P098 A novel flow cytometric assay to simultaneously detect ATG concentration and cytotoxic effect in renal transplant patient

Abstract

Aim To develop a new flow cytometry assay for detecting ATG concentration and immunokilling effect to monitor immunotherapy in transplantation. Methods CDC: ASHI standard protocol; Flow-ATG: 50ul patient sera or ATG spiked AB sera with and without complement inactivation were incubated with 0.2X106 PBMC for 15’ at 37 C. After 3 washes, the cells were resuspended in 110 ul stain mix containing PE-anti Rabbit IgG, FITC–CD3, PE-Cy7-CD19, and 7-AAD and continuously incubated at RT for an additional 10’. The cells were washed again and acquired on a Canto II flow cytometer. ATG binding (MCS) and cell death % were calculated on total lymphocyte, T, B, and non-T/B lymphocyte populations. Results ATG exerted a dose dependent effect in cell binding and killing on multiple immune cell populations including T, B lymphocytes, and non-T/B cells. T cells were more sensitive to ATG killing than non T cells (Fig. 1). In the binding assay, ATG at 0.4 ng/ml showed significant bindings on T and B lymphocytes and non-T/B cells. The killing effect of ATG was started at 0.25ug/ml and reached a maximum at 1.25ug/ml (100% death rate) on T and non-T cells. After complement inactivation (56C/30’), cytotoxic effect of ATG on immune cells was only slightly decreased (Fig. 2), which indicated the killing of ATG was mainly resulted from mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and apoptosis under our testing system. Five samples from kidney transplant patients undergoing ATG treatment were tested by both CDC and Flow-ATG. The quantitative dynamical changes of ATG binding and killing were observed by Flow-ATG. Compared to the traditional CDC assay Flow-ATG is much more sensitive and also able to measure the concentration of ATG in the same reaction. Conclusions The newly developed Flow-ATG is able to simultaneously detect ATG binding and killing effect on T, B cells, and non-T/B cells. The assay is simple, sensitive (0.4 ng/ml), quantitative, and can be used for monitoring clinical ATG immunotherapy in bone marrow and solid organ transplantations. Download : Download high-res image (263KB) Download : Download full-size image