*Purpose: A recent, multi-center immunosuppression withdrawal trial in liver transplant recipients unexpectedly revealed time post-transplant as an important predictor for operational tolerance. We hypothesize that T cell exhaustion promotes graft tolerance. The OPTIMAL study (ITN056ST, NCT02533180), a similarly structured immunosuppression withdrawal trial sponsored by the ITN, is being conducted to test this hypothesis. T cell exhaustion has a characteristic cell phenotype of expressing accumulated activation-induced inhibitory and costimulatory molecules. However, detecting these molecules on allo-reactive cells is challenging due to cell rarity and the dynamic nature of these markers. Here, we develop a novel assay to detect phenotypically exhausted allo-reactive cells using flow-based staining for a combination of early activation markers (CD154, CD137 and CD69) and select surface markers.

*Methods: PBMC from healthy, non-transplanted donors were stimulated by a mixed lymphocyte reaction (MLR) assay to activate allo-reactive responder cells. PBMC from healthy donors were CFSE labeled and served as stimulator cells. To enhance allo-reactivity, stimulator B cells were activated with CD40L multimer and rIL-4 for 2 days pre-assay, resulting in significantly higher expression of HLA class I, class II, and CD86 compared to non-activated B cells. Responder T cells were identified by exclusion of CFSE, CD14, CD16, and CD19 positive cells.

*Results: 12 hours of stimulation allowed for optimal expression of PD-1, TIM-3 and LAG-3, while expression of other activation/exhaustion marker candidates (2B4, TIGIT, CD28, and CD127) was too labile under stimulating conditions. At 12 hours, CD154⁺CD69⁺ and CD137⁺CD69⁺ gating detected 0.54±0.33% CD4⁺ and 0.40±0.17% CD8⁺ allo-reactive T cells, respectively (mean±SD, self-background 0.04±0.01% in CD4⁺, 0.08±0.05% in CD8⁺).These populations were significantly increased when compared to non-activated stimulators for both CD4⁺ (0.18±0.13%) and CD8⁺ (0.21±0.11%) T cells (p<0.05). PD-1, TIM-3, and LAG-3 were rarely detected in this primary allo-response.

*Conclusions: These studies were undertaken to demonstrate the feasibility of allo-stimulation, and the allo-specific detection of activation/exhaustion related markers. This novel assay allows for future work to assess whether allo-specific exhaustion is a relevant mechanism of operational tolerance following immunosuppression withdrawal. In the coming months, cells from operationally tolerant and non-tolerant patients will be assayed with this technique. Ultimately, this assay will be used prospectively to assess its predictive value as a biomarker of spontaneous operational tolerance in liver transplant recipients undergoing immunosuppression weaning.

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