Introduction: T cell allo-reactivity is limited by T cell receptor that recognize specific combination of allo-antigen and major histocompatibility complex on the antigen presenting cells. Understanding the characteristics and behavior of donor reactive T cells is crucial for assessing immune response after organ transplantation. We investigated the relevance of a novel assay that rapidly detects alloreactive T cells based on activating molecules (CD154 and CD137) expression after mixed lymphocyte stimulation using with activated donor B cells in a mouse skin transplant model.
Method: We assessed immune responses in a rejection model of MHC full-mismatched combination (BALB/c into C57BL/6) compared with those in a syngeneic combination on postoperative days (POD) 3 and 7. To observe immune response during tolerance induction, immune responses were also evaluated on POD 7 in a peripheral tolerance-induction model with CTLA-4 IgG and anti-CD40L antibody with MHC full-mismatched combinations (BALB/c into C3H and C57BL/6). Comprehensive alloreactive T cell detection assays were performed by co-culturing recipient T cells with CFSE-labeled and irradiated B cells that were activated for 48h prior to assay with multimeric CD40L and rIL-4. Monensin-supplemented medium was used to evaluate cytokine production. After 18-h incubation, CD4+ and CD8+ alloreactive T cells were detected using CD154+ and CD137+, respectively.
Result: In the rejection model, CD154+ and CD137+ donor-reactive T cell proportions were high among CD4+ and CD8+ T cells, respectively, on POD 7 (syngeneic vs rejection, median %alloreactive CD4+; 1.0% vs 1.4%, p< .05 and CD8+; 0.27% vs 0.53%, p< .05, respectively). The donor reactive CD8 T cells showed notable increase in interferon gamma and granzyme B production. With tolerance induction, long-term engraftment was observed only in the combination of BALB/c graft into C3H recipient. All BALB/c grafts into C57BL/6 recipients with tolerance induction were rejected within 20 days. Compare with the failed tolerance induction animals (BALB/c into C57BL/6), the proportion of donor reactive CD8++ T cells in tolerant animals (BALB/c into C3H) was significantly lower suggesting different immunogenicity resulting different tolerance outcome (C57BL/6 recipients vs C3H recipients, median % donor-reactive CD8+; 0.35% vs 0.18%, p< .05).
Conclusion: This assay effectively detected sensitized immune status in mice skin transplantation model, highlighting its potential for quantitative and qualitative monitoring of alloreactive T cells reflecting immune response. Monitoring donor reactive effectors could be ideal for managing immunosuppressive therapy and answering questions in translational research.