*Purpose: It is well known that certain infections can impair the stability of transplantation tolerance in recipients who had prolonged functional grafts, resulting in graft-loss. However, the underlying mechanism of pathogen-induced abrogation of tolerance is unclear. Cytomegalovirus (CMV) infection is highly prevalent and has long been associated with allograft-rejection. Herein, we aimed to investigate the impact of CMV infection on allograft tolerance in previously tolerant recipients, and the underlying mechanism of tolerance impairment.

*Methods: A fully mismatch mouse model of pancreatic islet transplantation (Balb/c to C57BL/6J) was used. Tolerance was induced by intravenous infusion of donor apoptotic splenocytes (treated with ethylene carbodiimide; ECDI-SP) on days -7 and +1. Tolerant recipients with normoglycemia for >90 days were infected with murine CMV strain Δm157 (10⁸ PFU). In some experiments, tolerant recipients were treated with anti-FR4 antibody (TH6) to deplete FR4⁺ anergic CD4 T cells prior to MCMV infection.

*Results: Infusion of ECDI-SP induced the allograft tolerance and these recipients carried functional grafts indefinitely. MCMV infection in these recipients however rescinded tolerance resulting in graft-loss within 2-4 weeks. Concurrently, allografts from uninfected tolerant recipients were enriched with CD44⁺CD4 T cells displaying an anergic phenotype (FR4^HICD73^HIPD1⁺CTLA4⁺). Interestingly, post-MCMV infection, the anergic phenotype of intragraft CD4 T cells was lost and this was corelated with tolerance impairment and graft-rejection. These data suggested that intragraft anergic CD4 T cells might contribute in tolerance maintenance in these recipients. However, depletion of anergic CD4 T cells (~80%) using anti-FR4 antibody did not impair allograft tolerance. In fact, depletion of anergic CD4 T cells from the tolerant recipients prior to MCMV infection prevented the disruption of transplantation tolerance resulting in prolonged allograft survival.

*Conclusions: Collectively, our data suggest that (1) tolerance promotes T cell-anergy, (2) intragraft anergic CD4 T cells in tolerant recipients might be alloreactive and contribute in graft-rejection by acquiring an effector phenotype post MCMV infection, and (3) depletion of anergic CD4 T cells using anti-FR4 antibody prevents tolerance impairment and graft-rejection in tolerant recipients infected by MCMV.

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