Polyomavirus-BK (BKV) associated nephropathy is a known cause of graft failure. No specific therapy is established so far and viral load monitoring accompanied by adjustment of immunosuppression is the only known effective therapeutic option. We previously demonstrated BKV-specific T-cells as a factor predicting BKV-clearance and disease recovery. However, data on the role and specificity of different T-cell subsets including cytolytic/helper CD4 and CD8 T-cells is scarce. Moreover, the impact and contribution of T-cells specific to early (LT, st) and late (VP1-3) viral antigens is also not defined.

We implemented a 17-parameter flow cytometry protocol and investigated the sensitivity and robustness of variable effector molecules (IFNg, TNFa, IL2, IL4, IL17, GranzymeB) and receptors (4-1BB, CD40L, TIM3, PD1) as BKV-specific activation markers under immunosuppression. By detecting BKV-specific T-cells according to the expression of the receptors CD137 and CD154 in combination with the effector molecule GranzymeB, we were able to detect specific T-cells more sensitively (compared to IFNg-based approaches) and categorized them into cytolytic/helper T-cells. Subsequently, antiviral immunity of 47 kidney transplant patients in clinical follow up and of 15 healthy volunteers was dissected into cytolytic and helper T-cell responses according to early (st, LT) and late (VP1-3) BKV-antigen specificity.

Our approach increased the sensitivity of detecting of BKV-specific T-cells by 4.9-fold (median) in comparison to previously used INFg-based detection by flow cytometry. Of importance, we showed that BKV clearance was observed only when both, cytolytic and helper, T-cells were simultaneously detected. Interestingly, cytolytic CD4 T-cells significantly contribute to viral clearance. Additionally we could assign cytolytic CD4 and CD8 T-cell responses to early and helper CD4 T-cell responses to late BKV-antigens.

By using surface markers together with the cytolytic molecule GranzymeB we showed for the first time that concerted action of cytolytic and helper T-cells directed against early and late BKV-antigens, respectively, is required for BKV clearance. This new approach allows a more sensitive and reliable monitoring of BKV-specific T-cells. It can prevent underestimation of BKV-specific immunity and improve the adjustment of immunosuppression, minimizing the risk of graft rejection.

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