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.
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.
Authors: Michael A Brehm; Julie Mangada; Thomas G Markees; Todd Pearson; Keith A Daniels; Thomas B Thornley; Raymond M Welsh; Aldo A Rossini; Dale L Greiner Journal: Blood Date: 2006-09-14 Impact factor: 22.113
Authors: Liisa K Selin; Michael A Brehm; Yuri N Naumov; Markus Cornberg; Sung-Kwon Kim; Shalyn C Clute; Raymond M Welsh Journal: Immunol Rev Date: 2006-06 Impact factor: 12.988