New tools to monitor the impact of viral infection on the alloreactive T‐cell repertoire

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Abstract
Accumulating evidence suggests that alloreactive memory T‐cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross‐reactivity of virus‐specific memory T‐cells is not available. We therefore aimed to develop a novel system for the detection of cross‐reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein‐Barr Virus (EBV) EBNA3A specific CD8 memory T‐cell clones (HLA‐B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon‐γ Elispot as readout. Generation of the T‐cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex‐specific tetramer. Monoclonality of the T‐cell clones was confirmed by T‐cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A‐specific T‐cell clones against SAL‐expressing HLA‐B*4402. Further screening against the entire panel of SALs also showed additional cross‐reactivity against SAL‐expressing HLA‐B*5501. Functionality of the cross‐reactive T‐cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross‐reactivity of viral‐specific CD8 memory T‐cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.

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