Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1,2,3, but dysfunction due to T cell exhaustion is an important barrier to progress4,5,6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7,8,9,10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
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The sequencing datasets generated in this publication have been deposited in NCBI Gene Expression Omnibus (GEO)60,61 and are accessible through GEO series accession numbers: bulk RNA-seq: GSE136891, scRNA-seq CD19/GD2-28z: GSE136874, scRNA-seq control/JUN-Her2-BBz TILs: GSE136805, ATAC-seq: GSE136796, ChIP–seq: GSE136853.
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This work was supported by a Stand Up To Cancer–St Baldrick’s–National Cancer Institute Pediatric Dream Team Translational Cancer Research Grant (C.L.M.), the Parker Institute for Cancer Immunotherapy (C.L.M., H.Y.C., Z.G.), the Virginia and D.K. Ludwig Fund for Cancer Research (C.L.M.), and NIH P50-HG007735 (H.Y.C.). H.Y.C. is an Investigator of the Howard Hughes Medical Institute. A.T.S. was supported by a Parker Bridge Scholar Award from the Parker Institute for Cancer Immunotherapy and a Career Award for Medical Scientists from the Burroughs Welcome Fund. R.C.L. was supported by the Emerson Collective Cancer Research Fund. The Illumina HiSeq 4000 used here was purchased with the NIH funds (award S10OD018220). Figure 4a was created by S. Knemeyer, SciStories LLC.
C.L.M., R.C.L., E.W.W. and E.S. are inventors on a Stanford University Provisional patent pending on modulating AP-1 to enhance function of T cells; 62/599,299; C.L.M. is a founder of, holds equity in and receives consulting fees from Lyell Immunopharma, which has licensed the technology. R.C.L. is employed by and E.W.W. and E.S. are consultants for Lyell Immunopharma.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Steven Albelda, Takeshi Egawa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 High-affinity 14g2a-GD2(E101K) CAR T cells manifest an exaggerated exhaustion signature compared with the original 14g2a-GD2 CAR T cells.
a, Surface inhibitory receptor expression in CD19, GD2 and HA-GD2(E101K) CAR T cells at day 10 of culture. High-affinity E101K mutation results in increased inhibitory receptor expression in CD4+ and CD8+ CAR T cells, compared with parental GD2 CAR T cells. b, IL-2 secretion after 24h co-culture of HA-GD2(E101K) or original GD2-28z CAR T cells with GD2+ target cells. The increased exhaustion profile of HA-GD2(E101K) CAR T cells corresponds to decreased functional activity, as measured by the ability to produce IL-2 after stimulation. Data are mean ± s.d. of triplicate wells; representative of four independent experiments. c, PCA of bulk RNA-seq demonstrates larger variance between HA-GD2(E101K) and CD19 CAR T cells, whereas GD2-28z (short hinge) CAR T cells are intermediary. Left, CD4+ T cells. Right, CD8+ CAR T cells, naive-derived. Number of replicates is indicated. d, e, HA-GD2(E101K) CAR expression causes enhanced inhibitory receptor expression (d) and decreased memory formation (e) in CD4+ CAR T cells. (See Fig. 1 for CD8+ data). f, IL-2 secretion from control CD19-28z CAR T cells or CD19 CAR T cells with bi-cistronic expression of tonically signalling HA-GD2(E101K) (19-HA-28z, blue) or bi-cistronic expression of Her2-28z (19-Her2-28z, grey) after 24-h stimulation with Nalm6 (CD19+GD2−Her2−) target cells to demonstrate that co-expression of HA-GD2-28z CAR induces T cell dysfunction in CD19-28z CAR T cells. Data are mean ± s.d. of triplicate wells; representative of three independent experiments. g, RNA-seq PCA from Fig. 1 showing PC2 separation is driven by central memory versus naive starting subset and PC3 separation driven by CD4 versus CD8. h, Gene set enrichment analysis (GSEA): gene sets upregulated in day-10 HA-28z CAR T cells versus CD19-28z CAR T cells showed significant overlap with genes upregulated in exhausted versus memory CD8+ cells (left), exhausted versus effector CD8+ cells (middle), and exhausted versus naive CD8+ cells (right) in a mouse model of chronic viral infection4. NES, normalized enrichment score. In b and f, P values determined by unpaired two-tailed t-tests. Source data
a, Venn diagram showing overlapping genes in differential expression analysis of single cell data (red) and the top 200 genes driving the separation of CD19 and HA-28z CAR T cells in bulk RNA-seq (yellow, Fig. 1f). In total, 79 out of the top 200 genes from bulk RNA-seq are differentially expressed by DESeq2 analysis in GD2-28z versus CD19-28z single cells. Highlighted genes from the intersection include inhibitory receptors (CTLA4, LAG3, GITR), effector molecules (CD25, IFNG, GZMB), cytokines (IL13 and IL1A) and bZIP/IRF family transcription factors (BATF3 and IRF4). b, Heat map clustering the top 50 differentially expressed genes in GD2-28z versus CD19-28z single-cell transcriptome analysis. Each row represents one cell. c, Violin plots depicting individual gene expression in CD8+ GD2-28z and CD19-28z single CAR T cells. Genes upregulated in GD2 CAR T cells include inhibitory receptors, effector molecules and AP-1 family transcription factors, whereas CD19 CAR T cells have increased expression of memory-associated genes. P values determined by unpaired two-tailed Wilcoxon–Mann–Whitney U test.
a, b, Insert length (a) and insert distance (b) from transcriptional start site (TSS) for combined (top) and individual (bottom) samples. c, Correlation between replicate samples. d, Location of mapped peaks in each sample by total number of peaks (top) and frequency of total (bottom).
a, Differentially accessible chromatin regions in CD4+ CD19-28z and HA-28z CAR T cells. Both the naive- and central-memory-derived subsets are incorporated for each CAR. b, PCA from Fig. 1h showing PC2 separation is driven by central memory versus naive, and PC3 separation driven by CD4 versus CD8. c, Top transcription factor motifs enriched in chromatin regions differentially accessible in HA-28z CAR T cells comprise AP-1–bZIP family factors in all starting T cell subsets. CD8+ naive subset is shown in Fig. 2. d, Peak clustering by shared regulatory motif (left) and enrichment heat map of transcription factor motifs (right) in each cluster. Ten different clusters including clusters associated with exhausted (EX1–EX4) or healthy (HLT1–HLT2) CAR T cells, central memory or naive starting subset, and CD4 or CD8 T cell subset. Genes of interest in each cluster are highlighted to the right. e, Bulk RNA-seq expression (fragments per kilobase of exon model per million reads mapped, FPKM) of indicated AP-1–bZIP and IRF family members in CD19-28z (black) and HA-28z (blue) CAR T cells. Data are mean ± s.e.m. from n = 6 samples across three donors showing paired CD19 versus HA expression for each gene. *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon matched-pairs signed rank test (see Supplementary Information for exact P values). f, Increased protein expression of c-Jun, JunB, BATF3 and IRF4 in HA-28z versus CD19-28z CAR T cells at day 10 of culture by immunoblotting. Densitometry measurements of the fold change in HA compared with CD19 (n = 2–5 experiments). P values determined by unpaired two-tailed t-tests. g, Correlation network of exhaustion-related transcription factor in naive-derived CD8+ GD2-28z CAR T cells using single-cell RNA-seq analysis. h, Violin plots depicting single-cell gene expression of FOS, JUN, BATF and IRF4 in CD8+ clusters associated with response (CD8.G) and non-response (CD8.B) in patients with metastatic melanoma after checkpoint therapy (CD8T-post-CD8G.B)29. P values determined by unpaired two-tailed Wilcoxon–Mann–Whitney U test. In i–m, AP-1-modified HA-28z CAR T cells exhibit enhanced functional activity. i–k, CAR T cells were co-transduced with (AP-1) or without (control) a lentiviral vector encoding AP-1 transcription factors Fos and c-Jun and a truncated NGFR (tNGFR) surface selection marker. i, Schematic of the lentiviral construct. j, Representative transduction efficiency of AP-1-modified CAR T cells as measured by NGFR surface expression in indicated CD4+ and CD8+ CAR T cells. k, IL-2 production in control (black) or AP-1-modified (red) CAR T cells after 24-h stimulation with 143B-CD19 target cells. AP-1-modified HA-28z CAR T cells show increased IL-2 production compared with control CAR T cells. Data are mean ± s.d. of triplicate wells; representative of two independent experiments. l, m, CAR T cells were co-transduced with lentiviral vectors encoding either c-Fos or c-Jun and a tNGFR surface selection marker. l, Schematics of the c-Fos and c-Jun lentiviral constructs. m, IL-2 production in control (blue), Fos (green) or c-Jun (red) modified CAR T cells after 24-h stimulation with Nalm6-GD2 target cells. Data are mean ± s.d. of triplicate wells; representative of two independent experiments. Asterisk in i and l denotes a stop codon. P values determined by unpaired two-tailed t-tests. ns, not significant (P > 0.05). Source data
JUN CAR T cells were produced as in Fig. 3. a, c-Jun overexpression does not affect the CD4:CD8 ratio in HA-28z CAR T cells at day 10 of culture (n = 14 independent experiments). Lines indicate paired samples from the same donor. P values determined by paired, two-tailed t-tests. b, c, Fold increase in IL-2 (b) and IFNγ (c) release after 24-h co-culture with the indicated target cells in JUN versus control CD19 and HA-28z CAR T cells. Each dot represents one independent experiment from different donors of n = 8 total experiments. d, Representative contour plots demonstrating increased intracellular cytokine production in both CD4+ and CD8+ JUN-HA-28z versus control HA-28z CAR T cells stimulated for 5 h with Nalm6-GD2 target cells. Representative of three independent experiments. e, Left, flow cytometry showing representative CD45RA and CD62L expression in control versus JUN CAR CD4+ T cells at day 10. Right, relative frequency of effector (CD45RA+CD62L−), stem-cell memory (CD45RA+CD62L+), central memory (CD45RA-CD62L+), and effector memory (CD45RA−CD62L−) cells in CD4+ control or JUN-HA-28z CAR T cells (n = 6 donors from independent experiments). Lines indicate paired samples from the same donor. P values determined by paired two-tailed t-tests. f, Extended in vitro expansion of control (blue) or JUN-modified (red) CD19 CAR T cells in five independent experiments with five different healthy donors. At the indicated time points, T cells were re-plated in fresh T cell media with 100 IU ml−1 IL-2. T cells were counted and fed to keep cells at 0.5 × 106 cells per ml every 2–3 days. For Donor-1, 5 × 106 viable T cells were re-plated on days 14 and 28. For Donor-2, 5 × 106 viable T cells were re-plated on days 14, 28, 42 and 56. For Donor-3, 5 × 106 viable T cells were re-plated on days 10, 17, 24 and 31. For Donor-4 and Donor-5, 5 × 106 viable T cells were re-plated on days 10, 17, 24 and 34. g, On day 42 of culture, 1 × 106 viable T cells from Donor-4 (top) and Donor-5 (bottom) were re-plated and cultured for 7 days with or without IL-2. h–j, Cell-surface phenotype of control or JUN-CD19-28z CAR T cells from Fig. 3g (Donor-3) on day 46 of culture. h, CD4 versus CD8 expression. i, Surface expression of CD45RA versus CD62L . j, Day 46 surface exhaustion marker expression in CD8+ T cells. Source data
Extended Data Fig. 6 c-Jun overexpression mediates transcriptional but not epigenetic reprogramming of exhausted HA-28z CAR T cells.
a, The log2-transformed fold change in HA versus JUN-HA ATAC-seq demonstrating no significantly different peaks between conditions. b, Gene expression of 319 genes differentially expressed in JUN versus HA-28z CAR T cells (log2-transformed fold change > 2, Padj < 0.05). Genes downregulated in JUN CAR T cells (blue) include exhaustion-associated genes such as BATF3, GZMB, LAG3, JUNB and ENTPD1 (encoding CD39). Genes upregulated in JUN CAR T cells (red) include genes associated with naive and memory differentiation such as IL7R, LEF1, SELL (CD62L), CD44, and KLF3. c, Venn diagrams showing overlap of the 319 genes differentially expressed in JUN versus HA-28z and the top 200 genes distinguishing exhausted (HA) and healthy (CD19) CAR T cells from PC1 in Fig. 1e, f. Genes downregulated in JUN CAR T cells overlap with exhaustion-associated (HA, PC1-exhausted) genes, and genes upregulated in JUN CAR T cells overlap with genes associated with healthy memory differentiation (CD19, PC1-healthy). d, DAVID bioinformatics analysis of transcription factor-binding sites within the 319 genes differentially expressed in JUN CAR T cells reveals that the top transcription factor binding motif belongs to the AP-1 family (269 out of 319 genes). e, Proposed mechanisms of c-Jun-mediated rescue of T cell exhaustion. AP-1i indicates an exhaustion-associated AP-1 complex. f, Immunoblot of total c-Jun and phosphorylated c-Jun (p-c-JunS73) in control, JUN-WT and JUN-AA HA-28z CAR T cells. g, Immunoblot analysis of c-Jun protein expression in control and indicated JUN-variant-expressing HA-28z CAR T cells in either soluble or chromatin-bound cellular lysate fractions. c-Jun variants with deletions in the C-terminal DNA binding and leucine zipper dimerization domains (basic, LeuZ and bZIP) cannot bind chromatin and do not rescue functional activity. h, Decrease in mRNA expression of BATF, BATF3 and JUNB in JUN HA-28z CAR T cells compared with HA-28z cells (n = 3 donors, normalized to CD19 mRNA). P values determined by ratio paired two-tailed t-test. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 7 c-Jun overexpression decreases chromatin binding and complexing of JunB–BATF–BATF3–AP-1 complexes.
a, Immunoblot analysis for the indicated AP-1–bZIP and IRF family member proteins in control and JUN CD19-28z and HA-28z CAR T cells (day 10). b, Immunoblot analysis for the indicated AP-1–bZIP and IRF family member proteins in control and JUN HA-28z CAR T cells (day 10) in either soluble or chromatin-bound cellular lysate fractions. c, c-Jun overexpression decreases JunB–BATF and JunB–BATF3 complexes by immunoprecipitation–immunoblot analysis. Input (left), immunoprecipitation for c-Jun (middle), or JunB (right) in control or JUN HA-28z CAR T cells. Levels of IRF4 protein and complexes with c-Jun are unchanged. d–h, ChIP–seq analysis for c-Jun and IRF4. d, Motif enrichment in IRF4-bound (left) or c-Jun-bound (right) loci. e, IRF4 signal genome-wide. Data shown for each transduction at all IRF4-bound sites. The x and y axes show log-transformed normalized count signal in control and JUN-overexpression cells, respectively. f, IRF4 and c-Jun ChIP–seq genome tracks in JUN or control HA-28z CAR T cells. c-Jun ChIP with reference exogenous genome (ChIP–Rx; top), with x axis representing genomic position and y axis representing reference-adjusted reads per million (RRPM). IRF4 ChIP (bottom), with x axis representing genomic position and y-axis representing reads per million (RPM). Arrows indicate peaks with increased c-Jun binding in HA-28z JUN cells at IRF4-bound sites within genes previously described to be regulated by IRF4 or BATF (TCF7, HAVCR2 and HIF1A)7. g, Overexpressed c-Jun is bound to IRF4-occupied sites in the genome. Enrichment plot of c-Jun ChIP–Rx signal (left) or IRF4 ChIP–seq signal (right) in either JUN overexpression (red) or control (blue) HA-28z CAR T cells at all JUN-bound sites. The x axis shows distance from centre of JUN-bound site, and y axis shows average RRPM across replicates for c-Jun ChIP or average RPM across replicates for IRF4 ChIP. h, Venn diagram showing number of genes bound by IRF4 and/or c-Jun (n genes expressed/n genes bound). GSEA analysis with genes bound only by IRF4 (top) and genes bound by c-Jun and IRF4 (bottom), comparing levels of expression in JUN versus control HA-28z CAR T cells (normalized P < 0.05, FDR < 25%) i, Immunoblot of indicated AP-1 and IRF protein in control or CRISPR-knockout HA-28z CAR T cells demonstrating productive knockout of target protein. j, IL-2 (top) and IFNγ (bottom) release in HA-28z CAR T cells with control or CRISPR-knockout of the indicated AP-1 or IRF4 gene after 24 h stimulation with Nalm6-GD2 or 143B target cells. Data are mean ± s.d. of triplicate wells; representative of six independent experiments. Fold change across all experiments in Fig. 4e. In k and l, NSG mice were inoculated with 1 × 106 Nalm6-GD2 leukaemia cells via intravenous injection. A stress-test dose of 1 × 106 mock, HA-28z control, JUN-WT, JUN-AA or JUN-ΔbZIP HA-28z CAR+ T cells was given intravenously on day 7. k, Tumour progression was monitored using bioluminescent imaging. l, JUN-WT and JUN-AA HA-28z CAR T cells enhanced long-term survival, and control and JUN-ΔbZIP HA-28z CAR T cells were almost non-functional compared with mock untransduced T cells at this dose. Data are mean ± s.e.m. of n = 5 mice per group. For gel source data see Supplementary Fig. 1. Source data
Extended Data Fig. 8 Functional rescue of exhausted HA-28z CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation.
a, Schematic of the destabilization domain (DD)-regulated JUN expression vector. b, Schematic of drug-induced stabilization of JUN-DD expression. Yellow diamond denotes trimethoprim (TMP)-stabilizing molecule. c, Kinetics of drug-induced c-Jun stability in JUN-DD CAR T cells as assessed by immunoblot. At time 0, 10 μM TMP was either added to untreated cells (ON) or washed out of previously treated cells (OFF). Cells were removed from each condition at 1, 2, 4, 8, 24 and 48 h and prepared for immunoblot analysis of c-Jun expression. The observed band corresponds to the size of JUN-DD. d, Densitometry analysis was performed on the blots from c and normalized to loading control. Expression was plotted against time and first-order kinetics curves were fit to the data to determine t1/2 for OFF and ON kinetics. e, Total c-Jun expression in control, JUN-WT and JUN-DD HA-28z CAR T cells at day 10 by intracellular flow cytometry (left) and immunoblot (right). f, IL-2 (left) and IFNγ (right) production in control (blue), JUN-WT (red) or JUN-DD (OFF-green, ON-purple) modified HA-28z CAR T cells 24 h after stimulation with Nalm6-GD2 or 143B target cells, or media alone (baseline) at day 10. In e and f, OFF indicates without TMP, ON indicates T cells cultured in the presence of 10 μM TMP from day 4 and during co-culture. In g and h, TMP was added either during T cell expansion (starting at day 4) or only during co-culture with tumour cells as indicated in g. For ON-to-OFF and OFF-to-ON conditions, TMP was removed or added 18 h before co-culture to ensure complete c-Jun degradation or stabilization, respectively, before antigen exposure. h, IL-2 expression in one representative donor (left, s.d. across triplicate wells) and fold increase in IL-2 (s.e.m. of n = 6 independent experiments representing three different donors, relative to OFF–OFF condition). P values determined by unpaired two-tailed t-tests. For gel source data, see Supplementary Fig. 1. Source data
Extended Data Fig. 9 c-Jun overexpression enhances CD19-BBz CAR T cells activity under suboptimal antigen stimulation and Her2 or GD2-BBz CAR T cell function in solid tumours.
a, CD19 surface expression on parental Nalm6-WT (green), CD19-knockout Nalm6 (19KO, black), and two different CD19-low Nalm6 clones (F and Z) Nalm6-19KO plus CD19low-F (F; blue), and Nalm6-19KO plus CD19 low-Z (red). b, IL-2 (left) and IFNγ (right) release after co-culture of control (blue) or JUN (red) CD19-BBz CAR T cells exposed to Nalm6-WT and Nalm6-19low clones F and Z. c, JUN versus control CD19-BBz CAR T cell lysis of GFP+ Nalm6-WT (top), Nalm6-F (middle) or Nalm6-Z (bottom) target cells at a 1:2 effector:target cell ratio, demonstrating enhanced activity of JUN CAR T cells at low antigen density. Data in b and c denote mean ± s.d. of triplicate wells; representative of four independent experiments. In d–f, NSG mice were inoculated with 1 × 106 Nalm6-19low clone F leukaemia cells. On day 1, 3 × 106 control or JUN CD19-BBz CAR+ T cells or 3 × 106 mock0transduced T cells were transferred intravenously. d, Tumour growth was monitored by bioluminescent imaging. e, JUN expression significantly improved the long-term survival of CAR-treated mice. f, Mice receiving JUN-CD19-BBz CAR T cells display increased peripheral blood T cells on day 20. Data in d–f denote mean ± s.e.m. of n = 5 mice per group; representative of three independent experiments. Long-term tumour-free survival is impeded in this model owing to outgrowth of CD19-negative disease. In g–i, NSG mice were inoculated with 1 × 106 143B osteosarcoma cells via intramuscular injection; then, 1 × 107 mock, Her2-BBz or JUN-Her2-BBz CAR T cells were given intravenously on day 7. g, Tumour growth was monitored by caliper measurements. h, Long-term survival. i, On day 20 after tumour implantation, peripheral blood T cells were quantified in mice treated as in g. Data are mean ± s.e.m. of n = 5 mice per group; representative of two independent experiments. j, Vector schematic of JUN-GD2-BBz retroviral vector construct. k, IL-2 (left) and IFNγ (right) production in JUN-modified (red) or control (blue) GD2-BBz CAR T cells after 24 h stimulation with Nalm6-GD2 or 143B target cells. l, GD2-BBz CAR T cell lysis of GFP+ Nalm6-GD2 target cells at 1:1 (left) or 1:4 (right) effector:target cell ratio. Data in k and l denote mean ± s.d. of triplicate wells; representative of four independent experiments. In m and n, NSG mice were inoculated with 0.5 × 106 143B-19 osteosarcoma cells via intramuscular injection; then, 1 × 107 mock, GD2-BBz or JUN-GD2-BBz CAR T cells were given intravenously on day 3. m, Tumour growth was monitored by caliper measurements. n, Peripheral blood CD4+ (left) or CD8+ (right) T cell counts at day 14 after tumour engraftment. Data are mean ± s.e.m. of n = 5 mice per group; representative of two independent experiments although early deaths (unrelated to tumour size) precluded survival curves in both models. P values in n were determined by a Mann–Whitney test. All other P values determined by unpaired two-tailed t-tests. Survival curves were compared using the log-rank Mantel–Cox test. Source data
Extended Data Fig. 10 c-Jun overexpressing CAR TILs demonstrate increased activity in osteosarcoma xenograft tumours.
Experimental design described in Fig. 6. a–c, Frequency (a), phenotype (b) and ex vivo functional activity (c) of CD4+ TILs from mice treated with Her2-BBz or JUN-Her2-BBz CAR T cells. a, Left, CD4+ levels as a proportion of total live tumour cells. Right, CAR+ as a frequency of total live CD4+. b, Percentage of PD-1+ (left) and PD-1 mean fluorescence intensity (MFI; middle) of total live CD4+ with representative flow histograms (right). Mock untransduced T cells were from spleens of tumour-bearing mice at the same time point. c, Frequency of indicated cytokine- or CD107a-producing cells after 5-h re-stimulation with Nalm6-Her2+ target cells. Gated on total, live CD4+ T cells (left) with representative contour plots (right). Data are mean ± s.e.m. of n = 6 mice per group. Each data point represents an individual mouse. P values determined by unpaired two-tailed t-tests. In d–g, dissociated tumour cell suspensions were labelled and sorted by FACS analysis to isolate live, human CD45+ TILs. Sorted cells from six mice per group were pooled and approximately 10,000 cells were processed for 3′ single-cell RNA-seq on the 10X Genomics platform. d, Volcano plot showing results of differential expression analysis comparing JUN-Her2-BBz CAR T cells with control Her2-BBz CAR T cells. Top three upregulated and downregulated genes are highlighted. e, Heat map of the top 20 most significantly upregulated and downregulated genes. f, Uniform manifold approximation and projection (UMAP) embedding analysis showing JUN-Her2-BBz and control Her2-BBz CAR T cells overlaid. g, Expression of indicated transcripts in JUN-Her2-BBz or control Her2-BBz CAR T cells showing localization of CD4+ and CD8+ subsets, activation marker (IL2RA), exhaustion marker (NR4A2), and maintenance of a small memory-like population (IL7RA, KLF2) in JUN-overexpressing Her2-BBz CAR T cells within the solid osteosarcoma tumour microenvironment. Source data
Supplementary Figure 1 . The original source images for all data obtained by electrophoretic separation (immune-western blots). The full scanned images show the uncropped form with molecular weight markers and loading controls. Gels are labeled according to the corresponding Figure panel within the main or Extended Data Figures.
Supplementary Table 1 Fig. 1f. List showing the gene name and FPKM value from RNA-seq of the indicated samples measured on day 10 of culture. These genes represent the top 200 most differentially expressed genes driving PCA separation of HA vs CD19 CAR across all samples.
Supplementary Table 2 . Table describing the exact statistical tests and providing exact associated values.
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Lynn, R.C., Weber, E.W., Sotillo, E. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019). https://doi.org/10.1038/s41586-019-1805-z
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