Autoantigen‐specific T and B Cell Cross‐reactivity to a Gut Commensal in Antiphospholipid Syndrome

The microbiota provides an enormous antigenic challenge to the adaptive immune system. We hypothesized that a homeostatic adaptive immune response to commensals can trigger and sustain autoreactive T and B cells via cross‐reactivity with autoantigens in an HLA‐susceptible host. We used antiphospholipid syndrome (APS), an autoimmune clotting disorder targeting the plasma protein b 2 ‐glycoprotein I (b 2 GPI), as a human model. Protein BLAST and Clustal Omega queries revealed that the human gut commensal Roseburia intestinalis contains peptide sequences with high homology to the immunodominant b 2 GPI T and B cell epitopes. Patient PBMCs proliferated significantly more to R. intestinalis protein extracts compared to phylogenetically closely related Eubacterium rectale , which lacks homologous sequences. We next established a MHCII tetramer for a major pathogenic T cell epitope in domain V of b 2 GPI, which shares a highly homologous region with an unrelated R. intestinalis protein. Sorting on tetramer‐positive CD4 memory T cells, we sought to determine if clones cross‐react with R. intestinalis proteins and peptides. Tetramer‐positive T cells proliferated significantly more to heat‐killed whole R. intestinalis bacteria in a dose‐responsive manner compared to controls. Cytokine measurements revealed a heterogeneous but pathogen‐associated phenotype. Tetramer positive clones showed an increase in GM‐CSF, TNF‐α IL‐9, IL‐13, and IL‐21. To demonstrate cross‐reactivity functionally in vivo, BALB/c mice were immunized with whole heat‐killed R. intestinalis and proliferation studies using splenocytes and lymphocytes were performed. Lymphocytes from draining lymph nodes and splenocytes showed significant proliferation in response to b 2 GPI compared to littermate mice immunized with adjuvant alone. Furthermore, ELISA revealed significant anti‐b 2 GPI titers in 1 of 3 R. intestinalis ‐immunized animals compared to controls. In vitro, immunoblotting using APS patient plasma and an RGGMR epitope‐specific monoclonal antibody showed strong reactivity with R. intestinalis proteins including one at 24 kDa at the size of the major mimic‐containing candidate protein. In summary, we have identified human epitope‐specific b 2 GPI‐reactive CD4 memory T cell clones, which have a pathogen‐associated phenotype and cross‐react to whole heat‐killed R. intestinalis . Additionally, immunization of BALB/c mice with R. intestinalis produces human b 2 GPI‐reactive lymphocytes and autoantibodies, the latter are also supported in vitro by an epitope‐specific monoclonal anti‐human b 2 GPI antibody binding to R. intestinalis protein. To our knowledge this work represents the first study to demonstrate b 2 GPI‐specific T and B cell cross‐reactivity to a common human‐commensal bacterium. Further studies to determine the R. intestinalis proteins and epitopes recognized by the patients' adaptive immune system are ongoing. Support or Funding Information NIH/NIAID