Fig 2: 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 3: Outcome of PD-1-PD-L1 Engagement at T Cell-APC Interface(A) Jurkat-PD-1OST cells were stimulated with Raji cells that have been preincubated in the absence (-) or presence (+) of 200 ng/mL SEE and lysed 2 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions probed with antibody to phosphorylated proteins (Anti-p-Tyr) or with phospho-tyrosine-specific antibodies directed against SLP76 pY128, ZAP70 pY493, LAT pY171 or VAV1 (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of three independent experiments.(B) Expression of CD3, CD28, PD-1, PD-L1, PD-L2, BTLA, and HVEM at the surface of Jurkat cells and Jurkat-PD-1OST cells, analyzed using flow cytometry.(C) Expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM at the surface of Raji cells and Raji-PD-L1 cells, analyzed using flow cytometry. In (B) and (C), gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.(D) IL-2 production by Jurkat and Jurkat-PD-1OST cells stimulated for 24 h with either Raji or Raji-PD-L1 cells in the absence (0) or presence of the specified amounts of SEE. Data are representative of three independent experiments, and mean and SEM are shown.(E) Jurkat-PD-1OST cells stimulated at 37°C with Raji or Raji-PD-L1 cells preincubated in the absence (–) or presence (+) of 200 ng/mL SEE and lysed 2 and 5 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions subjected to affinity purification on Strep-Tactin-Sepharose beads and probed with antibody to phosphorylated proteins (Anti-p-Tyr). Also shown are loading controls probed with anti-PD-1 antibody. Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent experiments.

Fig 4: 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 5: Either SHP-1 or SHP-2 Suffices for PD-1 Coinhibition in Jurkat T Cells(A) Immunoblot analysis of equal amounts of proteins from total lysates of representative clones of WT Jurkat, Jurkat-PD-1, Jurkat-PD-1OST, Jurkat-PD-1OST SHP-1–, Jurkat-PD-1OST SHP-2–, and Jurkat-PD-1OST SHP-1–SHP-2– cells probed with antibodies to SHP-1, SHP-2, or VAV1 (loading control). Left margin, molecular size in kilodaltons. Data are representative of two independent experiments.(B) Jurkat-PD-1OST, Jurkat-PD-1OST SHP-1–, Jurkat-PD-1OST SHP-2–, and Jurkat-PD-1OST SHP-1– SHP-2– cells were analyzed using flow cytometry for expression of CD3, CD28, PD-1, PD-L1, PD-L2, BTLA, and HVEM. Gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.(C) IL-2 production by Jurkat-PD-1OST, Jurkat-PD-1OST SHP-1–, Jurkat-PD-1OST SHP-2–, and Jurkat-PD-1OST SHP-1–SHP-2– clones stimulated with Raji cells or Raji-PD-L1 cells in the presence of 1 ng/mL SEE. Analysis of IL-2 production was performed 24 h after stimulation. Expression of IL-2 (ng/mL) using boxplot with the median, boxed interquartile range, and whiskers extending to the most extreme point up to 1.5 times the interquartile range. Each dot corresponds to a clone of the specified Jurkat cells. *p = 0.05, **p = 0.01, ***p = 0.001, and ****p = 0.0001; ns, non-significant (unpaired Student’s t test). Data are representative of two independent experiments.