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. 2000 Jul 17;192(2):295-302.
doi: 10.1084/jem.192.2.295.

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

S Read  1 , V Malmström, F Powrie
Affiliations

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

S Read et al. J Exp Med. .

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.

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Figures

Figure 1
Figure 1
CD4+ regulatory T cells that control intestinal inflammation are CD25+CD45RBlow. (a) Representative photomicrographs show severe colitis in mice that received CD45RBhighCD4+ cells alone. (b) Inhibition of colitis in mice given CD45RBhigh plus 2 × 105 unfractionated CD45RBlowCD4+ cells. (c) Regulatory T cell function is enriched within the CD25+CD45RBlow subset, as 105 CD25+CD45RBlow cells inhibited colitis when transferred with CD45RBhighCD4+ cells, whereas (d) mice restored with a mixture of 105 CD25−CD45RBlow and CD45RBhighCD4+ cells developed severe colitis. Severe colitis in mice restored with (e) CD45RBhigh and 2 × 105 CD45RBlowCD4+ cells or (f) CD45RBhigh and 105 CD25+CD45RBlowCD4+ cells and treated with anti-CTLA-4 mAb. Mice receiving CD45RBhigh and CD45RBlowCD4+ cells or CD45RBhigh and CD25+CD45RBlowCD4+ cells and treated with control hamster IgG were similar to those shown in b. Original magnification: ×80.
Figure 2
Figure 2
Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RBhighCD4+ cells alone (□) or in combination with 2 × 105 CD45RBlowCD4+ 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 CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus control IgG, P < 0.05. For CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RBhigh 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 CD45RBhighCD4+ cells (□), 2 × 105 CD45RBlow cells (⋄), both CD45RBhighCD4+ cells and 2 × 105 CD45RBlow cells (○), or CD45RBhighCD4+ cells in combination with 105 CD25+CD45RBlow 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 CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.05).
Figure 2
Figure 2
Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RBhighCD4+ cells alone (□) or in combination with 2 × 105 CD45RBlowCD4+ 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 CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus control IgG, P < 0.05. For CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RBhigh 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 CD45RBhighCD4+ cells (□), 2 × 105 CD45RBlow cells (⋄), both CD45RBhighCD4+ cells and 2 × 105 CD45RBlow cells (○), or CD45RBhighCD4+ cells in combination with 105 CD25+CD45RBlow 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 CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.05).
Figure 2
Figure 2
Anti–CTLA-4 treatment abrogates the function of regulatory T cells. (a) SCID mice were reconstituted with CD45RBhighCD4+ cells alone (□) or in combination with 2 × 105 CD45RBlowCD4+ 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 CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus control IgG, P < 0.05. For CD45RBhigh versus CD45RBhigh plus CD45RBlow cells plus anti–CTLA-4, results were not significant (Student's t test). (b) SCID mice were reconstituted with CD45RBhigh 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 CD45RBhighCD4+ cells (□), 2 × 105 CD45RBlow cells (⋄), both CD45RBhighCD4+ cells and 2 × 105 CD45RBlow cells (○), or CD45RBhighCD4+ cells in combination with 105 CD25+CD45RBlow 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 CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.01). Adminstration of anti–CTLA-4 mAb abrogated protection mediated by CD45RBlow cells (P < 0.01) and CD25+ cells (P < 0.05).
Figure 3
Figure 3
CTLA-4 is expressed constitutively on CD25+CD45RBlow CD4+ cells. (a–c) CTLA-4 staining on permeabilized (a) CD45RBhighCD4+, (b) CD25+CD45RBlowCD4+, and (c) CD25−CD45RBlowCD4+ splenocytes. SSC, side scatter. (d–f) CTLA-4 expression on the progeny of (e) CD25+CD45RBlowCD4+ and (d and f) CD45RBhighCD4+ cells after transfer into immunodeficient mice. C57BL/6 recombination activating gene (RAG)2−/− mice received CD45RBhighCD4+ cells from C57BL/6 mice (CD45.2) (d) alone or (e and f) in combination with 105 CD25+CD45RBlowCD4+ 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 (CD45RBhigh alone) and seven mice (CD45RBhigh plus CD25+) from two independent experiments.
Figure 4
Figure 4
Regulation of colitis by CD25+CD4+ cells is TGF-β dependent. SCID mice received CD45RBhighCD4+ 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).
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