). Also to prevent/treat transplantation reactions
such as GVHD (graft versus host disease) and graft rejections.
[0044] (F) The CD4.sup.+CD25.sup.+ T cells or expanded
CD4.sup.+CD25.sup.+ T cells of (1) or (3) above (as well as the
expansion method defined in (2) above, the pharmaceutical
composition of (4) above and the use of (5) and (7) to (9) above
can be used in conjunction with immature or mature dendritic cells
(DC) (or other antigen presenting cells including artificial ones)
that are or are not pulsed/loaded/fed with tissue or any other
antigen (defined or undefined) in order to
activate/stimulate/expan- d the CD4.sup.+CD25.sup.+ T cells in
vitro or in vivo, to generate/expand CD24.sup.+CD25.sup.+ T cells
with a particular desired antigen-specific TCR (T cell receptor).
Examples of defined antigens are autoantigens (e.g. desmoglein 3 in
the case of pemphigus vulgaris, melanA or tyrosinase in case of
vitiligo; thyreoglobulin in case of thyreoiditis) or foreign
antigens (e.g. pathogen-derived antigens such as Hepatitis C, etc.)
or alloantigens/transplantation antigens. Examples of undefined
antigens are tissue or cell-derived antigens (eg in the form of
necrotic or apoptotic cells or tissue derived RNA or hybrids
between cells of interest and dendritic cells/antigen presenting
cells or other forms of delivery of undefined antigens into
dendritic cells or other antigen presenting cells) or
pathogen-derived antigens. Autologous or non-autologous
CD4.sup.+CD25.sup.+ T cells and/or dendritic cells can be used.
[0045] (G) The agents specifically binding to the CD4 and/or CD25
and/or CTL-A4 (CD154) entities on the T cells as defined in (14)
above can be used to remove and to monitor removal of
CD4.sup.+CD25.sup.+ T cells in vivo e.g. by anti-CD25 and/or
anti-CTL-A4 mAb (unmodified or modified e.g. conjugated to toxins)
or by immunoadsorption (blood is flowing through columns and
CD4.sup.+CD25.sup.+ T cells are removed by solid phase-bound
antibodies directed to molecules expressed on the surface of
CD4.sup.+CD25.sup.+ T cells e.g. anti-CD25) in order to enhance
immune responses, e.g. dampen regulation by CD4.sup.+CD25.sup.+ T
cells in vivo, for example, to enhance tumor immunity.
[0046] (H) In another preferred embodiment of the invention the
expanded (i.e. activated) CD4.sup.+CD25.sup.+ T cells of (3) above
(and also the T cells of (1) and (12) above) may be fixated. Such
fixated T cells can be obtained by ex vivo treatment of the
(expanded) T cells with a suitable fixation agent including, but
not limited to, paraformaldehyde, etc. Preferably the fixation is
performed by suspending the cells in a 0.5 to 5% (w/w) aqueous
paraformaldehyde solution, most preferably in an about 2% (w/w)
paraformaldehyde solution, for 15 min to 3 h, most preferably for 1
h, followed by appropriate washing steps.
[0047] (I) The CD4.sup.+CD25.sup.+ T cells utilized in the
medicament of (5) above in the agents of (9) and (14) above, or the
method of (10) above can be autologous or non-autologous regulatory
T cells. In case of non-antologous regulatory T cells it is
preferred that the T cells are fixated as mentioned in (H)
above.
[0048] (J) The pharmaceutical composition of (4) and (13) above,
the medicament of (5) above, the agents of (7), (9) and (14) above
and the T cells utilized in the methods of (6), (8) and (10) above
may contain further ingredients such as
pharmaceutically/diagnostically suitable carriers, solvents,
diluents and additives known to the person skilled in the art. The
concentration of these ingredients are to be adapted for respective
purpose by the person skilled in the art.
[0049] The concept of suppressor or immunoregulatory T cells has
been revitalized during the past few years by the better
delineation of several regulatory cell types in rodents, the mutual
relationship of which is not yet finally defined. The so-called Tr1
and Th3 cells mediate bystander suppression--without need for
direct cell contact--by the secretion of high levels of IL-10 and
TGF-.beta., respectively (Groux, H. et al., Nature 389:737-742
(1997); Fukaura, H. et al., J. Clin. Invest. 98:70-77 (1996)). The
best characterized and apparently most important regulatory T cell
population identified so far are the CD4.sup.+CD25.sup.+ T cells.
They occur naturally in rodents (representing about 10% of
CD4.sup.+ cells in lymphoid organs), are characterized by
constitutive expression of CD25 (IL-2R-alpha), and are clearly of
crucial importance for maintaining tolerance and preventing
autoimmune disease in vivo. Surprisingly, a cell population
exhibiting equivalent properties has not been described in humans
to date. Here we have demonstrated, that the CD4.sup.+CD25.sup.+ T
cells in human blood that previously had been considered to
represent conventional memory T cells (Kanegane, H. et al., Int.
Immunol. 3:1349-1356 (1991); Taka, K. et al., Immunol., 72:15-19
(1990); Jackson, A. L. et al., Clin. Immunol. Innunopathol.
54:126-133 (1990)) in fact appear to be the exact human counterpart
of the unique CD4.sup.+CD25.sup.+ regulatory T cells that have been
known and studied for many years in rodents. We were able to
isolate the CD4.sup.+CD25.sup.+ T cells from adult blood in
sizeable quantities (average 6% of CD4.sup.+ T cells) so that a
detailed study and comparison to CD4.sup.+CD25.sup.- T cells could
be undertaken. It turned out that the human cells share the key
phenotypical and functional features with the murine
CD4.sup.+CD25.sup.+ immunoregulatory T cells. The most interesting
and previously unidentified phenotypical feature was that the
CTLA-4 molecule (CD152) was already constitutively expressed (at
high levels intracellularly, and at low levels at the surface) by
the human CD4.sup.+CD25.sup.+ T cells, was further upregulated
after stimulation via the TCR and maintained at high surface levels
for at least a week thereafter (in sharp contrast to
CD4.sup.+CD25.sup.- T cells that expressed CTLA-4 de novo upon
stimulation, and only very transiently as described (Thompson, C.
B., Allison, J. P., Immunity, 7:445-450 (1998); Chambers, C. A. et
al., Immunol. Rev. 153:27-46 (1996)). The expression pattern of
CTLA-4 by CD4.sup.+CD25.sup.- already supported their relationship
to the murine CD4.sup.+CD25.sup.+ regulatory T cells as these cells
constitutively express CTLA-4 as a molecule essential for their in
vivo suppressive activity (Read, S. et al., J. Exp. Med.
192:295-302 (2000); Salomon, B. et al., Immunity, 12:431-440
(2000)). Like their murine counterparts the human
CD4.sup.+CD25.sup.+ T cells showed almost no proliferation upon
stimulation, neither in response to polyclonal activation by
plate-bound anti CD3+ anti CD28 nor following (even repetitive)
stimulation with the most potent natural immunostimulatory cells,
i.e. mature (allogeneic) DC. When these stimuli were combined with
high doses of IL-2 (500 U/ml) anergy was partially reversed as
described in the mouse (Thornton, A. M., Shevach, E. M., J.
Immunol., 164:183-190 (2000)). A novel finding was that IL-15 at
high doses (50-100 ng/ml) induced comparable proliferation, and
that the combined action of IL-2 and IL-15 even at lower doses (10
U/ml and 10 ng/ml, respectively) had a strong synergistic action
and induced vigorous proliferation. This might proof important, as
expansion of CD4.sup.+CD25.sup.+ T cells is vital for potential
therapeutic applications and cloning of these cells for further
more detailed studies (including mechanistic and molecular ones).
Of interest was that neutralizing anti IL-10 mAb failed to promote
proliferation indicating that the release of IL-10 by these cells
was not causing anergy in an autocrine fashion. In coculture
experiments the CD4.sup.+CD25.sup.+ T cells displayed another key
feature in that they suppressed yet only upon activation via their
own TCR the proliferation of CD25.sup.-CD4.sup.+ or CD8.sup.+ T
cells in a contact- and dose-dependent, yet cytokine-independent
manner. Our ex vivo system has not allowed us to investigate
whether the suppression is completely antigen-nonspecific as has
recently been shown in the mouse by taking advantage of TCR
transgenic mice (Thornton, A. M., Shevach, E. M., J. Immunol.,
164:183-190 (2000)). Respective mechanistic studies might be
possible, however, by employing our IL-2+IL-15 approach for the
expansion of these cells.
[0050] It is most remarkable that a recent report has shown that T
cells with regulatory properties and a phenotype virtually
identical to the CD4.sup.+CD25.sup.+ T cells we have isolated ex
vivo from human blood can be generated in vitro by repetitive
stimulation of human naive T cells with immature DC (Jonuleit, H.
et al., J. Exp. Med., 192:1213-1222 (2000)). In the mouse
CD4.sup.+CD25.sup.+ regulatory T cell populations are continuously
generated in the thymus (Itoh, M. et al., J. Immunol. 162:5317-5326
(1999)), yet the maintenance of regulatory T cells in the periphery
requires the presence of tissue-specific antigens and IL-2 (25,
26). Based on the two supplementary findings (Jonuleit, H. et al.,
J. Exp. Med., 192:1213-1222 (2000)) it is certainly tempting to
speculate, that immature DC that have sampled peripheral tissues
via the uptake of apoptotic antibodies (Steinman, R. M. et al., J.
Exp. Med., 191:411-416 (2000); Roncarolo, M. G. et al., J. Exp.
Med. 193:F5-F9 (2001)), and present universal or tissue-specific
autoantigens, are responsible for the survival and possibly slight
proliferation of thymic regulatory T cell emigrants. It is believed
that the survival of the ex vivo isolated CD4.sup.+CD25.sup.+
regulatory T cells can be promoted by interaction with immature DC,
and that the recently reported "generation" of CD4.sup.+CD25.sup.+
T cells from nave T cells by immature DC in vitro (Jonuleit, H. et
al., J. Exp. Med., 192:1213-1222 (2000)) possibly rather represents
maintenance of survival of preexisting CD4.sup.+CD25.sup.+ in the
initial inoculum. It is also of note that it was found that
interaction of ex vivo isolated human CD4.sup.+CD25.sup.+ T cells
with syngeneic mature DC was insufficient to activate their
suppressive properties while allogeneic mature DC were potent
inducers of regulation. This observation again suggests testable
hypotheses. For example, will immature in contrast to mature
syngeneic DC activate CD4.sup.+CD25.sup.+ T cells (suggesting that
they carry some specific ligand for interaction), or will they do
so only after ingestion of apoptotic bodies (suggesting that
presentation of autoantigens is required)? Furthermore, can mature
DC that present nominal recall antigens (e.g. influenza
proteins/peptides) stimulate T cells both in the
CD4.sup.+CD25.sup.- T cell population and the CD4.sup.+CD25.sup.+ T
suggesting that besides autoantigens also the recognition of
foreign antigens could trigger regulation at inflammatory
sites.
[0051] In summary, it was shown that a sizeable population
(.about.6%) of CD4.sup.+CD25.sup.- T cells exists in the peripheral
blood of normal human adults that in contrast to previous belief do
not represent conventional memory but rather regulatory T cells
equivalent to the unique population of CD4.sup.+CD25.sup.-
"professional" suppressor/regulatory T cells that have been studied
for years in rodents. The identification and characterization of
the human CD4.sup.+CD25.sup.+ regulatory T cells will now allow for
their monitoring in various disease states, and has important
implications for understanding and treating autoimmunity, graft
rejection, and cancer.
[0052] The invention is further explained by the following figures
and examples which, however, are not intended to limit the
invention:
[0053] Figures
[0054] FIG. 1: CD4.sup.+CD25.sup.+ T Cells exhibits distinct
phenotypical differences to CD4.sup.+CD25.sup.- T Cells. CD4.sup.+
T cells were isolated from PBMC by negative MACS sorting, yielding
highly purified untouched CD4.sup.+ T cells. These cells were
labeled with anti CD25 magnetic beads and sorted.
[0055] (A): Sorting resulted in virtually pure CD25.sup.+ T cells.
A representative result out of 20 independent standardized
experiments is shown.
[0056] (B): The phenotype of CD4.sup.+CD25.sup.+,
CD4.sup.+CD25.sup.- and activated CD4.sup.+CD25.sup.- T cells was
analyzed as described in Materials & Methods. In addition,
CD4.sup.+CD25.sup.- T cells were activated with immobilized anti
CD3+ soluble anti CD28. After activation cells were labeled with
anti CD25 magnetic beads and sorted. Results were similar in 5
independent experiments.
[0057] (C): CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells
were stained with anti CTLA-4 antibody at 37.degree. C. for 2-h.
Staining was performed ex vivo and at different time points after
activation with immobilized anti CD3+ soluble anti CD28. One
representative result of 4 independent experiments is shown.
[0058] FIG. 2: CD4.sup.+CD25.sup.+ T cells are
nonproliferative/anergic to both allogeneic and polyclonal
stimulation, which is partially reversed by the addition of IL-2
and/or IL-15, but not by neutralizing anti IL-10 antibodies.
[0059] (A) CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells were
isolated from adult blood by MACS sorting as in FIG. 1.
1.times.10.sup.5 T cells/96 well were stimulated with different
numbers of mature allogeneic DC. Proliferation of T cells
(triplicate cultures) was determined by [.sup.3H]Tdr incorporation.
Results were similar in 6 independent experiments.
[0060] (B): Whole CD4.sup.+, CD4.sup.+CD25.sup.+ and
CD4.sup.+CD25.sup.- T cells were treated as described in (A).
Proliferation in triplicate cultures was determined by [.sup.3H]Tdr
incorporation (representative result of 5 independent
experiments).
[0061] (C): MACS sorted CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.-
T cells were primed and restimulated every week with mature
allogeneic DC from the same donor (DC:T cell ratio of 1:20).
Proliferation (1.times.10.sup.5 T cells/96 well) was determined by
[.sup.3H]Tdr incorporation. Similar results were obtained in 3
independent experiments.
[0062] (D): CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells
were stimulated with immobilized anti CD3 (10 .mu.g/ml) and soluble
anti CD28 (10 .mu.g/ml) [upper panels] or with mature allogeneic DC
[lower panels] as described in (A). 500 U/ml IL-2, 100 ng/ml IL-15,
a mixture of 10 U/ml IL-2+1 ng/ml IL-15 or 10 .mu.g/ml anti IL-10
were added at the onset of culture. [.sup.3H]Tdr incorporation was
measured after 5 days of culture. One of 3 independent experiments
is shown. The addition of IL-2 and/or IL-15 in the absence of a
polyclonal or allogeneic T cell stimulus did not induce significant
proliferation in the CD25.sup.+ or CD25.sup.- T cell subset (data
not shown).
[0063] FIG. 3: CD4.sup.+CD25.sup.+ T Cells if stimulated via the
TCR suppress the activation of CD4.sup.+ and CD8.sup.+ T Cells in a
cell contact- and dose-dependent manner.
[0064] (A, B): MACS sorted total CD4.sup.+ (A) and CD8.sup.+ (B) T
cells (105 T cells/96 well) were added to CD4.sup.+CD25.sup.+ T
cells at the ratios indicated, and stimulated with allogeneic DC at
a DC/CD4.sup.+ or CD8.sup.+ T cells ratio of 1:20. Proliferation
was determined by [.sup.3H]Tdr incorporation after 5 days. One of 5
independent experiments is shown.
[0065] (C): DC and CD4.sup.+CD25.sup.+ T cells were
generated/isolated from the same donor (donor I). In addition,
whole CD4.sup.+ T cells and CD4.sup.+CD25.sup.+ T cells were
isolated from another donor (donor II). 10.sup.5 whole CD4.sup.+ T
cells/96 well were cultured with 5.times.10.sup.3 DC/well (i.e.
DC:T ratio=1:20; results were comparable at a DC:T ratio of 1:100,
not shown). CD4.sup.+CD25.sup.+ T cells from donor I and donor II
were then added, respectively. Proliferation was determined by
[.sup.3H]Tdr incorporation after 5 days of culture. Results
representative of 3 independent experiments are shown as mean cpm
of triplicate cultures.
[0066] (D): Whole CD4.sup.+ T cells or CD4.sup.+CD25.sup.+ T cells
were (105 T cells/96 well) stimulated with 5.times.10.sup.3
allogeneic mature DC (DC:T ratio=1:20) (upper 2 panels). In
addition, whole CD4.sup.+ T cells were cocultured with
CD4.sup.+CD25.sup.+ T cells at a ratio of 1:1 (105 T cells/96 well
each) and stimulated with allogeneic DC again at a DC:T ratio of
1:20 in the presence or absence of 10 .mu.g/ml anti IL-10, 2
.mu.g/ml TGF-beta, 500 U/ml IL-2, 50 ng/ml IL-15 or a mixture of 10
U/ml IL-2, 1 ng/ml IL-15. In a parallel Transwell approach the
CD4.sup.+CD25.sup.+ T cells were stimulated with allogeneic DC (DC/
T cell ratio of 1:20) in a Transwell chamber, and whole CD4+ T cell
responders were put into the well together with allogeneic DC again
at a DC:T ratio of 1:20. Proliferation after 5 days of culture was
determined by [.sup.3H]Tdr incorporation. One of four
representative experiments is shown.
[0067] FIG. 4: Different cytokine profiles of CD4.sup.+CD25.sup.+
and CD4.sup.+CD25.sup.- T cells.
[0068] (A) MACS sorted CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.-
T cells were stimulated with PMA (20 ng/ml) and A23187 Ca.sup.2+
ionophore (500 .mu.g/ml) for 6 h. Monensin (2 .mu.M) was added for
the last 5 h. Staining of CD3 surface expression was performed.
Cells were washed, fixed, permeabilised and stained for detection
of intracellular cytokines using FITC- or PE-conjugated specific
antibodies. One of 5 independent experiments with similar results
is shown. Results were identical when T cells were stimulated with
platebound anti-CD3.sup.+ soluble anti-CD28 AB (not shown).
[0069] (B): CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells
were activated with platebound anti CD3+ soluble anti CD28. After
48 h of culture analysis of RNA expression was performed by RNase
Protection assay.
[0070] (C): Cytokines in the supernatant were measured by ELISA
(one of 5 independent experiments is shown).
[0071] Material and Methods
[0072] Culture Medium: RPMI 1640 (Bio Whittaker) supplemented with
1% heat-inactivated autologous plasma, 20 .mu.g/ml gentamicin
(Merck) and 2 mM glutamine (Bio Whittaker) was used for the
generation of dendritic cells (DC), X-VIVO-20 (Bio Whittaker)
supplemented with 1% heat-inactivated single donor human serum, 20
.mu.g/ml gentamicin (Merck) and 2 mM glutamine (Bio Whittaker) for
T cell culture.
[0073] Cytokines: All cytokines used in this study were recombinant
human proteins. Final concentrations were: GM-CSF 1,000 U/ml
(Leukomax.TM.; Novartis), IL-4 800 U/ml (Sandoz), IL-2 (Proleukin;
Chiron Corp.) and IL-15 (PeproTech) were used at the concentrations
indicated; for DC maturation we used a cocktail consisting of
IL-1.beta. 2 ng/ml (Sigma); IL-6 1000 U/ml (Sandoz); TNF-.alpha. 10
ng/ml (Bender, Vienna), and PGE.sub.2 1 .mu.g/ml (Sigma).
[0074] Antibodies: For immunostaining PE- and FITC-conjugated
Antibodies (Ab) (all from BD Pharmingen) against CD3, CD4, CD5,
CD8, CD14, CD19, CD25, CD28, CD45 RA, CD45 RO, CD56, CD62L, CD80,
CD83, CD86, CD95, CD95L, CD122, CD152, CD154, HLA-DR, and
respective mouse and rat isotype controls were employed. Ab used
for intracellular cytokine staining were FITC- and PE-conjugated
anti IL-2 (MQ1-17H12), anti IL-4 (8D4-8), anti IL-10 (JES3-19F1)
and anti IFN-.gamma. (4S.B3), all from BD Pharmingen. Unconjugated
anti IL-10 (JES3-19F1) (Pharmingen) and anti TGF-.beta. (R&D
Systems) were used for neutralization experiments, anti CD3 (UCHT1)
and anti CD28 (CD28.2) for polyclonal activation of T cells.
[0075] Cytokine Assays: T cells were stimulated with allogeneic DC
or with platebound anti CD3 (10 .mu.g/ml)+soluble anti CD28 (10
.mu.g/ml) in X-VIVO-20+1% serum. Cytokine analysis was performed at
different time points by analysis of supernatants with commercially
available ELISA kits for human IL-2, IL-4, IL-10, IFN-.gamma. and
TGF-.beta. (BD Pharmingen). For analysis of intracellular cytokine
production T cells were either stimulated with PMA 20 ng/ml and
Ca.sup.2+ ionophore A23187 500 .mu.g/ml (both from SIGMA) for 6
hours or with platebound anti-CD3 and soluble anti-CD28 Ab for 6
hours. Monensin, 2 .mu.M (SIGMA) was added for the last 4 hours of
culture. Cells were collected, washed, fixed and saponine
permeabilized (Fix/perm solution, BD Pharmingen) and stained with
cytokine specific Ab or isotype. For cytokine mRNA analysis T cells
were stimulated with platebound anti CD3 and soluble anti CD28 Ab.
Cells were analyzed by RNase Protection assay template sets (BD
Pharmingen).
[0076] Cell isolation and DC Generation: DC were generated from
buffy coats or leukapheresis products (both obtained from the
Department of Transfusion medicine from healthy donors after
informed consent was given) as described (18,19). In brief, PBMCs
were isolated by Ficoll density gradient centrifugation. Monocytes
were isolated by plastic adherence and cultured in RPMI Medium,
supplemented with IL-4 and GM-CSF. At day 6 a maturation cocktail
(IL-1, IL-6, PGE.sub.2 and TNF.alpha.) was added. At day 7
nonadherent cells were harvested and constituted mature DC that
were >90% double positive for costimulatory molecules (CD80,
CD86) and CD83.
[0077] CD4.sup.+ T cells were isolated from PBMC with a negative
CD4.sup.+ T cell isolation kit (Miltenyi Biotech).
CD4.sup.+CD25.sup.+ T cells were isolated from the pure, untouched
CD4.sup.+ T cells using CD25 Microbeads (Miltenyi Biotech).
Isolation of CD8.sup.+ T cells was performed using a negative
CD8.sup.+ T cell isolation kit (Miltenyi Biotech). Purity was
assessed by FACS.
[0078] Flow Cytometric Analysis: For immunofluorescence staining
cells were washed, stained for 20 min at 4.degree. C. with optimal
dilution of each Ab. Cells were washed again and analyzed by flow
cytometry (FACS Scan.TM. and CELLQuest.TM. software; Becton
Dickinson). For analysis of cell surface CD152 expression, cells
were stained with the appropriate antibody for 2 hours at
37.degree. C.
[0079] Proliferation Assays: To assess proliferation of different
CD4.sup.+ subtypes 105 sorted T cells were incubated in X-VIVO-20
with different numbers of DC or different concentrations of
platebound anti CD3+ soluble anti CD28 in 96-well plates. For
assessment of regulatory properties 10.sup.5 bulk CD4.sup.+ T cells
were cultured with 5.times.10.sup.3 (in some experiments also with
1.times.10.sup.3) DC in 96-well plates. Purified
CD4.sup.+CD25.sup.+ or CD4.sup.+CD25.sup.- T cells were added at
different concentrations. After 4-5 days of culture [3H]Tdr (37
KBq/well) was added for additional 16 h. Proliferation was measured
using a liquid scintillation counter.
[0080] Transwell Experiments: Transwell experiments were performed
in 24-well plates. 10.sup.6 bulk CD4.sup.+ T cells were stimulated
with 5.times.10.sup.3 DC. In addition, 10.sup.6 CD4.sup.+CD25.sup.+
or CD4.sup.+CD25.sup.- T cells were either added directly to the
culture or were placed in Transwell chambers (Millicell, 0.4 .mu.m;
Millipore). After 5 days of coculture T cells were transferred to
96-well plates (10.sup.5 cells/well) in triplicates. Proliferation
was measured after 16 h pulse with [3H]Tdr using a liquid
scintillation counter.
EXAMPLES
Example 1
CD4.sup.+CD25.sup.+ T Cells Show a Reduced Proliferative Response
to Both Allogeneic and Polyclonal Stimulation
[0081] A low proliferative potential is highly characteristic of
the well characterized regulatory CD25.sup.+CD4.sup.+ T cells in
the murine system (Sakaguchi S. et al., J. Immunol., 155:1151-1164
(1995)). To analyze the proliferative capacity of human CD4.sup.+
subpopulations CD4.sup.+ T cells were magnetically sorted for their
expression of CD25. By using a MACS CD4 negative selection kit and
afterwards a positive selection for CD25 a more then 95% pure
population of CD4.sup.+CD25.sup.+ T cells was obtained (FIG. 1A).
These cells comprised around 6% (2,8-17,2%, n=20) of peripheral
CD4.sup.+ T cells in the blood of the healthy adults we
studied.
[0082] Mature DC are known as the most powerful antigen presenting
cells (Bancherau, J., Steinman, R. M., Nature, 392:245-252 (1998)).
Nevertheless, the CD4.sup.+CD25.sup.+ T cells exhibited virtually
no proliferative response when stimulated in vitro with fully
mature allogeneic DC in sharp contrast to the CD4.sup.+CD25.sup.- T
cells (FIG. 2A, B) or the whole CD4.sup.+ population (FIG. 2B).
Interestingly, the CD4.sup.+ population depleted of CD25.sup.+ T
cells showed a higher proliferation when stimulated with allogeneic
DC compared to the whole CD4.sup.+ population (FIG. 2B).
[0083] It was next determined whether the CD4.sup.+CD25.sup.+ T
cells would possibly only proliferate upon repetitive stimulation
by mature DC. After restimulation the proliferative response of
CD25.sup.- T cells increased somewhat, whereas the response of
CD25.sup.+ T cells remained very low (FIG. 2C). Priming and
restimulation by allogeneic mature DC resulted in a 30- to 50-fold
expansion of the CD25.sup.- population after two rounds of
restimulation. In contrast, there was no significant increase of
the CD25.sup.+population (data not shown). A slightly (.about.10%)
decreased absolute number of CD4.sup.+CD25.sup.+ T cells was
harvested as compared to the initial inoculum after the repetitive
stimulation in the apparent absence of significant apoptosis or
necrosis (data not shown). The exceedingly low proliferative
response of CD4.sup.+CD25.sup.+ T cells was also apparent when
these cell populations were polyclonally stimulated with platebound
anti CD3.sup.+ soluble anti CD28 (FIG. 2D). To test whether the T
cell growth factors IL-2 and IL-15 could affect the proliferative
potential various doses were added to CD4.sup.+CD25.sup.+ and
CD4.sup.+CD25.sup.- T cells that were stimulated either with
immobilized anti CD3.sup.+ soluble anti CD28 (FIG. 2D, upper
panels) or with mature allogeneic DC (FIG. 2D, lower panels). A
series of pilot experiments revealed that IL-2 enhanced the
proliferation of CD25.sup.+ T cells only at high doses (100-1000
U/ml). IL-15 had a similar effect, again only at very high doses of
50-100 ng/ml. When both cytokines were mixed, they had strong
synergistic effects and doses of 10 U/ml IL-2 plus 10 ng/ml IL-15
were sufficient to promote the proliferation of CD4.sup.+CD25.sup.+
T cells. The addition of IL-2 and/or IL-15 in the absence of a
polyclonal or allogeneic T cell stimulus did not induce significant
proliferation in the CD25.sup.+ or CD25.sup.- T cell subset (data
not shown).
Example 2
CD4.sup.+CD25.sup.+ T Cells Exhibit Distinct Phenotypical
Differences to CD4.sup.+CD25.sup.- T Cells
[0084] To further characterize the CD25.sup.+CD4.sup.+ T cell
population, the expression of various surface molecules on
CD4.sup.+CD25.sup.+, CD4.sup.+CD25.sup.- was compared with that on
stimulated CD4.sup.+CD25.sup.- T cells (FIG. 1B). All three
populations showed homogenous expression of CD3 and CD4. No
contaminating cells such as monocytes, B cells, CD8.sup.+ T cells
or NK cells could be observed by FACS analysis (data not shown).
Without prior stimulation, i.e. ex vivo the CD25.sup.+ population
already expressed high levels of intracellular, and low levels of
cell surface CTLA-4 (CD152). Ex vivo isolated CD4.sup.+CD25.sup.+ T
cells furthermore constitutively expressed CD122 (IL-2R beta
chain), HLA-DR (.about.50%) and consisted primarily (.about.80%) of
CD45RO cells resembling a memory T cell phenotype. In sharp
contrast, the ex vivo isolated CD4.sup.+CD25.sup.- T cells did not
express CTLA-4 (neither intracellularly nor on the surface), CD122,
or HLA-DR, and more cells expressed CD45RA rather than CD45RO.
Following activation with plate bound anti-CD3+ soluble anti-CD28,
however, most CD4.sup.+CD25.sup.- became strongly CD25.sup.+ (the
level of CD25 expression was about 1 log higher, compared to the
CD4.sup.+CD25.sup.+ T cells, data not shown), and displayed high
levels of HLA DR and CD122 (again about 1 log higher compared to
CD4.sup.+CD25.sup.+ T cells) as to be expected. In addition, both
intracellular and surface CTLA-4 was upregulated within 24-48 h yet
quickly downregulated thereafter (FIG. 1C) as expected. The
kinetics of CTLA-4/CD152 expression proved strikingly different
when CD4.sup.+CD25.sup.+ T cells were stimulated. These cells also
upregulated their (constitutively already present albeit low) CD152
surface expression, yet the strong expression of CD152 remained
constant for a period of more than 1 week (FIG. 1C). Staining with
several other mAb such as anti CD28, CD62L, CD69, CD95, CD95L,
CD154 (CD40L) did not reveal reproducible and significant
differences between CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T
cells.
Examples 3
CD4.sup.+CD25.sup.+ T Cells if Stimulated via the TCR Suppress the
Activation of CD4.sup.+ and CD8.sup.+ T Cells in a Cell Contact-
and Dose-Dependent Manner
[0085] To analyze the putative regulatory properties of CD25.sup.+
T cells coculture experiments were performed. In a first series of
tests we isolated from a particular donor both the total CD4.sup.+
population and the CD25.sup.+ and CD25.sup.- fractions. Whole
CD4.sup.+ T cells were then mixed with CD4.sup.+CD25.sup.+ or
CD4.sup.+CD25.sup.- T cell subpopulations at indicated ratios, and
stimulated with allogeneic mature DC (FIG. 3A). CD4.sup.+CD25.sup.+
T cells significantly inhibited the proliferation of whole
CD4.sup.+ T cells, and at a 1:1 ratio virtually blocked it (cpm
then represented the background levels of CD25.sup.+ T cell
proliferation, see FIG. 2A-D). The addition of CD25.sup.- T cells
instead of CD25.sup.+ T cells slightly enhanced proliferation (not
shown). As CD4.sup.+CD25.sup.- rapidly expressed CD25 and CD122,
i.e. both chains of the IL-2R, upon polyclonal (see FIG. 1B) as
well stimulation by allogeneic DC (data not shown) this finding
indicated that the suppressive activity of the CD4.sup.+CD25.sup.+
T cell subset was not simply due to consumption or passive
adsorption of IL-2 via their IL-2R. CD4.sup.+CD25.sup.+ T cells
exerted also a suppressive activity on whole CD8.sup.+ T cells
albeit downregulation was less intense (FIG. 3B).
[0086] In a further set of experiments it was determined whether
activation of CD4.sup.+CD25.sup.+ T cells by syngeneic DC was
sufficient for induction of their regulatory properties. To this
end mature DC and CD4.sup.+CD25.sup.+ T cells were
generated/isolated from the same donor (donor I). In addition,
whole CD4.sup.+ T cells as well as the CD4.sup.+ CD25.sup.+ T cell
subset were isolated from another donor (donor II). The whole
CD4.sup.+ T cells (donor II) were then stimulated with allogeneic
mature DC (donor I) in the absence (FIG. 3C, CD4.sup.+ only) or
presence of various numbers of CD4.sup.+CD25.sup.+ T cells isolated
from either donor I or donor II (FIG. 3C). Whole CD4.sup.+ T cells
from donor II proliferated vigorously as expected when stimulated
with allogeneic, donor I-derived DC (FIG. 3C, CD4.sup.+ only). In
the presence of donor I-derived CD4.sup.+CD25.sup.+ T cells (i.e.
syngeneic to the DC used) the proliferation (i.e. alloreactivity)
of whole donor II-derived CD4.sup.+ T cells was not suppressed at
all (FIG. 3C). Potent suppression occurred, however, when donor
II-derived CD4.sup.+CD25.sup.+ T cells (i.e. allogeneic to the DC
used) were added (FIG. 3C). Suppression was also observed in
experiments where DC, whole CD4.sup.+ T cells, and CD4.sup.+CD25+ T
cells were derived from three different donors. These data
indicated that TCR-mediated activation of CD4.sup.+CD25.sup.+ T
cells was required to let them exert their regulatory function, and
that syngeneic DC were insufficient to induce their suppressive
activity.
[0087] Next, Transwell chamber experiments were performed to
investigate, whether the regulatory function of the
CD4.sup.+CD25.sup.+ T cells was mediated primarily by soluble
factors or required cell-cell contact (FIG. 3D). As shown in FIG.
3D the CD4.sup.+CD25.sup.+ T cells suppress proliferation of whole
CD4.sup.+ T cells almost completely in the presence of allogeneic
DC. Separation of the two populations in Transwell chambers
virtually abolished their suppressive effect. These observations
suggested that direct cell contact is essential for the inhibitory
capacity of CD4.sup.+CD25.sup.+ T cells, as the semipermeable
membrane of Transwell chambers allows free passage of soluble
factors, but excludes direct cell contact. The Transwell
experiments also confirmed that consumption of IL-2 by
CD4.sup.+CD25.sup.+ T cells was not the mechanism responsible for
suppression.
[0088] Despite the obvious requirement for close interaction
between regulatory and responding cells neither a targeting of the
antigen-presenting DC or a role of soluble factors was excluded by
the Transwell experiments. Therefore, a plate-bound anti-CD3 Ab (in
combination with soluble anti CD28 Ab) was also employed as an
antigen presenting cell-independent and polyclonal T cell stimulus.
Whole CD4.sup.+ T cells alone showed strong proliferation upon this
stimulation. As mentioned above (FIG. 2D) CD4.sup.+CD25.sup.+ T
cells did not proliferate. In coculture of both populations, there
was at least a 75% reduction at a 1:1 ratio compared to control
(data not shown). These data suggested, that regulation does not
primarily occur via modulation of APC function. Neutralizing
antibodies to the cytokines IL-10 and TGF-.beta. (critical for the
suppressive activities of the so-called Tr1 and Th3, respectively
(Groux, H. et al., Nature, 389:737-742 (1997); Fukaura, H. et al.,
J. Clin. Invest., 98:70-77 (1996)) did not abolish the regulatory
activity of the CD4.sup.+CD25.sup.+ T cells demonstrating that
these cytokines played no major suppressive role at least in the
assays we looked at. The addition of IL-2 and/or IL-15 to
cocultures at the high doses that promote the proliferation of
CD4.sup.+CD25.sup.+ T cells (see FIG. 2D) reduced their inhibitory
effects. The suppressive activity was, however, likely not
abolished as the significant proliferation of the
CD4.sup.+CD25.sup.+ T cells has to be taken into account when
interpreting the data FIG. 3D).
Example 4
CD4.sup.+CD25.sup.+ T Cells Predominantly Secrete IL-10
[0089] To analyze and compare the cytokine profiles freshly sorted
CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells were activated
with plate-bound anti CD3+ anti CD28. Supernatants were then
analyzed by ELISA, and RNA expression was studied by RNase
protection assays. In addition, intracellular cytokine staining was
performed to determine the percentage of cells releasing a certain
cytokine. As shown in FIG. 4 CD4.sup.+CD25.sup.- T cells
predominantly secreted IFN-.gamma. and IL-2, with little secretion
of IL-10 and IL-4, resembling a Th1 like profile.
CD4.sup.+CD25.sup.+ T cells on the other hand predominantly
produced IL-10 and only low levels of IL-2, IL-4 and IFN-.gamma.,
resembling Tr1 cells. Comparison of both subpopulations at the RNA
level revealed, that CD25.sup.+ T cells express more IL-10, less
IFN-.gamma. and similar levels of IL-2 mRNA compared to CD25.sup.-
T cells. IL-1 receptor antagonist (IL-1 Ra) mRNA was found
predominantly in CD4.sup.+CD25.sup.+ T cells, while significant
IL-1, mRNA levels were only present in CD4.sup.+CD25.sup.- T cells.
TGF-.beta. was expressed at similarly low levels in both cell
types.
Example 5
Activated and Afterwards Fixated CD4.sup.+CD25.sup.+ T Cells Still
Exhibit Regulatory Capacity
[0090] CD4.sup.+CD25.sup.- and CD4.sup.+CD25.sup.+ T cells were
isolated from whole CD4.sup.+ T cells from adult blood by MACS.RTM.
sorting as described. CD4.sup.+CD25.sup.+ T cells were divided into
three parts. One fraction was activated with 10 .mu.g/ml platebound
anti-CD3 and 10 .mu.g/ml soluble anti-CD28 antibodies overnight.
Next day this fraction and one part of the non activated
CD4.sup.+CD25.sup.+ T cells were fixed with 2% formaldehyde for 1
hour. The third part was left untreated. Cells were washed three
times.
[0091] Unfixed CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cells
alone and CD4.sup.+CD25.sup.+ T cells of each fraction, mixed at a
1:1 ratio with CD4.sup.+CD25.sup.- T cells, were activated with
immobilized anti-CD3 and soluble anti-CD28. After 5 days
proliferation was measured by [3H]Tdr incorporation. A
representative of 5 independent experiments is shown in FIG. 5
where the symbols represent the following:
[0092] CD4.sup.+CD25.sup.- unfixed CD4.sup.+CD25.sup.- cells
[0093] CD4.sup.+CD25.sup.+ unfixed CD4.sup.+CD25.sup.+ cells
[0094] reg. 1:1 unfixed CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.-
T cells at a 1:1 ratio
[0095] reg. 1:1
[0096] CD25+ stim fix activated fixed CD4.sup.+CD25.sup.+ T cells
and unfixed CD4.sup.+CD25.sup.- T cells at a 1:1 ratio
[0097] reg. 1:1
[0098] CD25+ fix non activated fixed CD4.sup.+CD25.sup.+ T cells
and unfixed CD4+CD25.sup.- t cells at a 1:1 ratio
* * * * *