Rapid Identification of Preformed Alloreactive T Cells for Use in a Clinical Setting

Abstract

Background. In clinical practice, HLA matching is generally applied to minimize the incidence of graft rejection after transplantation. Recently, graft rejection has been directly associated with the presence of preformed alloreactive T cells before transplantation. Despite this knowledge, assays to rapidly quantify preformed alloreactivity are not available for use in a clinical setting. In this study, such an assay was developed and evaluated in a large cohort to correlate alloreactive T-cell reactivity with HLA matching. Methods. Stimulator peripheral blood mononuclear cells were prestained with CD45-fluorescein isothiocyanate antibody and mixed with responder peripheral blood mononuclear cells. Activation-induced cytokine secretion was blocked using brefeldin A. After 6 hr, functionally active alloreactive responder CD4 and CD8 T cells were quantified among fluorescein isothiocyanate-negative cells by their expression of interferon-γ on flow cytometry. Results. Directly alloreactive CD4 and CD8 T cells among both stimulators and responders were easily distinguished after 6 hr of stimulation without being affected by bystander activation. Among 128 paired combinations, 23.4% of individuals had alloreactive CD8 T cells, 15.7% had alloreactive CD4 T cells, and 12.5% had alloreactivity in both T-cell subpopulations. Alloreactive T cells decreased from circulation within a few days after transplantation. In line with well-known clinical observations that associate HLA matching with graft outcome, the number of HLA-A and -B mismatches correlated with alloreactive CD8 T-cell frequencies in the whole study population, whereas it did not predict alloreactivity on an individual basis. Conclusion. Alloreactive T cells may rapidly be quantified after 6 hr of stimulation. Thus, the flow cytometric approach may be applied in a clinical setting to facilitate the individualization of immunosuppressive therapy and studies on the identification of patients who are at increased risk to develop graft rejection.