*Purpose: Chronic active antibody-mediated rejection (CAAMR) induced by de novo donor-specific antibodies (DSA) is the main cause of graft loss in the long-term. Currently, there is no effective treatment or predictive marker for CAAMR exists. Therefore, a reliable immunoresponse monitoring system to identify recipients who will develop CAAMR in the future is required to improve long-term allograft survival. A novel CFSE/MLR assay was developed to evaluate T cell responses in kidney transplant recipients.

*Methods: This assay was developed using isolated T cells as responders, which is much more sensitive to detect T cell responses compared with the standard MLR using PBMCs. In this study, 84 kidney transplant recipients were evaluated using this novel assay. The recipients were divided into two groups (stable; n=60, DSA+; n=24) and their T cell responses were compared.

*Results: In pathological analyses, all recipients in the stable group experienced any incidents of rejection, whereas 18 DSA+ recipients developed CAAMR and 6 DSA+ recipients still did not develop CAAMR (pre-CAAMR). The MLR assay revealed that the anti-donor CD4⁺/CD8⁺ T cell responses were significantly higher in DSA+ recipients than in stable recipients. Interestingly, in stable recipients, the anti-donor CD4⁺ T cell response was significantly lower than the anti-third-party response, and the donor-specific hyporesponsiveness was also observed in CD8⁺ T cells. In contrast, donor specific hyporesponsiveness of CD4⁺ T cells disappeared in DSA+ recipients. To elucidate the underlying mechanisms for DSA production, the levels of INF-γ, IL-4, IL-17 and FOXP3 in responder T cells were evaluated to identify which CD4⁺ T cell subsets were expanding. Proliferating CD4⁺ T cells showed a marked increase in the Th1 (CD4⁺INF-γ⁺) and Th17 (CD4⁺IL-17⁺) response in DSA+ recipients, whereas there was no difference in donor reactive Th2 (CD4⁺IL4⁺) and Treg (CD4⁺FOXP3⁺). Evaluation of the six pre-CAAMR recipients showed a similar CD4⁺ T cell landscape, suggesting that the novel MLR assay can predict the development of CAAMR.

*Conclusions: DSA+ recipients have a greater potential for immune responses against the donor tissue. CFSE/MLR using isolated T cells is a useful immunoresponse monitoring system to predict the development of CAAMR.

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