Characterization of a Novel CD8+ B Cell Subset in Monkeys Tolerant of Heart Allografts

Purpose We have previously reported that transient mixed hematopoietic chimerism induced via donor bone marrow transplantation can achieve tolerance to cardiac allografts in non-human primates (NHPs). The purpose of this study was to measure the frequency and properties of a unique subset of CD8+ B cells, recently identified in our laboratory, in tolerant monkeys. Methods Cynomolgus Macaques underwent organ transplantation followed by injection of donor bone marrow cells with mixed chimerism conditioning consisting of 3Gy TBI, 7Gy thymic irradiation, anti-thymocyte globulin, and a short course of anti-CD154 mAb and CyA therapy. The frequencies of different B cell subsets were measured in the peripheral blood of ten monkeys pre- and post-transplantation. In addition, PBMCs were isolated from peripheral blood of two tolerant animals (recipients of heart plus kidney or heart plus thymus cotransplants) 847 and 274 days post-bone marrow and FACS-sorted into five groups: T cells (CD3+), whole B cells (CD20+), mature B cells (CD20+CD8−CD27+), naive B cells (CD20+CD8−CD27−), and newly identified CD8+ B cells (CD20+CD8+). mRNA was extracted from lysates of each cell population and sequenced using an Illumina NextSeq 500. Results During the reconstitution phase of the conditioning protocol, CD8+ B cells exhibited a phenotype reminiscent of transitional B cells (CD20+CD27−IgMhiCD38hi) and the percentage of these cells among peripheral B cells was much higher than that observed in naive animals (23.4% vs 2.5% respectively; p Conclusion The prevalence of CD8+ B cells expressing genes involved in regulatory immunity after bone marrow transplantation suggests that these cells may play a role in tolerance induction to heart allografts in NHPs. Work is in progress to further characterize these novel cells.