ICF B-cells switch and secrete immunoglobulins after in vitro CD40L and IL-4 stimulation. (A) Enrichment of naive B cells from P1 and an HD. Naive CD19+CD27- B cells were enriched up to 95% by means of the StemSep B-cell enrichment system, and controls were depleted of memory B cells with the use of CD27 microbeads on a MiniMACS magnetic sorter. Results are representative of all patients and HDs. (B) (C) (D) Enriched naive B cells of patients and 4 HDs were stimulated in vitro with irradiated CD40L-transfected fibroblasts and rIL-4. Panel B (top) shows RT-PCR on VH-CH1γ and Igβ transcripts at time zero or after 10 days' stimulation. Bottom histogram in panel B shows total immunoglobulin production after 10 days' stimulation expressed in nanograms per milliliter. ▦ represents IgM fraction; ▪, IgG fraction. Panel C shows Iϵ-CH1ϵ and HPRT transcripts amplified by PCR from decreasing cDNA amounts (3, 2 and 1 μL) from patients and HDs. Panel D shows PCR amplification of AID, VH-CH1ϵ, and HPRT transcripts with the use of 3 μL cDNA. Histograms in panels C and D show transcription rate values normalized to HPRT values after quantification by means of MacBAS software (Fuji Film, Tokyo, Japan). Ratios are expressed on an arbitrary unit. Bars indicate SD. NS indicates unstimulated.
