Figure 2CD4+ T and DICER-KO B cells were co-cultured for 24 hours with or without OVA, and flow-cytometry sorted B cells were analyzed by small RNA next generation sequencing (NGS). Heat map showing microRNAs significantly upregulated in B cells after co-culture with T cells in the presence of OVA. Data are from 3 independent experiments (samples 1-3), and only microRNAs with an adjusted P-value < 0.02 are shown. Fold increase in the B cell content of upregulated microRNAs in the presence of OVA; *P<0.05, **P<0.01, ***P<0.001. Heat map showing NGS analysis of microRNAs differentially expressed in activated WT C57BL/6 CD4+ T cells and their secreted exosomes. Data are from 3 independent experiments (samples a-c), and only microRNAs with an adjusted P-value < 0.02 are shown. Mean tags per million (TPMs) for the differentially expressed microRNAs under study in exosomes from activated CD4+ T cells. Bars represent the mean ± SEM. Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with LPS+IL-4 Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G. Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with CD40+IgM Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G. Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with LPS+IL-4 Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH. Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with CD40+IgM Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH. Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of nucleofected microRNA mimics in FACS-sorted FAM+ B cells with normalization to RNU1A1 and RNU5G Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of Bcl2l11 and Pten mRNA targets in mimic-nucleofected B cells with normalization to GAPDH Flow cytometry analysis of mimic-nucleofected B lymphocytes. Dot-plots show B220+ IgG1+ B cells 48h after mimic nucleofection from at least 4 independent experiments and bar charts show mean values ± SEM of 4 independent experiments.