BackgroundCorrelation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.MethodsWe retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.ResultsOne-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMRhDSApos.ConclusionsIn conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

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