RESUMO
Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.
RESUMO
Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.
Assuntos
Linfócitos T CD8-Positivos , Diferenciação Celular , Tolerância Imunológica , Biossíntese de Proteínas , Receptores de Antígenos de Linfócitos T , Linfócitos T CD8-Positivos/imunologia , Animais , Diferenciação Celular/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância Imunológica/imunologia , Biossíntese de Proteínas/imunologia , Transdução de Sinais/imunologia , Camundongos Endogâmicos C57BL , Autoantígenos/imunologiaRESUMO
Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.
Assuntos
Proteína Forkhead Box O1 , Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Células-Tronco , Linfócitos T , Humanos , Camundongos , Linhagem Celular Tumoral , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Mitocôndrias/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/citologia , Microambiente Tumoral/imunologia , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapiaRESUMO
Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.
Assuntos
Células Matadoras Naturais , Fator de Transcrição AP-1 , Fator de Transcrição AP-1/genética , Células Matadoras Naturais/metabolismo , Receptores de Interleucina-15 , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismoRESUMO
There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.
Assuntos
Neoplasias , Animais , Camundongos , Citocinas/metabolismo , Imunidade , Imunoterapia , Linfócitos do Interstício Tumoral , Neoplasias/genética , Neoplasias/metabolismo , Microambiente TumoralRESUMO
CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.
Assuntos
Sistemas CRISPR-Cas , Inibidores de Checkpoint Imunológico , Retroalimentação , Proteínas Supressoras da Sinalização de Citocina/genética , Transdução de SinaisRESUMO
Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.
Assuntos
Linfócitos T CD8-Positivos , Células T de Memória , Camundongos , Animais , Linhagem da Célula , Memória ImunológicaRESUMO
Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.
Assuntos
Imunoterapia Adotiva , Neoplasias , Humanos , Imunoterapia Adotiva/métodos , Linfócitos T , Citocinas/metabolismo , Células-Tronco/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.
Assuntos
Mieloma Múltiplo , Animais , Camundongos , Humanos , Lenalidomida/farmacologia , Lenalidomida/uso terapêutico , Lenalidomida/metabolismo , Mieloma Múltiplo/metabolismo , Monócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células Matadoras Naturais , Dexametasona/uso terapêuticoRESUMO
There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
Assuntos
Proteínas F-Box , Leucemia Mieloide Aguda , Oxirredutases do Álcool , Proteínas de Ligação a DNA , Proteínas F-Box/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária , Proteína-Arginina N-Metiltransferases/metabolismo , Recidiva , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model. RESULTS: We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation. CONCLUSIONS: These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases.
Assuntos
Neoplasias da Mama , Interferon gama , Acetilação , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Metilação de DNA , Proteína p300 Associada a E1A , Feminino , Interferon gama/farmacologia , Janus Quinases , Proteínas de Membrana , Camundongos , Fosfoproteínas , Fatores de Transcrição STAT , Transdução de SinaisRESUMO
Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor ß (TGFß), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFß signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFß, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.
Assuntos
Interleucina-15 , Neoplasias , Animais , Linhagem Celular Tumoral , Interleucina-15/metabolismo , Células Matadoras Naturais , Camundongos , Neoplasias/patologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
In a recently published article, Melenhorst et al. performed a longitudinal analysis on chimeric antigen receptor (CAR) T cells isolated from patients over 10 years after therapy, revealing expansion of a long-lived CD4+ CAR T-cell population with a cytotoxic phenotype.
Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T CD4-Positivos , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos/genética , Linfócitos TRESUMO
Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αß T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.
Assuntos
Tecido Linfoide , Células T Invariantes Associadas à Mucosa , Células T Matadoras Naturais , Animais , Tecido Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Células T Invariantes Associadas à Mucosa/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionized cancer management. To date, these have focused on the blockade of cell-surface checkpoints such as PD-1. Herein we identify protein tyrosine phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that increased PTP1B limits T-cell expansion and cytotoxicity to contribute to tumor growth. T cell-specific PTP1B deletion increased STAT5 signaling, and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacologic inhibition of PTP1B recapitulated the T cell-mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively transferred chimeric antigen receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer. SIGNIFICANCE: Tumors subvert antitumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intratumoral T cells and that its deletion or inhibition enhances T-cell antitumor activity and increases CAR T-cell effectiveness against solid tumors. This article is highlighted in the In This Issue feature, p. 587.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Camundongos , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer immunotherapy utilizing checkpoint blockade antibodies or adoptive cellular therapy (ACT) with tumor-specific T cells has led to unprecedented clinical responses in patients with cancer and has been considered one of the most significant breakthroughs in cancer treatment in the past decade. Nevertheless, many cancers remain refractory to these therapies due to the presence of an immunosuppressive tumor microenvironment. This has led to the innovative idea of combining ACT with checkpoint inhibition. A landmark 2004 study by Blank and colleagues published in Cancer Research was one of the original demonstrations that adoptive transfer of T cells lacking the negative T-cell regulator, PD-1, was able to restore functional T-cell antitumor activity, resulting in rapid regression of established tumors in a preclinical model. This work was instrumental in not only driving clinical studies utilizing checkpoint inhibition but also a new wave of recent trials involving checkpoint blockade in the setting of ACT.See related article by Blank and colleagues, Cancer Res 2004;64:1140-5.
Assuntos
Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Humanos , Imunoterapia , Linfócitos T , Microambiente TumoralRESUMO
In this issue of Cancer Cell, Xue et al. demonstrate that adoptive transfer of tumor-specific Th9 cells can eradicate established tumors containing antigen-loss-variant cells (ALVs) through both direct killing and bystander effects mediated by intratumoral accumulation of extracellular ATP (eATP) that promotes monocyte infiltration and stimulation of IFNα/ß production.