Related Experiment Video
Updated: Aug 21, 2026
Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Published on: April 19, 2017
Rapid identification of preformed alloreactive T cells for use in a clinical setting
Urban Sester1, Stephan Thijssen, Kai van Bentum
1Medical Department IV, Nephrology, University of the Saarland, Homburg, Germany.
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-gamma 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.
