Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation

Abstract

It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25(+)CD45RB(low)CD4(+)) as those that control autoimmunity. Previous studies have failed to identify how CD25(+) Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor beta. Strikingly, constitutive expression of CTLA-4 among CD4(+) cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.

Figure 1

CD4⁺ regulatory T cells that control intestinal inflammation are CD25⁺CD45RB^low. (a) Representative photomicrographs show severe colitis in mice that received CD45RB^highCD4⁺ cells alone. (b) Inhibition of colitis in mice given CD45RB^high plus 2 × 10⁵ unfractionated CD45RB^lowCD4⁺ cells. (c) Regulatory T cell function is enriched within the CD25⁺CD45RB^low subset, as 10⁵ CD25⁺CD45RB^low cells inhibited colitis when transferred with CD45RB^highCD4⁺ cells, whereas (d) mice restored with a mixture of 10⁵ CD25⁻CD45RB^low and CD45RB^highCD4⁺ cells developed severe colitis. Severe colitis in mice restored with (e) CD45RB^high and 2 × 10⁵ CD45RB^lowCD4⁺ cells or (f) CD45RB^high and 10⁵ CD25⁺CD45RB^lowCD4⁺ cells and treated with anti-CTLA-4 mAb. Mice receiving CD45RB^high and CD45RB^lowCD4⁺ cells or CD45RB^high and CD25⁺CD45RB^lowCD4⁺ cells and treated with control hamster IgG were similar to those shown in b. Original magnification: ×80.

Figure 2

Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RB^highCD4⁺ cells alone (□) or in combination with 2 × 10⁵ CD45RB^lowCD4⁺ cells and treated with anti–CTLA-4 mAb (•) or purified hamster IgG (○). Asterisk indicates one of five mice killed on D32. Data represent the mean ± SEM for five mice per group. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus control IgG, P < 0.05. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RB^high cells alone and received anti–CTLA-4 mAb (▪) or control hamster IgG (□). Data represent the mean ± SEM for six mice per group. (c) SCID mice received CD45RB^highCD4⁺ cells (□), 2 × 10⁵ CD45RB^low cells (⋄), both CD45RB^highCD4⁺ cells and 2 × 10⁵ CD45RB^low cells (○), or CD45RB^highCD4⁺ cells in combination with 10⁵ CD25⁺CD45RB^low CD4⁺ cells (▵). Mice also received either anti–CTLA-4 (filled symbols) or control hamster IgG (open symbols). Data are pooled from three independent experiments. Significant protection was mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.05).

Figure 2

Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RB^highCD4⁺ cells alone (□) or in combination with 2 × 10⁵ CD45RB^lowCD4⁺ cells and treated with anti–CTLA-4 mAb (•) or purified hamster IgG (○). Asterisk indicates one of five mice killed on D32. Data represent the mean ± SEM for five mice per group. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus control IgG, P < 0.05. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RB^high cells alone and received anti–CTLA-4 mAb (▪) or control hamster IgG (□). Data represent the mean ± SEM for six mice per group. (c) SCID mice received CD45RB^highCD4⁺ cells (□), 2 × 10⁵ CD45RB^low cells (⋄), both CD45RB^highCD4⁺ cells and 2 × 10⁵ CD45RB^low cells (○), or CD45RB^highCD4⁺ cells in combination with 10⁵ CD25⁺CD45RB^low CD4⁺ cells (▵). Mice also received either anti–CTLA-4 (filled symbols) or control hamster IgG (open symbols). Data are pooled from three independent experiments. Significant protection was mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.05).

Figure 2

Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RB^highCD4⁺ cells alone (□) or in combination with 2 × 10⁵ CD45RB^lowCD4⁺ cells and treated with anti–CTLA-4 mAb (•) or purified hamster IgG (○). Asterisk indicates one of five mice killed on D32. Data represent the mean ± SEM for five mice per group. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus control IgG, P < 0.05. For CD45RB^high versus CD45RB^high plus CD45RB^low cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RB^high cells alone and received anti–CTLA-4 mAb (▪) or control hamster IgG (□). Data represent the mean ± SEM for six mice per group. (c) SCID mice received CD45RB^highCD4⁺ cells (□), 2 × 10⁵ CD45RB^low cells (⋄), both CD45RB^highCD4⁺ cells and 2 × 10⁵ CD45RB^low cells (○), or CD45RB^highCD4⁺ cells in combination with 10⁵ CD25⁺CD45RB^low CD4⁺ cells (▵). Mice also received either anti–CTLA-4 (filled symbols) or control hamster IgG (open symbols). Data are pooled from three independent experiments. Significant protection was mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RB^low cells (P < 0.01) and CD25⁺ cells (P < 0.05).

Figure 3

CTLA-4 is expressed constitutively on CD25⁺CD45RB^low CD4⁺ cells. (a–c) CTLA-4 staining on permeabilized (a) CD45RB^highCD4⁺, (b) CD25⁺CD45RB^lowCD4⁺, and (c) CD25⁻CD45RB^lowCD4⁺ splenocytes. SSC, side scatter. (d–f) CTLA-4 expression on the progeny of (e) CD25⁺CD45RB^lowCD4⁺ and (d and f) CD45RB^highCD4⁺ cells after transfer into immunodeficient mice. C57BL/6 recombination activating gene (RAG)2^−/− mice received CD45RB^highCD4⁺ cells from C57BL/6 mice (CD45.2) (d) alone or (e and f) in combination with 10⁵ CD25⁺CD45RB^lowCD4⁺ cells from C57BL/6.SJL.CD45 congenic mice (CD45.1). 4 wk after transfer, cells from the mesenteric lymph nodes were stained for CTLA-4 and congenic marker expression. Results are representative of four mice (CD45RB^high alone) and seven mice (CD45RB^high plus CD25⁺) from two independent experiments.

Figure 4

Regulation of colitis by CD25⁺CD4⁺ cells is TGF-β dependent. SCID mice received CD45RB^highCD4⁺ cells alone (□) or in combination with 10⁵ CD25⁺ cells (▵). Mice also received either anti–TGF-β (filled symbols) or PBS (open symbols). Significant protection mediated by CD25⁺ cells (P < 0.01) abrogated by treatment with anti–TGF-β mAb (P < 0.02).