OR35 A novel mixed lymphocyte culture method to evaluate donor HLA specific immunity in different HLA type mismatched donors and recipients

Abstract

Aim A novel flow cytometry based mixed lymphocyte culture assay (Flow Cellular Crossmatch: Flow-CXM) was developed for detecting antigen or donor specific T cell immunity. The preliminary study demonstrated that the donor specific T cell response in recipient was correlated with HLA mismatch (MM) numbers between donors & recipients. The aim of this study is to further evaluate the significance of Flow-CXM in different HLA type MM donors and recipients. Methods CXM (Fig. 1): 0.1 × 106 irradiated donor PBMCs were incubated with 0.1 × 106 recipient’s PBMCs and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for 16 h. The cells were lysed and the captured cytokines were detected by corresponding PE-labeled 2nd antibodies. The beads with different fluorescence were gated and PE stained beads (Flowspot %) were counted to represent the relative number of donor specific responding T cells. HLA Typing High-resolution HLA typing of all donor and recipient samples were performed by NGS or SSO. Results A total 109 paired bone marrow donor and recipient samples were tested by Flow-CXM. Compared to the HLA identical group, IL-2 but not IFNγ response significantly increased in HLA-class I (CI) (P Conclusions The newly developed Flow-CXM is able to detect earlier T cell activation of donor HLA specific immunity in recipient within 24 h. The multiple T cell activation markers (Th1/Th2 cytokines) can be measured in a single reaction. The methodology of Flow-CXM is simple, sensitive, and HLA specific. It hold a potential to be used in clinical transplantation for predicting and monitoring GVHD/GVL in BMT and cellular rejection in solid organ transplantation. Download : Download high-res image (404KB) Download : Download full-size image