Background and Objectives Antibodies encoded by the VH4–34 gene are inherently autoreactive, binding to red blood cell determinants. An idiotope present on the majority of immunoglobulins derived from the VH4–34 gene can be detected using the rat monoclonal antibody 9G4. 9G4 also allows the detection of B cells expressing B cell receptors derived from VH4–34 gene. We therefore determined the effect of B cell depletion therapy with rituximab (RTX) on B cell subpopulations expressing the 9G4 idiotype in peripheral blood of patients with rheumatoid arthritis (RA) as a means of following the fate of a specific autoreactive B cell population.
Materials and Methods B-cell subpopulations were characterised by flow cytometry using combinations of IgD, CD5, CD27 and CD38 in healthy controls (HC) (n = 7), patients with RA before (n = 10) and at clinical relapse after RTX (n = 17). The frequency of each 9G4+ B cell subpopulation was calculated after gating on 9G4+ cells.
Results No significant differences were observed in the frequency of total 9G4+ B cells between both patient groups and HC, although the levels of 9G4+ B cells tended to be higher at relapse after RTX treatment. After RTX treatment, repopulating total B cells were predominantly transitional (IgD+CD38++) and naïve (IgD+CD38+), with lower frequencies of most memory B-cell subpopulations. At relapse, RA patients had significantly more 9G4+IgD−CD38− memory B cells compared to HC. This B cell subset was positively correlated with a 9G4+CD5+CD27+ subpopulation. The frequency of 9G4+IgD–CD38+ memory B cells also tended to be increased after RTX compared to HC and pre-RTX. Lower levels of 9G4+IgD+CD38+ naïve, 9G4+IgD+CD38– memory and 9G4+CD5–CD27+ memory B cells were found after RTX treatment compared with HC and pre-RTX. No significant differences were observed in 9G4+IgD+CD38++ transitional, or 9G4+IgD–CD38++ plasmablasts in RA patients before and after RTX treatment in comparison with HC. However, there was a positive correlation between the frequency of 9G4+IgD–CD38++ plasmablasts and time after RTX treatment.
Conclusions Alterations in the frequency of memory B cell subpopulations expressing 9G4 idiotype occur in clinically relapsing RA patients after RTX treatment, particularly in IgD–CD38– memory B cell subset. It is possible that this 9G4+ B cell subpopulation escapes B cell depletion therapy, being rescued in niches such as secondary lymphoid organs, or in inflammatory sites. Our results also suggest that the normal tolerance mechanisms preventing 9G4+ B cells from becoming antibody producing cells is defective in patients with RA.