Fig 2: TLR-BCR linked coengagement induces CSR and plasma cell differentiation with increasing surface TLR4/TLR5 expression in mouse and human B cells.(A) Naïve IgM+IgD+ B cells from C57BL/6 mice were stimulated with nil (unstimulated), NP-LPS plus IL-4, NP-LPS plus TGF-ß, and retinoic acid (RA); S. Typhimurium flagellin plus IL-4; or flagellin plus TGF-ß and RA. At 96 hours, cultured cells were analyzed for surface CD19, TLR4, TLR5, and CD138; intracellular IgM, IgG, and IgA by single-cell flow imaging (scale bars, 10 µm); as well as for Aicda, Prdm1, Tlr4, Tlr5, germline IH-CH, and post-recombination Iµ-Cx (Cx being any switched isotype) transcripts, as measured by qRT-PCR and normalized to Gapdh expression (2-?Ct method). (B) Human naïve IgM+IgD+ B cells were stimulated with nil (unstimulated), NP-LPS plus huIL-2, huIL-4, and huIL-21 or S. Typhimurium flagellin plus huIL-2, huIL-4, and huIL-21. After 96 hours, cultured human B cells were analyzed for surface CD19, TLR4, TLR5, and CD138; intracellular IgM and IgG by single-cell flow imaging (scale bars, 10 µm); as well as for AICDA, PRDM1, TLR4, TLR5, germline IH-CH, and post-recombination Iµ-Cx (Cx being any switched isotype) transcripts, as measured by qRT-PCR and normalized to ß-ACTIN expression (2-?Ct method). *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired t test).

Fig 3: B cell TLR4-BCR linked coengagement induces generation of memory B cells and anamnestic response in Tcrß-/-Tcrd-/- mice.(A) C57BL/6 and Tcrß-/-Tcrd-/- mice were injected intraperitoneally with NP-LPS on days 0 and 21 (n = 5 mice per group) and euthanized on days 21 (primary response) or 35 (memory response) for analysis of the spleen and bone marrow cells. Class-switched NP-specific memory B cells (pregated CD19+CD38+IgG3+ and CD19+CD38+IgG2b+ cells) were identified in the spleens by binding of NP-PE, anti-CD19 and anti-CD38 mAbs, and anti-IgG3 and anti-IgG2b mAbs, as analyzed by flow cytometry (numbers are percentages of total mononuclear cells); negative control PE+ B cells were less than 0.5% (not shown). Histograms depict mean values of NP-specific CD19+CD38+IgG3+ and NP-specific CD19+CD38+IgG2b+ memory B cells ± SEM (% of IgG3+ or IgG2b+ memory B cells) in C57BL/6 and Tcrß-/-Tcrd-/- mice on days 21 and 35. Each dot represents an individual mouse. NP-specific IgM, IgG3, IgG1, IgG2b, and IgG2a ASCs as detected by ELISPOT in the bone marrow of C57BL/6 and Tcrß-/-Tcrd-/- mice, each dot representing an individual mouse; histograms depict mean numbers of ASCs per 105 plated cells ± SEM. (B) Elicitation of a class-switched anamnestic antibody response in Tcrß-/-Tcrd-/- mice. C57BL/6 and Tcrß-/-Tcrd-/- mice were injected intraperitoneally with NP-LPS or NP-CGG on days 0 and 21 (n = 8 mice per group). Sera were collected on days 0, 3, 5, 7, 14, 21, 28, 35, and 40 and analyzed for NP4-specific IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA by ELISA (titers expressed as RUs). Graphs depict NP4-specific antibody levels through day 40; data points are mean values ± SEM of eight mice at different times. *P < 0.05 (unpaired t test). MBCs, memory B cells.

Fig 4: S. Typhimurium flagellin TLR5-BCR linked coengagement–mediated antibody response entails GC-like B cell and plasma cell differentiation in Tcrß-/-Tcrd-/- mice.Tcrß-/-Tcrd-/- mice were injected intraperitoneally on day 0 with nil (alum, K207) or flagellin in alum (K210) and again at day 7 with PBS or flagellin in PBS, respectively. Mice were euthanized 7 days after the second injection (day 14). The spleen, mesenteric lymph nodes (MLNs), and Peyer’s patches mononuclear cells were analyzed by fluorescence-activated cell sorting for PNA+GL7+CD95+ GC-like B cells, as identified by staining with PNA and anti-CD19, anti-GL7, anti-CD95 (Fas), anti-Bcl6 (intracellular staining), anti-IgM, anti-IgG, and anti-IgA mAbs. Plasmablasts were identified by surface staining with anti-CD19 and anti-CD138 mAbs. AID and Blimp-1 expression were analyzed in B cells of the spleens, MLNs, and Peyer’s patches in nil-injected and flagellin-injected mice and were detected by intracellular staining with anti-AID and anti–Blimp-1 mAbs followed by flow cytometry analysis. Numbers are percentages of total mononuclear cells or total B cells. Data from additional nil-injected (n = 2 mice: K208 and K209) and flagellin-injected (n = 2 mice: K211 and K212) Tcrß-/-Tcrd-/- mice are displayed in fig. S15.

Fig 5: TLR5-BCR linked coengagement by S. Typhimurium flagellin induces SHM/CSR, clonal B cell expansion, and intraclonal diversification in Tcrß-/-Tcrd-/- mice.Tcrß-/-Tcrd-/- mice were injected intraperitoneally with flagellin on days 0 and 14 (n = 3 mice, one male and two females) and euthanized on day 28. VH1DJH-C?3, VH1DJH-C?2b, and VH1DJH-Ca transcripts from the Tcrß-/-Tcrd-/- mice were analyzed by Illumina MiSeq amplicon sequencing, as described in Fig. 4. (A) Sequence analysis allowed for identification of point mutations in the VH1 region of VH1DJH-C?3, VH1DJH-C?2b, and VH1DJH-Ca transcripts. Dots depict mutation frequencies in each sequence. Pie chart slices depict proportions of transcripts carrying given numbers of point mutations; slice gray gradients depict increasing numbers of point mutations (0 to more than 12) per transcript. Numbers below pie charts depict average overall point-mutation frequency (change per base) of all transcripts. Donut charts depict the nature of point mutations. (B) Each B cell clone was identified on the basis of segregation of VH1DJH-C?3, VH1DJH-C?2b, and VH1DJH-Ca transcripts consisting of the VH1 gene segment, the same and unique IgH CDR3 (depicted as translated amino acid sequence) together with the same JH sequence by MiSeq amplicon sequencing. Size and complex intraclonal SHM diversification (genealogical tree) of each discrete clone are depicted, as described in Fig. 5. (C) C57BL/6 mice and Tcrß-/-Tcrd-/- mice were injected intraperitoneally with flagellin on days 0 and 14 (n = 3 mice per group). Sera were collected on days 0, 7, 14, and 28 and analyzed for flagellin-specific IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA (titers expressed as RUs). Graphs depict flagellin-specific antibody levels through day 28; data points are mean values ± SEM of three mice at different times.