Background/Purpose B-cells utilizing the VH-region immunoglobulin gene VH4-34 produce natural autoreactive antibodies. The rat monoclonal antibody 9G4 recognizes B-cells with VH4-34-encoded B-cell receptors (9G4+ B-cells) and also secreted immunoglobulins. In healthy individuals, 9G4+ B-cells present in splenic marginal zones and tonsils are excluded from germinal centre (GC) reactions, whereas in autoimmune diseases such as systemic lupus erythematosus, 9G4+ B-cells expand in the post-GC memory compartment, suggesting defective 9G4+ B-cell censoring mechanisms. In rheumatoid arthritis (RA) patients, the distribution of tonsil 9G4+ B-cells is similar to healthy individuals, but only limited phenotypic analyses have examined 9G4+ B-cells in peripheral blood. The aim of this work was to perform an extensive phenotype characterization of 9G4+ B-cell sub-populations in peripheral blood in RA patients and healthy controls (HC).

Methods Blood samples were collected from established RA patients (n=12) and age and sex-matched HC (n=15). Peripheral blood mononuclear cells were isolated by density gradient centrifugation and 9G4+ B-cells (gated in CD19+ B-cells) were characterized by flow cytometry. 9G4+ B-cell subpopulations were defined using combinations of IgD, CD27 and CD38. The expression of CD5, CD24, IgM, BAFF-R and CXCR5 was analyzed in total 9G4+ B-cells. Statistical analysis was performed with Mann-Whitney test. 

Results The frequency of total 9G4+ B-cells in circulation was similar between RA patients and HC (median values: 3.83% and 4.68%, respectively). We found that RA patients had a higher frequency of 9G4+IgD-CD27- B-cells than HC (p=0.04). No significant differences were found in other 9G4+ subpopulations based on IgD/CD27 classification, although there was a tendency for higher levels of 9G4+ switched memory B-cells (IgD-CD27+). RA patients also had significantly lower levels of 9G4+ transitional B-cells (IgD+CD38+++) (p=0.004) and 9G4+ plasmablasts (IgD-CD27+++CD38+++) (p=0.004) and increased frequencies of 9G4+ naïve B-cells (IgD+38++) (p=0.04) when compared to HC. A tendency for higher levels of 9G4+ post-GC memory B-cells (IgD-CD38+) was also observed in RA patients. Furthermore, BAFF-R expression was significantly increased in 9G4+ B-cells from RA patients (both frequency and mean fluorescence intensity values) compared to HC (p=0.04 and p=0.006, respectively). There was a significant decrease in levels of 9G4+CD5+ B-cells (p=0.004) in RA patients, but no other significant differences were found in 9G4+ B-cells expressing CD24, IgM and CXCR5.

Conclusion RA patients have alterations in the frequency of 9G4+ B-cell subpopulations when compared to healthy individuals. The significant reduction in the frequency of 9G4+ transitional and 9G4+CD5+B-cells and the increased levels of 9G4+ naïve B-cells suggests a more rapid maturation of 9G4+ B-cells in RA patients following entry into the peripheral pool. This could be influenced by the antigen specificity of the B-cell receptor. Furthermore, the increased BAFF-R expression by 9G4+ B-cells observed in RA patients might support an increased survival mechanism for these already inherently autoreactive B-cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-arthritis-patients-have-alterations-in-inherently-autoreactive-9g4-b-cell-subpopulations-in-peripheral-blood/