Journal: Communications Biology

Article Title: PD-1 suppresses CAR signaling by forming the inhibitory signalosome colocalizing to CAR microclusters

doi: 10.1038/s42003-025-09415-8

Figure Lengend Snippet: a Representative confocal images of CD19CAR28.ζ (red) and PD-1 (green) in OT-I-TCR-expressing CD8 + T cell hybridomas. The cells were treated with the indicated anti-PD-1 antibody clones for 20 minutes at 37°C and plated onto an SLB containing CD19 and PD-L1. Intensity profiles of CD19CAR28.ζ (red) and PD-1 (green) at the dashed lines in the merged images are shown. b Percentages of CAR-T cells forming PD-1 microclusters in ( a ) ( n = 116, 58, 27, 17, 26, 30, 60, 64, and 34 for each condition, respectively). c PCC values between CAR and PD-1 in ( a ) ( n = 28, 32, 34, 28, 26, 56, 26, 22, and 18 for each condition, respectively). d The cells in ( a ) were cocultured with CD19 + or CD19 + PD-L1 + EL-4 cells in the absence or presence of each antibody at the indicated concentrations for 18 hours, and the concentration of IL-2 in the supernatant was measured by ELISA ( n = 3 per group). e OT-I-TCR-Tg Pdcd1 −/− CD8 + T cells transduced with CD19CAR28.ζ and PD-1 were cocultured with CD19 - PD-L1 - , CD19 + PD-L1 - or CD19 + PD-L1 + EL-4 cells and CD19 + PD-L1 + EL-4 cells in the presence of anti-PD-1 (clone: 29 F.1A12) for 18 h. The percentages of specific lysis at the indicated E/T ratios (left) were determined by RLuc8 intensity of surviving target cells after treatment with the RLuc8 substrate. The concentrations of IL-2 (middle) and IFN-γ (right) in the supernatant was measured by ELISA ( n = 3 per group). n.d., not determined. f Experimental timeline for CAR-T cell transfer into tumor-bearing mice in vivo. CD8 + T cells non-transduced ( n = 3) or transduced with CD19 CAR28.ζ ( n = 5) were intravenously injected into C57BL/6 Rag2 −/− mice transplanted with EL-4 cells expressing CD19 and PD-L1 at 6 days before CAR-T cell transfer. Anti-PD-L1 antibody was applied to the mice every 3 days after CAR-T cell inoculation ( n = 5). g Tumor growth curves (left) and Kaplan–Meier survival curves (right) for each group in ( f ). All data are representatives of 3 to 5 independent experiments. Statistical analysis was performed for each condition with the unpaired Student’s t -test ( c , e , g ), one-way ANOVA followed by Tukey-Kramer post hoc testing ( c , d , e ) and log rank test ( g ). *, p < 0.05, **, p < 0.01, ***, p < 0.001. Scale bars, 5 μm.

Article Snippet: The following antibodies and reagents were purchased: Alexa Fluor 647-anti-pCD3ζ (2.5 μg/ml, K25-407.69, 558489, RRID: AB_647152) from BD bioscience; anti-Erk (1:1000, 4695S, RRID: AB_390779) and anti-pErk (1:1000, 4370S, RRID: AB_2315112) from Cell Signaling Technology; anti-CD3ζ (1:200, 6B10.2, sc-1239, RRID:AB_627020), goat polyclonal anti-PD-1 (E-18), anti-SHP1 (1:500, C-19, sc-287, RRID:AB_2173829) and anti-SHP2 (1:500, C-18, sc-280, RRID:AB_632401) from Santa Cruz Biotechnology Inc.; anti-PD-1 (J43 (BE0033-2, RRID:AB_1107747), RMP1-14 (BE0146, RRID: AB_10949053) and 29 F.1A12 (CP159, RRID:AB_2927527)) from Bio X Cell; STELLA Fluor 650 (RRID: GCKA308-01) and TMR-labeled (RRID: G8252) HaloTag ligands from Promega; and SNAP-Cell647 (S9102S) from New England Biolabs.

Techniques: Expressing, Clone Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transduction, Lysis, In Vivo, Injection