*Purpose: In transplant recipients, disease severity after infection with Epstein-Barr virus (EBV) may range from asymptomatic DNAemia to PTLD. Host factors likely play a key role in this including the functionality and number of EBV-specific T-cells. Exhaustion markers expressed on T-cells such as PD-1, TIM-3, CTLA-4 and TOX may also be important indicators of overall functionality and may influence clinical parameters. We assessed expression of these T-cell exhaustion markers in patients with EBV DNAemia.

*Methods: We prospectively enrolled consecutive adult organ transplant recipients with EBV DNAemia (>10³ IU/mL). We performed flow cytometry on isolated PBMCs to simultaneously characterize cell-surface expression of T-cell-associated exhaustion markers (PD-1, CTLA-4, TIM-3 and TOX) on CD4 and CD8 T-cells. Functionality was evaluated by measuring EBV-specific cytokine (IFN-γ, IL-2, TNF-α)-producing T-cells following stimulation with an EBV-infected cell lysate at 10 µg/mL. Viral loads were measured clinically as close as possible to PBMC collection.

*Results: We enrolled 25 patients with median age 53 years (range 21-74). Types of transplants included liver (36%), heart (28%), kidney (24%), lung (8%), and small bowel (4%). The median time post-transplant to PBMC sampling was 2.0 years (range 0.2-21). Frequencies of T-cells expressing markers of exhaustion varied in the cohort, with PD-1 expression being highest on CD8 (median 6.2%, range 2.0-54%) and CD4 T-cells (median 10%, range 1.9-18%), followed by TOX, TIM-3 and CTLA-4. Patients were categorized according to high or low proportions of PD-1, TIM-3, CTLA-4 or TOX positive T-cells and compared according to the viral load contemporaneous with PBMC collection. Patients with higher proportions of TOX⁺ T-cells had comparatively lower viral loads (p=0.02 for CD4, p=0.03 for CD8). No other associations were observed with respect to exhaustion markers and viral load. In relation to EBV-specific T-cells, those with higher proportions of PD-1⁺ or TOX⁺ CD4 T-cells had higher frequencies of EBV-specific TNF-α producing CD4 T-cells (p=0.004 for both). Furthermore, patients with higher proportions of TIM-3⁺ CD8 T-cells had lower frequencies of polyfunctional CD8 T-cells (p=0.02), defined as those producing TNF-α, IL-2 and IFN-γ. Finally, those with higher proportions of CTLA-4⁺ T-cells had lower frequencies of EBV-specific polyfunctional CD4 T-cells (p=0.008) and TNF-α⁺ CD8 T-cells (p=0.01).

*Conclusions: We observed that many patients with EBV DNAemia exhibited markers of T-cell exhaustion. However, exhaustion markers were not necessarily correlated with negative virologic consequences and in some instances were actually associated with lower viral loads and higher EBV-specific T-cell responses. Future studies will assess the role of T-cell exhaustion as a risk for PTLD development.

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