Fig 2: Positional scanning library analysis confirms the G104βP mutation confers higher specificity. (A) The T96βK and G104βP/T96βK variants of 868-Z11 have EC50 values identical within error for the SL9 peptide in functional assays measuring cytokine release. Data points are averages and standard deviations of five separate titrations; values reported are the averages and standard deviations from the five experiments. Negative control data are for co-cultures with the irrelevant Tax11-19 peptide (sequence LLFGYPVYV). (B) Positional scanning library data for the T96βK and G104βP/T96βK variants of 868-Z11. For each peptide in the library, IL-2 production at 10 µM peptide in a co-culture experiment is normalized to that of the WT SL9 peptide as indicated by the scale on the right. Addition of the G104βP mutation results in fewer stimulatory peptides, particularly in the C-terminal half of the peptide. Data in each cell are the average of three separate co-culture experiments. (C) Fingerprint analysis from the data in panel B, showing the distribution of scores for all 1.28 billion peptides of the form XLXXXXXXL, where X is any of the 20 standard amino acids. The greater specificity conferred by the G104βP mutation is indicated by the left-shifted blue curve, further highlighted by the much smaller number of peptides with scores ≥ 0.8 as indicated by the inset.

Fig 3: BTN1A1 suppresses T-cell activation. (A, B) A bead-based T-cell proliferation assay was performed to test the ability of BTN1A1 to regulate T-cell activation, as outlined in (A). Beads were coated with anti-CD3, anti-CD28, and either human IgG (black), human PD-L1 extracellular domain (blue), or human BTN1A1 extracellular domain (red) prior to use for the stimulation of CFSE-stained T cells at the indicated T cell to bead ratios, with T-cell proliferation then being measured by flow cytometry based on CFSE dilution (B). (C, D) T cells were combined with parental or hBTN1A1-expressing PC3 cancer cells, and apoptotic cell death was monitored with a fluorescent caspase 3/7 substrate as a means of assessing the BTNA1A1-mediated inhibition of T cell-induced PC3 cell apoptosis. (E) Quantification of IFN-γ levels in PC3 cell culture supernatants was performed by ELISA. ***p<0.001 (Student’s t-test) (F) To assess the effect of mBTN1A1 on T-cell proliferation, the indicated numbers of 4T1 cells expressing mBTN1A1 were co-cultured with CFSE-labeled murine T cells, and T-cell proliferation was measured by flow cytometry based on CFSE dilution. (G) T cells were activated with anti-CD3 (1, 5, or 10 µg/mL) and IL-2 (10 ng/mL), and IFN-γ levels in culture supernatants were measured by ELISA. (H) T-cell clustering over time was measured using the real-time Incucyte ZOOM imaging system, with clustered T cells being counted with the Incucyte Image analysis software at the indicated time points. (I) Western blotting of primary T cells following T-cell receptor crosslinking using plate-bound OKT3 in the presence of BTN1A1-Fc, PD-L1, or control IgG-Fc (10 µg/mL) for 5 min. All data are representative of experiments that were repeated a minimum of three times. BTN1A1, butyrophilin 1A1; CFSE, carboxyfluorescein succinimidyl ester; EV, empty vector; hBTN1A1, human butyrophilin 1A1; IFN, interferon; IL, interleukin; mBTN1A1, murine BTN1A1; PD-L1, programmed cell death-ligand 1.

Fig 4: Treatment with the anti-BTN1A1 mAb STC810 mimics the effects of knocking out BTN1A1 and suppresses tumor growth. (A) A schematic diagram of the proposed mechanism through which STC810 inhibits signaling via the JAK-STAT pathway. (B) Western blotting analysis of whole-cell lysates derived from PC3 cells treated with 40 µg/mL STC810 or IgG isotype control. PD-L1, STAT1 (pTyr 701), STAT2 (pTyr 690), or IRF9 levels were assessed at 48 hours post-antibody treatment. (C) qRT-PCR was used to assess CXCL9 and CXCL10 messenger RNA levels in PC3 cells treated with 50 ng/mL IFN-γ, 5 µM ruxolitinib, 100 µg/mL STC810, or IgG isotype control at 48 hours post-treatment. qRT-PCR experiments were repeated three times. (D–F) Schematic overview of the live-cell imaging approach used to monitor the T cell-induced apoptosis of PC3 cells overexpressing BTN1A1 (D) with representative images of T cell-induced BTN1A1-overexpressing PC3 cell apoptosis (green) in the control IgG and hSTC810 groups (E). Dose-dependent increases in cytolysis were assessed for PC3 cells expressing hBTN1A1 that were co-cultured with activated effector T cells in the presence of hSTC810 at the indicated concentrations (F). (G–I) T-cell proliferation was examined by FACS based on CFSE dilution in CFSE-labeled primary T cells co-cultured with the indicated concentrations of anti-CD3/CD28 beads conjugated with control IgG1, hSTC810, anti-hPD-1, or anti-hCTLA4 (G). IFN-γ (H) and IL-2 release (I) from these cells were detected by ELISA using supernatant samples collected from cells in (G). All data are representative of experiments that were repeated a minimum of three times. CFSE, carboxyfluorescein succinimidyl ester; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; FACS, fluorescence-activated cell sorting; hBTN1A1, human butyrophilin 1A1; IFN, interferon; IL, interleukin; IRF9, interferon regulatory factor 9; JAK, Janus-activated kinase; mAb, monoclonal antibody; PD-1, programmed cell death protein 1; PD-L1, programmed cell death-ligand 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription.

Fig 5: TRAC usage does not improve m971-based ChTCR T cell functions.a. CD22 expression measured by flow cytometry on Nalm-6 WT or CD22ko. b. Schematic of anti-CD22 CAR and Full ChTCRs using m971 scFv in VH/VL or VL/VH orientation fused to TRAC. c. Representative flow plots showing expression of CD22-specific CAR and Full ChTCRs in primary CD8+ T cells (TCRα/β-BV421 and rCD22-APC). d. Geometric mean ± SD of rCD22-protein-APC bound to receptors (n = 2 independent experiments). e. and f. Concentration of IL-2 (e) or IFN-γ (f) in supernatant after overnight co-culture of CAR and ChTCR T cells with the indicated Nalm-6 cells (mean ± SD, n = 2 independent experiments). Source data