*Purpose: CTLA-4Ig blocks CD80/CD86, the ligands for both CD28 and CTLA-4. Thus, in addition to the intended effect of blocking CD28-mediated costimulation, CTLA4-Ig also has the unintended effect of blocking CTLA-4-mediated coinhibitory signaling. Recently, anti-CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA-4 signaling intact were shown to more effectively inhibit alloimmune responses and prolong graft survival in both murine models and non-human primates. However, the impact of selective CD28 blockade on protective immunity has not yet been investigated.

*Methods: We sought to compare the impact of CTLA-4Ig vs. anti-CD28dAb on murine CD8⁺ T cell immunity in the setting of a transplant-relevant pathogen, a murine homolog of Epstein-Barr virus. C57BL/6 mice were infected with 2×10³ PFU of a YFP-labeled recombinant murine gammaherpesvirus-68 (MHV-68) and treated with saline control, CTLA-4Ig, or anti-CD28 dAb. Splenocytes from infected mice were harvested at 14 and 28 days post infection and anti-viral immune responses and viral load were assessed via flow cytometry.

*Results: While anti-CD28dAb treatment resulted in a decrease in virus-specific CD8⁺ T cell frequencies and numbers as compared to CTLA-4Ig, effector function in the form of IFN-gamma production was not different between treatment groups. Similarly, viSNE analysis revealed that the expression of CXCR3 and CD27, surface markers indicating high-quality effector function, was also similar between the anti-CD28dAb and CTLA-4Ig treated groups. Additionally, CITRUS analysis of co-stimulatory and co-inhibitory marker expression (PD-1, TIM-3, and TIGIT) on MHV-specific CD8⁺ T cells demonstrated no difference between the two treatment groups. Importantly, MHV viral load was not significantly different between the anti-CD28dab and CTLA-4Ig treatment groups.

*Conclusions: We demonstrate that preserved CTLA-4 co-inhibition limits MHV-specific T cell accumulation, but the virus-specific CD8⁺ T cell population that remains retains sufficient effector function to control viral burden. These data indicate that use of selective CD28 blockade, in the form of non-crosslinking anti-CD28 dAb, is equivalent to CTLA-4Ig in its ability to control viral load during gammaherpesvirus infection, further highlighting the clinical promise of this therapy.

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