A novel regulatory region controls IgH locus transcription and switch recombination to a subset of isotypes

摘要

Role of the γ1E region in CSR and IgH locus transcription in CH12 and primary B cells. a RT-qPCR of γ1E transcript levels using primers located in the 5′ (top) or middle (bottom) region of the γ1E. b Surface expression of IgA analyzed by flow cytometry in γ1E+/− and γ1E−/− clones and pCH12 cells after 3 days in culture with TFG-β, IL-4, and anti-CD40 antibody. The percentage of switched cells is indicated. Representative dot plots of six experiments are shown. c Percentage of CSR in γ1E+/− and γ1E−/− clones relative to pCH12 cells. Data are pooled from six independent experiments. Statistical significance was determined by two-tailed Student’s t-test. d RT-qPCR for Iμ-Cμ (left panel) and Iα-Cα (right panel) transcripts. Triplicates were normalized to the abundance of Igβ, set as 1. Statistical significance was determined by a two-tailed Student’s t-test. (*p-value < 0.05; **p-value < 0.005). Data are representative of six experiments. e Flow cytometry analysis of surface Ig expression in CFSE-labeled primary B cells purified from γ1E+/+ or γ1E−/− mice and cultured in vitro for 3 days with LPS (CSR to IgG3 and IgG2b), LPS + IL4 (CSR to IgG1 and IgE), or LPS + IFNγ (CSR to IgG2a) or for 4 days with LPS + IL5 + TGFβ + retinoic acid (RA) (CSR to IgA). The percentage of switched cells is indicated. Representative dot plots from six experiments are shown. f CSR efficiency in primary B cells obtained from γ1E−/− (n = 13) or γ1E+/+ (n = 12) mice, shown as the percentage of CSR. The mean + SD from six experiments is presented relative to γ1E+/+ control B cells. Statistical significance was determined by two-tailed Student’s test (*p-value < 0.05; **p-value < 0.005). g RT-qPCR for donor (Iμ-Cμ; left panel) and acceptor (Iγ3-Cγ3, Iγ1-Cγ1, Iγ2b-Cγ2b, Iγ2a-Cγ2a, Iε-Cε, and Iα-Cα; right panel) GLT transcripts in primary B cells purified from γ1E+/+ or γ1E−/− mice and cultured as in e. Mean + SD of triplicate values was normalized to the abundance of Igβ and is shown relative to γ1E+/+ B cells, set as 1. Statistical significance was determined by a two-tailed Student’s t-test. (*p-value < 0.05; **p-value < 0.005). For biological replicates, refer to Fig. S1e