Fig 2: BTLA on T Cells Restrains GC B Cell Competitiveness(A) Flow-cytometric analysis of BTLA expression on B cells (left) and CD4+ T cells (right) compared with Btla-/- cells.(B) HVEM-overexpressing Btla-/- BM chimeras were made by transduction of empty vector (EV) or HVEM (WT) MSCV-Thy1.1 constructs into Btla-/- BM and reconstitution of CD4-depleted hosts. Chimeras were then immunized with SRBCs, and the participation of Thy1.1+ cells in GC versus Fo B cells was determined.(C) Contribution of Btla+/+ or Btla-/- CD45.2 cells to Fo and GC populations in the spleen of mixed BM chimeras made with ~40% CD45.2 and ~60% WT CD45.1 at day 7 after SRBC immunization.(D) Experimental scheme and timeline for experiments in (E) and (F).(E) Ratio of the frequency of CD45.1/2 Hvem-/- HEL+ B cells in BTLA-B-cell-deficient (Btlaf/fMb1Cre) hosts at day 5 to the pre-transfer frequency.(F) Ratio of CD45.1/2 Hvem+/+ or Hvem-/- HEL+ GC B cells to the pre-transfer mix in BTLA-T-cell-deficient (Btlaf/fCd4Cre) hosts at days 3–6. Data are pooled from four experiments.Unpaired two-tailed Student’s t test (B, C, E, and F): *p < 0.05, **p < 0.01, ***

Fig 3: BTLA Signaling into the T Cell through SHP1 Is Required for HVEM-Deficient GC B Cell Competitiveness(A) Model of murine BTLA truncation, termed Y3, in which all three cytoplasmic tyrosines have been removed.(B) Ratio of Hvem-/- GFP+HEL+ GC (IgDloFas+) B cells to the pre-transfer mix (~20% Hvem-/- GFP+ Hy10 to ~80% WT CD45.1 Hy10) in Btla+/+, Btla+/-, Btlay3/y3, and Btla-/- hosts. Data are pooled from two experiments.(C) Ratio of Hvem+/+ or Hvem-/- GFP+HEL+ GC B cells to the pre-transfer mix in Ptpn6f/f (SHP1) Cd4Cre or control hosts. Data are pooled from four experiments.(D) Ratio of Hvem+/+ or Hvem-/- GFP+HEL+ GC B cells to the pre-transfer mix in Ptpn11f/f (SHP2) Cd4Cre or control hosts. Data are pooled from three experiments.(E) Representative flow-cytometric analysis of CD40L surface mobilization on OT-II Tfh cells after a 30 min of incubation; B cells were pulsed with a titration of OVA peptide (0–10 µM).(F) CD40L expression on OT-II Tfh cells stimulated as in (E) (normalized to expression on unstimulated Tfh cells). Data are pooled from three experiments.(G) Representative flow-cytometric analysis of CD40L surface mobilization on OT-II Tfh cells after 60 min of stimulation on OVA-peptide-pulsed Hvem+/+ or Hvem-/- B cells.(H) Normalized CD40L mean fluorescence intensity on OT-II Tfh cells 30 or 60 min after stimulation with OVA-loaded Hvem+/+ or Hvem-/- B cells (2 or 10 µM). Data are pooled from two experiments.(I) Representative images of CD40 recruitment to the immunological synapse with or without HVEM and CD80 present in the lipid bilayer after 15 min of incubation with human Tfh cells; a standardized LUT is shown across panels so fluorescence can be directly compared. Upper images show immunofluorescence (IF), middle images show overlayed IF and IRM, and lower images show IRM.(J) Relative CD40 accumulation at synaptic interface in BTLA-transfected CD4+ T cell blasts and Tfh cells with or without HVEM and CD80 in the bilayer (normalized to the condition without HVEM and CD80). Data are pooled from three independent donors and experiments.Scale bars: 5 µm. Ordinary one-way ANOVA with Bonferroni’s multiple-comparisons test (B–D), unpaired two-tailed Student’s test (F), paired two-tailed Student’s test (H), or Mann-Whitney test (J): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S5.

Fig 4: BTLA-HVEM at the Immunological Synapse Recruits SHP1 to Inhibit Signaling in Tfh Cells(A) Example total internal reflection fluorescence (TIRF) and interference reflection microscopy (IRM) images of BTLA-transfected human CD4+ T cell blasts on supported lipid bilayers containing anti-CD3, ICAM1, and where indicated, HVEM and/or CD80. Staining for CD3 (UCHT1), ICAM1, and BTLA is shown.(B) Quantification of kinapse versus synapse state of cells imaged as in (A).(C) Model of human BTLA binding mutant Y257F/Y282F, designed to disrupt SHP1 and SHP2 recruitment to the last two of four cytosolic tyrosines.(D) Representative images of SHP1 and SHP2 recruitment to the synapse in relation to that of WT or mutant BTLA.(E and F) Quantification of SHP1 (E) and SHP2 (F) colocalization with WT or mutant BTLA with or without HVEM in the bilayer as determined with Pearson’s colocalization coefficient.(G) Representative images of pZAP70 in human Tfh cells in relation to HVEM in the bilayer after 15 min on the bilayer; a standardized lookup table (LUT) is shown across panels so fluorescence can be directly compared.(H) Relative amount of pZAP70 in BTLA-transfected CD4+ T cell blasts and Tfh cells with or without HVEM and CD80 in the bilayer (normalized to the condition without HVEM and CD80).(I) Representative images of pPKC? in human Tfh cells in relation to HVEM in the bilayer after 15 min on the bilayer; a standardized LUT is shown across panels so fluorescence can be directly compared.(J) Relative amount of pPKC? in BTLA-transfected CD4+ T cell blasts and Tfh cells with or without HVEM and CD80 in the bilayer (normalized to the condition without HVEM and CD80). Data are pooled from three independent donors and experiments.Scale bars: 5 µm. Chi-square and Fischer’s exact tests (B) or Mann-Whitney test (E, F, H, and J): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.