*Purpose: Identifying and phenotyping donor-reactive T cells in recipients following transplantation may further the understanding of interactions between the recipient’s immune system and donor antigen. Detecting donor-reactive T cells is difficult as they are sparse in number and potentially hyporesponsive, especially in operationally tolerant recipients.

*Methods: To this end, we have established and optimized a novel flow-based assay to detect and phenotype alloreactive T cells in cryogenically preserved PBMC samples.

*Results: By resting healthy donor responder PBMC for one day pre-assay, the inherent surface expression of activation-related surface markers was minimized (n=6). We found that activating stimulator cells with CD40L multimer+rIL-4 for 2 days pre-assay increased their ability to elicit T cell response. Responder PBMC were separately co-cultured with both stimulated self-cells (as a reference, n=12) and HLA-disparate, third party stimulator cells (total reaction, n=70). Reactive responder cells were defined by co-expression of the early activation markers CD69 and CD154 on CD4+ T cells, and CD69 and CD137 on CD8+ T cells. A time course experiment revealed that 12 hours of responder and stimulator co-culture was optimal for both detecting alloreactive T cells and markers of interest such as PD-1, LAG-3, and TIM-3 before they accumulate on the cell surface as a result of activation. Surface expression of KLRG-1and CD57 were found to be stable for at least 24 hours following allostimulation. Interestingly, the degree of HLA mismatch between the responder and stimulator cells did not significantly affect the percentage of responder CD4+ or CD8+ T cells that were alloreactive. Lastly, consistent with recent reports we observed FoxP3+ T cells to accumulate specifically in the CD137+CD154- fraction of CD4+ T cells, suggesting this assay may also detect alloreactive regulatory T cells.

*Conclusions: Our study suggests alloreactive T cells may be detected and phenotyped following a short-term co-culture with HLA-disparate cells by using the surface marker definition CD4+CD69+CD154+ and CD8+CD69+CD137+. Preliminary data suggest that this assay may also detect and phenotype donor-reactive cells in recipients following liver transplantation. Understanding the optimal conditions for detecting and phenotyping alloreactive T cells may allow for more in depth mechanistic studies of donor-reactive T cell responses following transplantation.

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