Expression of RAG1, RAG2 and VpreB genes in IgD+CD5± peripheral B cells during cyclophosphamide treatment in pediatric SLE

Peripheral B cells of adult SLE patients express recombination activating genes RAG1/2 and surrogate light chain (VpreB) suggesting secondary receptor revision. In pediatric SLE data on receptor revision is limited. We analyzed the expression of RAG1, RAG2 and VpreB mRNA in peripheral IgD + CD5 ± B cells of a juvenile SLE patient during cyclophosphamide therapy. Using FACS sorting and single cell RT-PCR, the frequency of IgD + CD5 ± B cells in peripheral mononuclear cells (PBMC) before (A), after one (B), two (C) and 6 cyclophosphamide pulses (D), in addition to the mRNA expression of RAG1, RAG2 and VpreB in individual B cells was assessed. Remission was achieved after 1 year (D). The percentage of IgD + B cells in PBMC decreased from 17% (A) to 10,22% (D). 19,7% (A) of IgD + B cells were CD5 + , changing to 11,2% (B), 17,4% (C) and 9,2% (D). Both IgD + CD5 ± B cell populations showed remarkably high RAG1 + RAG2 + (18%, 30%) expression before treatment. During cyclophosphamide, the two populations showed a different RAG expression: Fewer (1,4% (B), 8,8% (C)) of IgD + CD5 + B cells were RAG + during treatment. In contrast, more of IgD + CD5 – B cells were RAG + (40%; B and C). In both populations RAG expression reached low levels at D (4,6% in CD5 – , 6,3% in CD5 + ). At diagnosis 22%, 33% of RAG + IgD + CD5 + or – B cells were VpreB positive. After remission VpreB expression was absent in both (D). The frequency of VpreB in RAG + IgD + CD5 ± B cells correlated with disease activity in 3 SLE patients. RAG + B cells were absent in healthy controls. RAG1/2 and VpreB mRNA expression was upregulated at diagnosis and decreased during cytotoxic treatment in this pediatric SLE patient.