D. Mou1, J. R. Espinosa1, J. Kwun1, N. N. Iwakoshi1, A. D. Kirk2 1Emory University School Of Medicine,Surgery,Atlanta, GA, USA 2Duke University Medical Center,Surgery,Durham, NC, USA
Introduction:
Belatacept, a B7-specific fusion protein that blocks CD28-B7 costimulation to prevent kidney allograft rejection, is ineffective in a sizable minority of transplant recipients. Although T cell receptor and CD28 engagement initiates T cell activation, many human antigen-experienced memory T cells lose CD28, and can be activated without CD28 signals. We posit that these cells are central drivers of belatacept resistant rejection (BRR) and propose that they may arise from antigen exposure. CD28 loss is poorly described in mice, which are typically kept in pathogen-free conditions.
Methods:
To study mice in a clinically relevant scenario of viral exposure, we characterized T cell CD28 expression after sequential infections, 3 weeks apart, with Polyomavirus (PyV, BK virus homolog), murine CMV, and mHV68 (EBV homolog). 5 mice cohorts, each containing 10 C57BL/6 mice, were defined as mock infections, single PyV infection, single mCMV infection, single mHV68 infection, or ‘all 3’ infections. Flow analysis was performed on the day of infection, at peak infection, and at the memory time point. Mixed lymphocyte reactions (MLRs) of splenocytes were conducted to assess in vitro alloreactivity. Alloreactivity was further evaluated both in the presence and absence of costimulation blockade (CTLA4-Ig and MR1). We are currently performing heart transplants in infected and uninfected mice to assess the phenotypic role of CD28 loss in BRR.
Results:
CD28 MFI analysis of both CD4 and CD8 cells showed significant (P<0.05) CD28 down-regulation in the effector TMs all infected cohorts. MLRs revealed that the mHV68 and triply infected cohorts exhibited significantly (P<0.05) higher alloreactivity than the naïve cohorts. Interestingly, the triple infected cohort demonstrated significantly (P<0.05) more IFNy production than the mHV68 cohort. The increased alloreactivity is resistant to costimulation blockade.
Conclusions:
Clinically relevant viruses PyV, mCMV, and mHV68 induce CD28 down-regulation in mice, suggesting that Polyoma, CMV, and EBV infections may play a role in human BRR. Additionally, mHV68 exposure confers enhanced alloreactivity in MLRs, suggesting that viruses may lead to functionally relevant changes in the immune repertoire. The MLR observation that the triply infected cohort produced more IFNy than the mHV68 cohort is suggestive of an additive alloreactivity effect that is driven by multiple viral infections. Overall, the data suggest that clinically relevant viruses may play a significant role in determining how solid organ transplant patients respond to costimulation blockade therapies.