Fig 2: Vγ7+ IEL respond to different Btnl pairings.a Left: Alignment of the IgV-domain sequences of Btnl1, Btnl4 and Btnl6. Canonical Ig-fold β-strands [A, B, C, C′, C″, D, E, F, G] are indicated with arrows. CFG face motifs previously shown in Btnl6 to be critical for the response of Vγ7+ cells19 are highlighted in orange [AQPTP/SRFSE/SRFSA], blue [QF/HF/HF] and red [SQEVS/YDEAI/YEEAI]. Right: Cartoon representation of the IgV-domain of Btnl6, derived with 3D-JIGSAW from the crystal structure of BTN3A1 (PDB accession code 4F80), with the same annotation as in (a). Side chains are displayed for the two residues that differ in the CFG face motifs of Btnl6 versus Btnl4 (Ala versus Glu, Glu versus Asp). b TCR downregulation (left) and CD69 upregulation (right) by J76 cells expressing a Vγ7Vδ4 TCR and co-cultured with 293T transiently transfected with Btnl1 in combination with Btnl4 wild-type (L4WT) or mutated in the CFG region as indicated on the X-axis. Results are normalised to 293T transfected with empty vector (EV). Data are represented as mean ± SD of duplicate co-cultures, representative of n = 2 independent experiments. FC, fold change. c Experimental setup to analyse IEL from various KO strains in co-cultures with MODE-K cells overexpressing either Btnl1 and Btnl4 (L1L4) or Btnl1 and Btnl6 (L1L6). IELs are isolated from indicated mouse strains which can display distinct combinations of Btnl molecules on the epithelial surface during development. Following isolation, IEL were co-cultured o.n. with MODE-K cells displaying either Btnl1+4 or Btnl1+6 on their surface. MODE-K cells transduced with empty vector (EV) were used as control. d IEL response to MODE-K cells expressing different Btnl dimers (L1L4 or L1L6) was measured by analysing CD25+ cells gated on Vγ7+ cells in animals of indicated genotypes. Data are mean ± SEM of five independent experiments, nctrl: 13, nBtnl1KO: 8, nBtnl4KO: 9, nBtnl6KO: 10, nBtnl146Indel: 15. Statistical analysis: two-way ANOVA & Tukey’s multiple comparison post test. e IEL response to MODE-K cells expressing different Btnl dimers (L1L4 or L1L6) was measured as %CD25+ cells and further gated on Vγ7+Vδ4+ (left) or Vγ7+Vδ6.3+ (right) cells in animals of indicated genotypes. Data are mean ± SEM of five independent experiments, nctrl: 13, nBtnl4KO: 9, nBtnl6KO: 10. Statistical analysis: two-way ANOVA & Tukey’s multiple comparison post test.

Fig 3: GW9662 boosts anti-tumor immunity in E0771 TILs. Immunophenotyping of various tumor infiltrating lymphocytes (TILs) after treatments: (a) Total CD8+ cells, and in the CD8+ gate: (b) IFNγ+, (c) CD44+, (d) PD-1+, (e) CD4+ in CD3+, and in CD4+ gate: (f) CD25+FOXP3+, (g) IFNγ+, (h) CD44+, Sham: vehicle + anti-IgG (n = 5); α–PD-L1: vehicle + anti-PD-L1 (n = 4); GW: GW9662 + anti-IgG (n = 4); α–PD-L1 + GW: anti-PD-L1 + GW9662 (n = 5). Data are mean ± SD.

Fig 4: Human Intestinal ?d Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted ?d T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR V? chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR V?2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRV? chain usage (left) and cell surface TCR?d expression (right) in gut-derived ?d T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.

Fig 5: Co-expression of Btnl1 and Btnl6 and Their Impact on V?7+ IELs, Related to Figure 6(A) Cell surface expression of FLAG-Btnl1, HIS-Btnl4 or HA-Btnl6 co-transfected in MODE-K cells. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (B) Primary small intestinal IEL cultured for the indicated times with MODE-K cells transduced with constructs expressing an empty vector (EV) versus Btnl1+Btnl6 (L1+6) (n = 7). (C) Representative plots of cell surface CD122 and CD25 expression on V?7+ cells after the indicated overnight culture conditions (n = 21). (D) Cell surface CD25 expression in positively FACS-sorted V?7+ IEL after overnight co-culture with MODE-K cells expressing EV versus L1+6 (n = 4). (E) Cell surface CD25 expression in primary V?7+ IEL after overnight co-culture with the indicated MODE-K transductants in the presence PP2, PP3 or vehicle. Data are representative of representative of 2 (A,D), or > 5 (C) independent experiments. Some panels (B,E) present data pooled from 2 independent experiments. All error bars represent mean ± SD.