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Stimulation of CD25+CD4+ regulatory T cells through
GITR breaks immunological self-tolerance

Jun Shimizu, Ph.D.
Immunological Aging Research Group,

Accumulating evidence suggests that T cell-mediated control of self-reactive T cells contributes to the maintenance of natural immunological self-tolerance. For example, depletion of CD4+ CD25+ T cells, which constitute 5~10% of peripheral CD4+ T cells in normal naive mice (Fig. 1A), leads to the spontaneous development of various autoimmune diseases in genetically susceptible animals; reconstitution of the depleted population prevents the development of autoimmunity.
CD4+ CD25+ T cells have unique immunological characteristics compared with other regulatory or suppressive T cells. They do not proliferate in response to antigenic stimulation in vitro (that is, they are naturally anergic) and can potently suppress the activation and proliferation of other CD4+ (Fig. 1B, open symbols) or CD8+ T cells in an antigen-nonspecific manner through cell-to-cell interactions. Ligation of CD28 expressed on CD4+ CD25+ T cells, in conjunction with T cell receptor (TCR) stimulation, can break their anergic and suppressive states (Fig. 2). In addition, they constitutively express CTLA-4. Blockade of CTLA-4 abrogates CD4+ CD25+ T cell-mediated suppression in vitro (Fig. 2). This indicates that CTLA-4 may be a costimulatory molecule for activating CD4+ CD25+ T cells.
To investigate the molecular basis of CD4+ CD25+ T cell-mediated self-tolerance, we attempted to characterize cell surface molecules that are expressed on CD4+ CD25+ T cells and are engaged in their activation and proliferation or exertion of suppression. We raised monoclonal antibodies (DTA-1) capable of neutralizing in vitro CD4+ CD25+ T cell-mediated suppression and characterized the molecules recognized by the DTA-1. We showed that the glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR, also known as TNFRSF18) ---a member of the tumor necrosis factor-nerve growth factor (TNF-NGF) receptor family of proteins---is the molecules recognized by the DTA-1 and predominantly expressed on CD4+ CD25+ T cells (Fig. 1C) and on CD4+ CD8-CD25+ thymocytes in normal naive mice. We found that stimulation of GITR abrogated CD4+ CD25+ T cell-mediated suppression (Fig. 1B, closed symbols, and Fig. 2). In addition, removal of GITR-expressing T cells or administration of DTA-1 produced organ-specific autoimmune disease in otherwise normal mice.
Elimination or reduction of the number of CD4+ CD25+ T cells can also induce effective tumor immunity in otherwise nonresponding mice. Based on the role of GITR in attenuating the suppressive activity of CD4+ CD25+ T cells, it may be feasible to use anti-GITR treatment as an immunotherapy for cancer either alone or in combination with anti-CD25 and/or anti-CTLA-4 treatment. In addition to tumor immunity, there is accumulating evidence that CD4+ CD25+ T cells play a crucial role in maintaining transplantation tolerance.
Modulation of GITR-mediated signal transduction may break allograft tolerance as well as self-tolerance; alternatively, blockade of GITR-mediated signals may enable CD4+ CD25+ T cells to maintain allograft tolerance more stably. Thus, GITR may be a suitable molecular target for preventing or treating autoimmune disease, including tumor immunity, or establishing transplantation tolerance in humans.

Figure 1

Figure 1
DTA-1 mAb abrogates suppression mediated by CD4+ CD25+ T cells. (A) CD4+ CD25- or CD4+ CD25+ T cells (gated as a or b, respectively) were purified by cell-sorter from BALB/c spleen cells. (B) CD4+ CD25+ T cells (open square and closed square) or CD4+ CD25- T cells (open circle and closed circle) purified from 2-month-old BALB/c mice, or these two populations mixed in equal amounts (open triangle and closed triangle), were stimulated for 3 days along with MMC-treated spleen cells as APC in the absence or presence of graded amounts of DTA-1 mAb. Incorporation of [3H] thymidine by proliferating lymphocytes during the last 6 hr of the culture was measured. (C) Spleen cell suspensions prepared from 2-month-old BALB/c mouse were stained with anti-CD4, anti-CD25 and DTA-1. Expression of GITR (DTA-1) on CD4+ CD25+ T cells or CD4+ CD25- T cells is shown in the histogram. The dotted lines represent control staining with an irrelevant Ab.

Figure 2
CD4+ CD25+ T cell-mediated suppression of the activation of self-reactive T cells.


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