CAR-engineered lymphocyte persistence is governed by a FAS ligand-FAS autoregulatory circuit

Abstract

Chimeric antigen receptor (CAR)-engineered lymphocytes treat B cell malignancies; however, limited persistence can restrain the full therapeutic potential of this approach. FAS ligand (FAS-L)/FAS interactions govern lymphocyte homeostasis. Knowledge of which cells express FAS-L in patients with cancer and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancers to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG expression is limited primarily to endogenous T cells, natural killer (NK) cells and CAR-T cells, while tumor and stromal cell expression is minimal. To establish whether CAR-T and CAR-NK cell survival is FAS-L regulated, we performed competitive fitness assays using FAS-dominant negative receptor (ΔFAS)-modified lymphocytes. Following transfer, ΔFAS-expressing CAR-T/CAR-NK cells became enriched, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models in female mice, ΔFAS coexpression enhanced antitumor efficacy. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS autoregulatory circuit.

Fig. 1. FASLG is expressed by endogenous and CAR-expressing lymphocytes.

a, UMAP visualization of scRNA-seq from n = 244,809 immune and nonimmune cells obtained from the tumor and peripheral blood of patients with n = 10 hematologic cancers, n = 27 solid cancers and peripheral blood from n = 4 healthy donors. Each dot represents an individual cell assigned to one of 18 inferred cell types. CLL, chronic lymphocytic leukemia; Mono, monocyte; MP, macrophage; DC, dendritic cell; T_reg, regulatory T cell; T_con, conventional T cell; T_prolif, proliferating T cell. b, Log-transformed normalized gene expression values for FAS and FASLG overlaid on the UMAP coordinates defined in a. c, Comparison of the frequency and magnitude of FAS and FASLG expression by individual cells assigned to each inferred cell type identified in the UMAP. Bubble size represents the frequency of each cell type that expresses the indicated gene and color indicates the relative intensity of expression. d,e, Representative immunofluorescent confocal image (d) and summary violin plots (e) quantifying FASLG mRNA expression by endogenous and CAR-expressing T cells in the bone marrow of patients with B-ALL treated with a 1928ζ CAR. Samples were co-hybridized with DAPI (blue) and multiplexed RNA-FISH probes specific for the mRNA sequence of the CAR’s single-chain variable fragment (scFv) (green), CD3E mRNA (white) and FASLG mRNA (red). Data are derived from 52 annotated regions using samples from n = 3 patients. Violin distributions are centered around the median (red horizontal line) with quartiles ranges displayed above and below (dashed horizontal lines). The maxima and minima are represented by the top and bottom of each plot. Each dot represents mean FASLG mRNA expression within a particular cell type from an annotated region of interest. f, Summary scatter-plot demonstrating the frequency of FASLG⁺ T cells which either coexpress or do not express a 1928ζ CAR transgene in n = 16 patients with diffuse large B cell lymphoma (DLBC) measured using scRNA-seq. Each dot represents an individual patient. All P values were calculated using a two-sided Student’s t-test. a.u., arbitrary fluorescence units. Source data

Fig. 2. Cell-intrinsic disruption of FAS signaling blocks CAR-T cell apoptosis and protects against TCR-mediated rejection of allogeneic lymphocytes.

a, Schematic of multi-cistronic vectors encoding tEGFR and a 1928ζ CAR alone (EC) ± a FAS-dominant negative receptor (ΔFAS) (ECF). b, Representative FACS plots for activated caspase 3/7 in human T cells left untransduced (UT) or transduced with an indicated vector. Caspase activity was measured at rest and 4 h following stimulation with 100 ng ml⁻¹ of a recombinant FAS-L molecule (lzFAS-L). Median of n = 3 biologically independent samples is shown with mean ± s.e.m. of gated activate caspase 3/7⁺ lymphocytes. c, Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis representing cytokine polyfunctionality of T cells transduced with indicated CAR constructs and co-cultured with or without K562-CD19 cells. T cells exposed to PMA/I were used as a positive control. Concentric plots indicate the median expression of indicated cytokines from n = 3 biologically independent samples. d, Cytolytic activity of UT, EC or ECF-expressing T cells against Nalm6/mCherry. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. P values calculated using a one-way analysis of variance (ANOVA) with Welch’s correction. e,f, Representative FACS plots (e) and summary scatter-plot (f) measuring ZsGreen expression by Jurkat NFAT-ZsGreen reporter cells transduced with tEGFR alone (E), EC or ECF (n = 12 biologically independent samples). As a positive control, an aliquot of transduced Jurkat cells were exposed to PMA/I (n = 3 biologically independent samples). Data are shown as mean ± s.e.m. P values were calculated using a one-way ANOVA with a Šídák’s multiple comparisons test. g,h, Representative FACS plots (g) and summary graph (h) displaying the ratio of HLA-A*03:01⁺ T cells transduced with tLNGFR-1928ζ or tEGFR-1928ζ-ΔFAS tLNGFR and co-cultured with an indicated murinized (m)TCR transduced HLA-A*03:01⁻ T cell population. The anti-HLA-A*03 TCR recognizes HLA-A*03⁺ cells in a peptide agnostic manner while the Flu and PIK3CA (H1047L) TCRs recognize an HLA-A*03:01-restricted viral peptide and neopeptide, respectively. FACS plots were pre-gated on live⁺mTCR⁻HLA-A3⁺ cells. Data displayed as the mean ratio of tLNGFR/tEGFR HLA-A*03:01⁺ T cells ± s.e.m. using n = 3 biologically independent samples. Groups were compared using a one-way ANOVA with Šídák’s multiple comparisons test. Source data

Fig. 3. CAR-T derived FASLG auto-regulates cellular persistence in vivo.

a, Experimental design to test persistence of T cells expressing a 1928ζ CAR ± ΔFAS in tumor-bearing mice. T cells were co-transferred into NSG mice bearing Nalm6 B-ALL and tracked based on tLNGFR or tEGFR expression. i.v., intravenous. b, Distribution of memory T cell subsets before transfer. Bar graphs displayed as mean ± s.e.m. using n = 3 biologically independent samples. T_EM, effector memory T cell; T_EMRA, terminal effector memory T cell. c,d, Representative FACS (c) and summary scatter-plots (d) measuring the ratio of tEGFR⁺/tLNGFR⁺ T cells at the time of infusion (n = 3 biologically independent samples) and following adoptive transfer. Symbols represent individually evaluated mice (n = 10) and are displayed as mean ± s.e.m. P values calculated based on comparison to the infusion product using a two-sided Student’s t-test. e, Western blot for FAS-L protein from control or FASLG KO 1928ζ CAR-transduced T cells at rest or 48 h after anti-CD3/CD28 restimulation. The frequency of frameshift Indels in FASLG are displayed beneath each lane. Representative results from two independent experiments are shown. f, Relative antigen-driven in vitro expansion of control and FASLG KO 1928ζ CAR-T cells ± ΔFAS coexpression. CAR-T cells were combined ~1:1 and serially restimulated at indicated time points with K562-CD19 FASLG KO leukemia cells (left) or left unstimulated (right). Data are displayed as the mean ratio of tEGFR/tLNGFR T cells ± s.e.m. using n = 3 biologically independent samples. Groups compared using a paired two-tailed Student’s t-test for accumulated differences between each time point. g, Experimental design to test the influence of CAR-T-derived FASLG on in vivo persistence in mice bearing established Nalm6 B-ALL. Control or FASLG KO tLNGFR-1928ζ CAR-T cells were co-transferred ~1:1 with control or FASLG KO tEGFR-1928ζ-ΔFAS CAR-T cells into Nalm6 B-ALL-bearing NSG mice. h,i, Representative FACS (h) and summary scatter-plot (i) comparing the ratio of tEGFR to tLNGFR cells at the time of infusion (n = 2 biologically independent samples) and 4 weeks following transfer. Symbols represent values from individually evaluated mice (FASLG wild-type (WT), n = 5; FASLG KO, n = 7) and are displayed as mean ± s.e.m. Groups were compared using a two-sided Student’s t-test. NS, not significant (P > 0.05). Source data

Fig. 4. Disabling FAS signaling enhances CAR-T antitumor efficacy in vivo.

a, Experimental design to compare the in vivo antitumor efficacy of human T cells that express a 1928ζ CAR ± a FAS-dominant negative receptor (ΔFAS) against established Nalm6-luciferase (Luc) B-ALL. b–d, BLI (b), overall survival curves (c) and quantification of tumor burden (d) for Nalm6-Luc B-ALL-bearing NSG mice treated by i.v. injection with 5 × 10⁵ tEGFR⁺ T cells transduced with an indicated vector. Pooled survival data from identically performed experiments using T cells from two unique donors is shown in c and is plotted as a Kaplan–Meier survival curve (tEGFR alone, n = 10; tEGFR-1928ζ, n = 15; tEGFR-1928ζ-ΔFAS, n = 15). Statistical comparisons were made using a log-rank test. Quantification of tumor burden as a function of time in mice treated with transduced T cells from an indicated donor measured using BLI (total flux) (d). Source data

Fig. 5. Knockout of FASLG in 1928ζ CAR-T cells does not impair antitumor cytolytic activity across multiple B cell cancers.

a, Schematic for the CRISPR/Cas9-mediated KO of FASLG in human CD8⁺ T cells expressing a 1928ζ CAR. b, Scatter-plot of FAS RNA-seq values from n = 85 B cell cancer lines featured in the CCLE. Horizontal line represents the median and vertical bars represent the interquartile range. TPM, transcript per million. c, Correlation of FAS transcript counts to total FAS protein levels from n = 15 B cell malignancy lines featured in the CCLE with matched RNA-seq and quantitative proteomic data. All cell lines express WT FAS except for KARPAS-422, which contains a FAS (W176G) mutation located in the protein’s transmembrane domain. Line represents linear regression. d, Measurement of FAS expression on Nalm6 B-ALL cells using FACS and cytolytic activity of FASLG KO versus AAVS1 KO T cells transduced with tEGFR or tEGFR-1928ζ against Nalm6/NLS–mCherry cells. e, Same as d but using Raji B-NHL and Raji/NLS–mCherry B-NHL cells. f, Same as in d but using JVM2 B cell prolymphocytic leukemia (B-PLL) and JVM2/NLS–mCherry B-PLL cells. FAS mean fluorescence intensity (MFI) values compared to an isotype control using an unpaired Student’s t-test using n = 3 biologically independent samples (d–f). Cytolytic activity was measured at indicated E:T ratios using Incucyte with data shown as mean ± s.d. using n = 3 biologically independent samples (d–f). Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). The measured FASLG insertion/deletion frequency following FASLG KO was 93.0 ± 1.0% and 95.7 ± 1.5% in T cells transduced with tEGFR alone and tEGFR-1928ζ, respectively. Source data

Fig. 6. FAS-L/FAS signaling is dispensable for CAR-T antitumor efficacy.

a, Growth kinetics of Nalm6 B-ALL, Raji B-NHL or activated T cells in the presence or absence of lzFAS-L. Each cell type was transduced with mCherry. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. FC, fold change. b,c, Experimental design (b) and Kaplan–Meier survival curve (c) comparing the in vivo antitumor efficacy of human CD8⁺ T cells that express a 1928ζ CAR with CRISPR/Cas9-mediated KO of FASLG or AAVS1 against established Nalm6 B-ALL. tEGFR alone, n = 8; tEGFR-1928ζ AAVS1 KO, n = 15; tEGFR-1928ζ FASLG KO, n = 15. Statistical comparisons were made using a log-rank test. d, Scatter-plots displaying the enrichment or depletion of sgRNAs targeting indicated genes in the death receptor pathway by Cas9-expressing Nalm6 B-ALL cells. Tumor cells were placed under selection by T cells transduced with a 1928ζ CAR (left), a 41BBζ CAR (right) or left nontransduced as specificity controls (Ctrl). Data were reanalyzed from two published genome-scale CRISPR/Cas9 screens and are shown as mean log₂FC ± s.e.m. of sgRNAs targeting indicated genes. NTC, nontargeted control sgRNAs. n = 6 unique sgRNAs per gene for 1928ζ CAR experiment and n = 8 unique sgRNAs per gene for the 19BBζ experiment. Gene level significance was determined using a one-way ANOVA corrected for multiple comparisons by Dunnett’s test. e, Schematic for the CRISPR/Cas9-mediated KO of FAS using an individual sgRNA in Nalm6 B-ALL. f, Time-dependent cytolytic activity of 1928ζ CAR-T cells against FAS KO versus FAS-WT Nalm6/mCherry cells at a high (left) or low (right) E:T ratio. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). Source data

Fig. 7. CAR-NK survival is regulated by a FAS/FAS-ligand autoregulatory circuit.

a,b, Representative FACS (a) and summary scatter-plot (b) quantifying FAS expression by NK cells at rest and 5 d following activation. Data are shown as mean ± s.e.m. for n = 3 biologically independent samples. Statistical analysis was performed by two-sided Student’s t-test. c, Transduction efficiencies of T cells or NK cells displayed as median ± interquartile range from n = 26 T cell and n = 14 NK cell experiments. P values were calculated using an unpaired two-tailed t-test with Welch’s correction. d, Representative FACS plots quantifying lzFAS-L induced apoptosis in nontransduced NK cells or NK cells transduced with indicated vectors. Numbers indicate mean ± s.e.m. of activated caspase 3/7⁺/annexin V⁺ cells (n = 3 biologically independent samples). e, Western blot for FAS-L protein in lysates from FASLG KO or control (Ctrl) NK cells transduced with tEGFR-1928ζ-ΔFAS (ECF) or tLNGFR-1928ζ (LC). Frequency of frameshift Indels in FASLG are displayed beneath each lane. Representative results from two independent experiments are shown. f, Relative antigen-driven in vitro expansion of control and FASLG KO 1928ζ CAR-NK cells ± ΔFAS. CAR-NK cells were combined ~1:1 and serially restimulated at indicated time points with K562-CD19 FASLG KO cells or left unstimulated as controls. Data are displayed as the mean ratio of tEGFR⁺/tLNGFR⁺ cells ± s.e.m. (n = 3 biologically independent samples). Groups compared using a paired two-tailed Student’s t-test for accumulated differences between each time point. NS, not significant (P > 0.05). g, Cytolysis of Raji/mCherry cells co-cultured at indicated E:T ratios with FASLG KO versus FASLG-WT tEGFR-1928ζ CAR-NK cells. Data are shown as mean ± s.e.m. (n = 3 biologically independent samples). Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). NA, not applicable. h, Experimental design to test the in vivo persistence of NK cells that express a 1928ζ CAR ± ΔFAS in Raji B-NHL-bearing mice. i, Scatter-plot comparing the ratio of tEGFR⁺/tLNGFR⁺ CAR-NK cells before infusion and following adoptive transfer in the bone marrow. Symbols represent individually evaluated mice, n = 3 for baseline measurement and n = 10 per time point. P values compare infusion product to each time point using an unpaired, two-sided, Welch’s t-test. Source data

Fig. 8. Disabling FAS signaling enhances CAR-NK antitumor efficacy in vivo.

a, Comparison of the in vitro cytolytic efficiencies of NK cells transduced with a WT or 1XX version of the 1928ζ CAR against Nalm6/mCherry at high versus low E:T ratios. Data are shown as mean ± s.e.m. using n = 3 biologically independent samples. Statistical comparisons were performed using a one-way ANOVA. NS, not significant (P > 0.05). b, Experimental design to compare the in vivo antitumor efficacy of human NK cells expressing the 1XX 1928ζ CAR ± a FAS-dominant negative receptor (ΔFAS) against established Nalm6 B-ALL at a high (top) versus low (bottom) E:T ratio. All mice received a twice-weekly i.p. injection of 1 μg of IL-15 pre-complexed with IL-15Rα-Fc (1:1 M). c, Survival curves for high (top) versus low (bottom) E:T ratios (PBS, n = 5; nontransduced NK cells, n = 5; tEGFR alone, n = 5; 1XX 1928ζ-tEGFR, n = 10; 1XX 1928ζ-ΔFAS-tEGFR, n = 10). Data are plotted as Kaplan–Meier curves with groups compared using a log-rank test. NS, not significant (P > 0.05). d, Model for the dichotomous functions of FAS-L on CAR-T and CAR-NK cellular persistence and antitumor efficacy. Cells colored in red indicate FAS-L-induced apoptosis. Source data

Extended Data Fig. 1. Patient-level correlations between expression of T and NK cell-associated genes and FASLG using bulk RNA sequencing.

Correlation between CD3E, FCGR3A (the gene encoding CD16A), HBA1 (hemoglobin subunit α1), representing T, NK, and red blood cells as non-lymphocyte controls respectively, and FASLG using patient-level data obtained from The Cancer Genome Atlas (TCGA). Red line represents a simple linear regression through the data points. SKCM = skin cutaneous melanoma, BRCA = breast invasive carcinoma, OV = ovarian serous cystadenocarcinoma, PAAD = pancreatic adenocarcinoma. r = Pearson correlation coefficient, RSEM = RNA-Seq by Expectation Maximization, ns = not significant. P values calculated using a two-tailed F-test. Source data

Extended Data Fig. 2. Overview of patient samples used to generate a single-cell atlas of FASLG expression by endogenous and CAR-engineered cells.

Summary of (a) cell therapy naive patients with cancer, (b,c) patients with cancer treated with one of two distinct 1928ζ CARs, and (d) healthy donors analyzed using single-cell techniques to generate a FASLG expression atlas. Pictographs and tables display the sample size for each cancer cohort, gender distribution, age range, total number of single-cells analyzed, and reference to the primary datasets. B-ALL = B cell acute lymphoblastic leukemia; CLL = chronic lymphocytic leukemia; COAD = colon adenocarcinoma; DLBC = diffuse large B cell lymphoma; KIRC = kidney renal clear cell carcinoma; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; READ = rectal adenocarcinoma; SKCM = skin cutaneous melanoma; UM = uveal melanoma. n.d. = no data.

Extended Data Fig. 3. Quantification of FASLG expression by 1928ζ CAR-expressing T cells following in vitro co-culture with CD19⁺ leukemia cells using RNA in situ hybridization.

(a) Representative immunofluorescent confocal images and (b) summary violin plots quantifying FASLG mRNA expression by nontransduced or 1928ζ CAR-expressing T cells at rest or 24 h following in vitro co-culture with CD19⁺ Nalm6 B-ALL. Samples were co-hybridized with DAPI (blue) and multiplexed RNA-FISH probes specific for the mRNA sequence of the CAR’s single-chain variable fragment (scFv) (green), CD3E mRNA (white), and FASLG mRNA (red). Data shown is representative of results from n = 12, n = 13, and n = 15 gated regions from nontransduced T cells, CAR-T cells alone, and CAR-T cells co-cultured with Nalm6 cells, respectively. T cells were derived from n = 2 healthy donors. Violin distributions are centered around the median (red horizontal line) with quartiles ranges displayed above and below (dashed horizontal lines). The maxima and minima are represented by the top and bottom of each plot. Each dot represents mean FASLG mRNA expression within a particular cell type from a region of interest. P-values calculated using a one-way ANOVA with a Šídák’s multiple comparisons test. a.u. = arbitrary fluorescence units. Source data

Extended Data Fig. 4. Experimental design and reagents for testing whether ΔFAS protects against TCR-mediated rejection of allogeneic lymphocytes.

(a) Graphical overview of an experimental design to test whether ΔFAS coexpression protects CAR-T cells from elimination by allo-reactive T cells. CD8⁺ T cells from a non-HLA-A*03 donor were transduced either with control HLA-A*03:01-restricted TCRs or TCR (RG4382-5), an allo-reactive TCR that recognizes HLA-A*03⁺ cells in a peptide agnostic manner. TCR transduced T cells were co-cultured with a ~ 1:1 mixture of HLA-A*03:01⁺ T cells transduced with tLNGFR-1928ζ or tEGFR-1928ζ-ΔFAS at a 10:1 effector-to-target-ratio. (b) Table listing the TRAV, TRAJ, TRBV, TRBJ, and CDR3 sequences for a TCR (RG4382-5) that confers allogeneic recognition of HLA-A*03:01⁺ cells. (c) Representative FACS plots and (d) summary bar graphs of CD107a and TNFα expression by non-HLA-A*03 CD8⁺ T cells transduced with the RG4382-5 TCR and co-cultured with the indicated cells lines. The HLA-A haplotype of each cell line is listed as a table below. Numbers within each FACS plot and bar graph indicate the frequency of CD107a or TNFα producing T cells after pre-gating on live⁺mTCR⁺CD8⁺ cells. Bar graphs displayed as mean ± SEM (n = 3 biologically independent samples). Source data

Extended Data Fig. 5. Transduced human T cells do not upregulate FASLG when co-cultured with murine splenocytes and bone marrow cells.

Fold change in FASLG expression levels by human T cells transduced either with tEGFR alone (left panel) or tEGFR-1928ζ (right panel) and cultured alone or together either with Nalm6 cells or a 1:1 mixture of murine bone marrow (BM) cells and splenocytes. T cells were cultured for 24 h under indicated conditions before isolation by positive selection using an anti-EGFR antibody. FASLG expression was quantified by qPCR and normalized to GAPDH. Data shown as mean ± s.e.m. using donor cells obtained from n = 3 unique mice. P values calculated using a one-way ANOVA with a Šídák’s multiple comparisons test. Source data

Extended Data Fig. 6. Commercially available FACS antibodies fail to specifically detect FAS ligand expression on the surface of resting and activated human T cells.

A bulk population of human T cells was activated using ImmunoCult and left untreated as controls (mock KO) or underwent FASLG gene editing using CRISPR/Cas9 (FASLG KO). After one week, T cells were stained with the indicated fluorochrome-conjugated FAS-L antibody clone at rest or at indicated time points following re-activation. Anti-FAS-L antibody staining was performed in the absence or presence of a FcR blocking antibody. Data are representative of three individually cultured samples per donor and was repeated twice with similar results using two separate donors. Vertical lines define the negative staining T cell population at rest for each fluorescent channel.

Extended Data Fig. 7. ΔFAS enhances the competitive fitness of CAR-T cells following repetitive tumor cell exposure in a FASLG-dependent manner across donors and cancer types.

(a,b) Relative antigen-driven in vitro expansion of control and FASLG KO 1928ζ CAR-T cells ± ΔFAS coexpression following repetitive exposure to different hematologic malignancies. (a) CAR-T cells derived from donor 20240412 A that express (tEGFR⁺) or do not express (tLNGFR⁺) ΔFAS were combined in ~1:1 ratio on day 0 and serially restimulated at indicated time points with K562-CD19 FASLG KO leukemia cells (left panel) or left unstimulated as controls (right panel). (b) Same as (a) but CAR-T cells were generated from donor 20240518 A and restimulated with wild-type (WT) Nalm6 leukemia cells. (c,d) Relative antigen-driven in vitro expansion of control versus FASLG KO (PSMA)28ζ or (MSLN)28ζ CAR-T cells ± ΔFAS coexpression following repetitive exposure to different solid malignancies. (c) (PSMA)28ζ CAR-T cells that express (tEGFR⁺) or do not express (tLNGFR⁺) ΔFAS were combined in ~1:1 ratio on day 0 and serially restimulated at indicated time points with PC3-PSMA (left panel), a human prostate cancer cell line PC3 transduced with PSMA, or left unstimulated as controls (right panel). (d) Same as (c) but using a (MSLN)28ζ CAR serially restimulated with AsPC1, a human pancreatic cancer cell line that naturally expresses mesothelin. Data in all panels reflect the ratio of tEGFR/tLNGFR T cells measured by FACS at indicated time points and is displayed as the mean ratio ± s.e.m. (n = 3 biologically independent samples). Groups were compared using a paired two-tailed Student’s t-test for accumulated differences between each time point. ns, not significant (P > 0.05). Source data

Extended Data Fig. 8. Disabling FAS signaling restrains terminal differentiation of CAR-T cells following serial antigen encounter.

(a) Experimental design for testing the impact of ΔFAS coexpression on the distribution of memory T cell subsets following serial engagement of 1928ζ CAR-T cells with Nalm6 B-ALL cells. T cells transduced either with tLNGFR-1928ζ or tEGFR-1928ζ-ΔFAS were individually co-cultured at a 1:2 ratio with Nalm6 cells. Beginning nine days after the first exposure to tumor cells, Nalm6 cells were re-added to the co-culture every five to six days for a total of three rounds of stimulation. Following each round, an aliquot of cells was harvested and analyzed by FACS. (b) Summary of the distribution of T_CM (CD45RA⁻CD45RO⁺CCR7⁺) and T_EMRA (CD45RA⁺CD45RO⁺CCR7⁻) phenotype CAR-T cells following one, two, or three rounds of stimulation with Nalm6 cells. (c) Experimental design for testing the impact of T cell-derived FASLG on the distribution of memory T cell subsets following serial engagement of tEGFR-1928ζ-ΔFAS CAR-T cells with Nalm6 B-ALL cells. FASLG was knocked out (KO) of transduced T cells using CRISPR/Cas9 or left untreated as controls. Time points were identical to those shown in (a). (d) Summary of the distribution of T_CM and T_EMRA phenotype CAR-T cells following one, two, or three rounds of stimulation with Nalm6 cells. Data in panels (b) and (d) is displayed as the mean ± s.e.m. of the indicated memory subset (n = 3 biologically independent samples after gating on transduced T cells identified by EGFR or LNGFR expression). Groups were compared using a paired two-tailed t-test. ns = not significant. Source data

Extended Data Fig. 9. Disabling FAS signaling enhances CAR-T cell persistence and antitumor efficacy in the setting of B cell Non-Hodgkin lymphoma and a solid malignancy.

(a) Experimental design to compare the in vivo antitumor efficacy and persistence of human T cells expressing a 1928ζ CAR ± a FAS dominant negative receptor (ΔFAS) against four day established Raji-luciferase (Luc) B cell non-Hodgkin lymphoma (B-NHL). (b) Bioluminescence imaging, (c) individual, and (d) summary curves of tumor burden as a function of time using n = 5 (tEGFR), n = 10 mice (tEGFR-1928ζ), or n = 9 (tEGFR-1928ζ-ΔFAS) mice per group. Data in (d) displayed as mean ± standard deviation. Tumor burden at the final time point (d21) was compared using an unpaired one-tailed Mann-Whitney test. (e) Quantification of the absolute number of circulating CAR-T cells (cells mL⁻¹) in the peripheral blood as a function of time in Raji B-NHL bearing mice. Mice received by IV adoptive transfer 5e⁶ tEGFR-1928ζ or tEGFR-1928ζ-ΔFAS CAR-T cells. Each symbol represents values from individually evaluated mice. P values calculated based on comparison of ΔFAS-expressing Vs. not expressing CAR-T cells at each time point using an unpaired, two-sided, t-test. (f) Experimental design for comparing the in vivo antitumor efficacy of human T cells transduced with a (MSLN)28ζ CAR ± ΔFAS against subcutaneous (s.c.) 14 day established AsPC1-luciferase (Luc) pancreatic cancer tumors. All mice received twice-weekly intraperitoneal (i.p.) injections of 1 μg of IL-15 pre-complexed with IL-15Rα-Fc (1:1 M) and a single intravenous (i.v.) injection of 1e⁶ human CAR⁺ T cells. (g) Survival curves and (h) bioluminescent imaging of treated mice. Survival data is plotted as a Kaplan–Meier survival curve with groups statistically compared using a log-rank test. wk = week. Source data

Extended Data Fig. 10. Knockout of TRAIL in 1928ζ CAR-T cells significantly impairs antitumor cytolytic activity.

(a) RNA-seq values for CD19, CD3E, TNFRSF10A (the gene encoding DR4) and TNFRSF10B (the gene encoding DR5) in Nalm6, Raji, and JVM2 B cell malignancy cell lines. (b) Scatter-plot of TNFRSF10A and TNFRSF10B RNA-seq values from n = 83 B cell lines featured in the Cancer Cell Line Encyclopedia (CCLE). Horizontal line represents the median value while the vertical bars represent the interquartile range. P value calculated using an unpaired two-tailed Student’s t-test. TPM = transcript per million. (c) Schematic for the CRISPR/Cas9-mediated knockout (KO) of TRAIL in human CD8⁺ T cells expressing a 1928ζ CAR. (d) Cytolytic activity of TRAIL KO versus wild-type (WT)-TRAIL 1928ζ CAR-T cells or T cells transduced with tEGFR alone against Nalm6/NLS–mCherry at indicated effector-to-target (E:T) ratios. Data shown as mean ± s.e.m. (n = 3 biologically independent samples). Statistical comparisons performed using a one-way ANOVA. ns = not significant, P > 0.05. (e) Table displaying the measured frameshift insertion-deletion (Indel) frequency in TRAIL for each T cell group used in the cytolytic assay. Data shown as mean ± s.e.m. (n = 3 technical replicates from 1 experiment, repeated with similar results using n = 2 donors). Source data