The Transcription Factor Bhlhe40 Programs Mitochondrial Regulation of Resident CD8+ T Cell Fitness and Functionality

•Bhlhe40 is required for Trm cell and TIL fitness and function•Bhlhe40 is critical for TIL reinvigoration following anti-PD-L1 blockade•Bhlhe40 programs Trm cell and TIL mitochondrial metabolism and active chromatin state•Epigenetic targeting Trm cell and TIL functional program promotes tumor control Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function. Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function. Tissue-resident memory CD8+ T (Trm) cells are a recently described population of CD8+ memory T (Tmem) cells, which permanently reside in non-lymphoid tissues (NLTs) and rapidly respond to pathogen reinvasion (Ariotti et al., 2014Ariotti S. Hogenbirk M.A. Dijkgraaf F.E. Visser L.L. Hoekstra M.E. Song J.Y. Jacobs H. Haanen J.B. Schumacher T.N. T cell memory. 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Generation and maintenance of Trm cells are regulated by a more distinct set of transcription factors than those required for circulating Tmem cells, including Runx3, Notch, Blimp-1, Hobbit, and Nur77 (Hombrink et al., 2016Hombrink P. Helbig C. Backer R.A. Piet B. Oja A.E. Stark R. Brasser G. Jongejan A. Jonkers R.E. Nota B. et al.Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells.Nat. Immunol. 2016; 17: 1467-1478Crossref PubMed Scopus (279) Google Scholar, Mackay et al., 2016Mackay L.K. Minnich M. Kragten N.A. Liao Y. Nota B. Seillet C. Zaid A. Man K. Preston S. Freestone D. et al.Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes.Science. 2016; 352: 459-463Crossref PubMed Scopus (549) Google Scholar, Milner et al., 2017Milner J.J. Toma C. Yu B. Zhang K. Omilusik K. Phan A.T. Wang D. Getzler A.J. Nguyen T. Crotty S. et al.Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.Nature. 2017; 552: 253-257Crossref PubMed Scopus (316) Google Scholar, Skon et al., 2013Skon C.N. Lee J.Y. Anderson K.G. Masopust D. Hogquist K.A. Jameson S.C. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells.Nat. Immunol. 2013; 14: 1285-1293Crossref PubMed Scopus (496) Google Scholar). These transcription factors instruct a tissue-residency program that allows for the long-term retention and maintenance of Trm cells within NLTs. Trm cells have an elevated expression of a number of effector molecules, including interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), and granzyme B (GzmB), which enable Trm cells to rapidly respond to stimulation and orchestrate protective immunity (Gebhardt et al., 2009Gebhardt T. Wakim L.M. Eidsmo L. Reading P.C. Heath W.R. Carbone F.R. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.Nat. Immunol. 2009; 10: 524-530Crossref PubMed Scopus (812) Google Scholar, Jiang et al., 2012Jiang X. Clark R.A. Liu L. Wagers A.J. Fuhlbrigge R.C. Kupper T.S. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.Nature. 2012; 483: 227-231Crossref PubMed Scopus (629) Google Scholar). Currently, the transcriptional regulation of in situ Trm cell functionality (rapid production of a range of effector molecules) is largely unknown. Of note, Trm cells and CD8+ tumor-infiltrating lymphocytes (TILs) share a common core residency gene program (Milner et al., 2017Milner J.J. Toma C. Yu B. Zhang K. Omilusik K. Phan A.T. Wang D. Getzler A.J. Nguyen T. Crotty S. et al.Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.Nature. 2017; 552: 253-257Crossref PubMed Scopus (316) Google Scholar). Furthermore, a Trm cell gene signature has been associated with improved patient survival in lung, breast, and melanoma cancers (Ganesan et al., 2017Ganesan A.P. Clarke J. Wood O. Garrido-Martin E.M. Chee S.J. Mellows T. Samaniego-Castruita D. Singh D. Seumois G. Alzetani A. et al.Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.Nat. Immunol. 2017; 18: 940-950Crossref PubMed Scopus (298) Google Scholar, Guo et al., 2018Guo X. Zhang Y. Zheng L. Zheng C. Song J. Zhang Q. Kang B. Liu Z. Jin L. Xing R. et al.Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing.Nat. Med. 2018; 24: 978-985Crossref PubMed Scopus (637) Google Scholar, Savas et al., 2018Savas P. Virassamy B. Ye C. Salim A. Mintoff C.P. Caramia F. Salgado R. Byrne D.J. Teo Z.L. Dushyanthen S. et al.Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab)Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.Nat. Med. 2018; 24: 986-993Crossref PubMed Scopus (466) Google Scholar). However, the mechanisms by which Trm cell and TIL function and fitness are programmed or maintained are incompletely understood. CD8+ T cell activation, effector differentiation, and memory cell formation are closely associated with changes in the cellular metabolic programs. While effector T (Teff) cells mainly use aerobic glycolysis to fuel their expansion and effector function, mitochondrial metabolism and oxidative phosphorylation (OXPHOS) are important in supporting Tmem cell maintenance and function (Bantug et al., 2018Bantug G.R. Fischer M. Grahlert J. Balmer M.L. Unterstab G. Develioglu L. Steiner R. Zhang L. Costa A.S.H. Gubser P.M. et al.Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8(+) T Cells.Immunity. 2018; 48: 542-555.e6Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, Borges da Silva et al., 2018Borges da Silva H. Beura L.K. Wang H. Hanse E.A. Gore R. Scott M.C. Walsh D.A. Block K.E. Fonseca R. Yan Y. et al.The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells.Nature. 2018; 559: 264-268Crossref PubMed Scopus (149) Google Scholar, Buck et al., 2016Buck M.D. O’Sullivan D. Klein Geltink R.I. Curtis J.D. Chang C.H. Sanin D.E. Qiu J. Kretz O. Braas D. van der Windt G.J. et al.Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming.Cell. 2016; 166: 63-76Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, Pearce et al., 2009Pearce E.L. Walsh M.C. Cejas P.J. Harms G.M. Shen H. Wang L.S. Jones R.G. Choi Y. Enhancing CD8 T-cell memory by modulating fatty acid metabolism.Nature. 2009; 460: 103-107Crossref PubMed Scopus (1084) Google Scholar, Sena et al., 2013Sena L.A. Li S. Jairaman A. Prakriya M. Ezponda T. Hildeman D.A. Wang C.R. Schumacker P.T. Licht J.D. Perlman H. et al.Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling.Immunity. 2013; 38: 225-236Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Similarly, Trm cells require mitochondria-dependent lipid oxidation for their long-term maintenance (Pan et al., 2017Pan Y. Tian T. Park C.O. Lofftus S.Y. Mei S. Liu X. Luo C. O’Malley J.T. Gehad A. Teague J.E. et al.Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.Nature. 2017; 543: 252-256Crossref PubMed Scopus (376) Google Scholar). Mitochondrial energy metabolism and fitness are also required for TIL fitness and function (Scharping et al., 2016Scharping N.E. Menk A.V. Moreci R.S. Whetstone R.D. Dadey R.E. Watkins S.C. Ferris R.L. Delgoffe G.M. The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction.Immunity. 2016; 45: 374-388Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, Zhang et al., 2017Zhang Y. Kurupati R. Liu L. Zhou X.Y. Zhang G. Hudaihed A. Filisio F. Giles-Davis W. Xu X. Karakousis G.C. et al.Enhancing CD8+ T Cell Fatty Acid Catabolism within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy.Cancer Cell. 2017; 32: 377-391.e9Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). However, the transcriptional regulation of mitochondrial metabolism in Trm cells and TILs are largely elusive. In addition to energy generation, mitochondria produce a variety of macromolecules that are important in cell physiological responses, including the regulation of gene expression (Mehta et al., 2017Mehta M.M. Weinberg S.E. Chandel N.S. Mitochondrial control of immunity: beyond ATP.Nat. Rev. Immunol. 2017; 17: 608-620Crossref PubMed Scopus (221) Google Scholar). For instance, as a critical substrate of histone acetylated modification, acetyl-coenzyme A (acetyl-CoA) is produced following fatty acid or pyruvate oxidation in mitochondria (Peng et al., 2016Peng M. Yin N. Chhangawala S. Xu K. Leslie C.S. Li M.O. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism.Science. 2016; 354: 481-484Crossref PubMed Scopus (423) Google Scholar). Importantly, the increased presence of histone acetylation (e.g., H3K9 or H3K27 acetylation) and active chromatin state are tightly correlated with the function of CD8+ T cells (Henning et al., 2018Henning A.N. Roychoudhuri R. Restifo N.P. Epigenetic control of CD8+ T cell differentiation.Nat. Rev. Immunol. 2018; 18: 340-356Crossref PubMed Scopus (216) Google Scholar). To this end, the suppressive tumor microenvironment (TME) promotes the TIL acquisition of a dysfunctional chromatin state in advanced tumor stages (Philip et al., 2017Philip M. Fairchild L. Sun L. Horste E.L. Camara S. Shakiba M. Scott A.C. Viale A. Lauer P. Merghoub T. et al.Chromatin states define tumour-specific T cell dysfunction and reprogramming.Nature. 2017; 545: 452-456Crossref PubMed Scopus (460) Google Scholar, Sen et al., 2016Sen D.R. Kaminski J. Barnitz R.A. Kurachi M. Gerdemann U. Yates K.B. Tsao H.W. Godec J. LaFleur M.W. Brown F.D. et al.The epigenetic landscape of T cell exhaustion.Science. 2016; 354: 1165-1169Crossref PubMed Scopus (513) Google Scholar). The dysfunctional epigenetic programming may limit the efficacy of immunotherapies (Ghoneim et al., 2017Ghoneim H.E. Fan Y. Moustaki A. Abdelsamed H.A. Dash P. Dogra P. Carter R. Awad W. Neale G. Thomas P.G. Youngblood B. De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation.Cell. 2017; 170: 142-157.e19Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Thus, to achieve effective cancer immunotherapy, it is necessary to re-program the unresponsive T cell epigenetic state within the TME into a functional anti-tumor state. Bhlhe40 is a stress-responsive transcription factor that is important in a number of cell physiological responses (Ma et al., 2013Ma W. Shi X. Lu S. Wu L. Wang Y. Hypoxia-induced overexpression of DEC1 is regulated by HIF-1α in hepatocellular carcinoma.Oncol. Rep. 2013; 30: 2957-2962Crossref PubMed Scopus (18) Google Scholar). Bhlhe40 expression in T helper (Th) cells modulates the effector and pathogenic activities of Th1 and Th17 cells (Lin et al., 2016Lin C.C. Bradstreet T.R. Schwarzkopf E.A. Jarjour N.N. Chou C. Archambault A.S. Sim J. Zinselmeyer B.H. Carrero J.A. Wu G.F. et al.IL-1-induced Bhlhe40 identifies pathogenic T helper cells in a model of autoimmune neuroinflammation.J. Exp. Med. 2016; 213: 251-271Crossref PubMed Scopus (54) Google Scholar, Yu et al., 2018Yu F. Sharma S. Jankovic D. Gurram R.K. Su P. Hu G. Li R. Rieder S. Zhao K. Sun B. Zhu J. The transcription factor Bhlhe40 is a switch of inflammatory versus antiinflammatory Th1 cell fate determination.J. Exp. Med. 2018; 215: 1813-1821Crossref PubMed Scopus (68) Google Scholar). Bhlhe40 overexpression in in vitro-generated “memory” CD8+ T cells results in attenuated recall responses (Hu and Chen, 2013Hu G. Chen J. A genome-wide regulatory network identifies key transcription factors for memory CD8+ T-cell development.Nat. Commun. 2013; 4: 2830Crossref PubMed Scopus (83) Google Scholar), but the physiological roles of Bhlhe40 in regulating CD8+ Teff and/or Tmem responses remain unclear. Here, we demonstrate that Bhlhe40 is specifically required for the development, fitness, and polyfunctionality of Trm cells and TILs. Bhlhe40 deficiency leads to the impaired production of metabolites required for acetyl-CoA synthesis, resulting in decreased Trm cell and TIL histone acetylation for the proper expression of functional molecules. Building on the findings, we have identified a regimen that can enhance wild-type (WT) and Bhlhe40−/− CD8+ T cell functionality and tissue residency gene programs through the in vitro screening of an epigenetic library. Our results provide mechanistic insights into the local regulation of Trm cell and TIL functionality and offer a viable strategy for developing cancer immunotherapeutic strategies. To explore Bhlhe40 function in CD8+ T cells, we first determined Bhlhe40 expression in WT CD8+ T cells following activation. We found that Bhlhe40 was potently upregulated in CD8+ T cells following activation (Figure S1A). Bhlhe40 was required for sustained expansion and effector molecule production by activated CD8+ T cells in vitro (Figures S1B–S1D). Furthermore, there were pronounced differences in the transcriptional profiles between activated WT and Bhlhe40−/− CD8+ T cells (4 days post-activation [d.p.a.]) (Figure 1A). Gene set enrichment analysis (GSEA) showed that Bhlhe40 modulated a core set of tissue signature genes that were recently discovered to be shared by Trm cells and TILs (Milner et al., 2017Milner J.J. Toma C. Yu B. Zhang K. Omilusik K. Phan A.T. Wang D. Getzler A.J. Nguyen T. Crotty S. et al.Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.Nature. 2017; 552: 253-257Crossref PubMed Scopus (316) Google Scholar) but not gene programs differentiating effector versus memory or SLECs (short-lived effector cells) versus MPECs (memory precursor effector cells) (Sarkar et al., 2008Sarkar S. Kalia V. Haining W.N. Konieczny B.T. Subramaniam S. Ahmed R. Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates.J. Exp. Med. 2008; 205: 625-640Crossref PubMed Scopus (458) Google Scholar) (Figures 1B, S1E, and S1F; Table S1). Furthermore, analysis of single-cell RNA sequencing (scRNA-seq) data from human colorectal TILs (Zhang et al., 2018Zhang L. Yu X. Zheng L. Zhang Y. Li Y. Fang Q. Gao R. Kang B. Zhang Q. Huang J.Y. et al.Lineage tracking reveals dynamic relationships of T cells in colorectal cancer.Nature. 2018; 564: 268-272Crossref PubMed Scopus (411) Google Scholar) found that BHLHE40hi TILs exhibited enrichment of the core tissue-residency gene signature relative to BHLHE40low TILs (Figure 1C). We next used an acute influenza virus infection model and a melanoma (B16-F10-expressing chicken ovalbumin [OVA], B16-OVA) transplantation model to study Bhlhe40 function in Trm cell and TIL responses, respectively. RNA-seq and prime-flow staining showed that Trm cells and TILs expressed higher Bhlhe40 compared to their splenic counterparts (Figures 1D and S1G). Moreover, the top 20 Bhlhe40-associated genes predicted by the GIANT (Genome-Scale Integrated Analysis of Gene Networks in Tissues) database (Greene et al., 2015Greene C.S. Krishnan A. Wong A.K. Ricciotti E. Zelaya R.A. Himmelstein D.S. Zhang R. Hartmann B.M. Zaslavsky E. Sealfon S.C. et al.Understanding multicellular function and disease with human tissue-specific networks.Nat. Genet. 2015; 47: 569-576Crossref PubMed Scopus (485) Google Scholar) (Figure S1H) were enriched in both Trm cells and TILs compared to their splenic counterparts (Figure 1E). We compared BHLHE40 expression in tumor-reactive CD8+ T cells (CD45RO+PD-1+CD11a+) (Dronca et al., 2016Dronca R.S. Liu X. Harrington S.M. Chen L. Cao S. Kottschade L.A. McWilliams R.R. Block M.S. Nevala W.K. Thompson M.A. et al.T cell Bim levels reflect responses to anti-PD-1 cancer therapy.JCI Insight. 2016; 1: e86014Crossref PubMed Scopus (58) Google Scholar) within TILs or peripheral blood mononuclear cells (PBMCs) from renal cell carcinoma (RCC) patients using prime-flow analysis. Tumor-reactive TILs expressed higher BHLHE40 compared with their paired circulating counterparts (Figures 1F, left, and S1I). Similarly, human lung Trm (CD103+) cells had greater BHLHE40 expression than Tmem cells in the PBMCs (Hombrink et al., 2016Hombrink P. Helbig C. Backer R.A. Piet B. Oja A.E. Stark R. Brasser G. Jongejan A. Jonkers R.E. Nota B. et al.Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells.Nat. Immunol. 2016; 17: 1467-1478Crossref PubMed Scopus (279) Google Scholar) (Figure 1F, right). In addition, BHLHE40-associated genes were enriched in human TILs from colorectal cancer, hepatocellular carcincoma (HCC), and non-small-cell lung cancer (NSCLC) compared to CD8+ T cells in the PBMCs (Guo et al., 2018Guo X. Zhang Y. Zheng L. Zheng C. Song J. Zhang Q. Kang B. Liu Z. Jin L. Xing R. et al.Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing.Nat. Med. 2018; 24: 978-985Crossref PubMed Scopus (637) Google Scholar, Zhang et al., 2018Zhang L. Yu X. Zheng L. Zhang Y. Li Y. Fang Q. Gao R. Kang B. Zhang Q. Huang J.Y. et al.Lineage tracking reveals dynamic relationships of T cells in colorectal cancer.Nature. 2018; 564: 268-272Crossref PubMed Scopus (411) Google Scholar, Zheng et al., 2017Zheng C. Zheng L. Yoo J.K. Guo H. Zhang Y. Guo X. Kang B. Hu R. Huang J.Y. Zhang Q. et al.Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.Cell. 2017; 169: 1342-1356.e16Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar) (Figure 1G). These data suggest that Bhlhe40 and its associated genes are highly expressed in both mouse and human resident CD8+ T cells in the NLTs or tumors compared to their lymphoid or circulating counterparts. We infected WT or Bhlhe40−/− mice with influenza A X31 (H3N2) strain and examined influenza-specific effector and memory CD8+ T cell responses against MHC-I H2-Db-restricted nucleoprotein 366–374 peptide (NP366–374). Bhlhe40−/− mice exhibited comparable frequencies and numbers of NP366–374 Teff cells in the lungs (Teff-Lung) or spleens (Teff-SPL) as those of WT mice at 10 days post-infection (d.p.i.) (Figures 2A and S2A). However, Bhlhe40 deficiency caused a marked decrease in frequencies and numbers of lung CD8+ Trm cells (Figures 2B and S2C). The magnitude of the CD8+ lung circulating (Tmem-Cir) or splenic Tmem (Tmem-SPL) cells in Bhlhe40−/− mice was comparable to those of WT mice (Figure 2B). A similar decrease in lung NP366–374 and PA224–233 (H2-Db-restricted polymerase 224–233 peptide) Trm cell responses was observed in Bhlhe40−/− mice following influenza PR8 (H1N1) virus infection (Thomas et al., 2006Thomas P.G. Keating R. Hulse-Post D.J. Doherty P.C. Cell-mediated protection in influenza infection.Emerg. Infect. Dis. 2006; 12: 48-54Crossref PubMed Scopus (364) Google Scholar) (Figures 2C, 2D, and S2D). CD69+CD103+ Trm cells also diminished following X31 or PR8 infection in Bhlhe40−/− mice (Figures S2C and S2D). The decreased Trm cell magnitude in Bhlhe40−/− mice was associated with enhanced Trm cell apoptosis (Figure 2E). We next 1:1 mixed WT OTI (MHC-class-I-restricted, ovalbumin-specific CD8+ T cells [CD90.1+] and Bhlhe40−/− OTI [CD90.1+CD90.2+]) and transferred the mixed cells into WT mice. We then infected the mice with influenza PR8 expressing OVA (PR8-OVA) and followed the ratio of WT versus Bhlhe40−/− OTI cells at 10 and 35 d.p.i. (Figure 2F). The ratio of WT to Bhlhe40−/− OTI cells remained at ∼1:1 in the spleen at the two time points, suggesting that CD8+ T cell-intrinsic Bhlhe40 was dispensable for the generation of splenic Teff and Tmem cells (Figure 2F). Bhlhe40 deficiency caused a modest decrease in OTI effector cells in the lung at 10 d.p.i. but resulted in a marked impairment in the generation of lung Trm cells at 35 d.p.i. (Figures 2F and 2G). Moreover, Bhlhe40−/− Trm cells exhibited impairment in the production of IFN-γ (Figures 2H and S2E). Notably, the depletion of circulating CD8+ T cells with low-dose anti-CD8 or long-term FTY720 treatment at the memory stage did not alter the ratio of WT versus Bhlhe40−/− Trm cells in the lungs (Figures S2F–S2H). Thus, Bhlhe40 likely acts on tissue-resident CD8+ T cells to promote Trm cell formation, but probably not on recirculating memory T cells recently entering the lungs. To examine whether activated Bhlhe40−/− T cells also exhibit defects in Trm cell formation and/or maintenance, we stimulated WT or Bhlhe40−/− OTI cells in vitro and transferred the effector WT OTI (CD90.1+) and the Bhlhe40−/− OTI (CD90.1+CD90.2+) cells at the ratio of 1:1 into WT mice followed by PR8-OVA infection. Bhlhe40 deficiency in activated CD8+ T cells greatly diminished Trm cell formation, but it only modestly affected Tmem-SPL responses (Figure S2I). We also generated T cell-specific Bhlhe40-deficient mice (Bhlhe40ΔT) and infected the mice with PR8. T cell-specific deficiency of Bhlhe40 resulted in a decreased magnitude of total or CD69+CD103+ NP366–374 and PA224–233 lung Trm cells as well as diminished per cell expression of CD103 (Figures 2I, 2J, and S2J–S2L). In addition, both Bhlhe40−/− and T cell-specific Bhlhe40 deficiency led to a marked decrease in the production of effector molecules (IFN-γ, GzmB, and TNF) by Trm but not Tmem-SPL cells (Figures 2K and S2M). Thus, CD8+ T cell-autonomous Bhlhe40 is specifically required for Trm cell formation, fitness, and functionality. As a result, Bhlhe40−/− mice that were previously infected with X31 exhibited defects in Trm-cell-mediated protection against a lethal secondary PR8 challenge in the presence of FTY720, which blocked circulating memory T cell migration to the lungs (Iborra et al., 2016Iborra S. Martínez-López M. Khouili S.C. Enamorado M. Cueto F.J. Conde-Garrosa R. Del Fresno C. Sancho D. Optimal Generation of Tissue-Resident but Not Circulating Memory T Cells during Viral Infection Requires Crosspriming by DNGR-1+ Dendritic Cells.Immunity. 2016; 45: 847-860Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) (Figure 2L). Furthermore, Bhlhe40ΔT mice that were previously infected with PR8 had defects in Trm-cell-mediated protection against secondary X31 challenge (Figure 2M). Trm cells and TILs share a core tissue residency and function gene signature (Milner et al., 2017Milner J.J. Toma C. Yu B. Zhang K. Omilusik K. Phan A.T. Wang D. Getzler A.J. Nguyen T. Crotty S. et al.Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.Nature. 2017; 552: 253-257Crossref PubMed Scopus (316) Google Scholar). To this end, we subcutaneously implanted B16-OVA into WT or Bhlhe40−/− mice and checked TIL responses. Bhlhe40−/− mice showed decreased total and antigen-specific (H2-Kb OVA257–264 tetramer+) TILs at days 14 and 18 post-tumor transplantation (d.p.t.i.) (Figures 3A, 3B, and S3A). Bhlhe40−/− TILs had increased apoptosis and decreased IFN-γ and GzmB production (Figures 3C and 3D) yet showed proliferative responses comparable to those of WT TILs (Figure S3B). In contrast, Bhlhe40−/− splenic CD8+ T cells exhibit levels of apoptosis, IFN-γ, or GzmB production similar to their WT counterparts (Figures 3C and 3D). T cells capable of producing multiple effector molecules are polyfunctional and associated with better cancer control (Yuan et al., 2008Yuan J. Gnjatic S. Li H. Powel S. Gallardo H.F. Ritter E. Ku G.Y. Jungbluth A.A. Segal N.H. Rasalan T.S. et al.CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit.Proc. Natl. Acad. Sci. USA. 2008; 105: 20410-20415Crossref PubMed Scopus (287) Google Scholar, Zhao et al., 2016aZhao E. Maj T. Kryczek I. Li W. Wu K. Zhao L. Wei S. Crespo J. Wan S. Vatan L. et al.Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction.Nat. Immunol. 2016; 17: 95-103Crossref PubMed Scopus (267) Google Scholar). We thus evaluated the polyfunctionality of WT and Bhlhe40−/− TILs by measuring five effector molecules in TILs (IFN-γ, TNF-α, GzmB, C-C motif chemokine ligand 3 (CCL3), and CCL4). Bhlhe40−/− TILs showed decreased frequencies of cells simultaneously producing multiple effector molecules (Figures 3E and S3C). Importantly, Bhlhe40−/− TILs did not have increased PD-1, TIM-3, or CTLA4 compared to WT TILs (Figure S3D). Notably, “stem-like” TILs (PD-1int TCF1+ CXCR5+) expressed much lower Bhlhe40 than PD-1hi TILs and “exhausted” PD-1hi TIM-3+ TILs (Figure S3E), which is consistent with the reported findings that TCF1− TILs have an enrichment of Trm cell signature genes and had higher effector molecule expression compared to TCF1+ stem-like TILs (Kurtulus et al., 2019Kurtulus S. Madi A. Escobar G. Klapholz M. Nyman J. Christian E. Pawlak M. Dionne D. Xia J. Rozenblatt-Rosen O. et al.Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1(-)CD8(+) Tumor-Infiltrating T Cells.Immunity. 2019; 50: 181-194.e6Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, Siddiqui et al., 2019Siddiqui I. Schaeuble K. Chennupati V. Fuertes Marraco S.A. Calderon-Copete S. Pais Ferreira D. Carmona S.J. Scarpellino L. Gfeller D. Pradervand S. et al.Intratumoral Tcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy.Immunity. 2019; 50: 195-211.e10Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). Within the PD-1hi TIM-3+ TIL population, Bhlhe40hi TILs had higher Ifng and an enriched core Trm cell and TIL residency gene signature relative to Bhlhe40low TILs (Figures S3F–S3I) (Singer et al., 2016Singer M. Wang C. Cong L. Marjanovic N.D. Kowalczyk M.S. Zhang H. Nyman J. Sakuishi K. Kurtulus S. Gennert D. et al.A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells.Cell. 2016; 166: 1500-1511.e9Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) (Zhang et al., 2018Zhang L. Yu X. Zheng L. Zhang Y. Li Y. Fang Q. Gao R. Kang B. Zhang Q. Huang J.Y. et al.Lineage tracking reveals dynamic relationships of T cells in colorectal cancer.Nature. 2018; 564: 268-272Crossref PubMed Scopus (411) Google Scholar), suggesting that the amount of Bhlhe40 expression in this TIL population may correlate with their residual function in situ. Altogether, our data indicate that Bhlhe40 is key for maintaining TIL function. As a result, Bhlhe40−/− mice had an increased tumor burden following transplantation with multiple tumor cell lines, including B16-OVA, MC-38 (murine colon adenocarcinoma cells), and LLC (Lewis lung carcinoma cells) (Figures 3F–3H). We then 1:1 mixed WT and Bhlhe40−/− OTI cells and transferred them into B16-OVA-bearing mice. While Bhlhe40−/− OTI cells competed well with WT OTI cells inside the spleen, Bhlhe40 deficiency resulted in progressive and drastic OTI cell loss inside the tumor, suggesting that intrinsic Bhlhe40 is critical for TIL maintenance during tumor progression (Figures 3I and