Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy

doi:10.1038/s41586-019-1821-z

. 2019 Dec;576(7787):471-476.

doi: 10.1038/s41586-019-1821-z. Epub 2019 Dec 11.

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy

Jun Wei¹, Lingyun Long¹, Wenting Zheng², Yogesh Dhungana¹, Seon Ah Lim¹, Cliff Guy¹, Yanyan Wang¹, Yong-Dong Wang³, Chenxi Qian^{1

3}, Beisi Xu³, Anil Kc¹, Jordy Saravia¹, Hongling Huang¹, Jiyang Yu³, John G Doench⁴, Terrence L Geiger², Hongbo Chi⁵

Affiliations

¹ Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.
² Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA.
³ Department of Computational Biology, St Jude Children's Research Hospital, Memphis, TN, USA.
⁴ Broad Institute of Harvard and MIT, Cambridge, MA, USA.
⁵ Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA. hongbo.chi@stjude.org.

PMID: 31827283
PMCID: PMC6937596
DOI: 10.1038/s41586-019-1821-z

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy

Jun Wei et al. Nature. 2019 Dec.

. 2019 Dec;576(7787):471-476.

doi: 10.1038/s41586-019-1821-z. Epub 2019 Dec 11.

Authors

Affiliations

¹ Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.
² Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA.
³ Department of Computational Biology, St Jude Children's Research Hospital, Memphis, TN, USA.
⁴ Broad Institute of Harvard and MIT, Cambridge, MA, USA.
⁵ Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA. hongbo.chi@stjude.org.

PMID: 31827283
PMCID: PMC6937596
DOI: 10.1038/s41586-019-1821-z

Abstract

Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells¹. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8⁺ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8⁺ T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8⁺ T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8⁺ T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.

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Conflict of interest statement

Competing interests

H.C. and J.W. are authors of a patent application related to Regnase-1 and BATF.

Figures

**Extended Data Figure 1.. Validation of the effect of Regnase-1 deletion on CD8⁺ T cell accumulation in tumor immunity using the *in vivo* dual transfer system.**
(a) Diagram of the *in vivo* dual transfer system. OT-I cells transduced with sgRNA viral vectors expressing distinct fluorescent proteins were mixed and transferred into the same tumor-bearing hosts where further analyses were performed. (b) Gating strategy for sgRNA-transduced OT-I cell analysis. (c, d) OT-I cells transduced with non-targeting control sgRNA (mCherry⁺) were mixed at a 1:1 ratio with either cells transduced with control sgRNA (Ametrine⁺) (c (n = 2), d (n = 5), upper left) or two different sgRNAs targeting *Regnase-1* (sgRegnase-1, Ametrine⁺, c (n = 4), lower left; or sgRegnase-1 #2, Ametrine⁺, d (n = 5), lower left), and transferred into tumor-bearing hosts. Mice were analyzed at 7 days after adoptive transfer for the proportion of OT-I cells in CD8α⁺ cells (c, d, left), and quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to input in the spleen and TILs (c, d, right). Numbers in plots indicate frequencies of OT-I cells. (e) OT-I cells transduced with control sgRNA (Ametrine⁺) were mixed at a 1:1 ratio with cells transduced with sgRegnase-1 (mCherry⁺), and transferred into tumor-bearing hosts (n = 5). Mice were analyzed at 7 days after adoptive transfer for the proportion of OT-I cells in CD8α⁺ cells (left), and quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to input in the spleen and TILs (right). Numbers in plots indicate frequencies of OT-I cells. (f) Indel mutations after CRISPR targeted disruption in OT-I cells transduced with either control sgRNA or sgRegnase-1, via deep sequencing analysis of indels generated at the exonic target site of *Regnase-1* gene, including 97.3% indel events in sgRegnase-1-transduced cells isolated from tumors as compared to 1.3% in control sgRNA-transduced cells. Mean ± s.e.m. in c–e. ***P < 0.001; two-tailed unpaired Student’s t-test in d, e. Data are representative of two (e) independent experiments.

**Extended Data Figure 2.. Tumor-infiltrating and peripheral Regnase-1-null CD8⁺ T cells show distinct immune signatures.**
(a, b) GSEA enrichment plots of antigen-specific CXCR5⁺ and CXCR5^– exhausted CD8⁺ T cells from chronic infection using gene targets repressed by Regnase-1 (i.e. top 100 upregulated genes in TIL sgRegnase-1- compared to control sgRNA-transduced OT-I cells as identified by RNA-Seq). (c) Representative images (left) and quantification of mean fluorescence intensity (MFI; right) of TCF-1 expression (pink) in control sgRNA- (mCherry⁺; red) and sgRegnase-1-transduced OT-I cells (Ametrine⁺; green) in the whole tumor section (n = 4 mice). Scale bars, 20 μm. (d, e) Gene expression heat maps normalized by row (z-score) for the naïve or memory T-cell−associated transcription factors (d) or effector or exhausted T-cell−associated transcription factors (e) in control sgRNA- (n = 4) and sgRegnase-1- (n = 5) transduced OT-I cells isolated from TILs. Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (f) Real-time PCR analysis of *Irf4* mRNA expression in control sgRNA- (n = 4 samples) and sgRegnase-1- (n = 5 samples) transduced OT-I cells isolated from TILs. (g) Summary of ATAC-Seq motif enrichment data showing log₂ (odds ratio) and –log₁₀ (FDR) of cells from control sgRNA- and sgRegnase-1-transduced OT-I cells isolated from TILs (n = 4 samples each group). Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for ATAC-Seq analysis. (h) Tn5 insert sites from ATAC-Seq analysis were aligned to motifs for transcription factors from the TRANSFAC database, and the binding profiles of TCF-1, Bach2, Bcl6 and IRF4 are shown. (i) Venn diagram showing the overlap of significantly upregulated (left, sgRegnase-1- (n = 5 samples) versus control sgRNA-transduced OT-I cells (n = 4 samples)) or downregulated genes (right, sgRegnase-1- versus control sgRNA-transduced OT-I cells) by RNA-Seq profiling between TIL and peripheral lymph node (PLN) OT-I cells. Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (j) GSEA enrichment plots of PLN sgRegnase-1- (n = 5) versus control sgRNA- (n = 4) transduced OT-I cells using gene sets of four different tumor-infiltrating CD8 T-cell activation states. Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and PLN OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (k) OT-I cells transduced with control sgRNA (mCherry⁺) and sgRegnase-1 (Ametrine⁺) were mixed and transferred into tumor-bearing mice (n = 5 mice), and OT-I cells in the spleen were analyzed at day 7 for expression of TCF-1 (upper), and quantification of frequency of TCF-1⁺ cells (lower). Numbers in graphs indicate frequencies of cells in gates. Mean ± s.e.m. in c, f, k. *P < 0.05; **P < 0.01; ***P < 0.001; Kolmogorov-Smirnov test followed by Benjamini-Hochberg correction in a, b, j, two-tailed unpaired Student’s t-test in c, f, k, two-sided Fisher’s exact test followed by Benjamini-Hochberg correction in g, and two-sided Fisher’s exact test in i. Data are representative of two (c, f, k) independent experiments.

**Extended Data Figure 3.. Upstream signals regulate Regnase-1 expression and Regnase-1-null cell phenotypes.**
(a) Immunoblot analysis of Regnase-1 expression in control sgRNA-transduced OT-I cells isolated from PLN and TILs at 7 days after adoptive transfer (n = 4 samples each group) (upper). Quantification of relative intensity of Regnase-1 expression (lower). β-actin is loading control. (b) GSEA enrichment plots of PLN and TIL control sgRNA-transduced OT-I cells (n = 4) used in (a) by using gene targets repressed by Regnase-1 (i.e. top 100 upregulated genes in TIL sgRegnase-1- compared to control sgRNA-transduced cells as identified by RNA-Seq). (c) OT-I cells were stimulated with αCD3 and αCD28 for overnight before viral transduction, and then cultured in IL-7 and IL-15-containing medium for another 3 days *in vitro*. Pre-activated OT-I cells were then stimulated with αCD3, IL-2 or IL-21 for 0, 1 and 4 h (n = 5 samples each group) for immunoblot analysis of full length and cleaved Regnase-1 (upper), and quantification of relative intensity of cleaved Regnase-1 expression (lower). β-actin is loading control. (d, e) OT-I cells transduced with control sgRNA (mCherry⁺) and sgRegnase-1 (Ametrine⁺) were mixed at a 1:1 ratio and transferred into mice bearing B16-Ova (n = 6 mice) or B16-F10 (n = 6 mice) tumors. Mice were analyzed at day 7 after adoptive transfer for quantification of relative OT-I cell percentage in total cells normalized to input in the TILs (d), and expression of TCF-1 (e, left), and quantification of frequency of TCF-1⁺ cells (e, right) in tumor-infiltrating OT-I cells. (f, g) OT-I cells were stimulated with αCD3 and αCD28 for overnight before viral transduction, and then cultured in IL-2, IL-7 and IL-15-containing medium for another 3 days *in vitro*. Pre-activated OT-I cells were then continuously cultured in normoxia (21% O₂) or hypoxia (1% O₂) condition for 48 h for immunoblot analysis of expression of HIF1α, Regnase-1 and BATF (f), and for flow cytometry analysis of expression of BATF, CD69, GzmB, CD25 and TCF-1 (g). Numbers in graphs indicate MFI (g). β-actin is loading control. Mean ± s.e.m. in a, c–e. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test in a, Kolmogorov-Smirnov test followed by Benjamini-Hochberg correction in b, and one-way ANOVA in c–e. Data are representative of two (c, f, g) independent experiments, or pooled from two (d, e) independent experiments.

**Extended Data Figure 4.. Proliferation and survival analyses of Regnase-1-null CD8⁺ T cells in tumor immunity.**
(a) List of the top 10 significantly (FDR < 0.05) upregulated and downregulated pathways in TIL sgRegnase-1-transduced OT-I cells, as revealed by performing GSEA using “Hallmark” gene sets. Specifically, control sgRNA- (n = 4) and sgRegnase-1- (n = 5) transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (b) GSEA enrichment plots of TIL sgRegnase-1-transduced OT-I cells using cell cycling-associated gene sets, including E2F targets (upper), G2M checkpoint (middle) and mitotic spindle (lower). (c–g) OT-I cells transduced with control sgRNA (mCherry⁺) and sgRegnase-1 (Ametrine⁺) were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were analyzed at day 7 (d–g) (n = 6 mice) and day 14 (c) (n = 5 mice) by flow cytometry for Ki-67 expression (c, left; e, left), BrdU incorporation (d, upper; pulse for 18 h), active caspase-3 expression (f, left), Ser139 phosphorylation of histone variant H2A.X (g, upper), and quantification of MFI of Ki-67 (c, right; e, right), frequency of BrdU⁺ cells (d, lower), frequency of active caspase-3⁺ cells (f, right) and frequency of Ser139 phosphorylated histone variant H2A.X⁺ cells (g, lower). Numbers in graphs indicate MFI of Ki-67 (c, right; e, left). Numbers in plots indicate frequencies of BrdU⁺ cells (d, upper), active caspase-3⁺ cells (f, left) and Ser139 phosphorylated histone variant H2A.X⁺ cells (g, upper). (h) List of the top 15 significantly (FDR < 0.05) upregulated and top 4 significantly downregulated pathways in PLN sgRegnase-1-transduced OT-I cells, as revealed by performing GSEA using “Hallmark” gene sets. Specifically, control sgRNA- (n = 4) and sgRegnase-1- (n = 5) transduced OT-I cells were mixed and transferred into tumor-bearing mice, and PLN OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (i, j) OT-I cells transduced with control sgRNA (mCherry⁺) and sgRegnase-1 (Ametrine⁺) were mixed and transferred into tumor-bearing mice, and OT-I cells in the spleen were analyzed at day 7 (i, j) (n = 6 mice) by flow cytometry for BrdU incorporation (i, upper; pulse for 18 h) and active caspase-3 expression (j, upper), and quantification of frequencies of BrdU⁺ cells (i, lower) and active caspase-3⁺ cells (j, lower). Numbers in plots indicate frequencies of BrdU⁺ cells (i, upper) and active caspase-3⁺ cells (j, upper). Mean ± s.e.m. in c–g, i, j. *P < 0.05; **P < 0.01; ***P < 0.001; Kolmogorov-Smirnov test followed by Benjamini-Hochberg correction in a, b, h, two-tailed unpaired Student’s t-test in c–g, i, j. Data are representative of two (c) independent experiments, or pooled from two (d–g, i, j) independent experiments.

**Extended Data Figure 5.. Effector molecule expression of tumor-infiltrating Regnase-1-null CD8⁺ T cells.**
(a, b) OT-I cells transduced with control sgRNA (mCherry⁺) or sgRegnase-1 (Ametrine⁺) were mixed at a 5:1 ratio and transferred into tumor-bearing mice (n = 5 mice), and tumor-infiltrating OT-I cells were analyzed at day 7 for the expression of CD69, CD25, CD49a, KLRG1, ICOS, Lag3, PD-1 and CTLA4 (a, upper) and CD44 and CD62L (b, upper), and quantification of MFI of CD69, CD25, CD49a, KLRG1, ICOS, Lag3, PD-1 and CTLA4 (a, lower) and frequency of CD44⁺CD62L^– cells (b, lower). Numbers in graphs indicate MFI (a, upper). Numbers in plots indicate frequency of CD44⁺CD62L^– cells (b, upper). (c–f) OT-I cells transduced with control sgRNA (mCherry⁺) or sgRegnase-1 (Ametrine⁺) were mixed at a 5:1 ratio and transferred into tumor-bearing mice, and analyzed at days 7 (n = 10 mice) or 14 (n = 10 mice). Flow cytometry analysis of expression of IFN-γ (c, upper), GzmB (c, lower), TNF-α (e, upper) and IL-2 (e, lower) in TIL OT-I cells, and quantification of the numbers of IFN-γ⁺ cells (d, upper), GzmB⁺ cells (d, lower), TNF-α⁺ cells (f, left) and IL-2⁺ cells (f, right) normalized to input per gram tissue. Numbers adjacent to outlined areas indicate frequency of IFN-γ⁺ cells and MFI of IFN-γ in IFN-γ⁺ cells (c, upper), and frequency of GzmB⁺ cells and MFI of GzmB in GzmB⁺ cells (c, lower), and frequencies of TNF-α⁺ cells (e, upper), or IL-2⁺ cells (e, lower). Mean ± s.e.m. in a, b, d, f. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test in a, b, and two-tailed paired Student’s t-test in d, f. Data are representative of two (a–c, e) independent experiments, or pooled from two (d, f) independent experiments.

**Extended Data Figure 6.. scRNA-Seq and flow cytometry analyses of tumor-infiltrating Regnase-1-null OT-I cells.**
(a–e) scRNA-Seq analysis of control sgRNA- and sgRegnase-1-transduced OT-I cells isolated from TILs. Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by scRNA-Seq. tSNE visualization of *Pdcd1* (a, upper), *Havcr2* (a, lower), *Ifng* (c, upper), *Gzmb* (c, lower), *Batf* (d) and *Id2* (e) gene expression, and “CXCR5⁺ exhausted CD8 (Ahmed)” (b, upper) and “CXCR5⁺ exhausted CD8 (Yu)” (b, lower) gene signatures in individual cells. (f) OT-I cells transduced with control sgRNA and sgRegnase-1 were mixed and transferred into tumor-bearing mice (n = 5 mice; data from one representative mouse were shown), and tumor-infiltrating OT-I cells were analyzed at day 7 for the expression of TOX, Slamf6, CD127, KLRG1, Tim3 and PD-1 in TCF-1⁺ and TCF-1^– cells of control sgRNA- and sgRegnase-1-transduced OT-I cells. Numbers in graphs indicate mean ± s.e.m. of MFI of markers on the x-axis after gating on TCF-1⁺ or TCF-1^– subsets. Data are representative of two (f) independent experiments.

**Extended Data Figure 7.. Genome-scale CRISPR screening identifies BATF as an important Regnase-1 functional target in tumor immunity.**
(a) Scatterplot of the enrichment of each gene versus its adjusted P value in genome-scale CRISPR screening. Gene enrichment was calculated by averaging the enrichment of their sgRNAs (n = 4 for each gene) in tumor-infiltrating OT-I cells relative to input (log₂ ratio (TIL/input)), with the most extensively enriched (red) and selectively depleted (blue) genes (adjusted P < 0.05), as well as ‘dummy’ genes (green; generated by random combinations of 4 out of 1,000 non-targeting control sgRNAs per ‘dummy’ gene). (b) Venn diagram showing the overlap of genes between top depleted genes in genome-scale CRISPR screening (by less than −3.5 log₂ (TIL/input) fold change; adjusted P < 0.05) and top upregulated genes in TIL sgRegnase-1- versus control sgRNA-transduced OT-I cells as identified by RNA-Seq (by greater than 1.5 log₂ fold change; P < 0.05). (c) Tn5 insert sites from ATAC-Seq analysis were aligned to motifs for transcription factors from the TRANSFAC database, and the binding profiles of BATF are shown. (d) Enrichment of BATF-binding motifs in the genomic regions with upregulated accessibility in Regnase-1-null cells. First, we analyzed common regions in our Regnase-1-null ATAC-Seq data and published BATF ChIP-Seq peaks (GSE54191). Next, we scanned these common regions with TRANSFAC motifs for BATF, and numbers of motif matches and associated Fisher’s exact test P values and log₂ (odds ratios) are shown (a positive log₂ (odds ratio) value indicates that a motif is more likely to occur in Regnase-1-null cells than in wild-type samples; ‘E − x’ denotes ‘×10^−x’). (e) Luciferase activity of HEK293T cells measured at 48 h after transfection with *Il2* mRNA 3’ UTR (upper) or *Il4* mRNA 3’ UTR (lower) luciferase reporter plasmid, together with control (mock), wild-type or D141N Regnase-1-expressing plasmid (n = 3 samples each group). (f) OT-I cells transduced with control sgRNA (mCherry⁺; spike) were mixed at a 1:1 ratio with cells transduced with control sgRNA (Ametrine⁺), sgRegnase-1 (Ametrine⁺), sgBatf (GFP⁺) or sgBatf/*Regnase-1* (GFP⁺ and Ametrine⁺), and transferred into tumor-bearing hosts individually (n = 4 mice each group). Mice were analyzed at 5 days after adoptive transfer for quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to spike in the spleen (f, left) and TILs (f, right), and quantification of relative MFI of BATF normalized to spike in the tumor-infiltrating OT-I cells (f). (g) Immunoblot analysis of Regnase-1 and BATF expression in *in vitro* cultured OT-I cells 3 days after transduction with control sgRNA or sgBatf/*Regnase-1*. Hsp90 is loading control. (h–k) The same transfer system as in (f) was used. Five days after adoptive transfer, mice were analyzed for quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to spike in the spleen (h, left, n = 4) and TILs (h, right, n = 4). Tumor-infiltrating OT-I cells were analyzed at day 5 (n = 4 mice each group) for quantification of relative frequency of active caspase-3⁺ cells normalized to spike (i), and quantification of relative frequency of TCF-1⁺ cells normalized to spike (j), or at day 7 (n = 6 mice each group) for quantification of relative frequency of IFN-γ⁺ cells normalized to spike (k). (l) 4 × 10⁶ pmel-1 cells transduced with sgRegnase-1 (Ametrine⁺) (n = 10 recipients) or sgBatf/*Regnase-1* (GFP⁺ and Ametrine⁺) (n = 10 recipients) were transferred into mice at day 12 after B16-F10 melanoma engraftment, followed by analysis of tumor size. Mean ± s.e.m. in f, h–k. Mean ± s.d. in e. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s paired t-test followed by Bonferroni correction in a, two-sided Fisher’s exact test in d, one-way ANOVA in e, f, h–k, and two-way ANOVA in l. Data are representative of two (e) or three (h) independent experiments, or pooled from two (f, g, i–l) independent experiments.

**Extended Data Figure 8.. BATF overexpression markedly enhances CD8⁺ T cell antitumor responses.**
(a) OT-I cells were stimulated with αCD3 and αCD28 for overnight before viral transduction, and then cultured in IL-7 and IL-15-containing medium for another 3 days *in vitro*. Control sgRNA- and sgRegnase-1-transduced OT-I cells were then stimulated with αCD3, IL-2 or IL-21 for overnight for flow cytometry analysis of BATF expression (upper), and quantification of the MFI of BATF (lower) (n = 6 samples each group). Numbers in graphs indicate MFI (upper) and fold change between comparisons (lower). (b–h) OT-I cells transduced with control retrovirus (mCherry⁺) were mixed at a 1:1 ratio with cells transduced with *Batf*-overexpressing retrovirus (GFP⁺), and transferred into tumor-bearing hosts. Mice were analyzed at day 4 (e) (n = 4 mice), day 5 (b, h) (n = 4 mice), day 7 (c, d, f, g) (n = 6–8 mice) or day 14 (c, d) (n = 6 mice) for the expression of BATF (b, left), active caspase-3 (f, left), IFN-γ, GzmB, TNF-α and IL-2 (g, left), and TCF-1 (h, upper) in TIL OT-I cells, and quantification of MFI of BATF in TIL OT-I cells (b, right), and quantification of frequencies of active caspase-3⁺ cells (f, right), IFN-γ⁺, GzmB⁺, TNF-α⁺ and IL-2⁺ cells (g, right), and TCF-1⁺ cells (h, lower) in TIL OT-I cells, and analysis of the proportion of donor-derived OT-I cells in total CD8α⁺ cells in TILs and spleen (c), and quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to input in the spleen (d), and the dilution of CellTrace Violet (CTV) in TIL OT-I cells (e, left), and quantification of MFI of CTV in TIL OT-I cells (e, right). Numbers in graphs indicate MFI (b, left; e, left), frequencies of OT-I cells in gates (c), frequency of active caspase-3⁺ cells (f, left), frequencies of IFN-γ⁺, GzmB⁺, TNF-α⁺ or IL-2⁺ cells (g, left), and frequency of TCF-1⁺ cells (h, upper). Mean ± s.e.m. in a, b, d–h. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test in a, b, d–h. Data are representative of two (a, c) independent experiments, or pooled from two (b, d–h) independent experiments.

**Extended Data Figure 9.. Genome-scale CRISPR screening identifies mitochondrial metabolism as an important downstream pathway of Regnase-1 and BATF.**
(a) Chromatin accessibility heat maps normalized by row (z-score) for 7,480 genes with significantly increased chromatin accessibility (by |log₂ FC| > 0.5; P < 0.05) in sgRegnase-1-transduced OT-I cells as compared to control sgRNA-transduced cells. Specifically, OT-I cells transduced with control sgRNA (mCherry⁺) (n = 4), sgRegnase-1 (Ametrine⁺) (n = 4), sgBatf (GFP⁺) (n = 2) or sgBatf/*Regnase-1* (GFP⁺ and Ametrine⁺) (n = 4) were transferred into tumor-bearing hosts individually. OT-I cells were isolated from TILs at day 7 for ATAC-Seq analysis. We annotated the differential accessibility (DA) regions in ATAC-Seq for the nearest genes, and identified 7,480 genes with significantly increased chromatin accessibility in Regnase-1-null cells as compared to wild-type cells. BATF co-deletion reversed the upregulated chromatin accessibility for a large proportion of these genes (5,052 in total). Also, 2,527 among these 5,052 genes showed significantly downregulated chromatin accessibility in BATF-null cells as compared to wild-type cells. (b) Functional enrichment plots of the top 10 significantly (FDR < 0.05) enriched pathways in top-ranking depleted genes (n = 4 sgRNAs for each gene) identified in the genome-scale CRISPR screening (by less than −3.5 log₂ (TIL/input) fold change; adjusted P < 0.05). (c) GSEA enrichment plots of TIL sgRegnase-1-transduced OT-I cells using the OXPHOS “Hallmark” gene set. Specifically, control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by RNA-Seq. (d) Representative images (upper) and quantification of mitochondrial volume (stained with Tom20, white) per cell (lower) in control sgRNA- (mCherry⁺; red) and sgRegnase-1-transduced OT-I cells (Ametrine⁺; green) in tumors at 7 days after adoptive transfer (n = 4 mice). (e) Oxygen consumption rate (OCR) bioenergetic profiling of control sgRNA- and sgRegnase-1-transduced OT-I cells cultured *in vitro* for basal (left) and maximal (right) OCR (n = 9 samples each group). (f) List of the top 2 significantly (FDR < 0.05) upregulated and top 8 significantly downregulated pathways in TIL sgBatf/*Regnase-1*- (n = 3 samples) versus sgRegnase-1-transduced (n = 3 samples) OT-I cells isolated from TILs, as revealed by performing GSEA using “Hallmark” gene sets. Specifically, sgRegnase-1- and sgBatf/*Regnase-1*-transduced OT-I cells were mixed and transferred into tumor-bearing mice, and tumor-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by microarray. (g) GSEA enrichment plots of TIL sgBatf/*Regnase-1*- (n = 3 samples) versus sgRegnase-1-transduced OT-I cells (n = 3 samples) using OXPHOS gene set. (h) OT-I cells transduced with control sgRNA (mCherry⁺; spike) were mixed at a 1:1 ratio with cells transduced with control sgRNA (Ametrine⁺), sgRegnase-1 (Ametrine⁺), sgBatf (GFP⁺) or sgBatf/*Regnase-1* (GFP⁺ and Ametrine⁺), and transferred into tumor-bearing hosts individually (n = 4 mice each group). Mice were analyzed at 5 days after adoptive transfer for quantification of relative MFI of TMRM (left) and Mitotracker (right) normalized to spike in tumor-infiltrating OT-I cells. (i) Chromatin accessibility heat maps normalized by row (z-score) for mitochondrial genes with significantly increased chromatin accessibility (by |log₂ FC| > 0.5; P < 0.05) in sgRegnase-1-transduced OT-I cells as compared to control sgRNA-transduced cells as determined by ATAC-Seq as described in a. We annotated the DA regions in ATAC-Seq for the nearest genes, and superimposed these genes with 1,158 mitochondrial genes defined in MitoCarta 2.0 database. A total of 341 mitochondrial genes showed significantly upregulated chromatin accessibility in the absence of Regnase-1, 214 of which were blocked by BATF co-deletion in BATF/Regnase-1-null cells. Moreover, 96 among these 214 genes showed significantly downregulated chromatin accessibility in BATF-null cells as compared to wild-type cells. Mean ± s.e.m. in d, e, h. *P < 0.05; **P < 0.01; ***P < 0.001; two-sided Fisher’s exact test in a, i, right-tailed Fisher’s exact test in b, Kolmogorov-Smirnov test followed by Benjamini-Hochberg correction in c, f, g, two-tailed unpaired Student’s t-test in d, e, and one-way ANOVA in h. Data are representative of two (d, e) independent experiments, or pooled from two (h) independent experiments.

**Extended Data Figure 10.. PTPN2 and SOCS1 deletion efficiency and expression in Regnase-1-null cells, and model of Regnase-1 functions in tumor-specific CD8⁺ T cells.**
(a) Immunoblot analysis of Regnase-1, PTPN2 and SOCS1 expression in *in vitro* cultured OT-I cells 3 days after transduction with control sgRNA, sgPtpn2/*Regnase-1* (left), or sgSocs1/*Regnase-1* (right). Hsp90 is loading control. (b) Immunoblot analysis of Regnase-1, BATF, SOCS1 and PTPN2 expression in control sgRNA- and sgRegnase-1-transduced OT-I cells cultured *in vitro* for 3 days after viral transduction. β-actin is loading control. (c) Regnase-1 is a major negative regulator of CD8⁺ T cell antitumor responses, and TCR and IL-2 inhibit its expression and activity. Deletion of Regnase-1 unleashes potent therapeutic efficacy of engineered tumor-specific CD8⁺ T cells against cancers by coordinating transcriptional and metabolic programs to achieve greatly improved cell accumulation and function. As a key functional target of Regnase-1, excessive BATF drives robust cell accumulation and effector function, in part through enhancing mitochondrial metabolism, in Regnase-1-null CD8⁺ T cells. Regnase-1 deletion also reprograms cells to acquire increased naïve/memory cell-associated gene signatures and gain survival advantage, which contribute to the improved persistence of Regnase-1-null effector CD8⁺ T cells. Targeting PTPN2 and SOCS1 (not depicted here) acts in coordination with Regnase-1 inhibition to promote CD8⁺ T cell antitumor responses. Data are representative of three (a, b) independent experiments.

**Figure 1.. *In vivo* CRISPR screening identifies Regnase-1 as a major negative regulator of CD8⁺ T cell antitumor responses.**
(a) Diagram of CRISPR screening for metabolic regulators of ACT. (b) Scatterplot of the enrichment of candidates (n = 6 sgRNAs per gene) with the most extensively enriched (red) and selective depleted (blue) genes, as well as ‘dummy’ genes (green; generated by random combinations of 6 out of 1,000 non-targeting control sgRNAs per ‘dummy’ gene) highlighted. (c) Representative images (left) and quantification of relative OT-I cell number per area (μm²) normalized to input (right) in the tumor section (n = 4). OT-I cells transduced with control sgRNA (red) and sgRegnase-1 (green) were mixed at a 10:1 ratio and transferred into tumor-bearing mice, and analyzed at day 7. Scale bars, 500 μm. (d, e) Control sgRNA- and sgRegnase-1-transduced OT-I cells were mixed at a 10:1 ratio and transferred into tumor-bearing mice, followed by analyses of the proportion of OT-I cells in total CD8α⁺ cells (d), and quantification of normalized OT-I cell number relative to input (e) at days 7 (n = 10), 14 (n = 10) and 21 (n = 6). Cell number in the tumor indicates per gram tissue. Mean ± s.e.m. in c, e. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed paired Student’s t-test followed by Bonferroni correction in b, two-tailed unpaired Student’s t-test in c, e. Data are representative of two (c, d), or pooled from two (e) independent experiments.

**Figure 2.. Deletion of Regnase-1 enhances efficacy of ACT against solid and blood cancers.**
OT-I (a, b), pmel-1 (c, d) or CD8⁺ CAR-T (e, f) cells (5 × 10⁶) transduced with non-targeting control sgRNA or sgRegnase-1 were transferred into mice at day 12 after B16-Ova (a, b) or B16-F10 (c, d) melanoma engraftment, or at day 7 after Ph⁺ B-ALL cell engraftment (e, f), followed by analyses of tumor size (a, c), mouse survival (b, d, e) and tumor burden via Xenogen imaging of bioluminescent signal intensities (f). Non-treatment control mice received no T cell transfer. *P < 0.05; **P < 0.01; ***P < 0.001; two-way ANOVA in a, c, and Log-rank (Mantel-Cox) test in b, d, e. Data are representative of two (a, b, e, f) or four (c, d) independent experiments.

**Figure 3.. Deletion of Regnase-1 reprograms tumor-infiltrating CD8⁺ T cells to long-lived effector cells.**
(a) GSEA enrichment plots of RNA-Seq from sgRegnase-1- (n = 5) versus non-targeting control sgRNA- (n = 4) transduced OT-I cells isolated from TILs from the dual transfer system, using gene sets of tumor-infiltrating CD8⁺ T cell activation states. (b–d) Tumor-infiltrating sgRNA-transduced OT-I cells from the dual transfer system (n = 5) were analyzed at days 7 (b) and 14 (c, d) for the quantification of frequencies of TCF-1⁺ (b), BrdU⁺ (c) and active caspase-3⁺ (d) cells. (e–g) Diagram of *in vivo* persistence assay (e): sgRNA-transduced OT-I cells were isolated from TILs, mixed at a 1:1 ratio (1 × 10⁵ each) and transferred into tumor-bearing hosts (f) or naïve mice (g). Quantification of normalized OT-I cell frequency in TILs of tumor-bearing hosts (n = 6) (f) or in the spleen of naïve hosts (n = 6) (g). (h–k) Tumor-infiltrating sgRNA-transduced OT-I cells from the dual transfer system were analyzed at days 7 (n = 10) and 14 (n = 10) for the quantification of frequencies of IFN-γ⁺ cells (h, left), GzmB⁺ cells (h, right), TNF-α⁺ cells (j, left), IL-2⁺ cells (j, right) and polyfunctional IFN-γ⁺TNF-α⁺IL-2⁺ cells (k, left) in OT-I cells, and mean fluorescence intensity (MFI) of IFN-γ and GzmB in IFN-γ⁺ and GzmB⁺ cells, respectively (i), and cell number (normalized to input) per gram tissue (k, right) of polyfunctional IFN-γ⁺TNF-α⁺IL-2⁺ OT-I cells. (l–n) scRNA-Seq analysis of tumor-infiltrating sgRNA-transduced OT-I cells isolated from the dual transfer system at day 7. tSNE visualization of OT-I cells indicating genotypes (l, left), *Tcf7*^hi and *Tcf7*^lo cells (l, right), and *Tcf7* (m, left), *Slamf6* (m, right) and *Tox* (n) gene expression in individual cells. Mean ± s.e.m. in b–d, **f–k**. *P < 0.05; **P < 0.01; ***P < 0.001; Kolmogorov-Smirnov test followed by Benjamini-Hochberg correction in a; two-tailed unpaired Student’s t-test in b–d, f, g; two-tailed paired Student’s t-test in **h–k**. Data are representative of three (b), or pooled from two (c, d, **f–k**) independent experiments.

**Figure 4.. BATF is a key Regnase-1 functional target to mediate mitochondrial fitness and effector responses.**
(a) Diagram of secondary genome-scale CRISPR screening. (b) Tumor-infiltrating sgRNA-transduced OT-I cells from the dual transfer system (n = 6) were analyzed at day 7 for BATF expression (left), and quantification of BATF MFI (right). (c) Luciferase activity of HEK293T cells after transfection with *Batf* mRNA 3’ UTR reporter, together with control (mock), wild-type or D141N Regnase-1-expressing plasmid (n = 3). (d, e) *In vivo* accumulation of sgRNA- and double sgRNA-transduced OT-I cells (d) or *Batf*-overexpressing retrovirus-transduced OT-I cells (e) in the dual transfer system (n = 6). OT-I cell percentage in CD8α⁺ cells was normalized to co-transferred non-targeting control sgRNA-transduced spike cells (d). (**f–h**) Tumor-infiltrating OT-I cells transduced with sgRNA (f; n = 6), double sgRNA (g; n = 6) and *Batf*-overexpressing retrovirus (h; n = 8) from the dual transfer system were analyzed at day 7 for the quantification of MFI of TMRM (left) and Mitotracker (right). MFI of TMRM and Mitotracker was normalized to those of co-transferred control sgRNA-transduced spike cells (g). Mean ± s.e.m. in b, d–h. Mean ± s.d. in c. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed paired Student’s t-test in f, two-tailed unpaired Student’s t-test in b, e, h, and one-way ANOVA in c, d, g. Data are representative of two (c), or pooled from two (b, d–h) independent experiments.

**Figure 5.. Genome-scale CRISPR screening identifies PTPN2 and SOCS1 as additional targets to enhance Regnase-1-null CD8⁺ T cell antitumor activity.**
(a–e) OT-I cells transduced with non-targeting control sgRNA (spike) were mixed at a 1:1 ratio with cells transduced with non-targeting control sgRNA, sgRegnase-1, sgPtpn2, sgPtpn2/*Regnase-1*, sgSocs1 or sgSocs1/*Regnase-1*, and transferred into tumor-bearing hosts individually (n = 6). Tumor-infiltrating OT-I cells were analyzed at day 7 for quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to spike (a), quantification of relative MFI of TMRM (b), Mitotracker (c) and BATF (d) normalized to spike, and quantification of relative frequency of TCF-1⁺ cells normalized to spike (e). (f) sgRNA- or double sgRNA-transduced pmel-1 cells (4 × 10⁶) were transferred into mice at day 12 after B16-F10 melanoma engraftment, followed by analysis of tumor size. Non-treatment control mice received no T cell transfer. Mean ± s.e.m. in a–e. *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA in a–e, and two-way ANOVA in f. Data are pooled from two (a–f) independent experiments.

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Antitumour T cells stand the test of time.
Reina-Campos M, Goldrath AW. Reina-Campos M, et al. Nature. 2019 Dec;576(7787):392-393. doi: 10.1038/d41586-019-03731-w. Nature. 2019. PMID: 31844255 No abstract available.

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References

1. Lim WA & June CH The Principles of Engineering Immune Cells to Treat Cancer. Cell 168, 724–740, doi:10.1016/j.cell.2017.01.016 (2017). - DOI - PMC - PubMed
1. Gattinoni L et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 115, 1616–1626, doi:10.1172/JCI24480 (2005). - DOI - PMC - PubMed
1. Kishton RJ, Sukumar M & Restifo NP Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metab 26, 94–109, doi:10.1016/j.cmet.2017.06.016 (2017). - DOI - PMC - PubMed
1. Muri J et al. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation. Nat Commun 9, 1851, doi:10.1038/s41467-018-04274-w (2018). - DOI - PMC - PubMed
1. Peng M et al. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science 354, 481–484, doi:10.1126/science.aaf6284 (2016). - DOI - PMC - PubMed

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[1] Lim WA & June CH The Principles of Engineering Immune Cells to Treat Cancer. Cell 168, 724–740, doi:10.1016/j.cell.2017.01.016 (2017). - DOI - PMC - PubMed

[2] Lim WA & June CH The Principles of Engineering Immune Cells to Treat Cancer. Cell 168, 724–740, doi:10.1016/j.cell.2017.01.016 (2017). - DOI - PMC - PubMed

[3] Gattinoni L et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 115, 1616–1626, doi:10.1172/JCI24480 (2005). - DOI - PMC - PubMed

[4] Gattinoni L et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 115, 1616–1626, doi:10.1172/JCI24480 (2005). - DOI - PMC - PubMed

[5] Kishton RJ, Sukumar M & Restifo NP Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metab 26, 94–109, doi:10.1016/j.cmet.2017.06.016 (2017). - DOI - PMC - PubMed

[6] Kishton RJ, Sukumar M & Restifo NP Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metab 26, 94–109, doi:10.1016/j.cmet.2017.06.016 (2017). - DOI - PMC - PubMed

[7] Muri J et al. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation. Nat Commun 9, 1851, doi:10.1038/s41467-018-04274-w (2018). - DOI - PMC - PubMed

[8] Muri J et al. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation. Nat Commun 9, 1851, doi:10.1038/s41467-018-04274-w (2018). - DOI - PMC - PubMed

[9] Peng M et al. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science 354, 481–484, doi:10.1126/science.aaf6284 (2016). - DOI - PMC - PubMed

[10] Peng M et al. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science 354, 481–484, doi:10.1126/science.aaf6284 (2016). - DOI - PMC - PubMed

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Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy

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Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy

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