Abstract-CD8ϩ cytotoxic T lymphocytes contribute to viral and autoimmune myocarditis and cardiac allograft rejection. The role of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 as a negative regulator of CD4 ϩ T cells is well defined, yet CTLA-4 regulation of CD8 ϩ T cells is less clear. We studied CTLA-4 regulation of cytotoxic T lymphocytes in a transgenic model of CD8 ϩ T-cell-mediated myocarditis. We generated CTLA-4 Ϫ/Ϫ Rag 2 Ϫ/Ϫ OT-1 mice, the CD8 ϩ T cells of which express an ovalbumin (OVA) peptide-specific, class I major histocompatibility complex-restricted T-cell receptor.
CTLA-4Ϫ/ϪTc12 OT-1 effectors, differentiated with interleukin-12 present, are hyperproliferative in vitro, compared with CTLA-4 ϩ/ϩTc12 OT-1 controls. Transfer of low doses of CTLA-4 Ϫ/ϪTc12 OT-1 cells to cMy-mOVA mice, which express OVA on cardiac myocytes, causes severe myocarditis, with 99% mortality, compared with no mortality after transfer of low doses of CTLA-4 ϩ/ϩTc12 OT-1 cells. High doses of CTLA-4 ϩ/ϩTc12 cells cause lethal myocarditis in cMy-mOVA mice, but high doses of CTLA-4 ϩ/ϩTc0 CTL, generated without interleukin-12, are hypoproliferative within the cardiac-draining lymph node and do not significantly infiltrate the heart. In contrast, CTLA-4 Ϫ/ϪTc0 cytotoxic T lymphocytes do proliferate in the cardiac-draining lymph node and diffusely infiltrate the heart. Nonetheless, high doses of CTLA-4 Ϫ/ϪTc0 cells cause only limited tissue damage, and the disease is not lethal. These data show that CTLA-4 regulates myocarditic CD8 ϩ T cell responses and that CTLA-4 deficiency partly overcomes a differentiation block that exists when naïve CD8 ϩ T cells are stimulated without interleukin-12. Therefore, targeting CTLA-4 solely or in conjunction with interleukin-12 could influence effector CD8 ϩ T cell responses in therapeutically beneficial ways. ϩ T-cell activation and differentiation by members of the B7 and CD28 regulatory molecules remain incompletely understood. Cytotoxic T-lymphocyte antigen (CTLA)-4 is a CD28 family member that is expressed on the surface of lymphocytes after T-cell receptor (TCR) stimulation. 1 CTLA-4 and CD28 share specificity for the B7 molecules CD80 (B7-1) and CD86 (B7-2). It is well documented that CD80 and CD86 binding to CTLA-4 on CD4 ϩ T cells results in downregulation of cell-cycle progression and interleukin (IL)-2 production. 2,3 In contrast, there are conflicting studies regarding the effects of CTLA-4 ligation on CD8 ϩ T-cell regulation. 4,5 Several reports indicated that CTLA-4 blockade does not alter cytotoxic T lymphocyte (CTL) responses to lymphocytic choriomeningitis virus, mouse mammary tumor virus, or Leishmania major infection in mice. 4,6,7 Yet experiments with CTLA-4 -deficient class I major histocompatibility complex-restricted TCR transgenic mice indicate that CTLs are hyperresponsive to antigen on restimulation, suggesting a negative regulatory role in CTL activation. 4,5,8 Furthermore, blockade of CTLA-4 with neutralizing antibody augments CTL-mediated responses against tu...