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1.
Nature ; 630(8018): 968-975, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38867043

RESUMO

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.


Assuntos
Neoplasias , Obesidade , Receptor de Morte Celular Programada 1 , Macrófagos Associados a Tumor , Animais , Feminino , Humanos , Masculino , Camundongos , Apresentação de Antígeno/efeitos dos fármacos , Antígeno B7-2/antagonistas & inibidores , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Obesidade/imunologia , Obesidade/metabolismo , Fagocitose/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/efeitos dos fármacos
2.
J Immunol ; 211(4): 563-575, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37341499

RESUMO

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.


Assuntos
Diazo-Oxo-Norleucina , Neoplasias , Animais , Camundongos , Diazo-Oxo-Norleucina/farmacologia , Glutamina/metabolismo , Neoplasias/terapia , Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo
4.
Lab Invest ; 103(8): 100174, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37169083

RESUMO

We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors' peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.


Assuntos
Citotoxicidade Imunológica , Neoplasias , Leucócitos Mononucleares , Células Matadoras Naturais , Linfócitos T , Técnicas de Cocultura , Citometria de Fluxo , Neoplasias/metabolismo
5.
Blood ; 137(23): 3212-3217, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33720354

RESUMO

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4 , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Ipilimumab/administração & dosagem , Proteínas de Neoplasias , Células Alógenas , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Feminino , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/terapia , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-38438220

RESUMO

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies.


Assuntos
Neoplasias , Microambiente Tumoral , Microambiente Tumoral/imunologia , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Imunoterapia
7.
J Clin Invest ; 134(8)2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38618956

RESUMO

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Camundongos , Carcinogênese/genética , Carcinoma de Células Renais/genética , Transformação Celular Neoplásica , Rim , Neoplasias Renais/genética , Microambiente Tumoral , Proteína Supressora de Tumor Von Hippel-Lindau/genética
8.
STAR Protoc ; 3(2): 101362, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35573480

RESUMO

With the increasing use of mass cytometry in clinical research, a simplified and standardized protocol for immunophenotyping human peripheral blood mononuclear cells (PBMCs) in clinical trials is needed. We present a simplified in-plate staining protocol for up to 80 samples, for laboratories of all mass cytometry expertise levels, aimed to generate reproducible datasets for large clinical cohorts. In this protocol, we provide details on the requirements to obtain meaningful results, spanning from sample quality, barcoding, and batch-freezing of stained samples.


Assuntos
Criopreservação , Leucócitos Mononucleares , Criopreservação/métodos , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Coloração e Rotulagem
9.
J Immunother Cancer ; 10(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35264434

RESUMO

BACKGROUND: The combination of antiangiogenic agents with immune checkpoint inhibitors could potentially overcome immune suppression driven by tumor angiogenesis. We report results from a phase IB study of ziv-aflibercept plus pembrolizumab in patients with advanced solid tumors. METHODS: This is a multicenter phase IB dose-escalation study of the combination of ziv-aflibercept (at 2-4 mg/kg) plus pembrolizumab (at 2 mg/kg) administered intravenously every 2 weeks with expansion cohorts in programmed cell death protein 1 (PD-1)/programmed death-ligand 1(PD-L1)-naïve melanoma, renal cell carcinoma (RCC), microsatellite stable colorectal cancer (CRC), and ovarian cancer. The primary objective was to determine maximum tolerated dose (MTD) and recommended dose of the combination. Secondary endpoints included overall response rate (ORR) and overall survival (OS). Exploratory objectives included correlation of clinical efficacy with tumor and peripheral immune population densities. RESULTS: Overall, 33 patients were enrolled during dose escalation (n=3) and dose expansion (n=30). No dose-limiting toxicities were reported in the initial dose level. Ziv-aflibercept 4 mg/kg plus pembrolizumab 2 mg/kg every 2 weeks was established as the MTD. Grade ≥3 adverse events occurred in 19/33 patients (58%), the most common being hypertension (36%) and proteinuria (18%). ORR in the dose-expansion cohort was 16.7% (5/30, 90% CI 7% to 32%). Complete responses occurred in melanoma (n=2); partial responses occurred in RCC (n=1), mesothelioma (n=1), and melanoma (n=1). Median OS was as follows: melanoma, not reached (NR); RCC, 15.7 months (90% CI 2.5 to 15.7); CRC, 3.3 months (90% CI 0.6 to 3.4); ovarian, 12.5 months (90% CI 3.8 to 13.6); other solid tumors, NR. Activated tumor-infiltrating CD8 T cells at baseline (CD8+PD1+), high CD40L expression, and increased peripheral memory CD8 T cells correlated with clinical response. CONCLUSION: The combination of ziv-aflibercept and pembrolizumab demonstrated an acceptable safety profile with antitumor activity in solid tumors. The combination is currently being studied in sarcoma and anti-PD-1-resistant melanoma. TRIAL REGISTRATION NUMBER: NCT02298959.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Melanoma , Anticorpos Monoclonais Humanizados , Carcinoma de Células Renais/tratamento farmacológico , Feminino , Humanos , Neoplasias Renais/tratamento farmacológico , Masculino , Melanoma/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão
10.
STAR Protoc ; 1(2): 100055, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-33111099

RESUMO

As mass cytometry (MC) is implemented in clinical settings, the need for robust, validated protocols that reduce batch effects between samples becomes increasingly important. Here, we present a streamlined MC workflow for high-throughput staining that generates reproducible data for up to 80 samples in a single experiment by combining reference sample spike-in and palladium-based mass-tag cell barcoding. Although labor intensive, this workflow decreases experimental variables and thus reduces technical error and mitigates batch effects.


Assuntos
Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunofenotipagem/métodos , Espectrometria de Massas/métodos , Anticorpos , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/classificação , Leucócitos Mononucleares/citologia , Coloração e Rotulagem
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