2009 Volume 19 Issue 1 Pages 67-73
After organ transplantation, anti-donor alloreactivity, defined as the number and phenotype of alloreactive precursors in the recipient, could be used to monitor rejection or tolerance or for withdrawal of immunosuppression. For this purpose, we used intracellular fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixed lymphocyte reaction (CFSE-MLR). The CFSE-MLR enables determination of the number of proliferating cells in response to allogeneic stimulation and simultaneous determination of the phenotype of proliferating cells by using of multiparameter FCM analysis. In our previous study, remarkable proliferation of CD8+ T cells in association with CD25 expression on anti-donor CFSE-MLR reflected the greater susceptibility to acute cellular rejection after liver transplantation. In addition to the determination of the phenotype of the proliferating T cells in response to allogeneic stimulation, cytokine-secreting activity in those T cells could be simultaneously determined by combining intracellular cytokine immunofluorescence staining (ICIS). Quantification of cell populations expressing specific cytokine proteins and identification of the cytokinesecreting cell phenotype in MLR would be much more informative than just the detection of cytokines in culture supernatants. We have demonstrated the utility of a method combining CFSE-MLR, ICIS, and multiparameter FCM for the simultaneous determination of proliferation and cytokine-secreting activity in T cells responding to allogeneic stimulation in mice. In allogeneic MLR utilizing a mouse model, CFSE-labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In fully allogeneic combinations, interleukin (IL)-2 secreting cells and interferon (IFN)-γsecreting cells were identified predominantly in proliferating CD4+ and CD8+ T cell fractions, respectively. Thus, for monitoring the immune response after organ transplantation, the CFSE-MLR analysis combined with the ICIS assay would be an excellent tool to monitor the host T cell reactivity in response to allogeneic stimulation.
