Fig 2: Effect of EVs-DexP on IgA+lymphocytes immunophenotype in PPs. Flow cytometry analysis of the ratio of IgA+ B220+ cells in PPs from different intervention groups of IgAN mice. n=4 mice per group.* p<0.05 vs. IgAN mice; one-way ANOVA.

Fig 3: Btla expression in T cells limits serum IgA in aged animals(A) Spleen sections from 11-month-old Btlaflox and Btla?Cd4 animals stained for PNA and DAPI or B220, CD3, IgA, and DAPI. Scale bar equals 100 µm.(B) Graph of number of PNA+ GC in (A).(C) Plots and graph of IgDlowCD38lowGL7+Fas+ GC B cells in CD19+ B cells.(D) Plots of Cxcr4hiCD86lo DZ and Cxcr4loCD86hi LZ cells and graph of DZ/LZ cell ratio in spleens of aged Btlaflox and Btla?Cd4 animals.(E) Graph of serum IgA in aged Btlaflox, Btla?Cd4, and Btla?Cd19 animals.Data analyzed using multi-parameter linear modeling ANOVA. *p < 0.05; **p < 0.01. Representative of four separate experiments with n = 4 replicates for each condition. See also Figures S2 and S3J–S3M.

Fig 4: 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.

Fig 5: Btla expression in T and B cells regulates GC reactions in PPs(A and B) Plots and graphs of Foxp3−PD1+Cxcr5+ Tfh, Foxp3+PD1−Cxcr5− Treg, and Foxp3+PD1+Cxcr5+ Tfr cells (A) and IgDlowCD38hi memory B cells, IgDlowCD38lowGL7+Fas+ GC B cells, GC B cell cellularity, and PP cellularity (B) in PPs of 8-week-old Btlaflox, BtlaΔCd4, and BtlaΔCd19 animals.(C) PP sections from 8-week-old Btlaflox and BtlaΔCd4 animals stained with anti-CD4, anti-B220, anti-IgA, and DAPI. Scale bar equals 100 μm.(D) Plots of Cxcr4hiCD86lo DZ and Cxcr4loCD86hi LZ cells and graph of DZ/LZ cell ratio in PPs of 8-week-old Btlaflox, BtlaΔCd4, and BtlaΔCd19 animals.Data analyzed using multi-parameter linear modeling ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Representative of three separate experiments with n = 4 replicates for each condition. See also Figures S4A–S4H.