T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3,4,5, in part because they enter a hyporesponsive (‘exhausted’ or ‘dysfunctional’) state6,7,8,9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19+ tumour-bearing mice. CD8+CAR+ tumour-infiltrating lymphocytes and CD8+ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10,11,12. CD8+ T cells from humans with cancer or chronic viral infections13,14,15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.
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All data generated and supporting the findings of this study are available within the paper. RNA-seq and ATAC-seq data are available in the Gene Expression Omnibus (GEO) database under the SuperSeries reference number GSE123739. Source Data for Figs. 2, 4 and Extended Data Figs. 2, 4, 7, 8, 9 are provided in Supplementary Tables 1–5. Additional Source Data are provided in the online version of the paper. Additional information and materials will be made available upon request.
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We would like to thank C. Kim, L. Nosworthy, R. Simmons, D. Hinz and C. Dillingham of the LJI Flow Cytometry Core Facility for cell sorting; J. Day, S. Wlodychak and C. Kim of the LJI Next Generation Sequencing Facility for next-generation sequencing; S. Schoenberger for B16-OVA cells; A. W. Goldrath for MC38 cells; V. Wong, S. Trifari and G. Mognol for advice and discussions; B. Peters for statistics discussions; and the Department of Laboratory Animal Care (DLAC) and the animal facility for excellent support. This work was funded in part by the US National Institutes of Health (NIH) AI109842, AI040127, S10OD016262, S10 RR027366 (A.R.); NIH T32 GM007752 and PhRMA Foundation Paul Calabresi Medical Student Research Fellowship (J.C.); UC MEXUS-CONACYT Fellowship (I.F.L.-M.); AACR-Genentech Immuno-oncology Research Fellowship, 18-40-18-SEO (H.S.); Cancer Research Institute (CRI) Irvington Postdoctoral Fellowship (C.-W.J.L.); Fraternal Order of Eagles Fellow of the Damon Runyon Cancer Research Foundation, DRG-2069-11 (J.P.S.-B.); JSPS KAKENHI Scientific Research (B) 16KT0114 (T.S.); and JSPS KAKENHI (S) JP17H06175, Challenging Research (P) JP18H05376, and Advanced Research & Development Programs for Medical Innovation (AMED-CREST) JP18gm0510019, JP18gm1110009 (A.Y.).
Nature thanks Takeshi Egawa, Golnaz Vahedi and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The La Jolla Institute of Immunology has a pending patent, PCT/US2018/062354, covering the use and production of engineered immune cells to disrupt NFAT-AP1 pathway transcription factors, including the NR4A family members, with J.C., H.S., J.P.S-B. and A.R. listed as inventors. A.R. receives funding from Takeda for subsequent research related to this subject matter. None of the other authors has any competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Left three panels, EL4, MC38 and B16-OVA cell lines expressing hCD19. Grey, parental; black, hCD19-expressing cells. Right, B16-OVA-hCD19 cells recovered after growth in a C57BL/6J mouse followed by culture for 7 days. Grey, isotype control; black, anti-hCD19. Data from one biological replicate in each case. b, Left, growth curves (mean ± s.e.m., 15 mice per group) of 250,000 B16-OVA parental or B16-OVA-hCD19 tumour cells in vivo after inoculation into C57BL/6J mice. There is no significant difference at any time point (ordinary two-way ANOVA, P > 0.9999 at day 19). Right, growth curves (mean ± s.e.m.) of 250,000 (n = 5 mice) or 500,000 (n = 6 mice) B16-OVA-hCD19 tumour cells in vivo after inoculation (significant difference between the two groups at day 21; ordinary two-way ANOVA; *P = 0.0146). c, Diagram of the CAR construct. LS, leader sequence; SS, signal sequence; myc, myc epitope-tag; scFv, single chain variable fragment against human CD19; followed by the mouse (m) CD28 and CD3ζ signalling domains, the 2A self-cleaving peptide and the mouse Thy1.1 reporter. d, CAR surface expression monitored by myc epitope-tag and Thy1.1 expression. Mock-transduced CD8+ T cells were used as controls. e, Cytokine (TNF, IFNγ) production by CAR CD8+ T cells after re-stimulation with EL4-hCD19 cells or with PMA and ionomycin. f, Quantification of the data shown in e; P values (TNF: ****P < 0.0001, IFNγ: ***P = 0.0009) were calculated using a two-tailed unpaired t-test. g, In vitro killing assay (mean ± s.e.m.) of CD8+ CAR and mock-transduced T cells; data from two biologically independent experiments, each with three technical replicates. h, Inhibitory surface receptor expression on CAR- and mock-transduced CD8+ T cells cultured in vitro for 5 days; data representative of three biological replicates. Grey shading, isotype control; black line, mock or CAR. Data in d, e and h are representative of 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Extended Data Fig. 2 Adoptively transferred CD8+ CAR T cells infiltrating B16-OVA-hCD19 tumours exhibit phenotypes and gene expression profiles similar to those of OT-I and endogenous CD8+ TILs.
a, b, Experimental design to assess CD8+CD45.1+ OT-I and CD8+CD45.2+ endogenous TILs; 1.5 × 106 OT-I T cells were adoptively transferred into C57BL/6J mice 13 days after tumour inoculation. c, Tumour growth curves (mean ± s.e.m.) of mice adoptively transferred with CAR or OT-I CD8+ T cells; graph is a compilation of 3 independent experiments. At days 7 and 21, mouse numbers were: CAR, n = 24, 17; OT-I, n = 21, 20. d, Tumour growth curves (mean ± s.e.m.) of mice adoptively transferred with CAR or PBS; graph is a compilation of 3 independent experiments. At days 7 and 21, mouse numbers were: CAR n = 35, 35; PBS n = 8, 6. c, d, For tumour sizes on day 21 after tumour inoculation, P = 0.3527 for CAR compared to OT-I (c) and P = 0.6240 for PBS compared to CAR (d); P values were calculated using a two-tailed unpaired t-test with Welch’s correction, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. e, f, Flow cytometry gating scheme for CAR (e) and OT-I (f) CD8+ TILs. g, Mean average plots of genes differentially expressed in the indicated comparisons. Wald test was performed to calculate P values, as implemented in DESeq2; P values were adjusted using the Benjamini–Hochberg method. Genes differentially expressed (adjusted P < 0.1 and fold change (log2 scale) ≥ 1 or ≤ −1) are highlighted. Selected genes are labelled. Top row, comparisons of the CAR TIL populations amongst themselves and to endogenous PD-1loTIM3lo TILs; middle row, comparisons within the endogenous TIL populations; bottom row, comparisons of CAR and endogenous PD-1hiTIM3hi TILs (left), and CAR and endogenous PD-1hiTIM3lo TILs (right).
Extended Data Fig. 3 Adoptively transferred CD8+ CAR T cells infiltrating B16-OVA-hCD19 tumours exhibit chromatin accessibility profiles similar to those of endogenous CD8+ TILs.
a, Pair-wise euclidean distance comparisons of log2 transformed ATAC-seq density (Tn5 insertions per kilobase) between all replicates at all peaks accessible in at least one replicate. b, Scatterplot of pairwise comparison of ATAC-seq density (Tn5 insertions per kb) between samples indicated. c, Genome browser views of sample loci, Pdcd1 (left), Itgav (right); scale range is from 0–600 for all tracks and data are the mean of all replicates. CD8+ TIL populations are as indicated and defined in Fig. 1b, Extended Data Fig. 2b: (A) PD-1hiTIM3hi CAR, (B) PD-1hiTIM3lo CAR, (C) PD-1hiTIM3hi endogenous, (D) PD-1hiTIM3lo endogenous, (E) PD-1loTIM3lo endogenous, (F) PD-1hiTIM3hi OT-I.
a, b, Flow cytometry gating scheme for CAR (a) and endogenous (b) CD8+ TILs. c, Representative flow cytometry histograms of NR4A proteins in PD-1hiTIM3hi TILs, PD-1hiTIM3lo TILs, and PD-1loTIM3lo TILs and their corresponding fluorescence minus one controls (in off-white). Data are representative of 3 independent experiments in which the sample from each independent experiment is comprised of TILs pooled together from 9–14 mice. d, Representative flow cytometry histograms for NR4A protein expression, comparing CAR and endogenous TIL populations (A–E) defined in Fig. 1b. e–g, Plotting in single cells the expression of PDCD1 and HAVCR2 (x and y axis, respectively) and (displayed by the colour scale) the expression of the following: e, Genes differentially upregulated in PD-1hiTIM3hi TILs relative to PD-1loTIM3lo TILs. f, Genes coding for selected transcription factors showing differential expression in the comparison of PD-1hiTIM3hi TILs relative to PD-1loTIM3lo TILs. g, Genes differentially downregulated in PD-1hiTIM3hi TILs relative to PD-1loTIM3lo TILs. Each dot represents a single cell. Human CD8+ TILs data are from ref. 14.
Extended Data Fig. 5 Prolonged survival of immunocompetent tumour-bearing mice adoptively transferred with CD8+ Nr4a TKO CAR T cells compared to mice transferred with CD8+ wild-type CAR T cells.
a, CD8a only staining control (previously tested to be the same as fluorescence minus one controls for CAR+ expression and NGFR+ expression) of CAR T cells before adoptive transfer. b, CAR and NGFR expression of CD8+ wild-type CAR T cells before adoptive transfer. c, CAR and NGFR expression of CD8+ Nr4a TKO CAR T cells before adoptive transfer. Data in a–c are representative of 4 independent experiments for adoptive transfer into Rag1−/− recipient mice; preparation of adoptive transfer into immunocompetent mice was the same except for the use of GFP-expressing Cre and empty vector. d, 6 × 106 CAR T cells were adoptively transferred into C57BL/6J mice 7 days after tumour inoculation. e, Growth of B16-OVA-hCD19 (left; 13–15 mice per condition) and MC38-hCD19 (right; 10 mice per condition) tumours in individual mice. f, B16-OVA-hCD19 (left) and MC38-hCD19 (right) tumour sizes (mean ± s.d.) at day 21 and 19 post inoculation respectively. P values were calculated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test: B16-OVA-hCD19, no significant difference; MC38-hCD19, PBS versus Nr4a TKO ***P = 0.0001; PBS versus wild type, P = 0.3252; wild type versus Nr4a TKO, *P = 0.0120. g, Survival curves for mice bearing B16-OVA-hCD19 tumours (left) and MC38-hCD19 tumours (right). P values calculated using log-rank (Mantel–Cox) test. For B16-OVA-hCD19, surviving mouse numbers at day 7, day 21, day 90 were: PBS, n = 13, 11, 0; wild type, n = 15, 11, 0; Nr4a TKO, n = 14, 13, 2; *P = 0.0026. For MC38-hCD19, surviving mouse numbers at day 7 and day 19 were: PBS, n = 10, 9; wild type, n = 10, 7; Nr4a TKO, n = 10, 10; all mice died by day 23; *P = 0.0138. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Extended Data Fig. 6 Tumour-bearing mice adoptively transferred with CD8+ CAR T cells lacking all three NR4A family members exhibit prolonged survival compared to mice transferred with wild-type CD8+ CAR T cells or CD8+ CAR T cells lacking only one of the three NR4A family members.
a, Experimental design; 3 × 106 wild-type, Nr4a TKO, Nr4a1 KO, Nr4a2 KO or Nr4a3 KO CAR T cells were adoptively transferred into Rag1−/− mice 7 days after tumour inoculation. b, Growth of B16-OVA-hCD19 tumours in individual mice, comprised of 17 or more mice per condition (these data include the wild type and Nr4a TKO data from Fig. 3). c, Graph shows mean ± s.d. and the individual values of B16-OVA-hCD19 tumour sizes at day 21 after inoculation. P values were calculated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test; PBS versus wild type, *P = 0.0395; wild type versus Nr4a1 KO, P = 0.0511 (not significant); wild type versus Nr4a2 KO, **P = 0.002, wild type versus Nr4a3 KO, *P = 0.0161; and wild type versus Nr4a TKO, ****P < 0.0001. d, Survival curves. ****P < 0.0001, calculated using log-rank (Mantel–Cox) test. Surviving mouse numbers at day 7, day 21 and day 90 were n = 31, 14, 0 for PBS; n = 35, 25, 1 for wild type; n = 17, 12, 0 for Nr4a1 KO; n = 17, 15, 1 for Nr4a2 KO; n = 32, 22, 11 for Nr4a3 KO; and n = 39, 36, 27 for Nr4a TKO. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Extended Data Fig. 7 Phenotypic and genomic features of mouse CD8+ T cells expressing NR4A1, NR4A2 or NR4A3.
Mouse CD8+ T cells were isolated, activated, transduced with empty retrovirus or retroviruses encoding HA-tagged Nr4a1, Nr4a2 or Nr4a3 with human NGFR reporter, and assayed on day 5 post activation. a, Flow cytometry gating of CD8+NGFR+ empty vector control, NR4A1-, NR4A2- and NR4A3-expressing cells at a constant expression level of NGFR reporter. b, Quantification of surface receptor expression (data from 3 independent replicates), showing geometric MFI normalized across experiments to the average of all samples within each experiment. c, Representative flow cytometry plots of cytokine production upon re-stimulation with PMA and ionomycin. d, Quantification of the data in c, showing geometric MFI normalized across experiments to the average of all samples within each experiment. All P values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. e, PCA plot of RNA-seq data from in vitro resting mouse CD8+ T cells ectopically expressing NR4A1, NR4A2, NR4A3 and empty vector control. f, Mean average plots of genes differentially expressed in the comparisons of ectopic expression of NR4A1, NR4A2 or NR4A3 against empty vector (top row), and pairwise comparisons between the ectopic expression of various NR4A family members (bottom row). Wald test was performed to calculate P values, as implemented in DESeq2. P values were adjusted using the Benjamini–Hochberg method. Genes differentially expressed (adjusted P < 0.1 and fold change (log2 scale) ≥ 1 or ≤ −1) are highlighted using different colours as indicated in the PCA plot as in e. Selected genes are labelled. g, Scatterplot of pairwise comparison of ATAC-seq density (Tn5 insertions per kb) between the indicated samples. Data in a–d are from three independent experiments; data in e–g from two independent experiments, each with two technical replicates.
Extended Data Fig. 8 CD8+ Nr4a TKO CAR TILs show increased effector function compared to CD8+ wild-type CAR TILs.
a, Tumour growth curves (mean ± s.e.m.) after adoptive transfer of 1.5 × 106 CAR T cells into Rag1−/− mice on day 13 after tumour inoculation. Mouse numbers at day 7 and day 21 were: wild type, n = 47, 35; Nr4a TKO, n = 41, 32. P values were calculated using an ordinary two-way ANOVA with Tukey’s multiple comparisons test; for wild type versus Nr4a TKO, P = 0.5463. b, Flow cytometry gating scheme for surface markers, cytokines, and transcription factors expressed by wild-type (top) and Nr4a TKO (bottom) TILs. All samples are gated on cells with a set level of CAR expression (103–104) within the CAR+ NGFR+ population. c, Bar plots (mean ± s.d.) showing (left) number of wild-type and Nr4a TKO CAR TILs per g of tumour (5 independent experiments; P value was calculated using a two-tailed ratio paired t-test) and (right) mean fluorescence intensity of Ki67 of wild-type and Nr4a TKO CAR TILs (2 independent experiments). d, Top, representative flow cytometry plots for TIM3 and TCF1 expression in wild-type and Nr4a TKO CAR TILs (2 independent experiments). Bottom, bar plots (mean ± s.d.) of transcription factor expression by wild-type and Nr4a TKO CAR TILs (6 independent experiments). P values were calculated using two-tailed paired t-tests. For all P value calculations, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. e, PCA plot of RNA-seq data from Nr4a TKO or wild-type CAR TILs. f, Normalized enrichment scores (NES) of gene sets defined from pairwise comparisons of effector, memory and exhausted CD8+ T cells from LCMV-infected mice11. Enrichment score was calculated using a Kolmogorov–Smirnov test, as implemented in gene set enrichment analysis (GSEA). g, GSEA of RNA-seq data from Nr4a TKO and wild-type CAR TILs displayed as enrichment plots, ranking genes by fold change in expression between those conditions. The false discovery rate (FDR) for both (f, g) is controlled at a level of 5% by the Benjamini–Hochberg correction. For e–g, data are from two independent experiments each consisting of 1–2 technical replicates.
Extended Data Fig. 9 NR4A family members bind to predicted NR4A-binding motifs that are more accessible in wild-type CAR TILs compared to the Nr4a TKO CAR TILs, and regions more accessible in wild-type compared to Nr4a TKO CAR TILs are more accessible in CA-RIT-NFAT1- and NR4A1/2/3-transduced cells.
a, Top right, histogram view showing expression of NR4A in cells ectopically expressing HA-tagged versions of NR4A1, NR4A2, NR4A3; data are representative of 2 independent experiments. Middle, genome browser views of the Ccr7, Ccr6, Ifng loci for wild-type CAR TILs compared to Nr4a TKO CAR TILs, including binding motifs for NFAT, NR4A, bZIP, NFκB and the location of the qPCR primers used. Scale range is 0–600 for all tracks and data are mean of two independent experiments. Right, bar plots showing enrichment of NR4A at regions probed; data representative of 2 independent experiments consisting of three technical replicates each. b, Genome browser views of the Il21 (top), Tnf (bottom) loci incorporating wild-type CAR TILs compared to Nr4a TKO CAR TILs, including binding motifs for NFAT, NR4A, bZIP and NFκB. Scale range is 0–600 for Il21 and 0–1000 for Tnf; data are mean of two independent experiments. c, Top four panels, ATAC-seq data from Nr4a TKO and wild-type CAR TILs compared with data from cells ectopically expressing CA-RIT-NFAT1, NR4A1, NR4A2 or NR4A3. Bottom panel, ATAC-seq data from Nr4a TKO and wild-type CAR TILs compared with data from cultured cells re-stimulated with PMA and ionomycin.
Extended Data Fig. 10 Nr4a family members show a moderate decrease in mRNA expression in antigen-specific cells from LCMV-infected mice treated with anti-PDL1 or IgG control.
a, Mean average plots of genes differentially expressed in cells treated with anti-PDL1 compared to cells treated with IgG control, highlighting two different categories of differentially expressed genes: those with adjusted P < 0.1 and fold change (log2 scale) ≥ 0.5 or ≤ −0.5 (lighter colours); and those with adjusted P < 0.1 and fold change (log2 scale) ≥ 1 or ≤ −1 (darker colours). Selected genes are labelled. Displayed are the number of genes in each category. The sequencing data in this analysis were obtained from ref. 19. Wald test was performed to calculate P values, as implemented in DESeq2; P values were adjusted using the Benjamini–Hochberg method.
Comparisons of gene expression program in CAR vs endogenous CD8+ T cells infiltrating B16-OVA-hCD19 tumor.
Comparisons of human CD8+ TILs infiltrating a human melanoma.
Comparisons of cells ectopically expressing NR4A in CD8+ T cells in vitro. a) RNA-seq DESeq2 results comparing empty vector pMIN vs NR4A1; b) RNA-seq DESeq2 results comparing empty vector pMIN vs Nr4a2; c) RNA-seq DESeq2 results comparing empty vector pMIN vs NR4A3.
Comparison of gene expression program in Nr4a TKO vs wild-type CD8+ CAR T cells infiltrating B16-OVA-hCD19 tumor.
Comparison of gene expression program in antigen-specific cells from LCMV-infected mice treated with anti-PDL1 or IgG control.
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Chen, J., López-Moyado, I.F., Seo, H. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019). https://doi.org/10.1038/s41586-019-0985-x
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