RESUMO
T cell antigen receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we present a mass cytometric (CyTOF) approach to track T cell responses by combining antibodies for specific TCR Vα and Vß chains with antibodies against T cell activation and differentiation proteins in mice. This strategy identifies expansions of CD8+ and CD4+ T cells expressing specific Vß and Vα chains with varying differentiation states in response to Listeria monocytogenes, tumors and respiratory influenza infection. Expanded T cell populations expressing Vß chains could be directly linked to the recognition of specific antigens from Listeria, tumor cells or influenza. In the setting of influenza infection, we found that common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the TCR diversity and differentiation state of responding T cells. Thus, we present a method to monitor broad changes in TCR use paired with T cell phenotyping during adaptive immune responses.
Assuntos
Linfócitos T CD8-Positivos , Diferenciação Celular , Citometria de Fluxo , Listeria monocytogenes , Listeriose , Animais , Diferenciação Celular/imunologia , Camundongos , Listeria monocytogenes/imunologia , Linfócitos T CD8-Positivos/imunologia , Listeriose/imunologia , Citometria de Fluxo/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Ativação Linfocitária/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunidade Adaptativa , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
BACKGROUND: Treatment of salivary gland tumors (SGTs) remains challenging. Little is known about the immune landscape of SGTs. We aimed to characterize the tumor microenvironment in benign and malignant SGTs. METHODS: Eleven benign and nine malignant tumors were collected from patients undergoing curative intent surgery. Specimens were analyzed using mass cytometry by time-of-flight. Immune cell populations were manually gated, and T cells were clustered using the FlowSOM algorithm. Population frequencies were compared between high-grade and low-grade malignancies, corrected for multiple hypothesis testing. RESULTS: There were trends towards increased CD4+ and CD8+ T cells among malignant tumors. High-grade malignancies exhibited trends towards higher frequencies of CD8+ PD-1+ CD39+ CD103+ exhausted T cells, CD4+ FoxP3+ TCF-1+ CD127- Tregs, and CD69+ CD25- CD4+ T cells compared to low-grade malignancies. CONCLUSION: SGTs exhibit significant immunologic diversity. High-grade malignancies tended to have greater infiltration of exhausted CD8+ T cells and Tregs, which may guide future studies for immunotherapy strategies.
Assuntos
Neoplasias das Glândulas Salivares , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Neoplasias das Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/imunologia , Neoplasias das Glândulas Salivares/terapia , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T CD4-Positivos/imunologia , Citometria de FluxoRESUMO
T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR Vα- and Vß-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific Vß and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vß chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.
RESUMO
CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Animais , Camundongos , Linfonodos , Neoplasias/terapia , Neoplasias/patologia , Imunoterapia/métodos , Microambiente TumoralRESUMO
Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Animais , Proliferação de Células/fisiologia , Feminino , Glicólise/imunologia , Memória Imunológica/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação Oxidativa , Receptores de Antígenos Quiméricos/imunologia , Análise de Célula Única/métodosRESUMO
Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.
Assuntos
Infecções Bacterianas/imunologia , Neoplasias da Mama/imunologia , Melanoma Experimental/imunologia , Microambiente Tumoral/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Ativação Linfocitária/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Linfócitos T/imunologia , Microambiente Tumoral/genéticaRESUMO
Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.
Assuntos
Neoplasias da Mama/patologia , Leptina/sangue , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Proteínas Adaptadoras de Transdução de Sinal , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Animais , Índice de Massa Corporal , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Leptina/genética , Leptina/metabolismo , Glândulas Mamárias Humanas/metabolismo , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Obesidade/patologia , Lesões Pré-Cancerosas , Fuso Acromático/metabolismo , Fuso Acromático/patologiaRESUMO
Transforming growth factor ß (TGFß) is an effector of immune suppression and contributes to a permissive tumor microenvironment that compromises effective immunotherapy. We identified a correlation between TGFB1 and genes expressed by myeloid cells, but not granulocytes, in The Cancer Genome Atlas lung adenocarcinoma data, in which high TGFB1 expression was associated with poor survival. To determine whether TGFß affected cell fate decisions and lineage commitment, we studied primary cultures of CD14+ monocytes isolated from peripheral blood of healthy donors. We discovered that TGFß was a survival factor for CD14+ monocytes, which rapidly executed an apoptotic program in its absence. Continued exposure to TGFß in combination with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL6) amplified HLA-DRlowCD14+CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) at the expense of macrophage and dendritic cell (DC) differentiation. MDSCs generated in the presence of TGFß were more effective in suppressing T-cell proliferation and promoted the T regulatory cell phenotype. In contrast, inhibition of TGFß signaling using a small-molecule inhibitor of receptor kinase activity in CD14+ monocytes treated with GM-CSF and IL6 decreased MDSC differentiation and increased differentiation to proinflammatory macrophages and antigen-presenting DCs. The effect of autocrine and paracrine TGFß on myeloid cell survival and lineage commitment suggests that pharmacologic inhibition of TGFß-dependent signaling in cancer would favor antitumor immunity.
Assuntos
Comunicação Autócrina , Diferenciação Celular/imunologia , Imunomodulação , Monócitos/imunologia , Monócitos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma de Pulmão/etiologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Apresentação de Antígeno/imunologia , Biomarcadores , Sobrevivência Celular/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Humanos , Monócitos/citologia , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple-negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP-based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non-malignant breast epithelial cells. In contrast, TNBC cells and non-malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC-specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non-malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non-malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non-toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.