The B lymphocyte in rheumatoid arthritis: analysis of rearranged Vx genes from B cells infiltrating the synovial membrane

Abstract

The participation of the humoral immune system in rheumatoid arthritis (RA) is characterized by the production of rheumatoid factors (RF). RF are autoantibodies against the Fc part of IgG which are encoded by diverse germ-line genes. Most of the RF-encoding genes are unmutated, but in RA, a substantial quantity is encoded by somatically mutated genes. In addition, the synovial membranes (SM) of the diseased joints of RA patients are infiltrated by B lymphocytes which form germinal center-like aggregates. To analyze the local immune response, B cell foci from two RA SM were isolated by micromanipulation. From DNA of these foci, the rearranged kappa light chain variable region (V kappa) genes were amplified by polymerase chain reaction (PCR), cloned and sequenced. The amplification of different V kappa-J kappa combinations of different foci suggested oligoclonal expansion of B lymphocytes, which was confirmed by sequence analysis: each PCR product contained members of a single B cell clone. The sequence analysis of 29 different clones revealed rearrangements of diverse V kappa genes. Both frequent representatives of the V kappa 3 and the V kappa 1 family, as well as rarely used genes such as the L10 and B2 genes of the V kappa 2 and V kappa 5 families were found. Of the eleven potentially functional gene rearrangements, eight were significantly mutated, indicating their derivation from antigen-selected B cells. Intraclonal diversity in one of these clones may suggest ongoing mutation in the diseased synovial membrane of patients with RA.

Lineage tree analysis of immunoglobulin variable-region gene mutations in autoimmune diseases: Chronic activation, normal selection

“…In others, only sequences were published [16][17][18][19][20][21]; in these cases, germline genes were identified by alignment with published human IGV using DNAPLOT (http://vbase.mrc-cpe.cam.ac.uk/), and accordingly grouped and aligned using ClustalW (http://www.ebi.ac.uk/clustalw/). Trees were then generated using our program IgTree© (M. Barak et al, in preparation), which is specifically tailored to deal with IGV sequences.…”

“…Mutational lineage trees of B cell clones from patients with MG, RA, SS, and MS [6,[16][17][18][19][20][21], and from normal human GCs [25][26], were created from published and unpublished IgV sequence data; we also used published lineage trees [12,10,14]. Each dataset contained IGV sequences from different IGV groups and patients (Table 1).…”

Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA)

“…Individual B cell aggregates (foci) of about 50-300 cells were isolated by microdissection as previously described [12]. Half of the DNA was amplified by 35 cycles of PCR with a mixture of all V H -specific and J H -specific oligonucleotides.…”

“…An over-representation of one V H 4 gene, the V H 4.21, in the autoimmune repertoire has been described [15]. A high representation of members of the V H 4 family was also found within hybridomas derived from synovial Clin Exp Immunol 1996; 106: [5][6][7][8][9][10][11][12] tissue of one patient with RA [16]. Recently, it was postulated that a negative selection of V H 4 takes place in the peripheral blood lymphocytes (PBL) of healthy individuals as a means to avoid autoimmunity [17].…”

The B cell repertoire of patients with rheumatoid arthritis. II. Increased frequencies of IgG+ and IgA+ B cells specific for mycobacterial heat‐shock protein 60 or human type II collagen in synovial fluid and tissue

“…Interestingly, B cells in all stages of differentiation, from mature B cells to plasma cells, have been observed (16)(17)(18)(19)(20). There is some molecular evidence that B cells are specifically activated and clonally expanded in the synovium (21)(22)(23). Organized structures resembling germinal centers have been described, which contain follicular dendritic cells and CD4+ T cells and reveal clonal expansion of B cells exhibiting isotype switching and somatic hypermutation (19,22).…”