*Purpose: One of the major causes of kidney allograft loss is antibody mediated rejection. This process is mainly mediated by HLA antibodies(Abs) which are believed to be produced by long-lived plasma cells (LLPC). LLPCs study is limited due to accessibility, rarity(0.05-0.5% of bone marrow (BM) mononuclear cells (MNC)), and difficulty maintaining in vitro. The goal of this study was to develop an in-vitro culture model for the long-term maintenance of BM-derived LLPCs that produce HLA Abs.

*Methods: Bilateral BM aspirations were obtained from kidney transplant recipients prior surgery. BM MNCs were isolated after red blood cell lysis or ficoll-hypaque separation. LLPCs were enriched from BM MNC by positive selection with CD138+ microbeads. After isolation, the LLPCs were cultured on an irradiated mouse clonal stromal cell line (SC) for 2 or 4 weeks and the co-cultures supplemented weekly IL-6 (20ng/ml). Supernatants were collected weekly and concentrated using an Amicon Ultra-2 centrifugal filter to obtain a 25X concentration. Serum HLA Single Antigen Bead (SAB) results were obtained prior to transplant. Finally, by using Luminex technology assay for both HLA classes were obtained with double secondary antibody concentration from the manufacturer’s directions.

*Results: A total of 7 patients were studied of which 5 (3 sensitized and 2 not sensitized) were tested for Class I and Class II antibodies. The other 2 patients were tested for one of the HLA classes to which they were not sensitized. The comparison between the PC-SC co-culture supernatant SABs to their respective serum SABs resulted in AUC of 0.89 (Serum MFI>1000) and 0.94 (Serum MFI>2000). We obtained a high negative predictive value of 0.96 and accuracy of 0.95 when the serum MFI >2000 was considered as positive. Figure1 shows the detection of SABs from the supernatant co-culture of LLPC compared with the level of detection of the serum SABs by HLA class.

*Conclusions: Using a PC-SC culture model we were able to maintain LLPCs in vitro for 2-4 weeks and assay production of HLA Abs from the culture supernatant from a BM aspiration. These findings suggest that PCs isolated from BM and cultured in vitro can be maintained and evaluated for production of alloantibodies to HLA antigens. It might suggest that the higher the serum MFI, the more likely we are able to identify the PC that produces these HLA Abs in the BM. This method might help in the in vitro detection of specific classes of HLA Abs and serve as a potential model system in which to test new therapies.

« Back to 2019 American Transplant Congress