The dual specificities of B7-1 and B7-2 for CD28 and CTLA-4 have made it challenging to elucidate physiological interactions in the B7-CD28/CTLA-4 pathway. The uncontrolled T cell activation seen in the CTLA-4−/− mice provides a valuable experimental system for dissecting in vivo functions of B7-1 and B7-2, and for searching for additional members of this family of costimulators. To do this, we have generated mouse strains lacking CTLA-4 and either B7-1, B7-2, or both B7 molecules, and analyzed them for evidence of T cell activation in vivo and in vitro. The phenotypes of the CTLA-4/B7-1−/− and CTLA-4/ B7-2−/− strains demonstrate overlapping roles for B7-1 and B7-2 in CD28-mediated signaling, as the presence of either B7-1 or B7-2 is sufficient to produce the CTLA-4−/− phenotype. However, the CTLA-4/B7-1−/− mice have shorter survival and greater T cell activation than age-matched CTLA-4/B7-2−/− mice. These differences may reflect earlier expression and higher cell surface levels of B7-2 compared with B7-1. Since either B7 molecule is capable of producing lymphoproliferation and T cell activation in the CTLA-4−/− mice, it seems unlikely that B7-1 and B7-2 produce fundamentally different signals through CD28.
The striking absence of T cell activation in the CTLA-4/ B7-1/B7-2−/− TKO strain demonstrates that B7-1 or B7-2 is required to produce the CTLA-4−/− lymphoproliferative disease. The absence of CD28-mediated costimulation in the CTLA-4/B7-1/B7-2−/− TKO strain is demonstrated by the (a) lack of lymphoproliferation in vivo, as evidenced by the normal life-span and absence of splenomegaly, lymphadenopathy, and lymphocytic infiltrates; (b) naive phenotype of T cells in vivo; (c) lack of spontaneous proliferation of splenocytes in vitro; and (d) impaired proliferative response to CD3 stimulation in vitro. Similar results have been obtained by treating CTLA-4−/− mice with CTLA4Ig. Our results extend these studies and show that B7-1 or B7-2 is capable of activating CTLA-4−/− T cells.
This requirement for B7-1 or B7-2 in activating CTLA-4−/− T cells is most consistent with a role of unopposed signaling by CD28, the only known activating T cell receptor for B7 molecules. We have formally established this by showing that anti-CD28 antibody can reproduce disease in CTLA-4/B7-1/B7-2−/− TKO mice. Interestingly, the same anti-CD28 antibody has no detectable effect on T cell activation in wild-type mice in vivo. These findings demonstrate the critical balance between CD28-mediated T cell activation and CTLA-4–mediated downregulation, and suggest that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signals in vivo. These results may explain why many studies show no or inconsistent in vivo effects of anti-CD28 antibodies, which are known to be strong agonists when added to T cells in the absence of APCs in vitro.
We have not found evidence for additional stimulatory CD28 ligands in the CTLA-4/B7-1/B7-2−/− TKO mice. This strain provides an exquisitely sensitive means for examining whether additional stimulatory CD28 counterreceptors exist, since in the absence of CTLA-4, even the effects of weak CD28-mediated signaling would be apparent. The profound deficits observed in the CTLA-4/B7-1/ B7-2−/− TKO mice would suggest that if additional B7 ligands exist, they either do not stimulate T cell activation through CD28, or they are expressed in a restricted microenvironment. Such microenvironments might include organs such as the brain or kidney, which exhibit no inflammatory changes in CTLA-4−/− mice.
The CTLA-4/B7-1/B7-2−/− TKO strain has a phenotype that appears to be comparable to the B7-1/B7-2−/− strain both in vivo and in vitro. Therefore, in the absence of B7-1 and B7-2, the presence of CTLA-4 has no detectable downregulatory effect. Because T cells from the CTLA-4/B7-1/B7-2−/− TKO strain are mostly naive, unlike cells from CTLA-4−/− mice, CTLA-4/B7-1/B7-2−/− TKO mice also can serve as a useful tool to study the function of CTLA-4 during the initial activation of T cells.