Fig 2: 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.

Fig 3: 6PGD-/- CD8+ T cells display a TEFF phenotype(A) CD8+ T cells were isolated from spleen of 6PGD-/- and 6PGDfl/fl mice and stimulated for 48 h with aCD3 + aCD28 mAbs, followed by RNA extraction and RNA-sequencing analysis. Heatmap of gene expression for 6PGD-/- versus 6PGDfl/fl CD8+ T cells is shown.(B) For confirmation, naive (CD62Lhigh CD44low) CD8+ T cells were isolated and stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL). Expression of representative genes was confirmed by real-time PCR on day 0 and day 4.(C–M) Naive CD8+ T cells were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL) followed by assessment of IFN-? expression by intracellular staining (C) and quantification of IFN-? production by ELISA (D). Expression of granzyme B (E–F), T-bet (G and H), Fas (CD98) (I and J), FasL (CD178) (K and L), and CD69 (M) was assessed by flow cytometry. Results are representative of three independent experiments with n = 6 mice per group. Error bars represent ± SEM. ***p < 0.001; ****p < 0.0001.

Fig 4: CSR and SHM are impaired in ROD1exon3-/- mice. a The absence of ROD1 protein in ROD1exon3-/- B cells. Diagram of gRNAs and the major AUG start codon in the ROD1 loci (upper panel). ROD1 and AID expression were examined by Western blotting. ß-actin served as the loading control. b RT-qPCR examination of post-switch transcripts containing Iµ and each of the CH exons. The RNA levels were normalized to GAPDH (n = 3). c Flow cytometric analysis of IgG1 and IgG3 with ex vivo cultured splenic B cells activated either by LPS or LPS plus IL4. Splenic B cells were isolated from wild-type and ROD1exon3-/- mice. Exogenous ROD1 was transduced into ROD1exon3-/- B cells before stimulation to rescue CSR defects (n = 3). d Quantification of IgG1 and IgG3 CSR efficiency by normalizing to WT levels as shown in (c). *P < 0.05, **P < 0.01 (two-tailed Student’s t test). e Flow cytometric analysis of Peyer’s patch germinal center B cells in wild-type (WT) and ROD1 knockout (KO) mice. Percentage of GL-7+/Fas+ germinal center B cells amongst all B220+ cells is indicated. f SHM analysis of Peyer’s patch germinal center B cells at AID hypermutation substrates located at the JH4 intron. The mean values were determined by Sanger sequencing from 73 (WT) and 88 (-/-) sequenced clones

Fig 5: RNAi via miR‐AB driven by multiple eukaryotic promoters in different cell typesTwo top‐ranked SplashRNA‐designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus‐expressing miR‐AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression. The packaged lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control. After > 96 h of infection, FAS protein levels in the transduced Venus‐positive cells were determined by surface staining, shown in histograms (top panels) and quantified by MFI of APC (bottom panels).