Fig 2: Recombinant IFNß promotes apoptosis, activation, inhibitory receptor expression, and dysregulated expression of the CAR.a Annexin V expression on CD8 CAR T cells or untransduced (UTD) T cells cultured in IL2 (50 U/mL) in the presence or absence of recombinant murine IFNß and 1µM Ruxolitinib (JAK1/2 inhibitor) for 48 h. b Annexin V expression on CD8 CAR T cells cocultured with B16EGFRvIII cells at an E:T ratio of 1:5 in the presence or absence of recombinant murine IFNß and/or 1µM Ruxolitinib. Fas (c) or cleaved caspase (d) expression on CD8 CAR T cells or UTD cells cultured in IL2 with IFNß. e Median fluorescence intensity (MFI) of CAR expression measured using Biotin-Protein L and streptavidin- PE (Protein L + SA PE) compared to Annexin V on CAR T cells cultured in IL2 with or without IFNß (10 ng/mL). CAR expression on CD8 CAR T cells cultured in IFNß for 48 h in the absence (f) or presence of target tumor cells (g). Representative (h) and mean (i) expression of CD25 and CD69 for CAR T cells cultured in IL2 with IFNß. Statistical comparisons are indicated for CD25- CD69- populations in (i). j Co-expression of the CAR and activation markers on CD8 CAR T cells. k Mean expression of PD1, LAG3, TIM3 on CD8 CAR T or UTD cells cultured in IL2 or in the presence of B16EGFRvIII cells (E:T 1:5). l Fold change in inhibitory receptor and CAR expression is shown for cells grown in IL2 with additional recombinant IFNß relative to culture in IL2 alone. m WT or transgenic IFNAR1 KO CAR T cells were cocultured with B16EGFRvIII (E:T ratio of 1:5) which were mock infected or infected with VSVmIFNß 6 h prior to coculture. Surface CAR expression is represented as fold change relative to coculture with mock infected tumor cells. n Inhibitory receptor expression was quantified on WT or IFNAR1 KO CD8 CAR T cultured as in (m). Data are representative of two independent experiments (panels a–l). Experiments in panels (m, n) were performed once. Means ± SD of n = 4 (panel a) or n = 3 (panels b–d, f, g, i, k, m, n) technical replicates are shown. P-values were determined using a two-way ANOVA with a Tukey multiple comparisons post-test (a–d, f, g, i). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.

Fig 3: TCRTg101 expand and acquire a dysfunctional phenotype in leukemia-bearing animals(A and B) Survival of C57BL/6 mice challenged i.v. with C1498 cells (106) and transferred or not with in vitro activated (A) or naive (B) TCRTg101 (4 × 106) 3 days later. Data are pooled from 2 independent experiments with 3–4 mice/group.(C–G) 2 × 106 CTV-labeled TCRTg101 (CD45.2) were transferred into B6.SJL mice (CD45.1) followed by an i.v. challenge with 106 C1498 leukemia cells 1 day later.(C) Representative FACS plots showing CTV dilution of adoptively transferred TCRTg101 in spleens and livers of leukemia-bearing mice at the indicated time points.(D–G) Quantitative data showing the percentage of divided TCRTg101 (D and F) and TCRTg101 number (E and G) in spleens (D and E) or livers (F and G) of leukemia-bearing mice over time. Data are pooled from 2 to 3 independent experiments with 2 to 4 mice/group including data from leukemia-free control mice.(H) Representative FACS plots showing expression of PD-1, LAG-3, TIM3, and TIGIT on TCRTg101 in livers of tumor-free or leukemia-bearing mice.(I) Quantitative data from (H) as mean ± SD.(J–Q) Mononuclear cells isolated from livers of mice 13 to 14 days (J–M) or 17 to 18 days (N–Q) after C1498 cell inoculation were restimulated with PMA + ionomycin (P+I) (J–M) or anti-CD3 and anti-CD28 antibodies (N–Q) for 4 h. Cytokine production was analyzed by flow cytometry. Gating was performed on TCRβ+CD8+CD45.1 cells (endogenous CD8+ T cells) or TCRβ+CD8+CD45.2 cells (TCRTg101).(J and N) Representative FACS plots showing TNFα and IFNγ production by endogenous CD8+ T cells or TCRTg101.Quantitative data are shown in (K)–(M) and (O)–(Q) as mean ± SD.(R) TCRTg101 killing assay. CTVhi and CTVlo TCRTg101 (CD45.1.2) were FACS-purified from livers of mice 17 to 18 days after C1498 inoculation and were co-cultured for 20 h with equal numbers of C1498 cells and EL4 cells (both CD45.2) labeled with different CTV concentrations. Naive TCRTg101 purified from control Tg101 mice, or those activated in vitro with anti-CD3 and anti-CD28 antibodies or with C1498.B7.1 cells served as negative and positive controls for C1498 cell lysis, respectively. Specific lysis was analyzed by flow cytometry. Gating was performed on live CD8−CD45.2 cells.Data are representative (K–M) of or pooled (I and O–R) from 2 independent experiments with 2 to 4 mice/group and shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant; NT, no tumor.

Fig 4: Recombinant IFNß promotes human CAR T cell apoptosis and inhibitory receptor expression.CAR and untransduced (UTD) T cells from three healthy donors were cultured in the absence or presence of human IFNß (0, 0.1, 1, or 10 ng/mL) for 48 h and analyzed for the expression of (a) cleaved caspase 3, (b) TIM3, (c) LAG3, and (d) PD1. e Average expression of individual and combinations of inhibitory receptors for three donors. Data are representative of two independent experiments. Data points indicate the average of two technical replicates for each donor and P-values were determined for all three donors using a two-way repeated measures ANOVA with a Tukey multiple comparisons post-test. Source data are provided in the Source Data File.

Fig 5: Type I IFN resistant CAR T cells provide enhanced therapy with VSVmIFNß in lymphodepleted mice.a CAR T cells were genetically modified using CRISPR Cas9 one day after transduction by nucleofection of an RNP complex consisting of Cas9 duplexed with tracrRNA and two IFNAR1 specific or two negative control crRNAs. 48 h following modification, expression of the CAR (Thy1.1) and the IFNAR1 is shown. b Two days after modification, CAR T cells were cultured in IL2 (50 U/mL) in the absence or presence of additional recombinant mouse IFNß. CAR expression is shown for representative CD8 CAR T cells (left) and quantified in three replicates in CD8 and CD4 CAR T cells (right). c The percent of CRISPR IFNAR1 KO or control CD8 and CD4 CAR T cells expressing Fas is shown. d Inhibitory receptor expression (PD1, LAG3, TIM3) quantified on CRISPR IFNAR1 KO or control CD8 CAR T cells cultured in IL2 in the absence or presence of additional IFNß. Data shown are representative of two independent experiments. Technical replicates are shown ± SD (n = 3 (b) n = 4 (c)). e Mice bearing B16EGFRvIII tumors were treated with 5 × 107 pfu VSVmIFNß or PBS 6 h prior to administration of 1 × 107 IFNAR1 KO EGFRvIII CAR T cells on day 7. Select groups received two additional doses of 5 × 107 pfu VSVmIFNß on days 10 and 13. Select groups received a lymphodepleting dose of radiation (5 Gy TBI) on day 6. n = 6/group. f Tumor growth is shown in the left panel and overall survival is shown in right panel. g Mice bearing B16EGFRvIII tumors received a lymphodepleting dose of radiation (5 Gy TBI) on day 6 and were treated with 5 × 107 pfu VSVmIFNß or PBS 6 h prior to administration of 1 × 107 WT or IFNAR1 KO EGFRvIII CAR T cells on day 7. Select groups received six additional doses of 5 × 107 pfu VSVmIFNß on days 10,13,17,20,23, and 27. n = 6/group. Experiments in (e) and (g) were performed once. P-values were determined using the Log-rank Mantel-Cox test (f, h) and a two-way ANOVA with a Tukey multiple comparisons post-test (b, c). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.