ABSTRACT
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.
ABSTRACT
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.
Subject(s)
CD8-Positive T-Lymphocytes , Cell Differentiation , Immune Tolerance , Protein Biosynthesis , Receptors, Antigen, T-Cell , CD8-Positive T-Lymphocytes/immunology , Animals , Cell Differentiation/immunology , Mice , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Immune Tolerance/immunology , Protein Biosynthesis/immunology , Signal Transduction/immunology , Mice, Inbred C57BL , Autoantigens/immunologyABSTRACT
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.
Subject(s)
Forkhead Box Protein O1 , Immunotherapy, Adoptive , Neoplasms , Receptors, Chimeric Antigen , Stem Cells , T-Lymphocytes , Humans , Mice , Cell Line, Tumor , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Mitochondria/metabolism , Phenotype , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/cytology , Tumor Microenvironment/immunology , Stem Cells/cytology , Stem Cells/immunology , Stem Cells/metabolism , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapyABSTRACT
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.
Subject(s)
Neoplasms , Animals , Mice , Cytokines/metabolism , Immunity , Immunotherapy , Lymphocytes, Tumor-Infiltrating , Neoplasms/genetics , Neoplasms/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Immune Checkpoint Inhibitors , Feedback , Suppressor of Cytokine Signaling Proteins/genetics , Signal TransductionABSTRACT
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.
Subject(s)
Immunotherapy, Adoptive , Neoplasms , Humans , Immunotherapy, Adoptive/methods , T-Lymphocytes , Cytokines/metabolism , Stem Cells/metabolism , Receptors, Antigen, T-Cell/metabolismABSTRACT
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.
Subject(s)
Multiple Myeloma , Animals , Mice , Humans , Lenalidomide/pharmacology , Lenalidomide/therapeutic use , Lenalidomide/metabolism , Multiple Myeloma/metabolism , Monocytes/metabolism , Leukocytes, Mononuclear/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Killer Cells, Natural , Dexamethasone/therapeutic useABSTRACT
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.
Subject(s)
F-Box Proteins , Leukemia, Myeloid, Acute , Alcohol Oxidoreductases , DNA-Binding Proteins , F-Box Proteins/genetics , HLA Antigens/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Lymphocyte Activation , Protein-Arginine N-Methyltransferases/metabolism , Recurrence , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
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.
Subject(s)
Breast Neoplasms , Interferon-gamma , Acetylation , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , DNA Methylation , E1A-Associated p300 Protein , Female , Interferon-gamma/pharmacology , Janus Kinases , Membrane Proteins , Mice , Phosphoproteins , STAT Transcription Factors , Signal TransductionABSTRACT
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.
Subject(s)
Interleukin-15 , Neoplasms , Animals , Cell Line, Tumor , Interleukin-15/metabolism , Killer Cells, Natural , Mice , Neoplasms/pathology , Transforming Growth Factor beta/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Receptors, Chimeric Antigen , CD4-Positive T-Lymphocytes , Humans , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell , Receptors, Chimeric Antigen/genetics , T-LymphocytesABSTRACT
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.
Subject(s)
Lymphoid Tissue , Mucosal-Associated Invariant T Cells , Natural Killer T-Cells , Animals , Lymphoid Tissue/metabolism , Mice , Mice, Transgenic , Mucosal-Associated Invariant T Cells/metabolism , Natural Killer T-Cells/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolismABSTRACT
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.
Subject(s)
Neoplasms , Programmed Cell Death 1 Receptor , Animals , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Humans , Immunotherapy, Adoptive , Mice , Neoplasms/metabolism , Programmed Cell Death 1 Receptor/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
B7-H1 Antigen , Programmed Cell Death 1 Receptor , Humans , Immunotherapy , T-Lymphocytes , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Neoplasms , Adoptive Transfer , Humans , Neoplasms/genetics , Neoplasms/therapyABSTRACT
The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.
Subject(s)
Immunity, Cellular , Killer Cells, Natural/immunology , Mucosal-Associated Invariant T Cells/immunology , Neoplasms/immunology , Animals , Antineoplastic Agents , Cell Line, Tumor , Cytokines , Histocompatibility Antigens Class I/genetics , Humans , Immunity , Mice , Mice, Inbred C57BL , Mice, Knockout , Minor Histocompatibility Antigens/genetics , Neoplasm Metastasis , Neoplasms/pathologyABSTRACT
CAR T cell therapy successes are challenged by several mechanisms of resistance including the development of dysfunctional states such as exhaustion. The features of CAR T cell exhaustion, its role in limiting the efficacy of CAR T therapy in both liquid and solid malignancies, and potential strategies to overcome it are discussed.