Conclusions
The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8(+) T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard.
Methods
Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8(+) T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined.
Results
The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not. Conclusions: The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8(+) T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard.