*Purpose: T cell-mediated rejection results in graft dysfunction and contributes to premature failure. Due to the complexity of the TCR:pMHC interaction, prediction of alloreactive T cells is difficult, and there are no widely available methods to measure alloreactive T cell responses at the allele level in a given recipient.

*Methods: We developed an in vitro system to detect and expand HLA-A2-alloreactive CD8+ T cells using K562 cells that express HLA*A02:01 and the costimulatory molecule CD86.

*Results: Using proliferation identified by CellTrace Violet (CTV) as a surrogate for alloreactivity, we identified that HLA-A2-negative individuals harbor a mean precursor frequency of 1.53% (n=5) of alloreactive CD8+ T cells. Monoclonal antibody treatment blocking either anti-HLA-A2 (clone BB7.2) or CD86 abrogated proliferation of CD8+ T cells (25% divided vs 2% with αHLA-A2 and 1% with αCD86), confirming HLA-A2 allospecificity and a reliance on costimulation. Stimulating HLA-A2-negative individuals with K562-A2+ cells over several weeks leads to a narrowing of the repertoire, as identified by progressive clonality and an increase in the proportion of the top 500 rearrangements overtime through analysis of TCRβ sequences. Following four stimulations, a single clone expanded to take up as much as 27% of the repertoire.

To query the immunopeptidome recognized within allogenic HLA, we developed a bioinformatic pipeline to analyze a public HLA-A2 ligandome data set identified from B-LCL cells and then intersected those with genes expressed in human kidney, focusing on peptides from proteins with cytosolic and wide tissue expression. By assessing strength of binding and estimated pMHC copy number, we generated a list of the top 480 self-peptides within HLA-A2, reasoning that the combination of self-peptides within allogenic HLA-A2 would elicit an allospecific CD8+ T cell against both peptide and MHC. After generating peptide pools, we have begun preliminary experiments using T2 cells that lack TAP and express little endogenous peptides on HLA-A2. Following stimulation of HLA-A2-negative individuals with peptide-pulsed T2 cells, we found a 4- and 6-fold increase in proliferated CD8+ T cells from two donors compared with pulsing with a single HLA-A2 peptide from CMV (NLVPMVATV), demonstrating the importance of peptide for allorecognition.

*Conclusions: Herein, we describe a novel assay to assess CD8+ T cell alloreactivity and a bioinformatic method to develop peptide pools to interrogate pMHC allorecognition. Continued research identifying the peptides recognized within allo-MHC by alloreactive T cells is critical to track and mitigate alloreactive responses in vivo.

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