title: Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium.
abstract: BACKGROUND
  Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
  
  
  RESULTS
  Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
  
  
  CONCLUSIONS
  Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.
authors.affiliation: William Harvey Research Institute, Barts and the London School of Medicine, Charterhouse Square, London, United Kingdom.
authors.forename: Frances
authors.initials: F
authors.lastname: Humby
authors.forename: Michele
authors.initials: M
authors.lastname: Bombardieri
authors.forename: Antonio
authors.initials: A
authors.lastname: Manzo
authors.forename: Stephen
authors.initials: S
authors.lastname: Kelly
authors.forename: Mark C
authors.initials: MC
authors.lastname: Blades
authors.forename: Bruce
authors.initials: B
authors.lastname: Kirkham
authors.forename: Jo
authors.initials: J
authors.lastname: Spencer
authors.forename: Costantino
authors.initials: C
authors.lastname: Pitzalis
mesh_terms: D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology* / Q000378:metabolism; D001323:Autoantibodies / Q000096:biosynthesis* / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism; D003564:Cytidine Deaminase / Q000378:metabolism; D020566:Dendritic Cells, Follicular / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D017578:Immunoglobulin Class Switching; D051379:Mice; D016513:Mice, SCID; D013583:Synovial Membrane / Q000201:enzymology / Q000276:immunology*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D001323:Autoantibodies; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase