Download Progress Report

Progress Report
Aditya Murthy
991368337
May 12, 2017
Does CTLA-4 cross-linking on activated CD4+CD25- cells convert them to a
regulatory phenotype in vitro?
Aditya Murthy
Abstract
The subset of T cells that are CD4+CD25+ regulate the activity of other T cells and play
an important role in controlling response to self vs. non-self antigens (Ags). The ability of
these Regulatory T cells (Treg) to suppress an immune response via educating other T
cells has potential utility in treating various autoimmune diseases such as immune
deficient diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), and
experimental autoimmune thyroiditis (EAT) among others. However, the function of the
CD4+CD25- cells, termed Effector T cells (Teff), in regulating an immune response has
not been fully investigated. Both Treg and Teff cells, once activated, express the negative
costimulatory receptor Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4;
CD152). CTLA-4 is perhaps the most well documented signalling molecule involved in
the suppression of an immune response by Treg cells. The mechanism of CTLA-4 activity
and its consequences have not been fully elucidated. In this study we analysed the ability
of CD4+CD25+ and CD4+CD25- cells to convert naïve T cells to a regulatory phenotype
via CTLA-4 cross-linkage in vitro. Thus far, the project’s experimental data shows that
activated Treg cells and Teff cells display similar levels of cell surface CTLA-4. Therefore
the functional roles of CTLA-4 in these T cell subsets need to be compared in order to
elucidate the mechanism(s) involved in CTLA-4 mediated conversion of naïve T cells to
a regulatory phenotype.
1
Progress Report
Aditya Murthy
991368337
May 12, 2017
Regulatory T Cells
Treg cells constitute 5-10% of the T cell population and control other T cells, suppressing
their activity and thereby preventing harmful autoimmune responses to self Ags. Treg cells
have been categorised as those T cells that express CD4, CD25 (IL-2R α-chain), and
constitutively express CTLA-4 on the cell surface (1-3). On the other hand, Teff cells are
those that are CD4+CD25- and do not constitutively express CTLA-4 (4-6). These cells
are under the control of the Treg subpopulation, and exhibit pro- or anti- inflammatory
effects, either through humoral or cell-mediated mechanisms depending on the stimulus
provided. It is well established that the Treg cells have suppressive functions and
downregulate an immune response by relaying anti-proliferative signals to the Teff
population (1, 7). Consequently, the Teff cells respond by developing a regulatory
phenotype themselves (8, 9). Additionally, a forkhead-winged-helix transcription factor
(foxp3) has been the subject of great interest in the development and maintenance of Treg
cells. Several groups (7, 10-12) have shown that foxp3 is not only a specific marker for
Treg cells, but is needed to convert naïve T cells to a regulatory phenotype. However these
findings require further investigation, since T cell phenotypes change drastically as
periods of stimulation, activation and anergy affect gene expression patterns over time.
2
Progress Report
Aditya Murthy
991368337
May 12, 2017
CTLA-4
Several attempts to explain the suppressive function of Treg cells, ranging from direct
cell-cell contact to release of regulatory cytokines (1, 7-9), list CTLA-4 as the most
potent costimulatory molecule involved in Treg mediated suppression of Teff activity.
However a specific mechanism of CTLA-4 function that affects Teff phenotype has yet to
be elucidated. The well established two signal theory states that TCR engagement with its
respective antigen-MHC complex and CD28 engagement with its ligands B7.1 (CD80)
and B7.2 (CD86) are required for T cell activation (13-15). This activation results in the
expression of CTLA-4 on CD4+CD25- cells that otherwise have biologically insignificant
levels of surface CTLA-4. Since Treg cells constitutively express greater levels of surface
CTLA-4, activation results in enhanced potency of Treg function (16).
CTLA-4 is a negative costimulator of T cell function and has high affinity for its ligands
B7.1 and B7.2 on APCs (17, 18). It is a homologue of CD28, which is a positive
costimulator of T cell activity and has higher T cell surface expression levels than CTLA4, but lower affinity for ligands B7.1 and B7.2 (19-23). Thus the two costimulatory
receptors compete for the same ligand, and a dynamic balance of Teff activation or
suppression is maintained via the ratio of CTLA-4 and CD28 concentrations, and their
subsequent interaction with B7 ligands. The cross-linking of CTLA-4 with B7.1 leads to
immunosuppressive consequences, downregulating T cell proliferation and increasing
secretion of regulatory cytokines such as TGF-β and IL-10 (4, 13, 20). Several in vivo
studies have clearly shown that CTLA-4 deficiency results in severe lymphoproliferative
disorders (2, 4, 24). Thus the importance of CTLA-4 in immunosuppression has been
well established.
3
Progress Report
Aditya Murthy
991368337
May 12, 2017
Progress
Cell surface CTLA-4 expression levels on inactive and active Treg and Teff cells were
compared by flow cytometry. It was observed that, when activated, CD4+CD25- (Teff)
cells exhibit CTLA-4 levels similar to that of activated CD4+CD25+ (Treg) cells. These
findings may suggest functional similarity between active Treg and Teff populations. If
similarity does indeed exist (and is otherwise absent in Teff populations not expressing
high levels of CTLA-4), then one may conclude that Treg cells can convert Teff cells to a
regulatory phenotype, and that CTLA-4 is a critical factor in this conversion.
Future
To test for functional similarity, the following assays will be performed: Comparison of
the cytokine profiles (IL-10, TGF-β) of inactive Treg and Teff cells cells activated by
CTLA-4 cross-linking with plate-bound anti-CTLA-4 mAbs. ELISA will be used to
compare the cytokine concentrations. This will answer questions pertaining to the cellautonomous effects of CTLA-4 cross-linking. Finally to test the suppressive effect of
activated Teff cells, naïve T cells will be combined with activated Teff and Treg cells in
separate assays in varying ratios. An MTT proliferation assay will be performed, and the
two populations will be compared for their ability to suppress T cell proliferation in vitro.
4
Progress Report
Aditya Murthy
991368337
May 12, 2017
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Zheng S.G., Wang J.H., Gray J.D., Soucier H., Horwitz D.A. (2004). Natural and Induced CD4+CD25+ Cells
Educate CD4+CD25- Cells to Develop Suppressive Activity: The Role of IL-2, TGF-, and IL-10. Journal of
Immunology, 172:5213-5221.
Takahashi T., Tagami T., Yamazaki S., Uede T., Shimizu J., et al. (2000). Immunologic Self-Tolerance
Maintained by CD25+CD4+ Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyteassociated Antigen 4. Journal of Experimental Medicine, 192(2):303-309.
Read, S. Malmstrom V., Powrie F. (2000). Cytotoxit T Lymphocyte-associated Antigen 4 Plays an Essential
Role in the Function of CD25+CD4+ Regulatory Cells that Control Intestinal Inflammation. Journal of
Experimental Medicine, 192(2):295-302.
Chen W., Jin W., Wahl S.M. (1998). Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4) Induces Transforming Growth Factor  (TGF-) Production by Murine CD4+ T Cells. Journal of
Experimental Medicine, 188(10):1849-1857.
Alegre M-L., Frauwirth K.A., Thompson C.B. (2001). T-Cell Regulation by CD28 and CTLA-4. Nature
Reviews Immunology, 1: 220-228.
Viola A., Lanzavecchia A. (1996). T Cell Activation Determined by T Cell Receptor Number and Tunable
Thresholds. Science, 273:104-106.
Nishimura E., Sakihama T., Setoguchi R., Tanaka K., Sakaguchi S. (2004). Induction of antigen-specific
immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising
Foxp3+CD25+CD4+ regulatory T cells. International Immunology, 16(8):1189-1201.
Fantini M.C., Becker C., Monteleone G., Pallone F., Galle P.R., Neurath M.F. (2004), Cutting edge: TGF-
induces a Regulatory Phenotype in CD4+CD25- T Cells through Foxp3 Induction and Down-Regulation of
Smad7. Journal of Immunology, 172(9):5149-5153.
Liang S., Alard P., Zhao Y., Parnell S., Clark S.L., Kosiewicz M.M. (2005). Conversion of CD4+CD25- cells
into CD4+CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. Journal of
Experimental Medicine, 201(1)127-137.
Ochs H.D., Ziegler S.F., Torgerson T.R. (2005). FOXP3 acts as a rheostat of the immune response.
Immunological Reviews, 203:156-164.
Karagiannidis C., Akdis M., Holopainen P., Woolley N.J., Hense G., Ruckert B., et al. (2004).
Glucocorticoids upregulate FOXP3 expression and regulatory T cells in astma. Journal of Allergy and
Clinical Immunology, 114(6):1425-1433.
Hori S., Nomura T., Sakaguchi S. (2003). Control of Regulatory T Cell Development by the Transcription
Factor Foxp3. Science, 299:1057-1061.
Vasu C., Gorla S.R., Prabhakar, B.S., Holterman M.J. (2003). Targeted engagement of CTLA-4 prevents
autoimmune thyroiditis. International Immunology, 15(5):641-654.
Linsley P.S. (1995). Distinct roles for CD28 and Cytotoxic T Lymphocyte-associated Molecule-4 Receptors
during T Cell Activation? Journal of Experimental Medicine, 182(2):289-292.
Bretscher P., Cohn M. (1970). A Theory of Self-Nonself Discrimination. Science, 169:1042-1049.
Tang Q., Boden E.K., Henriksen K.J., Bour-Jordan H., Bi M., Bluestone J.A. (2004). Distinct roles of CTLA4 and TGF- in CD4+CD25+ regulatory T cell function. European Journal of Immunology, 34:2996-3005.
Chambers C.A., Allison, J.P. (1999). Costimulatory regulation of T cell function. Current Opinion in Cell
Biology, 11:203-210.
Peach, R.J., Bajorath J., Brady W., Leytze G., Greene J., Naemura J., et al. (1994). Complementarity
Determining Region 1 (CDR1)-and CDR3-analogous Regions in CTLA-4 and CD28 Determine the Binding
to B7-1. Journal of Experimental Medicine, 180(6):2049-2058.
Allison J.P., Krummel M.F. (1995). The Yin and Yang of T Cell Costimulation. Science, 270:932.
Prud’homme G.J. (2004). Altering immune tolerance therapeutically: the power of negative thinking. Journal
of Leukocyte Biology, 75(4):586-599.
Bluestone J.A. (1997). Is CTLA-4 a Master Switch for Peripheral T Cell Tolerance? Journal of Immunology,
158:1989-1993.
Haspot F., Villemain F., Laflamme G., Coulon F., Olive D., Tiollier J., Soulillou J-P., Vanhove B. (2002).
Differential effect of CD28 versus B7 blockade on direct pathway of allorecognition and self-restricted
responses. Blood, 99:2228-2234.
Chen W., Jin W., Hargeden N., Lei K., Li L., Marinos N., McGrady G., Wahl S.M. (2003). Conversion of
Peripheral CD4+CD25- Naïve T Cells to CD4+CD25+ Regulatory T Cells by TGF- Induction of
Transcription Factor Foxp3. Journal of Experimental Medicine, 198(12);1875-1886.
Fowler S., Powrie F. (2002). CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required
for oral tolerance. European Journal of Immunology, 32:2997-3006.
5