B7-1 or B7-2 is required to produce the lymphoproliferative phenotype in mice lacking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)

Abstract

The costimulatory molecules B7-1 and B7-2 regulate T lymphocyte activation by delivering activating signals through CD28 and inhibitory signals through cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The importance of CTLA-4-mediated inhibition was demonstrated by the uncontrolled T cell activation and lymphoproliferative disease that develops in CTLA-4-deficient (-/-) mice. To examine the role of B7 signaling in the activation of CTLA-4-deficient T cells, we bred CTLA-4(-/-) mice with mice lacking B7-1, B7-2, or both B7 molecules. The CTLA-4/B7-1(-/-) and the CTLA-4/B7-2(-/-) mice develop lymphoproliferation and enhanced T cell activation. Mice lacking CTLA-4, B7-1, and B7-2 have a normal life-span, and do not have lymphocytic infiltrates in any organs, or increased T cell activation. Therefore, the two B7 molecules have overlapping functions, since either B7-1 or B7-2 alone can cause the CTLA-4(-/-) phenotype. Elimination of both B7-1 and B7-2 from the CTLA-4- deficient mouse abrogates the lymphocyte activation and disease, and does not reveal evidence for additional stimulatory CD28 ligands. The CTLA-4(-/-) phenotype can be reproduced with anti-CD28 antibody in mice lacking CTLA-4, B7-1, and B7-2, but wild-type mice are unaffected by the same treatment. This suggests that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signaling in vivo.

Figure 1

Either B7-1– or B7-2–mediated signaling is sufficient to produce fatal lymphoproliferative disease in mice lacking CTLA-4. Mice were observed daily, and survival was recorded. Survival of CTLA-4^−/−, CTLA-4/B7-1^−/−, and CTLA-4/B7-2^−/− strains is shown. Each mouse is indicated by a different symbol, and mean life-span is shown as a horizontal bar. Wild-type and CTLA-4/B7-1/B7-2^−/− TKO mice consistently live longer than 300 d.

Figure 2

Removal of B7-1– and B7-2–mediated signaling prevents the lymphocytic infiltration and necrosis of organs observed in CTLA-4^−/− mice. Heart (A, B, and C) and pancreas (D, E, and F) are shown (original magnification: ×50). CTLA-4^−/− mice (B and E), as well as CTLA-4/B7-1^−/− and CTLA-4/B7-2^−/− mice (not shown), killed at 2 wk of age show severe pancreatitis and myocarditis, in contrast to wild-type littermates (A and D). CTLA-4/B7-1/B7-2^−/− TKO mice killed at 2 mo show no evidence of inflammatory infiltrates (C and F).

Figure 3

In the absence of CTLA-4, in vivo T cell activation requires B7-1 or B7-2. Splenocytes were isolated from 2-wk-old mice and double stained with PE–anti-CD3 and FITC– anti-CD69 for analysis by flow cytometry. The percentage shown next to each dot plot indicates the proportion of CD3⁺ cells that are CD69⁺. Results are representative of five mice of each strain.

Figure 4

Proliferation of CTLA-4^−/− T cells requires B7-1– or B7-2– mediated costimulation. (A) Proliferative responses of unstimulated splenocytes from 2-wk-old mice of the indicated strain at 24 h are shown. Data are representative of three experiments, and the mean of two mice of each strain are shown. (B) Proliferative responses of anti-CD3-stimulated splenocytes from 2-mo-old mice at 48 h are shown. Data are representative of four experiments. The mean of two mice of each strain are shown. Proliferation at all time points was assayed in triplicate, with SD < 15% of the mean.

Figure 4

Proliferation of CTLA-4^−/− T cells requires B7-1– or B7-2– mediated costimulation. (A) Proliferative responses of unstimulated splenocytes from 2-wk-old mice of the indicated strain at 24 h are shown. Data are representative of three experiments, and the mean of two mice of each strain are shown. (B) Proliferative responses of anti-CD3-stimulated splenocytes from 2-mo-old mice at 48 h are shown. Data are representative of four experiments. The mean of two mice of each strain are shown. Proliferation at all time points was assayed in triplicate, with SD < 15% of the mean.

Figure 5

Administration of anti-CD28 antibody to CTLA-4/B7-1/ B7-2^−/− TKO mice activates T cells. CTLA-4/B7-1/B7-2^−/− TKO and wild-type mice received either anti-CD28 or control hamster IgG. At days 14–19, freshly isolated splenocytes were stained for CD3 and the activation marker CD69, and analyzed by flow cytometry. The percentage shown next to each dot plot indicates the proportion of CD3⁺ cells that are CD69⁺. Data are representative of at least three mice of each strain.