*Purpose: SOT recipients are at high risk for EBV-associated PTLD. EBV viremia is a risk factor for PTLD. In some patients viremia is self-limited while others develop a chronic viremia phenotype. An in-depth profiling of the adaptive immune response associated with chronic EBV viremia could provide insight into the pathogenesis of EBV-associated PTLD, and could potentially inform the identification of biomarkers of clinical illness.

*Methods: We performed a cross-sectional study of patients with EBV viremia (≥ 1,000 copies/mL). Viral loads (VL) were checked either due to routine monitoring for high-risk (EBV D+/R-) patients or in patients with suspected EBV disease/PTLD. Patients meeting the criteria had PBMCs cryopreserved. Patients (n=28) were organized into two a priori defined groups: self-limiting viremia (SLV=11) and chronic viremia (CV=17). Chronic EBV viremia was defined by a whole blood VL ≥1000 copies/mL on at least 3 consecutive readings over one year. Mass cytometry (CyTOF) was performed on PBMCs stimulated with EBV lysate, then stained with lanthanide-conjugated antibodies targeting 29 cell-surface and intracellular markers associated with adaptive immunity.

*Results: Demographics, transplant type, and immunosuppression were similar between SLV and CV groups. Using conventional manual gating strategies, we measured significantly higher frequencies of CD4+ central memory (Tcm) (30.1% vs 16.2%, p=0.017) and effector memory (Tem) T-cells (39.4% vs 25.5%, p=0.041) in patients with chronic EBV viremia compared to SLV group. In contrast, significantly lower frequencies of naïve T-cells (Tn) were measured in patients with CV (22.0% vs 40.3%, p=0.011) than SLV. No differences in CD8+ T-cells or B-cells were measured. Among T-cells expressing stimulatory (CD27, CD28, CD40L, OX40) or inhibitory (PD-1, Fas, CTLA4, Tim3) checkpoints, we detected increased frequencies of Fas+ CD4+ T-cells in those with chronic viremia (65.1% vs 48.0%, p=0.048). Lower frequencies of EBV-associated IFNγ-producing CD4+ T-cells were also detected in the CV vs SLV group (1.05% vs 1.26%, p=0.014). We used the unsupervised CITRUS algorithm with the SAM correlative association model to identify T-cell clusters that were differentially expressed between groups. In concordance with our supervised, manual gating approach, CITRUS analysis revealed a CD4+ T-cell cluster at significantly higher frequency in the chronic viremia group (FDR = 1%). This cluster had high CD95; intermediate CD28, CD127 and CD45RO; low HLA-DR, CD69, CD40L, OX40, CD27, PD1 and CCR7; with negative CD57, CD8, CD45RA, CTLA4, CD38 and Tim-3, suggestive of Tem or Tcm phenotype.

*Conclusions: Our study shows the differential immune response between patients with CV and SLV and suggests that CD4+ naive T-cells and EBV-specific IFNγ producing T-cells are important for the control of viremia whereas central and effector memory T-cells may also play a role in pathogenesis.

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