OR5 Phenotypic and functional evaluation of KIR-regulated natural killer cells responding to Epstein – Barr virus: Implications on post-transplant lymphoproliferative disease (PTLD) after allogeneic HCT

Aim Recipients of allogeneic HCT remain vulnerable to a heightened risk of reactivation of otherwise latent viral infections owing to a compromised immune system early after transplantation. Reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disease (PTLD) is one of such major complications after T-cell depleted HCT. We have determined that copy number variation in donor KIR genes and motifs influences NK Cells’ response to EBV and modifies the risk of developing PTLD after HCT. Here we set out to elaborate the NK cell phenotypes and transcripts associated with their response to EBV targets. Methods Next generation sequencing based KIR profiles of 30 healthy volunteer donors were obtained. NK cells negatively selected from PBMNCs of these donors were stimulated with EBV-transformed target cells. Flow cytometry based enumeration of NK cells that were either degranulating (CD107a expression) or producing cytokine (IFN- γ ), as well as their simultaneous KIR-based phenotypic characterization was performed. RNA was extracted before and after co-stimulation of NK cells with EBV targets, on which the transcript analysis was performed using a custom NanoString codeset containing probes for 53 NK cell function related genes. NK cells responding to targets and their phenotypes were expressed as percentages of parent and the correlations were performed using Mann–Whitney Wilcoxon test. Empirical Bayes statistics for differential gene expression were performed to identify associated NK cell transcripts using R statistical packages. Results Twenty-four distinct NK cell subsets were identified based on the expression of NKG2A and various combinations of KIR proteins that differentially correlated with response to EBV targets. Significant differences in expression of NK cell signaling proteins (DAP10, FYN, PKB) and effectors (GM-CSF, IFN- γ , TNF) were observed, which correlated with KIR tA01, a motif associated with higher functional NK response to EBV, and protection from PTLD after HCT. Conclusions Our findings provide vital mechanistic clues to NK cells’ response to EBV. Phenotypic and functional reconstitution kinetics of identified subsets and transcripts after HCT can identify patients at a high risk for developing PTLD and help enable a pre-emptive intervention.