*Purpose: CMV remains a threat for kidney transplant recipients (KTR). Deciphering the impact of CMV on the immune system of KTR has helped improving CMV outcomes and management. In this study, we combined phenotype and functional response analyses to investigate the early immune response to CMV of KTR.

*Methods: In a population of 28 CMV positive KTR, we sampled PBMC on 5 occasions: pre-transplantation, then 1-month (M1), 3-months (M3), 12-months (M12). For each sample, we simultaneously assessed a CyTOF (Cytometry by Time Of Flight) extended T phenotype using 35 antibodies and the CMV-specific T-ELISpot functional response against IE1 and pp65 antigens. We also investigated the CMV replication events. Using semi-supervised clustering of the phenotypes, we identified Natural Killer T cells (NKT) subsets and gamma-delta T cells subsets and evaluated their association with CMV replication events and with both functional responses.

*Results: Among the 28 patients, 12 patients underwent a CMV-replication event, at a median time of 1.8 (IQ 1-3) months. Overall, the whole populations of NKT or gamma-delta cells were not associated with the outcome nor the functional response. On the other hand, senescent CD4 and exhausted CD8 populations were positively associated with both functional responses. In the semi-supervised CyTOF analysis, there were 12 NKT and 12 gamma-delta clusters. We show heterogeneity within NKT and gamma-delta cells. In a Projection to Latent Structures – Discriminant Analysis (PLS-DA), we combined clusters in meta-clusters associated with a CMV replication event. A subset of 3 NKT clusters was inversely associated with both CMV replication events and with concomitant functional responses, when compared to the 9 other clusters that had the inverse behavior (figure 1). These 3 NKT clusters were defined by CD11b (ITGAM) expression. Similarly, a subset of gamma-delta T cells was positively correlated with the pp65 response, expressing CD11c. The other cluster expressing CD11c but negatively correlated with the functional response expressed PD1, a known exhaustion marker.

*Conclusions: Using the extended phenotyping allowed by CyTOF analysis, this study shows the added value of subpopulations delineation among known lymphocytes populations, when compared to traditional flow cytometry. This suggests investingating CD11b on NKT cells and CD11c on gamma-delta cells.

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