OR33 A novel method for HLA antigen specific B cell detection
Aim
HLA-Ag specific B cells play an important role in the allograft humoral immunity through producing donor specific HLA-Abs and presenting donor HLA-Ag to regulate allograft cellular immunity. The aim of the present study was to develop a HLA antigen specific B cell multiplex detection system through B cell receptor capture approach by flow cytometry beads array platform(Flow-BCR).
Methods
LabScreen Single Antigen Beads (LSAB; LMX-IgG): One Lambda protocol; Flow-BCR: LSAB were co-incubated with 1.0 × 106 patient PBMC for 1 h, the cells were lysed and the beads were washed 3 times. The beads were resuspended in stain buffer containing PE-anti-hIgG and incubated for another 30 min. After washes. the final beads were acquired in a Canto-II flow cytometer. The PE positive and negative beads of each population were gated and counted.
Results
A hybridoma cell line with HLA-A2 membrane BCR was used to evaluate the method sensitivity. Flow-BCR was able to detect one A2-BCR hybridoma cell spiked into 1000,000 PBMC cells (Fig. 1). A total of 55 patient PBMCs including 20 circulating HLA-Ab negative and 35 circulating HLA-Ab positive patient samples were tested by multiplex Flow-BCR. Compared to Non-sensitized group, significantly higher frequencies of HLA-Ag specific BCR cells were identified in HLA antigen class I and class II (Fig. 2a, b) sensitized patient samples.
Conclusions
The novel Flow-BCR is able to detect HLA-Ag specific memory B cells in sensitized patients and opens a new window of HLA antigen specific allograft immunity. The procedure of Fow-BCR is simple, specific, and very sensitive (1/1000,000 cells). It may provide a new tool to assess HLA sensitization before transplantation and monitor graft rejection after transplantation.