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: Induction of T-independent B cell–intrinsic class-switched antibody responses to NP and flagellin in NSG/B mice.(A) NSG, NSG/B, and BALB/c mice (n = 3 per group) were injected intraperitoneally with NP-LPS; peripheral blood and spleen B and T cells were identified (percentages of total mononuclear cells) by anti-CD19 and anti-CD3 mAbs. Spleen mononuclear cells were analyzed for CD3, CD19, CD11b, CD11c, and cytokine transcripts by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (normalized to Gapdh, 2-?Ct method). NSG, NSG/B, and BALB/c mice (n = 4) were injected intraperitoneally with NP-LPS at days 0, 2, and 4. NP4-specific IgM, IgG3, IgG1, IgG2b, and IgA were measured by ELISA (titers are RUs) at day 14. Histograms depict mean values ± SEM. Each dot represents an individual mouse. (B) NSG/B, NSG/B Rosa26fl-STOP-fl-Luc, NSG/B Aicda-creRosa26fl-STOP-fl-Luc, and NSG/B Aicda-cre Rosa26+/+ mice were constructed by grafting NSG mice with B cells (2.0 × 107) from C57BL/6, Rosa26fl-STOP-fl-Luc, Aicda-cre Rosa26fl-STOP-fl-Luc, or Aicda-cre Rosa26+/+ mice. NSG/B mice were injected intraperitoneally with NP-LPS (n = 5) or S. Typhimurium flagellin (n = 3) at days 0, 2, and 4. Serum NP4-specific and flagellin-specific IgM, IgG3, IgG1, IgG2b, and IgA were measured by ELISA (titers are RUs) at day 14. Histograms depict mean values ± SEM. Dots are individual mice. NSG/B Rosa26fl-STOP-fl-Luc, NSG/B Aicda-cre Rosa26fl-STOP-fl-Luc, and NSG/B Aicda-cre Rosa26+/+ mice (n = 2 per genotype) were injected intraperitoneally with NP-LPS or flagellin under the same schedule. AID was identified in the spleen, intestines, and splanchnic district by luciferase bioluminescence imaging. Ab, antibody.