*Purpose: T cell-mediated rejection is a significant factor that leads to graft damage and loss in transplant recipients. 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: To understand the phenotype and function of alloreactive T cells, we developed an in vitro system to detect HLA-A2-alloreactive CD8+ T cells using K562 cells that express the HLA Class I molecule HLA-A2:01 and the costimulatory molecule CD86.

*Results: Using these K562-A2⁺ cells, we find that HLA-A2-negative individuals harbor a mean precursor frequency of 1.53% (n=5) of alloreactive T cells against HLA-A2-expressing K562 cells. Furthermore, these alloreactive HLA-A2-specific CD8⁺ T cells exhibited a ~30-fold expansion in culture, indicating robust divisional capacity. Monoclonal antibody treatment blocking anti-HLA-A2 (clone BB7.2) completely abrogated proliferation of CD8⁺ T cells by these K562-A2⁺ stimulator cells as detected by Cell Trace Violet (CTV) dilution (25% CTV^lo vs 2%), confirming HLA-A2 allospecificity. In addition to their specificity to HLA-A2, T cell proliferation depended on costimulatory molecule CD86, as therapeutic blockade with anti-CD86 completely abrogated proliferation in HLA-A2 negative individuals (1% CTV^lo). Phenotypically, we find that the majority of HLA-A2 allospecific cells detected by proliferation are CD45RA^–CCR7^– (Tem) or CD45RA^–CCR7⁺ (Tcm), and >90% express the IL-2Ra as detected by CD25. As these phenotypes are likely due to their activation and not reflective of their initial state, further sorting experiments will determine from which subset allospecific cells reside.

*Conclusions: Based on these data, we hypothesize that use of this in vitro system will allow for the detection and expansion of alloreactive T cells in transplant recipients. Further work elucidating the peptides presented by these K562-A2⁺ cells will contribute to novel tools that will aide our understanding of the presence, phenotype, and function of alloreactive T cells in transplant recipients.

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