Journal: bioRxiv
Article Title: T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors
doi: 10.64898/2025.12.23.696308
Figure Lengend Snippet: (A) Prf1-/- CAR-T cells were co-cultured at varying E:T ratios with B16 targets that are WT, TAK1 KO, TBK1/IKKε DKO and HOIP KO, and analyzed in the IncuCyte for 48 h. Cell death data for E:T of 1:16 is shown. (B) The experiment in (A) was independently performed 3 times. The mean confluency at 60 h of 9 wells from the 3 biological replicates is shown in the bar chart. (C) The indicated B16 targets were co-cultured with media or with Prf1-/- CAR-T cells at varying E:T ratios with control IgG or anti-TNF/IFNγ (50 ug/mL). Target killing was analyzed in the IncuCyte for 48 h and data for E:T of 1:16 is shown. (D) RFP-labeled B7H3 KO targets of the indicated genotypes were mixed with 10% unlabeled B7H3⁺ B16 WT cells and co-cultured with Prf1 -/- CAR-T cells at varying E:T ratios. Paracrine killing was quantified by RFP counts of the B7H3-negative targets in the IncuCyte. Data from E:T ratios of 1:16 and 1:256 are shown. (E) 2.5 x 10 5 B7H3 KO or B7H3/TBK1/IKKε TKO B16 cells that were unmixed or mixed with 10% WT B16 cells were implanted into B6 hosts. 5 days later, mice were administered Prf1-/- CAR-T cells against B7H3 via tail vein infusion and tumor growth measured. Spider plots shown were compiled from two independent experiments, each with N=5 per group. Thick red line indicates average tumor volume in each group of mice. Bar chart shows mean tumor volume ± SD for each group at day 23. p values from Mann-Whitney tests are shown for data in (B and E).
Article Snippet: For western blotting, primary antibodies used were TBK1 (Cell Signaling, 3013S), IKKε (Cell Signaling, 2690S), phospho-STAT1 (Tyr701) (Cell Signaling, 9167S), STAT1 (Cell Signaling, 9172S), cleaved caspase 8 (Cell Signaling, 8592S), cleaved caspase 3 (Cell Signaling, 9661S), cleaved PARP (Cell Signaling, 9541S), FADD (Abcam, ab124812), NFκB p65/RelA (Cell Signaling, 8242S), NFκB p105/p50 (Cell Signaling, 12540S), NFκB p100/p52 (Cell Signaling, 52583S), RelB (Cell Signaling, 4922S). β-Actin (Cell Signaling, 3700S) was used as a loading control for whole cell lysates, while HDAC1 (Cell Signaling, 5356T) was used as a loading control for nuclear lysates.
Techniques: Cell Culture, Control, Labeling, MANN-WHITNEY
Santa Cruz Biotechnology is a verified supplier 
