Combined Analysis of Multiple Inhibitory T Cell Receptors on Cytomegalovirus Specific CD4 T Cells Allows Identification of Patients at Risk for Viral Reactivation after Transplantation

Abstract

We have previously shown that an increase of PD-1 and loss of IL-2 expression characterises an anergic phenotype of CMV-specific CD4 T cells that precedes CMV-disease in patients after renal transplantation. In this study, we compared the potential of three inhibitory T cell receptors for predicting episodes of viremia in patients after renal transplantation. Furthermore a score combining all three markers was assessed as a parameter to identify patients at risk for CMV-reactivation. We cross-sectionally analysed 25 healthy controls, 36 hemodialysis patients, 35 renal transplant recipients with (n=18) and without (n=17) viremia (all CMV-seropositive), and 8 patients with CMV-primary infection after transplantation. Longitudinal analyses comprised CMV-seropositive renal transplant recipients with (n=11) and without (n=8) CMV-reactivation. CMV-specific CD4 T cells were identified using flow-cytometric intracellular cytokine staining. The specific phenotypic characteristics of CMV-specific CD4 T cells were assessed by combined analysis of the inhibitory T cell receptors TIM-3, CTLA-4 and PD-1. To evaluate the strength in predicting episodes of viremia we calculated an anergy score for each inhibitory receptor (MFI on CMV-specific/MFI on non-CMV-specific CD4 T cells) and performed ROC analysis of viremic compared to non-viremic patients. All three inhibtory T cell receptors were found to be significantly upregulated on CMV-specific CD4 T cells in both patients with CMV-primary infection and CMV-reactivation, compared to healthy controls, hemodialysis patients and non-viremic patients (p< 0.0001 for each PD-1, CTLA4 and TIM-3). Longitudinal analysis showed an increase in inhibitory receptors in both patients with and without subsequent viremia, but initial expression levels were higher and the subsequent increase was more pronounced in patients developing viremia. In patients with CMV-reactivation, PD-1 and TIM-3 levels before transplantation (PD-1 MFI=383, TIM-3 MFI=230) increased to MFI=551 (PD-1) and MFI=313 (TIM-3) before onset of viremia and reached MFI=578 (PD-1) and MFI=436 (TIM-3) during viremia (PD-1: p< 0.003; TIM-3: p< 0.007). Increase in CTLA-4 expression was even more pronounced from MFI=1095 before transplantation to MFI=1503 prior to onset of viremia and MFI=2828 during viremia (p< 0.0001). For PD-1 we found an anergy score of >3.00 to be predictive for episodes of viremia (specificity=93.8%, sensitivity=52.0%, AUC=0.74, p< 0.01), compared to a score of >1.13 for TIM-3 (specificity=75.0%, sensitivty=76.0%, AUC=0.80, p< 0.01). Sensitivity was remarkably higher for the CTLA-4 anergy score of >6.6 (specificity=85.7%, sensititvity=68.0%, AUC=0.82, p< 0.001). Combined analysis of all three markers yielded in an anergy score of >10.9 being predictive of viremia (specifity=92.9%, sensitivity=64.0%, AUC=0.82, p=0.001). In conclusion, all three analysed inhibitory T cell receptors correlated with episodes of viremia in patients after renal transplantation, yet differences were most pronounced for CTLA-4. A combined anergy score of all three inhibitory receptors had the highest specificity and sensitivity as compared to analysing scores of any of the receptors individually. Consequently analysis of the CTLA-4 expression alone or in combination with additional markers has the potential to serve as a tool to identify patients at highest risk for CMV reactivation, facilitating an individualised therapeutic decision.