Chronic antibody mediated rejection (CAMR) is a major cause of graft loss in renal transplant recipients. Previous work utilising an IFNγ ELISPOT assay revealed two broad patterns of B cell involvement in indirect anti-donor alloreactivity in response to whole donor antigens in CAMR: 1. B cell-dependent anti-donor reactivity, and 2. apparent suppression of anti-donor reactivity by B cells. We hypothesised that these different ELISPOT patterns were due to the presence of distinct phenotypes of alloantigen-specific B cells, and that an understanding of the phenotype of antigen-specific B cells is required if we are ever to achieve our goal of highly tailored treatment of CAMR in each individual.

Pure™ HLA proteins A*01:01 or A*02:01 were used in the indirect IFNγ ELISPOT assay and to detect HLA binding B cells in a cohort of patients with HLA A1 or A2 mismatched grafts respectively. IFNγ production in response to A1 or A2 was seen in 12/34 samples: when B cells were depleted, IFNγ spots reduced in 8/12 (=Bdep); increased in 2/12 (=Bsup) and were unaffected in 2/12 reactive and the remaining 22 non-reactive samples (=Bnon). Analysis of whole B cells revealed no differences in gross phenotype when comparing Bdep, Bsup or Bnon. HLA A1 binding B cells were detected in 4/33 samples from patients with A1 or A2 mismatched grafts Detailed phenotypic analysis was performed on 8/14 samples where the frequency of Ag-binding B cells was >50% above background. Ag-binding B cells were predominantly non transitional and IgM+ in all of these samples. 3/8 Bdep samples had HLA binding B cells that could be phenotyped in depth; IgMhiCD45RB+ (a memory phenotype) cells represented 76±1.5% of Ag-binding cells, whereas IgM+CD45RB- cells accounted for 13±5.4%. 1/2 Bsup and 4/24 Bnon had HLA-binding B cells that could be phenotyped. In these the phenotype was in contrast to the Bdep samples as IgMhiCD45RB+ and IgM+CD45RB- cells made up 45.9±13% and 38.8±8% respectively. These results complement previous data from our lab and indicate that ELISPOT reactivity correlates with the predominance of Ag-binding IgM+ memory cells in patients with CAMR.

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