Fig 2: 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 3: Cell-intrinsic maintenance of Treg cell homeostasis and function by Btla in PPs(A–C) Graphs of T cell subsets in PPs of control or anti-Btla-treated wild-type (A), Tnfrsfl14-/- (B) animals, or of untreated Btlaflox and Btla?Foxp3 animals (C).(D) Plots and graph of FoxP3+YFP+/- Treg cells in PPs of untreated wild-type Foxp3+/cre and Btlaflox/floxFoxp3+/cre animals.(E and F) Plots and graphs of Foxp3-PD1+Cxcr5+ Tfh, Foxp3+PD1-Cxcr5- Treg, and Foxp3+PD1+Cxcr5+ Tfr cells (E) and IgDlowCD38lowGL7+Fas+ GC B cells (F) in PPs of untreated wild-type, Btla-/-, and tamoxifen-treated Btlaflox and Btla?Foxp3ERT2 animals. Tam, yamoxifen.Data analyzed using multi-parameter linear modeling ANOVA. *p <0.05; **p<0.01; ***p < 0.001; ****p < 0.0001. Representative of six(wild-type) experiments, with at least n = 5 replicates for each condition; two (Tnfrsf14-/-) experiments, with at least n = 7 replicates for each condition in antibody treatment experiments; and four (constitutive) experiments, with at least n = 3 replicates for each condition and two (induced) Treg cells Btla deletion experiments, with at least n = 8 replicates for each condition. See also Figures S5 and S6.

Fig 4: Anti-Btla inhibits inflammatory signaling in T and B cells(A) Setup for RNAseq experiment with C57BL/6 animals.(B) Volcano plots indicating genes significantly altered in anti-Btla-treated cells compared with control in anti-CD3-activated CD4+ T cells or anti-IgM/IgG activated.(C) Heatmaps of significantly altered genes in CD4+ T or B cells with GSEA hallmark pathways shown.(D) Hallmark GSEA pathways in CD4+ T and B cells.(E) Graphs of gene expression in germinal center B cells in SRBC-immunized wild-type or Btla−/− hosts.Data analyzed using Student’s t test. *p < 0.05; ****p < 0.0001. Representative two experiments with n = 4 replicates for each condition. See also Figure S1.

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.