Fig 2: Status of Eμ enhancer in DP thymocytes. (A) Enhancer elements and transcription factors that bind to these sites are shown below the schematic of the DH-CH part of the IgH locus. Arrows originating from the enhancer represent bi-directionally transcribed eRNAs named as Iμ sense and Iμ antisense. (B) Enhancer-associated histone modifications were scored by ChIP using anti-H3K4me1 and anti-H3K27ac antibodies in CD19+ pro-B cells and DP thymocytes derived from TCRβ × Rag2−/− transgenic mice. Y-axis represents fold enrichment of the indicated amplicon in the immunoprecipitate compared to an equal amount of input DNA. For each independent experiment PCR was done in triplicate. The data shown is the mean of two independent ChIP experiments. Error bars represent standard error of the mean (n = 2). γ-actin was used as a positive control, TCF7 enhancer was used as a positive control for T-lineage-specific gene and Cγ3 was used as a negative control. (C) Levels of eRNAs that originate within the enhancer in DP thymocytes and pro-B cells. Reverse transcription was carried out with strand specific primers, or no primer (np), followed by amplification with primers that score for sense and antisense Iμ transcripts. RNA amounts were calculated based on a standard curve obtained from serial dilutions of 1 kb DNA spanning both sense and antisense transcribed region. (D) Transcription factor binding to Eμ was assayed by chromatin immunoprecipitation using antibodies directed against the indicated factors. Enrichment of specific amplicons in co-precipitated DNA was calculated relative to an equal amount of input DNA (Y-axis). Error bars represent standard error of the mean (n = 2). Positive controls for each transcription factor correspond to the first set of bars in each graph. IgG served as additional negative control. (E) YY1, E2A, Ets-1, and RUNX1 expression in DP thymocytes and Rag2−/− pro-B cells were assayed by immunoblotting with the respective antibodies.

Fig 3: Reconstitution of B cell population after depletion. (A) Flow cytometry analysis of total number of B (CD19+) and T cells (CD3+) in single cell spleen preparations from C57Bl/6 mice colonized with S. pneumoniae 3 days after B cell depletion treatment. B cell reconstitution has been assessed 26 days after aCD20 antibody IP administration (3 mice/group). Undepleted mice are shown in black, depleted in white. (B, C) Reconstituted B cell subset populations in splenocytes after one (B) or two (C) depletion treatments were analyzed by flow cytometry 31 and 26 days after depletion, respectively. Marginal zone (CD5-CD21+CD23-IgM+), follicular (CD5-CD23+IgM-), and regulatory (CD5+CD21+IgM+) B cells populations are shown. Depleted/vaccinated (white bars), undepleted/vaccinated (black bars) mice were analyzed (n=6/group). Bars represent mean values for each group, error bars indicate SDs and Mann Whitney U test was used for statistical analysis (*P < 0.05, ns, not significant).

Fig 4: Immunogenicity of GAd-ZIKV Vaccines in WT C57BL/6 Mice(A) Schematic representation of the transgene cassettes. GAd-control has no transgene insert, whereas GAd-Eecto and GAd-Zvp contain ZIKV (strain H/PF/2013) a full prM-E cassette or prM-E ectodomain, respectively.(B) A549 cells were infected with GAd-Eecto or GAd-Zvp followed by concentration of the supernatants by ultracentrifugation, negative staining, and transmission electron microscopy imaging to confirm the production of VLPs. High-power images are shown and compared to ZIKV VLP preparation generated by transfection of prM-E genes. Scale bar, 100 nm.(C) ZIKV E protein expression in GAd-Eecto or GAd-Zvp. A549 cells were infected with GAd-Eecto, GAd-Zvp, or mock-infected, and supernatants were prepared and run on SDS-PAGE under reducing (upper blot) or non-reducing (lower blot) conditions along with recombinant ZIKV E ectodomain protein followed by detection using ZV-48. One representative blot from three experiments is shown.(D-H) C57BL/6 mice were immunized with 109 viral particles of GAd-control, GAd-Eecto, or GAd-Zvp via intramuscular inoculation and boosted 3 weeks later.(D and E) Humoral responses were evaluated in the sera of immunized mice at day 21 post-prime and day 21 post-boost. An ELISA measured ZIKV E-specific binding IgG (D), and a FRNT determined neutralization activity (EC50 values are shown) (E). Data for humoral responses are pooled from two experiments with n = 10–18 per group (ANOVA with a Dunnett’s post-test comparing vaccine and control groups: ****p < 0.0001).(F) Localization of GAd-Zvp and GAd-Eecto-induced polyclonal antibodies from sera. Binding to ZIKV Eecto, DIII, or DIII-LR mutant (A310E and T335K) was determined by ELISA from sera obtained post-boost at day 42 (n = 17 per group, ANOVA with a Tukey’s post-test: ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001).(G-J) Cell-mediated immune responses were analyzed at 90–110 days post-initial immunization. Splenocytes were assayed for IFN-γ+ ZIKV E-specific CD8+ T cells using intracellular staining after peptide restimulation. Representative flow cytometry plots of four experiments are shown with percentages of IFN-γ+ ZIKV-specific CD8+ T cells (G). Summary of frequencies of IFN-γ+ ZIKV-specific CD8+ T cells are shown (H). ZIKV E-specific MBCs per million CD19+ cells obtained from the spleen were determined by ELISA and Poisson distribution analysis (see STAR Methods) (I). An ELISPOT determined the number of ZIKV-specific IgG producing LLPCs per million bone marrow cells (J). Bars indicate median values per vaccine group. Data for cell mediated responses are pooled from four experiments with n = 6–17 animal per group (ANOVA with a Dunnett’s post-test comparing vaccine and control groups: ns, not significant; **p < 0.01; ***p < 0.001). In this figure, dotted lines represent the limit of detection (LOD) of the assays.

Fig 5: BLP+ M?s Have Distinct Transcriptomic Profile and Express CD11c as a Specific Marker(A) Single-cell RNA-seq experiment workflow. M?s were isolated from proximal/distal colon by FACS and sequenced. 2,106 M?s were identified, n = 1176 for proximal and n = 930 for distal colon, and shown on a t-distributed stochastic neighbor embedding (tSNE) representation; cells isolated from proximal (orange), cells from distal colon (blue) (top 22 PCs computed on the top 1,000 variable genes, vst method).(B) Selection of clustering parameters was based on the silhouette score and expected fraction of BLP+ M?s in proximal and distal colon obtained from IHC (mean fraction of BLP+ M?s out of all M?s; data pooled from two independent experiments). The selected clustering solution (graph-based clustering, 30 neighbors, resolution = 0.3) revealed the presence of two M? subpopulations, displayed on the same tSNE as in (A) and colored accordingly: cluster 0, containing 964 cells in proximal and 390 cells in distal colon, and cluster 1, containing 212 cells in proximal and 540 cells in distal colon. Cluster 1 expresses CD11c as a specific marker.(C) Co-staining of MHC class II and CD11c of the distal colon from C57B6J mouse. Maximum z-projection of 30 µm; scale bar: 20 µm.(D) Percentage of CD11c+ M?s out of all M?s in proximal and distal colon determined by imaging (top, % of CD11c+ M?s out of F4/80+ CD103- MHCII+ cells) or flow cytometry (bottom, % of CD11c+ ??s out of CD45+CD3-CD19-CD11b+CD103-CD64+F4/80+Ly6C-MHCII+ cells); dots represent average number per individual mice; three independent experiments. Mean ± SEM, Mann-Whitney test, **p < 0.01.(E) Heatmap of the top 20 significantly up- and downregulated genes between BLP+ M?s (cluster 1) and BLP- M?s (cluster 0), respectively, sorted by non-decreasing p value; entries represent the scaled (Z score) normalized expression, values <-2.5 or >2.5 are clipped; genes found respectively up- and downregulated in Kang et al. (2020) are highlighted in bold (see F).(F) (Top) Comparison of significantly upregulated genes in BLP+ M?s (cluster 1, 88 genes, in blue) and in cluster 4 from Kang et al. (2020) (166 genes, in gray). Fisher’s exact test using 10,768 detected genes in the 2,106 M?s as background. (Bottom) Comparison of significantly upregulated genes in BLP- M?s (cluster 0, 121 genes, in pink) and cluster 6 from Kang et al. (2020) (168 genes, in gray). See also Figures S3 and S4 and Tables S1 and S2.