U.S. patent application number 10/681627 was filed with the patent office on 2004-04-15 for methods for modulating t cell responses by manipulating intracellular signal transduction.
This patent application is currently assigned to The United States of America as represented by the Secretary of Navy, The United States of America as represented by the Secretary of Navy. Invention is credited to June, Carl H..
Application Number | 20040072766 10/681627 |
Document ID | / |
Family ID | 22926047 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072766 |
Kind Code |
A1 |
June, Carl H. |
April 15, 2004 |
Methods for modulating T cell responses by manipulating
intracellular signal transduction
Abstract
Methods for modulating T cell responses by manipulating
intracellular signals associated with T cell costimulation are
disclosed. The methods involve inhibiting or stimulating the
production of at least one D3-phosphoinositide in a T cell.
Production of D3-phosphoinositides can be manipulated by contacting
a T cell with an inhibitor or activator of phosphatidylinositol
3-kinase. Inhibitors of phosphatidylinositol 3-kinase for use in
the methods of the invention include wortmannin and quercetin, or
derivatives or analogues thereof. The methods of the invention can
further comprise modulating other intracellular signals associated
with costimulation, such as protein tyrosine phosphorylation, for
example by modulating the activity of a protein tyrosine kinase or
a protein tyrosine phosphatase in the T cell. Inhibition of a T
cell response in accordance with the disclosed methods is useful
therapeutically in situations where it is desirable to inhibit an
immune response to an antigen(s), for example in organ or bone
marrow transplantation and autoimmune diseases. Alternatively,
stimulation of a T cell response in accordance with the disclosed
methods is useful therapeutically to enhance an immune response to
an antigen(s), for example to stimulate an anti-tumor response in a
subject with a tumor, to stimulate a response against a pathogenic
agent or increase the efficacy of vaccination. Novel screening
assays for identifying inhibitors or activators of
phosphatidylinositol 3-kinase, which can be used to inhibit or
stimulate a T cell response, are also disclosed.
Inventors: |
June, Carl H.; (Rockville,
MD) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
The United States of America as
represented by the Secretary of Navy
|
Family ID: |
22926047 |
Appl. No.: |
10/681627 |
Filed: |
October 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10681627 |
Oct 8, 2003 |
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08245282 |
Apr 29, 1994 |
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6632789 |
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Current U.S.
Class: |
514/27 ; 514/183;
514/468 |
Current CPC
Class: |
A61K 31/365 20130101;
A61P 35/00 20180101; A61P 37/04 20180101; A61K 31/00 20130101; A61K
31/35 20130101; A61P 37/06 20180101; A61P 37/02 20180101; A61P
43/00 20180101 |
Class at
Publication: |
514/027 ;
514/183; 514/468 |
International
Class: |
A61K 031/7048; A61K
031/33 |
Claims
1. A method for inhibiting a response by a T cell expressing a cell
surface receptor which binds a costimulatory molecule, comprising
contacting the T cell with an agent which inhibits production of
D-3 phosphoinositides in the T cell.
2. The method of claim 1, wherein the agent is an inhibitor of
phosphatidylinositol 3-kinase.
3. The method of claim 2, wherein the inhibitor of
phosphatidylinositol 3-kinase is selected from a group consisting
of wortmannin, quercetin and LY294002, and derivatives or analogues
thereof.
4. The method of claim 1, wherein the response by the T cell
comprises production of at least one lymphokine.
5. The method of claim 4, wherein the lymphokine is
interleukin-2.
6. The method of claim 1, wherein the response by the T cell
comprises proliferation.
7. The method of claim 1, further comprising contacting the T cell
with a second agent which inhibits protein tyrosine phosphorylation
in the T cell.
8. The method of claim 7, wherein the second agent is an inhibitor
of a protein tyrosine kinase.
9. The method of claim 8, wherein the inhibitor of a protein
tyrosine kinase is herbimycin A or a derivative or analogue
thereof.
10. The method of claim 7, wherein the second agent is a tyrosine
phosphatase or an activator of a tyrosine phosphatase.
11. The method of claim 10, wherein the tyrosine phosphatase is a
cellular tyrosine phosphatase.
12. The method of claim 11, wherein the cellular tyrosine
phosphatase is CD45 or Hcph.
13. The method of claim 12, wherein the second agent is a molecule
which binds to and activates CD45.
14. The method of claim 13, wherein the second agent is an
anti-CD45 antibody, or fragment thereof.
15. A method for inducing unresponsiveness to an antigen in a T
cell expressing a cell surface receptor which binds a costimulatory
molecule, comprising contacting the T cell with the antigen and an
agent which inhibits production of D-3 phosphoinositides in the T
cell.
16. The method of claim 15, wherein the agent is an inhibitor of
phosphatidylinositol 3-kinase.
17. The method of claim 16, wherein the inhibitor of
phosphatidylinositol 3-kinase is selected from a group consisting
of wortmannin, quercetin and LY294002, and derivatives or analogues
thereof.
18. The method of claim 15, wherein the antigen is an
alloantigen.
19. The method of claim 15, wherein the antigen is an
autoantigen.
20. The method of claim 15, wherein the T cell is contacted with
the antigen and the agent in vitro and the method further comprises
administering the T cell to a subject.
21. A method of claim 20, wherein the antigen is on a surface of an
allogeneic or xenogeneic cell and the subject is a recipient of an
allogeneic or xenogeneic cell.
22. A method of claim 20, wherein the subject is suffering from an
autoimmune disease or a disorder associated with an inappropriate
or abnormal immune response.
23. A method for stimulating a response by a T cell which has
received a primary activation signal and expresses a surface
receptor that binds a costimulatory molecule, comprising contacting
the T cell with an agent which stimulates production of D-3
phosphoinositides in the T cell.
24. The method of claim 23, wherein the agent is an activator of
phosphatidylinositol 3-kinase.
25. The method of claim 23, wherein the response by the T cell
comprises production of at least one lymphokine.
26. The method of claim 25, wherein the lymphokine is
interleukin-2.
27. The method of claim 23, wherein the response by the T cell
comprises proliferation.
28. The method of claim 23, further comprising contacting the T
cell with a second agent which stimulates protein tyrosine
phosphorylation in the T cell.
29. The method of claim 28, wherein the second agent is an
activator of a protein tyrosine kinase.
30. The method of claim 28, wherein the second agent is an
inhibitor of a cellular tyrosine phosphatase.
31. The method of claim 30, wherein the cellular tyrosine
phosphatase is CD45.
32. A method for stimulating a response to an antigen by a T cell
expressing a cell surface receptor which binds a costimulatory
molecule comprising contacting the T cell with the antigen and an
agent which stimulates production of D-3 phosphoinositides in the T
cell.
33. The method of claim 32, wherein the agent is an activator of
phosphatidylinositol 3-kinase.
34. The method of claim 32, wherein the antigen is a
tumor-associated antigen.
35. The method of claim 32, wherein the antigen is from a pathogen
selected from the group consisting of a bacteria, a virus, a fungus
and a parasite.
36. The method of claim 32, wherein the T cell is contacted with
the antigen and the agent in vitro and the method further comprises
administering the T cell to a subject.
37. A method of claim 36, wherein the antigen is expressed by a
tumor cell present in the subject.
38. A method of claim 36, wherein the antigen is expressed by a
pathogen present in the subject.
39. A method for identifying an inhibitor of a phosphatidylinositol
3-kinase comprising: a) providing a T cell which expresses a
receptor that binds a costimulatory molecule; b) stimulating an
intracellular signal transduction pathway in the T cell associated
with ligation of the receptor in the presence of an agent to be
tested; and c) determining an amount of at least one D-3
phosphoinositide produced in the T cell, wherein a reduced amount
of at least one D-3 phosphoinositide produced in the T cell in the
presence of the agent relative to an amount produced in the T cell
in the absence of the agent indicates that the agent is an
inhibitor of a phosphatidylinositol 3-kinase.
40. The method of claim 39, wherein the receptor is CD28.
41. The method of claim 40, wherein the T cell is contacted with a
ligand for CD28.
42. The method of claim 40, wherein the ligand for CD28 is a
membrane-bound form of a B lymphocyte activation antigen selected
from the group consisting of B7-1 and B7-2.
43. The method of claim 39, wherein production of at least one D-3
phosphoinositide in the T cell is measured by high pressure liquid
chromatography.
44. A method for identifying an activator of phosphatidylinositol
3-kinase comprising: a) contacting a T cell which expresses a
receptor that binds a costimulatory molecule with an agent to be
tested; and b) determining an amount of at least one D-3
phosphoinositide produced in the T cell, wherein an increased
amount of at least one D-3 phosphoinositide produced in the T cell
in the presence of the agent relative to an amount produced in the
T cell in the absence of the agent indicates that the agent is an
activator of a phosphatidylinositol 3-kinase.
45. The method of claim 44, wherein production of at least one D-3
phosphoinositide in the T cell is measured by high pressure liquid
chromatography.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 08/245,282, which claims priority to U.S. application Ser. No.
08/245,282, filed Apr. 29, 1994, entitled "Methods for Modulating T
Cell Responses by Manipulating Intracellular Signal Transduction";
this application is related to PCT/US95/05213, filed May 1, 1995,
entitled "Methods for Modulating T Cell Responses by Manipulating
Intracellular Signal Transduction". The entire contents of each of
these applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The induction of antigen-specific T cell responses involves
multiple interactions between cell surface receptors on T cells and
ligands on antigen presenting cells (APCs). The primary interaction
is between the T cell receptor (TCR)/CD3 complex on a T cell and a
major histocompatibility complex (MHC) molecule/antigenic peptide
complex on an antigen presenting cell. This interaction triggers a
primary, antigen-specific, activation signal in the T cell. In
addition to the primary activation signal, induction of T cell
responses requires a second, costimulatory signal. In the absence
of proper costimulation, TCR signalling can induce a state of
anergy in the T cell. Subsequent appropriate presentation of
antigen to an anergic T cell fails to elicit a proper response (see
Schwartz, R. H. (1990) Science 248:1349).
[0003] A costimulatory signal can be triggered in a T cell through
a T cell surface receptor, such as CD28. For example, it has been
demonstrated that suboptimal polyclonal stimulation of T cells
(e.g. by anti-CD3 antibodies or phorbol ester, either of which can
provide a primary activation signal) can be potentiated by
crosslinking of CD28 with anti-CD28 antibodies (Linsley, P. S. et
al. (1991) J. Exp. Med. 173:721; Gimmi, C. D. et al. (1991) Proc.
Natl. Acad Sci. USA 88:6575). Moreover, stimulation of CD28 can
prevent the induction of anergy in T cell clones (Harding, F. A.
(1992) Nature 356:607-609). Natural ligands for CD28 have been
identified on APCs. CD28 ligands include members of the B7 family
of proteins, such as B7-1(CD80) and B7-2 (B70) (Freedman, A. S. et
al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al. (1989)
J. Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J. Exp.
Med. 174:625-631; Freeman, G. J. et al. (1993) Science 262:909-911;
Azuma, M. et al. (1993) Nature 366:76-79; Freeman, G. J. et al.
(1993) J. Exp. Med. 178:2185-2192). In addition to CD28, proteins
of the B7 family have been shown to bind another surface receptor
on T cells related to CD28, termed CTLA4, which may also play a
role in T cell costimulation (Linsley, P. S. (1991) J. Exp. Med.
174:561-569; Freeman, G. J. et al. (1993) Science 262:909-911).
[0004] The elucidation of the receptor:ligand relationship of
CD28/CTLA4 and the B7 family of proteins, and the role of this
interaction in costimulation, has led to therapeutic approaches
involving manipulation of the extracellular interactions of surface
receptors on T cells which bind costimulatory molecules. For
example, a CTLA4Ig fusion protein, which binds to both B7-1 and
B7-2 and blocks their interaction with CD28/CTLA4, has been used to
inhibit rejection of allogeneic and xenogeneic grafts (see e.g.,
Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA
89:11102-11105; Lenschow, D. J. et al. (1992) Science 257:789-792).
Similarly, antibodies reactive with B7-1 and/or B7-2 have been used
to inhibit T cell proliferation and IL-2 production in vitro and
inhibit primary immune responses to antigen in vivo (Hathcock K. S.
et al. (1993) Science 262:905-907; Azuma, M. et al. (1993) Nature
366:76-79; Powers, G. D. et al. (1994) Cell. Immunol. 153:298-311;
Chen C. et al. (1994) J. Immunol. 152:2105-2114). Together, these
studies indicate that T cell surface receptors which bind
costimulatory molecules such as B7-1 and B7-2 are desirable targets
for manipulating immune responses.
[0005] While the extracellular interactions between CD28/CTLA4 with
their ligands have been characterized in some detail, little is
known regarding the intracellular events that occur in a T cell
following ligation of these molecules. T cell costimulation is
thought to involve an intracellular signal transduction pathway
distinct from signalling through the TCR since the costimulatory
pathway is resistant to the inhibitory effects of cyclosporin A
(see June, C. H. et al. (1990) Immunology Today 11:211-216) Protein
tyrosine phosphorylation has been shown to occur in T cells upon
CD28 ligation and it has been demonstrated that a protein tyrosine
kinase inhibitor, herbimycin A, can inhibit CD28-induced IL-2
production (Vandenberghe, P. et al. (1992) J. Exp. Med.
175:951-960; Lu, Y. et al. (1992) J. Immunol. 149:24-29).
SUMMARY OF THE INVENTION
[0006] This invention relates to the regulation of T cell responses
by manipulation of intracellular signal transduction. In
particular, intracellulal signalling events which occur upon
costimulation of a T cell are manipulated. The invention
encompasses methods for inhibiting or stimulating T cell responses
by inhibiting or stimulating one or more intracellular signals
which result from ligation of a surface receptor on a T cell which
binds a costimulatory molecule. It has now been discovered that
CD28 receptor stimulation leads to the production of
D3-phosphoinositides within a T cell. Moreover, it has been
discovered that inhibition of the activity of phosphatidylinositol
3-kinase in a T cell can inhibit T cell responses, such as
lymphokine production and cellular proliferation. These discoveries
indicate a functional role for D3-phosphoinositides in a
costimulatory signal transduction pathway and provide
phosphatidylinositol 3-kinase as an intracellular target for
modulation of T cell responses. Accordingly, intracellular
signalling events involving D3-phosphoinositides can be modulated
either to inhibit a costimulatory signal and thereby induce T cell
unresponsiveness, or to trigger a costimulatory signal and thereby
generate a T cell response. In addition, novel screening assays for
identifying inhibitors or activators of phosphatidylinositol
3-kinase, which can be used to inhibit or stimulate a T cell
response, are within the scope of the invention.
[0007] One aspect of the invention pertains to methods for
inhibiting a response by a T cell which expresses a surface
receptor that binds a costimulatory molecule. These methods involve
contacting the T cell with an agent which inhibits production of
D3-phosphoinositides in the T cell. In one embodiment, the agent is
an inhibitor of a phosphatidylinositol 3-kinase, such as the fungal
metabolite wortmannin or the bioflavenoid quercetin, or derivatives
or analogues thereof (e.g LY294002). In another embodiment of the
method of the invention, the T cell is contacted with at least one
additional agent which inhibits a different intracellular signal
associated with costimulation, such as protein tyrosine
phosphorylation. For example, the T cell can be contacted both with
an inhibitor of phosphatidylinositol 3-kinase and with an inhibitor
of a protein tyrosine kinase. A preferred inhibitor of a protein
tyrosine kinase is herbimycin A. Alternatively, protein tyrosine
phosphorylation can be inhibited in a T cell by a tyrosine
phosphatase or an activator of a tyrosine phosphatase. In this
embodiment, the T cell can be contacted with an inhibitor of
phosphatidylinositol 3-kinase and with a molecule, e.g., an
antibody, which binds to and activates a cellular tyrosine
phosphatase, such as CD45 or Hcph.
[0008] The invention also provides methods for inducing
unresponsiveness to an antigen in a T cell by triggering a primary,
antigen-specific signal in a T cell while interfering with an
intracellular signal associated with costimulation in the T cell.
As a result of interfering with costimulatory signal transduction,
the T cell fails to receive a proper costimulatory signal in the
presence of the antigen and antigen-specific unresponsiveness is
induced in the T cell. To induce T cell unresponsiveness, an
antigen-specific T cell is contacted with the antigen in a form
suitable for stimulation of a primary activation signal in the T
cell, together with an agent which inhibits production of D-3
phosphoinositides in the T cell. For example, a T cell can be
contacted with an antigen presented by an APC together with an
inhibitor of phosphatidylinositol 3-kinase, such as wortmannin or
quercetin or derivatives or analogues thereof (e.g LY294002).
Additionally, other intracellular signals associated with
costimulation, such as protein tyrosine phosphorylation, can be
inhibited in the T cell.
[0009] Methods for inhibiting T cell responses and for inducing T
cell unresponsiveness are useful in situations where it is
desirable to down-modulate an immune response, for example in a
transplant recipient (e.g., of an organ graft or bone marrow graft
etc.) or a subject suffering from an autoimmune disease or other
disorder associated with an abnormal immune response. An agent
which inhibits signal transduction associated with costimulation
(e.g., an inhibitor of inositol phosphate 3-kinase) can be
administered to a subject or, alternatively, T cells can be
obtained from the subject, treated in vitro as described herein and
administered to the subject.
[0010] Another aspect of the invention pertains to methods for
stimulating a response by a T cell which has received a primary
activation signal and expresses a surface receptor that binds a
costimulatory molecule. These methods involve contacting the T cell
with an agent which stimulates production of D-3 phosphoinositides
in the T cell, such as an activator of phosphatidylinositol
3-kinase. In another embodiment, the T cell is contacted with an
agent which stimulates production of D-3 phosphoinositides and at
least one additional agent which stimulates a different
intracellular signal associated with costimulation, such as protein
tyrosine phosphorylation. For example, the T cell can be contacted
with an activator of phosphatidylinositol 3-kinase together with an
activator of a protein tyrosine kinase, such as pervanadate.
Alternatively, an inhibitor of a cellular phosphatase, such as CD45
or Hcph, can be used in conjunction with a PI3K activator. In yet
another embodiment of the invention, an antigen-specific T cell
response is stimulated by contacting an antigen-specific T cell
with the antigen together with an agent which stimulates production
of D-3 phosphoinositides in the T cell, thereby stimulating both a
primary activation signal and a costimulatory signal in the T
cell.
[0011] Methods for stimulating T cell responses are useful in
situations where it is desirable to up-regulate an immune response.
For example a response against a tumor in a tumor-bearing subject
can be stimulated or a response against a pathogen (e.g., a
bacteria, a virus, such as HIV, fungus, parasite etc.) in a subject
can be stimulated. Additionally, the methods can be used to enhance
the efficacy of vaccination. An agent which stimulates an
intracellular signal associated with costimulation (e.g., an
activator of inositol phosphate 3-kinase) can be administered to a
subject or, alternatively, T cells can be stimulated in vitro and
then administered to a subject.
[0012] Another aspect of the invention pertains to screening assays
for identifying inhibitors or activators of a phosphatidylinositol
3-kinase. In one embodiment, a T cell which expresses a cell
surface receptor (e.g., CD28) which binds a costimulatory molecule
is utilized. To identify an inhibitor, an intracellular signal
transduction pathway associated with the receptor in the T cell is
stimulated in the presence of an agent to be tested and an
inhibitor is identified based upon its ability inhibit production
of at least one D-3 phosphoinositide in a T cell. To identify an
activator, the T cell is contacted with an agent to be tested and
an activator is identified based upon its ability to stimulate
production of at least one D-3 phosphoinositide in a T cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graphic representation of the production of
phosphatidylinositol(3,4,5)-triphosphate in CD28.sup.+ T cells
(Jurkat cells) following stimulation of the cells with medium, an
anti-CD3 antibody or an anti-CD28 antibody, demonstrating distinct
kinetics of phosphatidylinositol 3-kinase activation upon
stimulation through CD3 or CD28.
[0014] FIG. 2 is a grahic representation of the production of
phosphatidylinositol(3,4)-bisphosphate in CD28.sup.+ T cells
(Jurkat cells) following stimulation of the cells with CHO cells
transfected to express B7-1 or B7-2, demonstrating distinct
kinetics of phosphatidylinositol 3-kinase activation upon
stimulation with B7-1 or B7-2.
[0015] FIG. 3 is a graphic representation of the effect of various
concentrations of wortmannin (0-100 .mu.M) on production of
phosphatidylinositol(3,4,5)-triphosphate in CD28.sup.+ T cells
(Jurkat cells_following stimulation of the cells with CHO cells
transfected to express B7-2.
[0016] FIG. 4 is a graphic representation of the effect of
wortmannin or herbimycin on calcium influx in T cells induced by
ligation of CD28 with an anti-CD28 antibody.
[0017] FIG. 5 is a series of flow cytometric profiles from a
cell-conjugate assay in which Jurkat cells were incubated either
CHO-neo, CHO-B7-1 or CHO-B7-2 cells. Calcium flux is indicated on
the Y-axis and cell conjugation is indicated on the X-axis.
[0018] FIG. 6 is a graphic representation of the effect of
membrane-bound B7-1 and B7-2, in combination with an anti-CD3
antibody, on IL-2 production by purified human peripheral blood T
cells, demonstrating a dose dependent increase in IL-2 production
by costimulation with either B7-1 or B7-2.
[0019] FIG. 7A is a graphic representation of the effect of
wortmannin treatment (1 nM to 1 .mu.M) on IL-2 production by
resting human T cells 24 hours after stimulation of the cells with
media, immobilized anti-CD3+CHO-B7-1, immobilized
anti-CD3+CHO-B7-2, PMA+CHO-B7-1 or PMA+CHO-B7-2.
[0020] FIG. 7B is a graphic representation of the percent
inhibition by wortmannin (1 to 100 nM) of IL-production by human T
cells stimulated for 24 hours with anti-CD3 antibody (OKT3)
together with CHO cells expressing B7-1, B7-2 or both B7-1 and
B7-2, or T cells stimulated with PMA together with CHO cells
expressing B7-1 or B7-2.
DETAILED DESCRIPTION OF THE INVENTION
[0021] This invention features methods for regulating T cell
responses by modulating intracellular signals generated in a T cell
upon costimulation, e.g. by binding of a surface receptor on the T
cell to a costimulatory molecule. In particular, the invention
pertains to modulation of the production of D-3 phosphoinositides
in a T cell to inhibit or stimulate a costimulatory signal to
thereby inhibit or stimulate T cell responses. Inhibition of a
costimulatory signal by interfering with signal transduction
associated with costimulation can further be used to induce T cell
unresponsiveness. The invention is based, at least in part, on the
discovery that stimulation of a T cell through the CD28 surface
receptor leads to the production of D-3 phosphoinositides in a T
cell and that an inhibitor of a phosphatidylinositol 3-kinase (also
referred to herein as PI3K) inhibits production of D-3
phosphoinositides in the T cell upon CD28 ligation. The invention
is further based, at least in part, on the discovery that
inhibition of PI3K activity in a T cell inhibits T cell responses,
such as cytokine production and cellular proliferation.
[0022] Accordingly, one aspect of the invention pertains to methods
for inhibiting a response by a T cell by interfering with
intracellular signal transduction associated with signal
transduction. In one embodiment, an intracellular signal is
inhibited by contacting a T cell expressing a cell surface receptor
that binds a costimulatory molecule with an agent which inhibits
production of D-3 phosphoinositides in the T cell. The term "a T
cell expressing a cell surface receptor that binds a costimulatory
molecule" is intended to encompass T cells expressing CD28 and/or
CTLA4, or other receptor capable of binding a costimulatory
molecule such as B7-1, B7-2 or other B7 family member.
[0023] A "response" by a T cell is intended to encompass T cell
responses that occur upon triggering of a primary activation signal
and a costimulatory signal in the T cell, and includes lymphokine
production (e.g., IL-2 production) and T cell proliferation.
Inhibition of a T cell response may involve complete blocking of
the response (i.e., a lack of a response) or a reduction in the
magnitude of the response (i.e., partial inhibition of the
response).
[0024] The term "D-3 phosphoinositides" is intended to include
derivatives of phosphatidylinositol that are phosphorylated at the
D-3 position of the inositol ring and encompasses the compounds
phosphatidylinositol(3)-m- onophosphate (PtdIns(3)P),
phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P.sub.2), and
phosphatidylinositol(3,4,5)-trisphosphate
(PtdIns(3,4,5)P.sub.3).
[0025] D-3 phosphoinositides are generated intracellularly by the
activity of a phosphatidyl-inositol 3-kinase (PI3K). Accordingly,
in one embodiment, the agent which inhibits production of a D-3
phosphoinositide in the T cell is an agent which inhibits the
activity of a PI3K. A preferred agent which inhibits PI3K activity
in a T cell is the fungal metabolite wortmannin, or derivatives or
analogues thereof. Wortmannin derivatives or analogues include
compounds structurally related to wortmannin which retain the
ability to inhibit PI3K and T cell responses. Examples of
wortmannin derivatives and analogues are disclosed in Wiesinger, D.
et al. (1974) Experientia 30:135-136; Closse, A. et al. (1981) J.
Med. Chem. 24:1465-1471; and Baggiolini, M. et al. (1987) Exp. Cell
Res. 169:408-418. Another inhibitor of PI3K activity that can be
used is the bioflavenoid quercetin, or derivatives or analogues
thereof. Quercetin derivatives or analogues include compounds
structurally related to quercetin that retain the ability to
inhibit PI3K and inhibit T cell responses. Examples of quercetin
derivatives and analogues are disclosed in Vlahos, C. J. et al.
(1994) J. Biol. Chem. 269:5241-5284. A preferred quercetin
derivative which inhibits PI3K activity is LY294002 (described in
Vlahos et al. cited supra). Alternatively, other inhibitors of
PI3K, for example those identified by methods described below, can
be used.
[0026] Another aspect of the invention involves inhibiting a
response by a T cell by interfering with two or more intracellular
signalling events associated with costimulation. For example, CD28
stimulation has been shown to result in protein tyrosine
phosphorylation in the T cell (see e.g., Vandenberghe, P. et al.
(1992) J. Exp. Med. 175:951-960; Lu, Y. et al. (1992) J. Immunol.
149:24-29). Accordingly, in one embodiment, a T cell response is
inhibited by contacting a T cell with a first agent which inhibits
production of at least one D-3 phosphoinositide in the T cell and
with a second agent which inhibits tyrosine phosphorylation in the
T cell. For example, the T cell can be contacted both with an agent
which inhibits PI3K activity and with an agent which inhibits
protein tyrosine kinase activity. A preferred protein tyrosine
kinase inhibitor is one which inhibits src protein tyrosine
kinases. In one embodiment, the src protein tyrosine kinase
inhibitor is herbimycin A, or a derivative or analogue thereof.
Derivatives and analogues of herbimycin A include compounds which
are structurally related to herbimycin A and retain the ability to
inhibit the activity of protein tyrosine kinases. In another
embodiment, the agent which inhibits protein tyrosine
phosphorylation is a protein tyrosine phosphatase or an activator
of a protein tyrosine phosphatase. By increasing the tyrosine
phosphatase activity in a T cell, the net amount of protein
tyrosine phosphorylation is decreased. The protein tyrosine
phosphatase can be a cellular protein tyrosine phosphatase within
the T cell, such CD45 or Hcph. The activity of a cell surface
tyrosine phosphatase on a T cell can be activated by contacting the
T cell with a molecule which binds to the phsophatase and
stimulates its activity. For example, an antibody directed against
CD45 can be used to stimulate tyrosine phosphatase activity in a T
cell expressing CD45 on its surface. Accordingly, in one
embodiment, the agent which inhibits protein tyrosine
phosphorylation within the T cell is an anti-CD45 antibody, or a
fragment thereof which retains the ability to stimulate the
activity of CD45. Examples of antibody fragments include Fab and
F(ab')2 fragments. Antibodies, or fragments thereof, can be
provided in a stimulatory form, for example multimerized or
immobilized etc.
[0027] Other intracellular signals associated with costimulation
can be inhibited together with inhibition of D-3 phosphoinositide
production to inhibit T cell responses. For example, CD28 ligation
has been associated with increased phospholipase C activity (see
e.g., Nunes, J. et al. (1993) Biochem. J. 293:835-842) and
increased intracellular calcium levels (see e.g. Ledbetter, J. A.
et al. (1990) Blood 75:1531-1539 and the Examples). Accordingly, T
cells can be contacted with both a first agent which inhibits PI3K
activity and a second agent which inhibits phospholipase C activity
and/or inhibits increases in intracellular calcium levels. As
demonstrated in the Examples, the tyrosine kinase inhibitor
herbimycin A also inhibits CD28-induced calcium flux in T
cells.
[0028] T cell responses can be inhibited according to the methods
of the invention either in vitro or in vivo. For example, an agent
which inhibits D-3 phosphoinositide production in a T cell can be
administered to a subject at a dose and for a period of time
sufficient to inhibit T cell responses. Alternatively, T cells can
be obtained from a subject, contacted with the agent in vitro and
readministered to the subject. The term subject is intended to
include animals in which immune responses occur, e.g., mammals,
including humans, monkeys, dogs, cats, rabbits, rats, mice, and
transgenic species thereof. Subjects in which T cell responses can
be inhibited include subjects in which it is desirable to
downmodulate an immune response. Downmodulation is intended to
encompass both partial and complete inhibition of T cell responses,
such as lymphokine production and T cell proliferation. The methods
are applicable, for example, to a subject suffering from an
autoimmune disease or other disorder associated with an abnormal
immune response, or a transplant recipient, such as a recipient of
a bone marrow transplant or other organ transplant.
[0029] In one embodiment of the invention, a costimulatory signal
is inhibited in a T cell to induce antigen-specific T cell
unresponsiveness. Accordingly, another aspect of the invention
pertains to methods for inducing T cell unresponsiveness to an
antigen. The term "T cell unresponsiveness" as used herein refers
to a reduction in or lack of a T cell response (e.g.,
proliferation, lymphokine secretion or induction of effector
functions) by a T cell upon exposure to an antigen (or antigenic
portion) to which the T cell has been rendered unresponsive. The
terms "T cell unresponsiveness" and "T cell anergy" are used
interchangeably herein. T cell unresponsiveness to an antigen can
be induced by triggering an antigen-specific primary activation
signal in the T cell (e.g., activation through the TCR/CD3 complex)
in the absence of a costimulatory signal. In the method of the
invention, a costimulatory signal is blocked in a T cell by
contacting the T cell with an agent which interferes with an
intracellular signal associated with costimulation. Specifically, T
cell unresponsiveness to an antigen can be induced by contacting an
antigen-specific T cell (i.e., a T cell expressing a TCR which
recognizes the antigen) with the antigen in a form suitable to
trigger a primary activation signal in the T cell in the presence
of an agent which inhibits production of D-3 phosphoinositides in
the T cell to inhibit a costimulatory signal. The antigen can be,
for example, an autoantigen which stimulates an autoimmune reaction
or an alloantigen which stimulates rejection of transplanted cells.
Preferably, the agent which inhibits production of D-3
phosphoinositides inhibits the activity of PI3K in the T cell, such
as wortmannin or quercetin, or a derivative or analogue thereof
(e.g., LY294002). Additional agents which inhibit other
intracellular signals associated with costimulation (as discussed
above) can also be used in conjuction with an agent which inhibits
production of D-3 phosphoinositides in the T cell. For example, the
T cell can be contacted with a PI3K inhibitor together with a
protein tyrosine kinase inhibitor, such as herbimycin A.
[0030] To induce T cell unresponsiveness, an antigen-specific T
cell is contacted with an antigen in a form suitable to trigger a
primary activation signal in the T cell, which means that the
antigen is presented to the T cell such that a signal is triggered
in the T cell through the TCR/CD3 complex. For example, the antigen
can be presented to the T cell by an antigen presenting cell in
conjuction with an MHC molecule. A syngeneic antigen presenting
cell, such as a B cell, macrophage, monocyte, dendritic cell,
Langerhan cell, or other cell which can present antigen to a T
cell, can be incubated with the T cell in the presence of the
antigen such that the antigen presenting cell presents the antigen
to the T cell. Alternatively, to induce anergy to alloantigens, the
T cell can be incubated with an allogeneic cell, which presents
alloantigens to the T cell.
[0031] To induce T cell unresponsiveness to an antigen in vivo, an
agent which inhibits production of D-3 phosphoinositides in a T
cell is administered to a subject at a dose and for a period of
time sufficient to induce T cell unresponsiveness to the antigen.
Following administration of the agent, antigen-specific T cells are
contacted with the antigen endogenously (for example, an
autoantigen expressed by cells endogenously). Alternatively, to
induce T cell unresponsiveness to an antigen in vitro,. In this
case, T cells are obtained from a subject, contacted in vitro with
the antigen together with the agent to induce antigenic
unresponsiveness, and then readministered to the subject. For
example, T cells obtained from a transplant recipient can be
contacted with allogeneic cells from a graft donor together with an
agent which inhibits D-3 phosphoinositide production in the T cells
(e.g., wortmannin, quercetin, LY294002) prior to transplantation of
the graft into the recipient to induce alloantigen-specific T cell
unresponsiveness. The recipient T cells which have been rendered
unresponsive to the donor antigens are then readministered to the
recipient. Alternatively, in the case of bone marrow
transplantation, bone marrow to be transplanted (including any
residual T cells) can be contacted in vitro with allogeneic cells
from the bone marrow recipient together with an agent which
inhibits D-3 phosphoinositide production to induce unresponsiveness
in the donor T cells to recipient alloantigens. This pretreatment
can be performed to inhibit graft versus host disease.
[0032] The methods for inducing T cell unresponsiveness can be
applied therapeutically in situations where it is desirable to
downmodulate an immune response, such as transplantation, including
organ transplants and bone marrow transplants (as discussed above),
and autoimmune diseases and other disorders associated with an
abnormal immune response. Examples of autoimmune diseases or
disorders associated with an inappropriate or abnormal immune
response include rheumatoid arthritis, juvenile rheumatoid
arthritis, psoriatic arthritis, allergies, contact dermatitis,
psoriasis, leprosy reversal reactions, erythema nodosum leprosum,
autoimmune uveitis, multiple sclerosis, allergic encephalomyelitis,
systemic lupus erythematosus, acute necrotizing hemorrhagic
encephalopathy, idiopathic bilateral progressive sensorineural
hearing loss, aplastic anemia, pure red cell anemia, idiopathic
thrombocytopenia, polychondritis, scleroderma, Wegener's
granulomatosis, chronic active hepatitis, myasthenia gravis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's
disease, Graves ophthalmopathy, sarcoidosis, primary biliary
cirrhosis, primary juvenile diabetes, dry eye associated with
Sjogren's syndrome, uveitis posterior, and interstitial lung
fibrosis.
[0033] Another aspect of the invention pertains to stimulating a T
cell response by providing a costimulatory signal to a T cell.
Delivery of a costimulatory signal, in conjuction with a primary
activation signal, can generate a T cell response. In the method of
the invention, a costimulatory signal is provided by contacting a T
cell which has received a primary activation signal with an agent
which stimulates production of D-3 phosphoinositides in the T cell.
A T cell "response" is intended to encompass production of at least
one lymphokine by the T cell (e.g., IL-2) and/or proliferation by
the T cell. A primary activation signal can delivered to a T cell
by stimulating the T cell through the TCR/CD3 complex, for example
by anti-CD3 antibodies or by an MHC/antigen complex, or by use of
an agent which mimics this stimulation, for example a phorbol ester
(e.g., PMA). The term "agent", as used herein, is intended to
encompass chemicals and other pharmaceutical compounds which
stimulate a costimulatory signal in a T cell without the
requirement for an interaction between a T cell surface receptor
and a costimulatory molecule. For example, the agent may act
intracellularly to stimulate a signal associated with
costimulation. In one embodiment, the agent is a non-proteinaceous
compound. As the agent used in the method is intended to bypass the
natural receptor:ligand costimulatory mechanism, the term agent is
not intended to include a cell expressing a natural ligand of
CD28/CTLA4 (e.g., expressing B7-1 and/or B7-2).
[0034] Preferably, production of D-3 phosphoinositides in the T
cell is stimulated by contacting the T cell with an activator of
PI3K. Activators of PI3K can be identified, for example, by methods
described below. Additional agents which stimulate one or more
other intracellular signals associated with costimulation can be
used in conjuction with an inhibitor of D-3 phosphoinositide
production. For example, the T cell can be contacted both with a
first agent which stimulates PI3K activity and a second agent which
stimulates protein tyrosine phosphorylation within the T cell.
Protein tyrosine phosphorylation can be stimulated in the T cell,
for example, by contacting a T cell with an activator of protein
tyrosine kinases, such as pervanadate (see O'Shea, J. J. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:10306-103101; and Secrist, J.
P. (1993) J. Biol. Chem. 268:5886-5893). Alternatively, the T cell
can be contacted both with an activator of PI3K and with an agent
which inhibits the activity of a cellular protein tyrosine
phosphatase, such as CD45, to increase the net amount of protein
tyrosine phosphorylation in the T cell. The method also encompasses
stimulation of other intracellular signals associated with
costimulation of a T cell, such as stimulation of phospholipase C
activity and/or increases in intracellular calcium levels.
[0035] Another embodiment of the invention provides a method for
stimulating a specific response to an antigen by an
antigen-specific T cell. To stimulate a T cell response, an
antigen-specific T cell is contacted with the antigen together with
an agent which stimulates production of D-3 phosphoinositides in
the T cell, thereby triggering a costimulatory signal in the T
cell. Preferably, the agent which stimulates production of D-3
phosphoinositides in the T cell is an activator of PI3K. The T cell
is contacted with the antigen in a form suitable for stimulating a
primary activation signal in the T cell (e.g., through the TCR/CD3
complex), such as in conjuction with an MHC molecule. An antigen
presenting cell (e.g., a B cell, macrophage, monocyte, dendritic
cell, Langerhan cell, or other cell which can present antigen to a
T cell) can be incubated with the T cell in the presence of the
antigen (e.g., a soluble antigen). Alternatively, a cell expressing
an antigen of interest can be incubated with the T cell. For
example, a tumor cell expressing tumor-associated antigens can be
incubated with a T cell together with an agent which induces an
intracellular costimulatory signal to induce a tumor-specific
response. Alternatively, a cell infected with a pathogen, e.g. a
virus, which presents antigens of the pathogen can be incubated
with a T cell in the presence of the agent. In addition to
stimulating production of D-3 phosphoinositides in the T cell, the
T cell can be contacted with one or more other agents which
stimulate one or more additional intracellular signals associated
with CD28 ligation, for example an activator of a protein tyrosine
kinase, such as pervanadate.
[0036] An agent which stimulates a CD28-associated intracellular
signal in T cells, e.g., an activator of PI3K, can be administered
to a subject in vivo, or alternatively, a T cell can be obtained
from a subject, stimulated in vitro, and readministered to the
subject. The methods for stimulating T cell responses are useful in
therapeutic situations where it is desirable to upregulate an
immune response (e.g., induce a response or enhance an existing
response). For example, the method can be used to enhance a T cell
response against tumor-associated antigens. Tumor cells from a
subject typically express tumor-associated antigens but may be
unable to stimulate a costimulatory signal in T cells (e.g.,
because they lacks expression of costimulatory molecules). Thus,
tumor cells can be contacted with T cells from the subject together
with an agent which stimulates D-3 phosphoinositides in the T cell
to trigger a costimulatory signal in the T cell. Alternatively, T
cells can be stimulated as described herein to induce or enhance
responsiveness to pathogenic agents, such as viruses (e.g., human
immunodeficiency virus), bacteria, parasites and fungi.
Additionally, the efficacy of vaccines against such pathogenic
agents can be enhanced. For example, an agent which stimulates D-3
phosphoinositide production in T cells can be administered to a
subject infected with a pathogenic agent or can be coadministered
with a vaccine to enhance the responsiveness of T cells to antigens
of the vaccine. Alternatively, T cells can be cultured in vitro
which antigen presenting cells which express an antigen(s) from a
pathogenic agent together with an agent which stimulates an
intracellular signal associated with costimulation (e.g., an
activator of PI3K).
[0037] Another application of the method for stimulating T cell
responses pertains to patients who have impaired signal
transduction through CD28 and/or other cell surface molecule(s)
associated with costimulation (e.g., CTLA4). For example, a patient
with idiopathic thrombocytopenia has been reported to exhibit
defective CD28-mediated signal transduction, presumably due to a
congenital defect (see Perez-Blas, M. et al. (1991) Clin. Exp.
Immunol. 85:424-428). In patients having defective CD28 signalling
ability, it may be possible to bypass the defect and restore
CD28-dependent T cell activation by contacting T cells from the
patient with one or more agents which stimulate intracellular
signals generated upon normal CD28 ligation. For example, a patient
having a defect resulting in reduced or a lack of D-3
phosphoinositide production upon CD28 ligation can be treated by
contacting T cells from the patient with an agent which stimulates
production of D-3 phosphoinositides in the T cells.
[0038] Another aspect of the invention pertains to screening assays
for identifying inhibitors and activators of PI3K which can then be
used to inhibit or stimulate, respectively, T cell responses. PI3K
is a heterodimer consisting of a regulatory and a catalytic
subunit. Two forms of the enzyme which preferentially use
PtdIns(4,5)P.sub.2 as a substrate and are inhibitable by wortmannin
have been described (see Otsu, M et al. (1991) Cell 65:91-104; Hu,
P. et al. (1993) Mol. Cell. Biol. 13:7677-7688; and Hiles, I. D. et
al. (1992) Cell 70:419). Another form of the enzyme which
preferentially uses PtdIns as a substrate and is not inhibitable by
wortmannin has also been described (see Stephens, L. et al. (1994)
Curr. Biol. 4:203-214). It will be appreciated that identification
of specific inhibitors or activators of PI3K must be specific to
the appropriate intracellular form(s) of PI3K involved in
costimulatory signals to avoid unwanted or adverse side effects.
Thus, agents which specifically inhibit or activate a form(s) of
PI3K involved in costimulation (e.g., a form which is also
inhibitable by wortmannin) are preferable.
[0039] In one embodiment, a screening assay of the invention is
based upon the ability of an inhibitor or activator of a PI3K to
inhibit or stimulate, respectively, the production of at least one
D-3 phosphoinositides in a T cell (preferably
PtdIns(3,4,5)P.sub.3). To identify an inhibitor of a PI3K, a T cell
is stimulated through a cell surface receptor that binds a
costimulatory molecule (i.e., a T cell which has received a
costimulatory signal) in the presence and absence of a substance to
be tested. Preferably, a T cell which expresses CD28 is used in the
assay. Alternatively, a T cell which expresses CTLA4 can be used. A
costimulatory signal can be stimulated in the T cell by contacting
the T cell with a ligand for CD28 or CTLA4. Preferably, the ligand
is a physiologic ligand, such as membrane-bound B7-1 or B7-2,
rather than antibodies directed against the T cell surface
receptor. A cell which naturally expresses B7-1 and/or B7-2 can be
used or more preferably a cell (e.g., a CHO cell) which is
transfected to express a costimulatory molecule is used. In the
presence of an inhibitor of PI3K, stimulation of a T cell through a
surface receptor which binds a costimulatory molecule (e.g., CD28)
results in reduced production of D-3 phosphinositides in the T cell
relative to stimulation in the absence of the inhibitor. Production
of D-3 phosphoinositides in the T cell can be measured by any
suitable method known in the art. For example, production of D-3
phosphoinositides in the T cell can be measured by high pressure
liquid chromatography (as described in the Examples).
Alternatively, D-3 phosphoinositide production can be assessed by
thin layer chromatography, e.g. as described in Okada, T. et al.
(1994) J. Biol. Chem. 269:3563-3567. D-3 phosphoinositides whose
intracellular production can be assessed include PtdIns(3)P,
PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3. Preferably, production
of PtdIns(3,4,5)P.sub.3 in the T cell is detected in the presence
or absence of the substance to be tested.
[0040] To identify an activator of a PI3K, a T cell which expresses
a cell surface receptor which binds a costimulatory molecule is
contacted with a substance to be tested. An activator of a PI3K is
identified based upon its ability to stimulate production of at
least one D-3 phosphoinositides in a T cell (preferably
PtdIns(3,4,5)P.sub.3). Thus, in the presence of a PI3K activator,
the amount of a D-3 phosphoinositide in the T cell is increased
relative to the amount of the D-3 phosphoinositide in the T cell in
the absence of the substance. Production of D-3 phosphoinositides
(e.g., PtdIns(3)P, PtdIns(3,4)P.sub.2 and/or PtdIns(3,4,5)P.sub.3)
in the T cell can be assessed by standard methods, such as high
pressure liquid chromatography or thin layer chromatography, as
discussed above.
[0041] In another embodiment of the screening assays of the
invention, the ability of a substance to directly inhibit or
stimulate the activity of a PI3K isolated from a cell is assessed
and then a substance identified as an inhibitor or activator of the
PI3K is contacted with a T cell to determine the ability of the
substance to inhibit or stimulate a T cell response. For example,
an isolated PI3K is incubated with a substrate (e.g.
PtdIns(4,5)P.sub.2) in the presence of a radiolabeled phosphate
donor and a substance to be tested. An inhibitor of the kinase
activity of the PI3K will cause reduced phosphorylation of the
substrate (relative to phosphorylation in the absence of the
inhibitor), whereas an activator will cause increased
phosphorylation of the substrate (relative to phosphorylation in
the absence of the activator). An inhibitor or activator so
identified in vitro is then contacted with a T cell to determine
the ability of the inhibitor or activator to inhibit or stimulate,
respectively, a T cell response (e.g., IL-2 production).
Other Embodiments
[0042] Other cells types in addition to T cells have been described
which express CD28 on their surface. These cell types include
plasma cells (see Kozbor, D. et al. (1987) J. Immunol.
138:4128-4132) and bone marrow-derived mast cells. Stimulation of
other CD28.sup.+ cells types through CD28 may also lead to
production of D-3 phosphoinositides in the cells and generation of
specific cell responses. Inhibition or activation of D-3
phosphoinositide production in these cells, by the methods
described herein, may also be useful for inhibiting or stimulating
responses by other CD28.sup.+ cell types.
[0043] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLE 1
Ligation of CD28 Stimulates Production of D-3 Phosphoinositides
[0044] In this example, the production of D-3 phosphoinositides in
CD28.sup.+ Jurkat cells upon stimulation of the Jurkat cells
through CD28 or CD3 was examined. Jurkat cells were labeled with
carrier-free [.sup.32P]-orthophosphate ([.sup.32Pi]) as follows:
Jurkat cells were washed 3 times in phosphate-free media
(DMEM/RPMI) and incubated for 10 minutes at 37.degree. C. for 10
minutes between washes. The cells were resuspended in 10 ml
phosphate-free media containing 20 mM HEPES, pH 7.4 and 5% dialysed
fetal calf serum (dialysed overnight against saline). Carrier-free
[.sup.32Pi] was added to the cells (1 mCi/10 ml cells) and the
cells were incubated at 37.degree. C. for 4 to 6 hours. After
labeling, cells were washed 3 times with phosphate-free media and
resuspended in RPMI 1640 containing 20 mM HEPES.
[0045] In various experiments, aliquots of [.sup.32Pi]-labeled
Jurkat cells (0.15 ml; 2.times.10.sup.7 cells) were stimulated with
media alone, an anti-CD3 antibody (G19-4), an anti-CD28 antibody
(9.3), untransfected CHO cells, or CHO cells transfected to express
a CD28 ligand, either human B7-1 (CHO--B7-1) or B7-2 (CHO--B7-2).
[obtained from Drs. G. Freeman and L. Nadler; CHO cells were
transfected with a recombinant expression vector containing a cDNA
encoding human B7-1, the sequence of which is disclosed in Freeman,
G. J. et al. (1989) J. Immunol. 143:2714-2722, or a cDNA encoding
human B7-2, the sequence of which is disclosed in Freeman, G. J. et
al. (1993) Science 262:909-911, by standard techniques). For
stimulation with CHO cells, Jurkat cells were incubated with
10.sup.7 CHO cells and cell contact was achieved by low speed
centrifugation in a microfuge for 5 seconds. At various time
intervals following stimulation (ranging between about 1 and 25
minutes), the cells were lysed and phospholipids were extracted,
deacylated and separated by anion exchange HPLC basically as
described in Ward, S. G et al. (1992) J. Immunol. 22:45, with
modifications as described below.
[0046] The incubations were terminated by addition of 750 .mu.l
CHCl.sub.3/methanol/water (32.6%/65.3%/2.1% v/v/v). Once cell
reactions were quenched, the samples were kept on ice during
subsequent extractions. Phases were separated by addition of 725
.mu.l CHCl.sub.3 (containing 10 mg Folch lipids; e.g., from Sigma,
Catalogue No. B1502) and 172 .mu.l 2.4 M HCl, 5 mM
tetrabutylammonium sulphate to each sample. The samples were
vortexed and centrifuged for 5 minutes at 1000 rpm to separate
phases. The lower phase was removed and added to a tube containing
1/2 volume of 1 M HCl, 25 mM Na.sub.2EDTA, pH 7.0, 5 mM
tetrabutylammonium sulphate. The samples were recentrifuged to
separate phases, the bottom layer was removed and placed in a clean
tube, and the sample was dried down in vacuo. Samples were
deacylated by adding 1 ml methylamine reagent (40% in
water/methanol/n-butanol 4:4:1 v/v/v), vortexing and incubating at
53.degree. C. for 40 minutes. Samples were placed on ice and then
dried down in vacuo. The samples were resuspended in 0.5 ml sterile
distilled water and vortexed to mix. The samples were extracted
twice with 0.7 ml n-butanol:40-60% petroleum ether/ethyl fornate
(20:4:1 v/v/v). The bottom aqueous phase was dried in vacuo and
stored at -70.degree. C. prior to HPLC analysis.
[0047] HPLC was performed using a gradient based on buffers A
(water)/B [1.25 M (NH.sub.4).sub.2 HPO.sub.4] (adjusted to pH 3.8
with H.sub.3PO.sub.4 at 25.degree. C.) and a Partisphere SAX column
(commercially obtained from Whatman). Deacylated phospholipid
samples were resuspended in 0.1 ml distilled water and injected
onto the column. The eluate was fed into a Canberra Packard A-500
Flo-One on-line beta-radiodetector, where it was mixed with three
parts Flo-Scint IV scintillation cocktail and the results were
analyzed on the Flo-One data program (Radiomatic, USA). Eluted
peaks were compared to retention times for standards prepared from
[.sup.3H]PtdIns, [.sup.3H]PtdIns(4)P and
[.sup.3H]PtdIns(4,5)P.sub.2 (commercially obtained from Amersham
International). Standard [.sup.32P]PtdIns(3)P,
[.sup.32P]PtdIns(3,4)P.sub- .2 and [.sup.32P]PtdIns(3,4,5)P.sub.3
were prepared by incubating isolated phosphatidylinositol 3-kinase
with PtdIns (commercially obtained from Sigma) as described in
Ward, S. G. et al. (1992) J. Biol. Chem. 267:23862.
[0048] The production of PtdIns(3,4,5)P.sub.3 in Jurkat cells
following stimulation with anti-CD3 or anti-CD28 antibodies for 1-5
minutes is shown in FIG. 1. The results demonstrate that while
stimulation through either CD3 or CD28 induces PtdIns(3,4,5)P.sub.3
production, the induction kinetics for the two pathways are
distinct. Upon CD3 stimulation, PI3K activation (as assessed by
PtdIns(3,4,5)P.sub.3 production) increases early (i.e., within 2
minutes) and is transient (i.e., returns to baseline by 5 minutes).
In contrast, PI3K activation induced by CD28 stimulation is delayed
compared to CD3 stimulation (i.e., is not maximal until 5 minutes
or later) and persists longer than that induced by CD3 stimulation.
These results indicate that distinct mechanisms are involved in
PI3K activation mediated by either CD3 or CD28 ligation.
[0049] The production of PtdIns(3,4,5)P.sub.3 in Jurkat cells
following stimulation with CHO--B7-1 or CHO--B7-2 cells for 0-20
minutes is shown in FIG. 2. The results indicate that stimulation
of CD28 with either B7-1 and B7-2 induces potent activation of PI3K
(as assessed by PtdIns(3,4,5)P.sub.3 production). The induction
kinetics are slightly different for the two CD28 ligands: B7-1
stimulates activation earlier than B7-2, although both plateau to
the same level. Stimulation of PtdIns(3,4,5)P.sub.3 production by
either B7-1 and B7-2 is very strong and persistent (ie., continues
for more than 20 minutes).
[0050] This example demonstrates that stimulation of T cells
through CD28, either by its natural ligands B7-1 and B7-2 or by an
anti-CD28 antibody, induces the production of D-3 phosphoinositides
within T cells, indicating activation of PI3K upon CD28 ligation.
In addition, this example demonstrates that CD28 shares in common
B7-1 and B7-2 as physiological ligands, since Jurkat cells are
CD28.sup.+ but CTLA4.sup.- and cannot be induced to express CTLA4
(as shown in Lindsten, T. (1993) J. Immunol. 151:3489-3499). Thus,
CTLA4 apparently is not required for B7-induced signal transduction
and both B7-1 and B7-2 are physiologic ligands for CD28.
EXAMPLE 2
A Phosphatidylinositol 3-Kinase Inhibitor Can Inhibit Production of
D-3 Phosphoinositides Induced by CD28 Ligation
[0051] In this example, the effect of an inhibitor of
phosphatidylinositol 3-kinase on CD28-mediated production of D-3
phosphoinositides within Jurkat cells was examined. Jurkat cells
were labeled with orthophosphate and stimulated with CHO cells
transfected to express B7-2, as described in Example 1.
Additionally, during stimulation, the cells were incubated in the
presence of various concentrations (0-100 .mu.M) of the fungal
metabolite wortmannin, which is an inhibitor of
phosphatidylinositol 3-kinase. Wortmannin was obtained commercially
from Sigma Chemical Co. and stored as a 10 mM solution in DMSO at
-40.degree. C. It was diluted in medium immediately before addition
to cells in culture. Following stimulation in the presence or
absence of wortmannin, the amount of PtdIns(3,4,5)P.sub.3 produced
in the cells was measured by HPLC, as described in Example 1. The
results are shown in FIG. 3, wherein the amount of
PtdIns(3,4,5)P.sub.3 detected in wortmannin-treated Jurkat cells
upon stimulation with CHO--B7-2 is plotted graphically as a
percentage of the amount of PtdIns(3,4,5)P.sub.3 detected in
untreated Jurkat cells stimulated with CHO--B7-2. The results
demonstrate that treatment of Jurkat cells with increasing
concentrations of wortmannin decreases the amount of D-3
phosphoinositides produced in the cells upon ligation of CD28 with
B7-2. Accordingly, this example demonstrates that the generation of
D-3 phosphoinositides intracellularly as a result of stimulation of
T cells through CD28 can be inhibited by inhibiting the activity of
phosphatidylinositol 3-kinase within the T cells.
EXAMPLE 3
Effect of Pharmacological Inhibitors on CD28-Mediated Calcium
Flux
[0052] In this example, the effect of pharmacological inhibitors on
calcium flux in Jurkat cells induced by anti-CD28 antibodies was
examined. The pharmacological inhibitors studied were wortmannin,
which inhibits the activity of PI3K, and herbimycin A, which
inhibits the activity of protein tyrosine kinases. Jurkat cells
were stimulated with an anti-CD28 antibody, either in medium alone
or in the presence of wortmannin or herbimycin A, and the mean
calcium concentration (nM) in the cells was measured over several
minutes following stimulation. As illustrated in FIG. 4, herbimycin
A was capable of inhibiting CD28 antibody-induced calcium flux. In
contrast, wortmannin was unable to inhibit CD28-antibody induced
calcium flux. These results indicate that the effects of wortmannin
on T cells are not mediated by interfering with calcium flux.
Furthermore, given results described below in Example 5 showing
that wortmannin inhibits costimulation as measured by IL-2
production induced by B7-1 or B7-2, this data indicates that
measurement of CD28-induced calcium elevation is likely to be a
misleading read-out for assessing compounds that specifically
induce T cell unresponsiveness (i.e., anergy) or costimulation.
EXAMPLE 4
Induction of Calcium Flux by Natural CD28 Ligands
[0053] In this example, adhesion of CHO--B7-1 or CHO--B7-2 to
Jurkat cells and induction of calcium mobilization in Jurkat cells
in response to stimulation with CHO--B7-1 and B7-2 were examined. A
flow cytometic cell calcium-conjugate assay, as described in Abe,
R. et al. (1992) J. Exp. Med. 176:459-468, was used. In the cell
conjugate assay, T cells are loaded with the calcium sensitive
fluorescent probe indo-1 (which generates blue and green signals).
CHO cells transfected with a control plasmid (CHO-neo) or CHO cells
transfected to express B7-1 or B7-2 are loaded with the tracer dye
DilC22(3) (which generates red signals; obtained commercially from
Molecular Probes). Jurkat cell-CHO cell conjugates are analyzed by
flow cytometry. Conjugates consisting of T cells and CHO cells can
be measured by gating on red signals and calcium levels can be
measured in the T cells by gating on the blue and green signals.
The results are displayed as a series of two paramater dot plots,
shown in FIG. 5. Calcium (indo-1 ratio) is on the Y axis and cell
conjugates (red tracer) is on the X axis. Cells in the upper right
quadrant represent Jurkat cells having high levels of calcium
conjugated to CHO cells. Cells in the lower right quadrant
represent Jurkat cells having normal calcium levels conjugated to
CHO cells. Cells in the upper and lower left quadrants represent
non-conjugated Jurkat cells having high or low levels of calcium,
respectively.
[0054] The data indicates that both B7-1 and B7-2 can mediate
adhesion to Jurkat cells. However, both ligands are poor at causing
increases in calcium mobilization. Therefore, B7-1 and B7-2 are
much more efficient at inducing PI3K activation (see Example 1)
than calcium mobilization. In contrast, anti-CD28 antibodies are
capable of stimulating both PI3K activation (see Example 1) and
calcium flux (see Example 3). Thus, it appears that there are
differences in the intracellular signals generated through CD28
ligation, depending upon whether natural ligands (e.g., B7-1 or
B7-2) or antibodies are used for stimulation. It has previously
been described (Nunes, J. et al. (1993) Int. Immunol. 5:311-315)
that CD28 antibodies can have multiple and distinct effects on
biochemical aspects of T cell signal transduction and activation
(e.g., IL-2 production). These observations further indicate that
it was not possible to predict the biochemical effects of natural
ligands of CD28 (i.e., membrane-bound B7-1 and B7-2) on production
of D-3 metabolites, as described herein, based upon extrapolation
from previous results with CD28 antibodies.
EXAMPLE 5
A Phosphatidylinositol 3-Kinase Inhibitor Can Inhibit Production of
Interleukin-2 Induced by CD28 Ligation
[0055] In this example, the effect of an inhibitor of
phosphatidylinositol 3-kinase on CD28-dependent production of
interleukin-2 by T cells was examined. In a first series of
experiments, the effect of T cell stimulation through CD28, in
conjunction with stimulation through CD3, on IL-2 production was
assessed in the absence of wortmannin. Highly purified human
peripheral blood T cells were incubated for 24 hours with an
immobilized anti-CD3 antibody (OKT3) alone or together with either
an anti-CD28 antibody (9.3) or mitomycin C-treated CHO cells,
either untransfected or transfected to express B7-1 or B7-2.
Increasing numbers of CHO cells were tested (0.5.times.10.sup.6 to
4.times.10.sup.6). After culture for 24 hours, the culture
supernatants were assayed for IL-2 production by ELISA by standard
techniques. As shown in FIG. 6, cells incubated in medium alone,
OKT3 alone, or OKT3 together with untransfected CHO cells did not
produce IL-2. In contrast, culture of the cells with OKT3 together
with CHO cells expressing B7-1 or B7-2 stimulated IL-2 production
in a dose dependent manner. Culture with OKT3 and 9.3 antibodies
also stimulated IL-2 production. These results confirm that CD28
ligation, such as by B7-1 or B7-2 stimulation, can provide a
costimulatory signal for lymphokine production.
[0056] In a next series of experiments, resting human T cells were
stimulated with either: 1)immobilized OKT3+CHO--B7-1, 2)
immobilized OKT3+CHO--B7-2, 3) immobilized OKT3+CHO--B7-1+B7-2, 4)
PMA+CHO--B7-1 or 5) PMA+B7-2. Stimulation of T cells was performed
in media alone or in media containing wortmannin at concentrations
between 1 nM and 1 .mu.M. Twenty-four hours following culture, the
supernatants were assayed for IL-2 production by ELISA. The results
are shown in FIGS. 7A and 7B. The results indicate that wortmannin
can inhibit IL-2 production stimulated by either B7-1 or B7-2 in
conjunction with CD3 stimulation. Wortmannin-mediated inhibition of
IL-2 production was dose dependent. The ID.sub.50 for inhibition of
B7-2-mediated stimulation was approximately 10 nM. The ID.sub.50
for inhibition of B7-1-mediated stimulation was between 10 and 100
nM. These doses of wortmannin are not generally toxic to the cells
and do not inhibit IL-2 production by a non-specific mechanism, as
evidenced by the fact that IL-2 production stimulated by PMA
together with membrane-bound B7-1 or B7-2 was not inhibited by
wortmannin at concentrations as high as 1 .mu.M. This example
demonstrates that T cell activation, as assessed by production of
IL-2 in response to stimulation through the TCR/CD3 complex and
CD28, can be inhibited by treatment of the T cell with an agent
which inhibits the activity of phosphatidylinositol 3-kinase within
the T cell.
Equivalents
[0057] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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