RESUMO
Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.
Assuntos
Neoplasias/genética , Neoplasias/imunologia , Splicing de RNA/genética , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epitopos/imunologia , Etilenodiaminas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Inflamação/patologia , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Pirróis/farmacologia , Splicing de RNA/efeitos dos fármacos , Sulfonamidas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias/tratamento farmacológico , Proclorperazina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Biópsia , Cetuximab/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Xenoenxertos , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Trastuzumab/farmacologiaRESUMO
Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/ß-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Quimioterapia Combinada , Neoplasias Pulmonares/terapia , Evasão Tumoral/efeitos dos fármacos , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Imunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Camundongos , Linfócitos T/imunologia , Transcriptoma , Microambiente TumoralRESUMO
Sex differences in mammalian complex traits are prevalent and are intimately associated with androgens1-7. However, a molecular and cellular profile of sex differences and their modulation by androgens is still lacking. Here we constructed a high-dimensional single-cell transcriptomic atlas comprising over 2.3 million cells from 17 tissues in Mus musculus and explored the effects of sex and androgens on the molecular programs and cellular populations. In particular, we found that sex-biased immune gene expression and immune cell populations, such as group 2 innate lymphoid cells, were modulated by androgens. Integration with the UK Biobank dataset revealed potential cellular targets and risk gene enrichment in antigen presentation for sex-biased diseases. This study lays the groundwork for understanding the sex differences orchestrated by androgens and provides important evidence for targeting the androgen pathway as a broad therapeutic strategy for sex-biased diseases.
Assuntos
Androgênios , Células , Caracteres Sexuais , Análise de Célula Única , Transcriptoma , Animais , Feminino , Humanos , Masculino , Camundongos , Androgênios/metabolismo , Androgênios/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Imunidade Inata , Linfócitos/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Biobanco do Reino Unido , Células/efeitos dos fármacos , Células/imunologia , Células/metabolismoRESUMO
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 fármacosRESUMO
Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.
Assuntos
Adenocarcinoma/imunologia , Autofagia/imunologia , Carcinoma Ductal Pancreático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pancreáticas/imunologia , Evasão Tumoral/imunologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Evasão Tumoral/efeitos dos fármacosRESUMO
Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.
Assuntos
Antígenos CD/genética , Apirase/genética , Autoimunidade , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-17/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Animais , Anticorpos/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígenos CD/metabolismo , Apirase/metabolismo , Autoanticorpos/imunologia , Autoimunidade/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Camundongos , Camundongos Knockout , Bainha de Mielina/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Interleucina , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/análise , Benzimidazóis/farmacologia , Antígenos de Histocompatibilidade Classe I/imunologia , MAP Quinase Quinase 1/antagonistas & inibidores , Melanoma/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Neoplasias/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Células Tumorais CultivadasRESUMO
New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígeno CD47/genética , Molécula de Adesão da Célula Epitelial/genética , Neoplasias Mamárias Experimentais/terapia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Ligação a RNA/genética , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos Imunológicos/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/imunologia , Aptâmeros de Nucleotídeos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , Molécula de Adesão da Célula Epitelial/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Fagocitose/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/imunologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/imunologia , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia , Carga Tumoral/efeitos dos fármacosRESUMO
There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1ß and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.
Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/fisiologia , Resinas Acrílicas/química , Adjuvantes Imunológicos/farmacologia , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , NADPH Oxidase 2/fisiologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.
Assuntos
Dinoprostona/metabolismo , Vírus da Influenza A/fisiologia , Interferon Tipo I/metabolismo , Oxirredutases Intramoleculares/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células Cultivadas , Dinoprostona/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Imunidade/genética , Interferon Tipo I/genética , Oxirredutases Intramoleculares/genética , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Infecções por Orthomyxoviridae/imunologia , Prostaglandina-E Sintases , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Linfócitos T/imunologia , Linfócitos T/virologia , Replicação Viral/genéticaRESUMO
Fibrocytes, a distinct population of collagen-producing, monocyte-derived cells, are involved in wound healing as well as fibrotic diseases. Recently, fibrocytes have been revealed to play a role in the tumor microenvironment, particularly under antiangiogenic therapy. In addition, combination cancer immunotherapy with immune checkpoint inhibitor and antiangiogenic agents have been developed for various cancers in the clinical setting, although the immunological background is not clear. In the current study, we aimed to determine the function of fibrocytes in tumor immunity induced by immune checkpoint inhibitor therapy. Human and murine fibrocytes were generated from PBMCs and lungs, respectively. The expression of costimulatory and inhibitory molecules on fibrocytes was examined by flow cytometry. The stimulation of CD8+ T cells by fibrocytes was examined in MLRs with a 3H-thymidine incorporation assay. Fibrocytes expressed CD80low and CD86high as a costimulatory molecule, and expressed PD-L1high, but not PD-L2, as a coinhibitory molecule. Without any stimulation, fibrocytes strongly enhanced the proliferation of CD8+ T cells in mice and humans. Treatment with anti-CD86 and -CD54 Abs inhibited the growth of CD8+ T cells induced by fibrocytes. Anti-PD-L1 Ab further enhanced the proliferation of CD8+ T cells, even in the OVA-specific MLR with OT-1Rag-/- mice. Importantly, fibrocytes derived from PBMCs of patients with lung adenocarcinoma or murine MC38 tumors augmented the proliferation of CD8+ T cells with PD-L1 blockade. These results suggest that fibrocytes infiltrating tumor sites may play a role in the antitumor immunity mediated by CD8+ T cells when the activity is further enhanced by PD-L1/PD-1 blockade.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Apresentação de Antígeno/efeitos dos fármacos , Células do Tecido Conjuntivo/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Cultura Primária de Células , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Mimetismo Biológico/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Interferons/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , RNA de Cadeia Dupla/genética , Proteínas Repressoras/biossíntese , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Transcriptoma , Vírus/efeitos dos fármacos , Vírus/genética , Vírus/imunologiaRESUMO
Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly, which correlates with the changes seen in adults with asthma. However, whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here, we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism, we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR), mixed granulomatous airway inflammation comprising eosinophils and neutrophils, and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate, lung, and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes, which progressively declined with age and further by HDM-induced inflammation. Moreover, we found increased glycolytic reprogramming, maturation/activation of DCs, the proliferation of OT-II cells, and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity.
Assuntos
Envelhecimento/patologia , Glicoproteínas/metabolismo , Inflamação/patologia , Fosfoproteínas/metabolismo , Sistema Respiratório/patologia , Esteroides/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Dermatophagoides pteronyssinus/efeitos dos fármacos , Dexametasona/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glicólise/efeitos dos fármacos , Granuloma/patologia , Linfonodos/patologia , Mediastino/patologia , Modelos Biológicos , Sistema Respiratório/parasitologiaRESUMO
Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5-/- DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Autofagossomos/imunologia , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/imunologia , Proteína 5 Relacionada à Autofagia/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Wortmanina/farmacologiaRESUMO
Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.
Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Insulisina/metabolismo , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Anticorpos Bloqueadores/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Antígenos de Neoplasias/imunologia , Fracionamento Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Células Clonais , Citosol , Glicopeptídeos/farmacologia , Antígeno HLA-A1/metabolismo , Humanos , Insulisina/genética , Insulisina/imunologia , Interferon gama/metabolismo , Espectrometria de Massas , Metaloendopeptidases/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/imunologia , Fenantrolinas/farmacologia , Inibidores de Proteassoma , RNA Interferente Pequeno/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologiaRESUMO
BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Etanol/farmacologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Hepatócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Doença Hepática Terminal/virologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Antígeno HLA-A2/análise , Hepatócitos/transplante , Hepatócitos/virologia , Xenoenxertos , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/fisiologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that ßγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of ßγ-CAT. The cellular action of ßγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of ßγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Células Dendríticas , Endossomos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Linfócitos T , Imunidade Adaptativa , Animais , Anuros/metabolismo , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
BACKGROUND: Poly(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles have potential applications as a vaccine adjuvant and delivery system due to its unique advantages as biodegradability and biocompatibility. EXPERIMENTAL: We fabricated cationic solid lipid nanoparticles using PLGA and dimethyl-dioctadecyl-ammonium bromide (DDAB), followed by loading of model antigen OVA (antigen ovalbumin, OVA257-264) to form an OVA@DDAB/PLGA nano-vaccine. And we investigated the intracellular signaling pathway in dendritic cells in vitro and antigen transport pathway and immune response in vivo mediated by an OVA@DDAB/PLGA nano-vaccine. RESULTS: In vitro experiments revealed that the antigen uptake of BMDCs after nanovaccine incubation was two times higher than pure OVA or OVA@Al at 12 h. The BMDCs were well activated by p38 MAPK signaling pathway. Furthermore, the nano-vaccine induced antigen escape from lysosome into cytoplasm with 10 times increased cross-presentation activity than those of OVA or OVA@Al. Regarding the transport of antigen into draining lymph nodes (LNs), the nano-vaccine could rapidly transfer antigen to LNs by passive lymphatic drainage and active DC transport. The antigen+ cells in inguinal/popliteal LNs for the nano-vaccine were increased over two folds comparing to OVA@Al and OVA at 12 h. Moreover, the antigen of nano-vaccine stayed in LNs for over 7 days, germinal center formation over two folds higher than those of OVA@Al and OVA. After immunization, the nano-vaccine induced a much higher ratio of IgG2c/IgG1 than OVA@Al. It also effectively activated CD4+ T, CD8+ T and B cells for immune memory with a strong cellular response. CONCLUSION: These results indicated that DDAB/PLGA NP was a potent platform to improve vaccine immunogenicity by p38 signaling pathway in BMDCs, enhancing transport of antigens to LNs, and higher immunity response.
Assuntos
Apresentação de Antígeno , Células Dendríticas , Nanoestruturas/química , Transdução de Sinais , Vacinas , Adjuvantes de Vacinas/química , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Compostos de Amônio Quaternário/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Vacinas/química , Vacinas/imunologia , Vacinas/farmacocinética , Vacinas/farmacologiaRESUMO
Short-chain chlorinated paraffins (SCCPs) have been listed as a new class of persistent organic pollutants by the Stockholm Convention. SCCPs exhibit carcinogenic-, endocrine-, and metabolism-disrupting effects. However, the knowledge of the immunomodulatory effects of SCCPs and their underlying mechanisms, especially in specific immune cells, remains limited. In addition to SCCPs, C9-13-CPs have also been detected in humans. In this study, murine RAW264.7 macrophages were exposed to C9-13-CPs at environmentally relevant concentrations to investigate whether or how C9-13-CPs exhibit immunomodulatory effects. The results showed that the exposure of RAW264.7 cells to C9-13-CPs increased cell viability, as assayed by MTT analysis at 490 nm, and also promoted cell proliferation, as indicated by EdU uptake assay, which was measured at excitation and emission wavelengths of 488 and 512 nm, respectively. In addition, exposure to C9-13-CPs not only led to elevated ATP level and intracellular Ca2+ level but also caused AMPK signaling activation and NF-κB signaling inhibition. Moreover, molecular docking showed that the ß2-AR receptor could bind to C9-13-CPs. Taken together, these results suggest that the immune dysfunction of RAW264.7 cells caused by C9-13-CPs is closely related to the ß2-AR/AMPK/NF-κB signaling axis.