RESUMO
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Assuntos
Antígenos CD , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Proteína do Gene 3 de Ativação de Linfócitos , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Receptor de Morte Celular Programada 1 , Animais , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Citotoxicidade Imunológica , Proliferação de Células , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologiaRESUMO
Persistent inflammation driven by cytokines such as type-one interferon (IFN-I) can cause immunosuppression. We show that administration of the Janus kinase 1 (JAK1) inhibitor itacitinib after anti-PD-1 (programmed cell death protein 1) immunotherapy improves immune function and antitumor responses in mice and results in high response rates (67%) in a phase 2 clinical trial for metastatic non-small cell lung cancer. Patients who failed to respond to initial anti-PD-1 immunotherapy but responded after addition of itacitinib had multiple features of poor immune function to anti-PD-1 alone that improved after JAK inhibition. Itacitinib promoted CD8 T cell plasticity and therapeutic responses of exhausted and effector memory-like T cell clonotypes. Patients with persistent inflammation refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve the efficacy of anti-PD-1 immunotherapy by pivoting T cell differentiation dynamics.
Assuntos
Linfócitos T CD8-Positivos , Carcinoma Pulmonar de Células não Pequenas , Inibidores de Checkpoint Imunológico , Janus Quinase 1 , Inibidores de Janus Quinases , Neoplasias Pulmonares , Receptor de Morte Celular Programada 1 , Animais , Feminino , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Linfócitos T CD8-Positivos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Janus Quinase 1/antagonistas & inibidores , Inibidores de Janus Quinases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidoresRESUMO
Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.
Assuntos
Linfócitos T CD8-Positivos , Fatores de Transcrição , Fatores de Transcrição/genética , Interleucina-2 , Regulação da Expressão Gênica , Receptor de Morte Celular Programada 1/metabolismoRESUMO
CD8+ T cells are end effectors of cancer immunity. Most forms of effective cancer immunotherapy involve CD8+ T cell effector function. Here, we review the current understanding of T cell function in cancer, focusing on key CD8+ T cell subtypes and states. We discuss factors that influence CD8+ T cell differentiation and function in cancer through a framework that incorporates the classic three-signal model and a fourth signal-metabolism-and also consider the impact of the tumor microenvironment from a T cell perspective. We argue for the notion of immunotherapies as "pro-drugs" that act to augment or modulate T cells, which ultimately serve as the drug in vivo, and for the importance of overall immune health in cancer treatment and prevention. The progress in understanding T cell function in cancer has and will continue to improve harnessing of the immune system across broader tumor types to benefit more patients.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Imunoterapia , Ativação Linfocitária , Microambiente TumoralRESUMO
Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Exaustão das Células T , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Linfócitos T CD8-Positivos , Diferenciação Celular , EpigenômicaRESUMO
CD8+ T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell T cell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts TEX toward CX3CR1+ intermediates with robust enrichment of TKLR via clonal T cell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ TKLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ T cells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Animais , Camundongos , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
CD8+ T cell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused in vivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in Tex-cell differentiation. Depletion of the canonical SWI/SNF form, BAF, impaired initial CD8+ T cell responses in acute and chronic infection. In contrast, disruption of PBAF enhanced Tex-cell proliferation and survival. Mechanistically, PBAF regulated the epigenetic and transcriptional transition from TCF-1+ progenitor Tex cells to more differentiated TCF-1- Tex subsets. Whereas PBAF acted to preserve Tex progenitor biology, BAF was required to generate effector-like Tex cells, suggesting that the balance of these factors coordinates Tex-cell subset differentiation. Targeting PBAF improved tumor control both alone and in combination with anti-PD-L1 immunotherapy. Thus, PBAF may present a therapeutic target in cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Montagem e Desmontagem da Cromatina , Cromatina , Diferenciação Celular , Epigênese GenéticaRESUMO
Identifying novel molecular mechanisms of exhausted CD8 T cells (T ex ) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T ex can be costly and inefficient. In vitro models of T ex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo T ex . We leveraged this model of in vitro chronic stimulation in combination with pooled CRISPR screening to uncover transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate subsets of T ex . By developing and benchmarking an in vitro model of T ex , we demonstrate the utility of mechanistically annotated in vitro models of T ex , in combination with high-throughput approaches, as a discovery pipeline to uncover novel T ex biology.
RESUMO
T cell exhaustion (T EX ) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR + T cells may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrate that deleting the pseudokinase Trib1 shifts T EX towards CX3CR1 + intermediates (T INT ) with robust enrichment of KLR + CD8 + T cells (T KLR ) via clonal T cell expansion. These changes are associated with globally increased KLR gene expression throughout the exhaustion program. Further, Trib1 loss augments anti-PD-L1 blockade to improve viral clearance by expanding the T KLR population. Together, these data identify Trib1 as an important regulator of T cell exhaustion whose targeting enhances the KLR + effector state and improves the response to checkpoint inhibitor therapy.
RESUMO
Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.
Assuntos
Linfócitos T CD8-Positivos , Viroses , Humanos , Camundongos , Animais , Receptores de Antígenos de Linfócitos T/genética , Diferenciação Celular , Linfócitos do Interstício TumoralRESUMO
The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.
Assuntos
Epigenômica , Ativação Linfocitária , Linfócitos T CD8-Positivos , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Humanos , Ativação Linfocitária/genéticaRESUMO
SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , SARS-CoV-2/fisiologia , Células Th1/imunologia , Vacina de mRNA-1273 contra 2019-nCoV , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Vacina BNT162 , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Memória Imunológica , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Adulto JovemRESUMO
Exhausted CD8 T cells (TEX) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate TEX cells, but reinvigoration is not durable. A major unanswered question is whether TEX cells differentiate into functional durable memory T cells (TMEM) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, TEX cells partially (re)acquire phenotypic and transcriptional features of TMEM cells. These 'recovering' TEX cells originated from the T cell factor (TCF-1+) TEX progenitor subset. Nevertheless, the recall capacity of these recovering TEX cells remained compromised as compared to TMEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for TEX cell-targeted immunotherapies.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Memória Imunológica/imunologia , Coriomeningite Linfocítica/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Epigênese Genética/genética , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcrição Gênica/genética , Células VeroRESUMO
Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohorts, flow cytometric and serologic analyses demonstrated that patients with solid cancer and patients without cancer had a similar immune phenotype during acute COVID-19, whereas patients with hematologic cancer had impairment of B cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses. Despite the impaired humoral immunity and high mortality in patients with hematologic cancer who also have COVID-19, those with a greater number of CD8 T cells had improved survival, including those treated with anti-CD20 therapy. Furthermore, 77% of patients with hematologic cancer had detectable SARS-CoV-2-specific T cell responses. Thus, CD8 T cells might influence recovery from COVID-19 when humoral immunity is deficient. These observations suggest that CD8 T cell responses to vaccination might provide protection in patients with hematologic cancer even in the setting of limited humoral responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias/imunologia , Idoso , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , COVID-19/complicações , COVID-19/mortalidade , Estudos de Coortes , Feminino , Neoplasias Hematológicas/complicações , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunofenotipagem , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias/complicações , Modelos de Riscos Proporcionais , Estudos Prospectivos , SARS-CoV-2 , Taxa de SobrevidaRESUMO
Cancer patients have increased morbidity and mortality from Coronavirus Disease 2019 (COVID-19), but the underlying immune mechanisms are unknown. In a cohort of 100 cancer patients hospitalized for COVID-19 at the University of Pennsylvania Health System, we found that patients with hematologic cancers had a significantly higher mortality relative to patients with solid cancers after accounting for confounders including ECOG performance status and active cancer status. We performed flow cytometric and serologic analyses of 106 cancer patients and 113 non-cancer controls from two additional cohorts at Penn and Memorial Sloan Kettering Cancer Center. Patients with solid cancers exhibited an immune phenotype similar to non-cancer patients during acute COVID-19 whereas patients with hematologic cancers had significant impairment of B cells and SARS-CoV-2-specific antibody responses. High dimensional analysis of flow cytometric data revealed 5 distinct immune phenotypes. An immune phenotype characterized by CD8 T cell depletion was associated with a high viral load and the highest mortality of 71%, among all cancer patients. In contrast, despite impaired B cell responses, patients with hematologic cancers and preserved CD8 T cells had a lower viral load and mortality. These data highlight the importance of CD8 T cells in acute COVID-19, particularly in the setting of impaired humoral immunity. Further, depletion of B cells with anti-CD20 therapy resulted in almost complete abrogation of SARS-CoV-2-specific IgG and IgM antibodies, but was not associated with increased mortality compared to other hematologic cancers, when adequate CD8 T cells were present. Finally, higher CD8 T cell counts were associated with improved overall survival in patients with hematologic cancers. Thus, CD8 T cells likely compensate for deficient humoral immunity and influence clinical recovery of COVID-19. These observations have important implications for cancer and COVID-19-directed treatments, immunosuppressive therapies, and for understanding the role of B and T cells in acute COVID-19.
RESUMO
Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.
Assuntos
Linfócitos T CD8-Positivos/citologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Doença Crônica , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Epigênese Genética , Redes Reguladoras de Genes , Infecções/imunologia , Camundongos , Neoplasias/imunologiaRESUMO
Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.
Assuntos
Linfócitos B/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Subpopulações de Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19 , Citocinas/sangue , Feminino , Humanos , Memória Imunológica , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Plasmócitos/imunologia , SARS-CoV-2 , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Tempo , Adulto JovemRESUMO
CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.