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BACKGROUND: Olfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches. METHODS: Multispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed. RESULTS: A significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration. CONCLUSION: These data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.
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Quimiocina CXCL10 , Quimiocina CXCL9 , Estesioneuroblastoma Olfatório , Antígenos HLA-DR , Imunoterapia , Microambiente Tumoral , Humanos , Estesioneuroblastoma Olfatório/terapia , Estesioneuroblastoma Olfatório/patologia , Estesioneuroblastoma Olfatório/imunologia , Quimiocina CXCL10/metabolismo , Imunoterapia/métodos , Feminino , Masculino , Pessoa de Meia-Idade , Quimiocina CXCL9/metabolismo , Microambiente Tumoral/imunologia , Antígenos HLA-DR/metabolismo , Idoso , Neoplasias Nasais/terapia , Neoplasias Nasais/patologia , Neoplasias Nasais/imunologia , Adulto , Regulação Neoplásica da Expressão GênicaRESUMO
Whole-sporozoite vaccines confer sterilizing immunity to malaria-naive individuals by unknown mechanisms. In the first PfSPZ Vaccine trial ever in a malaria-endemic population, Vδ2 γδ T cells were significantly elevated and Vγ9/Vδ2 transcripts ranked as the most upregulated in vaccinees who were protected from Plasmodium falciparum infection. In a mouse model, absence of γδ T cells during vaccination impaired protective CD8 T cell responses and ablated sterile protection. γδ T cells were not required for circumsporozoite protein-specific Ab responses, and γδ T cell depletion before infectious challenge did not ablate protection. γδ T cells alone were insufficient to induce protection and required the presence of CD8α+ dendritic cells. In the absence of γδ T cells, CD8α+ dendritic cells did not accumulate in the livers of vaccinated mice. Altogether, our results show that γδ T cells were essential for the induction of sterile immunity during whole-organism vaccination.
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Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium falciparum/fisiologia , Esporozoítos/imunologia , Linfócitos T/imunologia , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Antígenos CD8/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Seguimentos , Humanos , Imunidade , Fígado/patologia , Malária/prevenção & controle , Mali , Camundongos , Fragmentos de Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , VacinaçãoRESUMO
Objective: The objective of the present study was to investigate pork quality from Hereford×Berkshire (HB) female and intact male pigs reared outdoors in an alternative production system. Methods: Berkshire purebred sows were artificially inseminated, once in the fall and again in the spring of the following year, with semen from Hereford boars and managed free of antibiotics in an outdoor hoop structure until the last month of pregnancy, after which they were moved to a pasture-based unit of 0.8 hectares with individual lots with a farrowing hut, shade, and water ad libitum. Piglets were weaned at 4 weeks of age and housed in a deep-bedded hoop structure, grouped by sex. Animals were harvested at market weight of 125 kg, approximately 200 days of age. Hot carcass weight was collected at the time of the harvest. After 24 hours of refrigeration, carcass characteristics were measured. Longissimus dorsi samples collected from the right side loin. Loins were cut into 2.54-cm thick chops and were used to measure marbling score, color score, drip loss, and ultimate pH. Sensory panel tests were conducted as well at North Carolina State University. For pork characteristics and sensory panel data, trial and sex were included in the statistical model as fixed effects. Hot carcass weight was included in the model as a covariate for backfat thickness. Results: Neither the subjective nor the objective color scores displayed any differences between the boars and the gilts. No difference was found for pH and marbling score between trials or sexes. Gilts had a thicker backfat measurement at the last lumbar and a narrower longissimus muscle area measurement when compared to the boars. The only difference in the sensory characteristics was found between the trials for texture and moisture scores. Conclusion: Consumers were not able to detect boar taint under the condition of this study, which is that the intact males were reared outdoors. Additional trials would be necessary; however, based on the results of the present study, outdoor rearing can be suggested as a solution to the issue of boar taint.
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OBJECTIVES: To characterize the distribution of immune cell subsets within laryngeal papillomas and to study the function of potentially immunosuppressive neutrophilic and regulatory T cells (Tregs). METHODS: Fresh clinical papilloma specimens were collected at the time of surgery and studied with multiparameter flow cytometry. Papilloma infiltrating neutrophilic cells and Tregs were sorted and studied functionally with ex vivo T cell suppression assays. RESULTS: Flow cytometric analysis of fresh laryngeal papillomas samples from 18 adult patients with recurrent respiratory papillomatosis revealed patterns in immune constituency between patients. Clearly divergent phenotypes based primarily on the degree of neutrophilic and T cell infiltration were identified. Relative neutrophilic cell enrichment and T cell depletion were observed in 50% of samples and neutrophilic cell depletion and T cell enrichment were observed in the others. Greater papilloma neutrophilic cell enrichment was positively associated with the number of clinically indicated interventions required in the 12 months prior to sample collection, linking papilloma neutrophil inflammation to disease severity. Functional assays revealed the ability of both papilloma infiltrating neutrophilic and Tregs to suppress T cell function at roughly equal magnitudes, but substantially increased infiltration of neutrophilic cells compared to Tregs across samples. CONCLUSION: Neutrophilic cells are an important contributor to immunosuppression within the respiratory papilloma microenvironment. Given these data and the association between greater neutrophilic cell infiltration and lack of clinical response to therapeutic vaccination, additional study of strategies aimed at limiting neutrophilic cell infiltration or function within papillomas is warranted. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:3238-3244, 2024.
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Citometria de Fluxo , Neutrófilos , Papiloma , Fenótipo , Linfócitos T Reguladores , Humanos , Neutrófilos/imunologia , Neutrófilos/patologia , Linfócitos T Reguladores/imunologia , Masculino , Adulto , Papiloma/patologia , Papiloma/imunologia , Feminino , Pessoa de Meia-Idade , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Neoplasias Laríngeas/patologia , Neoplasias Laríngeas/imunologia , Microambiente Tumoral/imunologia , IdosoRESUMO
Introduction: Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation. Methods: Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis. Results: In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma. Discussion: To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate.
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A tissue resident-like phenotype in tumor infiltrating T cells can limit systemic anti-tumor immunity. Enhanced systemic anti-tumor immunity is observed in head and neck cancer patients after neoadjuvant PD-L1 immune checkpoint blockade (ICB) and transforming growth factor ß (TGF-ß) neutralization. Using T cell receptor (TCR) sequencing and functional immunity assays in a syngeneic model of oral cancer, we dissect the relative contribution of these treatments to enhanced systemic immunity. The addition of TGF-ß neutralization to ICB resulted in the egress of expanded and exhausted CD8+ tumor infiltrating lymphocytes (TILs) into circulation and greater systemic anti-tumor immunity. This enhanced egress associated with reduced expression of Itgae (CD103) and its upstream regulator Znf683. Circulating CD8+ T cells expressed higher Cxcr3 after treatment, an observation also made in samples from patients treated with dual TGF-ß neutralization and ICB. These findings provide the scientific rationale for the use of PD-L1 ICB and TGF-ß neutralization in newly diagnosed patients with carcinomas prior to definitive treatment of locoregional disease.
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OBJECTIVES: Biomarkers are needed to identify patients likely to respond to neoadjuvant immunotherapy (NIT) prior to receiving definitive treatment. MATERIALS AND METHODS: We hypothesized that expression of tumor cell HLA class I would correlate with pathologic response (PR) following NIT for primary untreated head and neck cancer. Multispectral immunofluorescence of pre- and post-treatment biopsy specimens from a neoadjuvant study of bintrafusp alfa, a dual TGF-ß and PD-L1 inhibitor, was performed. RESULTS: Discordant expression of tumor cell HLA class I and PD-L1 measured by multispectral immunofluorescence was observed with most positive tumor cells expressing HLA class I or PD-L1 but not both. Spatial analysis revealed colocalization between tumor parenchyma T cells and HLA class I positive tumors cells, but no clear colocalization between T cells and PD-L1 positive tumor cells. Greater pre-treatment tumor cell HLA class I expression, but not PD-L1 expression or tumor T cell infiltration, correlated with the development of a PR. Additionally, increased tumor cell HLA class I expression after NIT compared to before NIT correlated with development of a PR, whereas inconsistent changes in PD-L1 and T cell infiltration were observed after treatment in all patients. CONCLUSIONS: These data provide the rationale for the study of tumor cell HLA class I expression in larger prospective studies powered to determine the performance of biomarkers of PR in newly diagnosed HNSCC patients receiving NIT.
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Neoplasias de Cabeça e Pescoço , Antígenos de Histocompatibilidade Classe I , Humanos , Terapia Neoadjuvante , Estudos Prospectivos , Biomarcadores , Imunoterapia , Antígeno B7-H1/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK-NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.
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Recurrent respiratory papillomatosis (RRP) is a rare, debilitating neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11 and characterized by growth of papillomas in the upper aerodigestive tract. There is no approved medical therapy, and patients require repeated debulking procedures to maintain voice and airway function. PRGN-2012 is a gorilla adenovirus immune-therapeutic capable of enhancing HPV 6/11-specific T cell immunity. This first-in-human, phase 1 study (NCT04724980) of adjuvant PRGN-2012 treatment in adult patients with severe, aggressive RRP demonstrates the overall safety and clinically meaningful benefit observed with PRGN-2012, with a 50% complete response rate in patients treated at the highest dose. Responders demonstrate greater expansion of peripheral HPV-specific T cells compared with nonresponders. Additional correlative studies identify an association between reduced baseline papilloma HPV gene expression, greater interferon responses and expression of CXCL9 and CXCL10, and greater papilloma T cell infiltration in responders. Conversely, nonresponders were characterized by greater HPV and CXCL8 gene expression, increased neutrophilic cell infiltration, and reduced T cell papilloma infiltration. These results suggest that papilloma HPV gene expression may regulate interferon signaling and chemokine expression profiles within the tumor microenvironment that cooperate to govern clinical response to therapeutic HPV vaccination in patients with respiratory papillomatosis.
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Papiloma , Infecções por Papillomavirus , Infecções Respiratórias , Adulto , Humanos , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/patologia , Microambiente Tumoral , Infecções Respiratórias/terapia , Interferons , Papiloma/terapia , Papiloma/patologia , VacinaçãoRESUMO
TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Inibidor de NF-kappaB alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/genética , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , NF-kappa B , Morte Celular , Linhagem Celular Tumoral , Ubiquitina Tiolesterase/genéticaRESUMO
Neoadjuvant immunotherapies (NITs) have led to clinical benefits in several cancers. Characterization of the molecular mechanisms underlying responses to NIT may lead to improved treatment strategies. Here we show that exhausted, tumor-infiltrating CD8+ T (Tex) cells display local and systemic responses to concurrent neoadjuvant TGF-ß and PD-L1 blockade. NIT induces a significant and selective increase in circulating Tex cells associated with reduced intratumoral expression of the tissue-retention marker CD103. TGF-ß-driven CD103 expression on CD8+ T cells is reversed following TGF-ß neutralization in vitro, implicating TGF-ß in T cell tissue retention and impaired systemic immunity. Transcriptional changes implicate T cell receptor signaling and glutamine metabolism as important determinants of enhanced or reduced Tex treatment response, respectively. Our analysis illustrates physiological and metabolic changes underlying T cell responses to NIT, highlighting the interplay between immunosuppression, tissue retention, and systemic anti-tumor immunity and suggest antagonism of T cell tissue retention as a promising neoadjuvant treatment strategy.
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Linfócitos T CD8-Positivos , Neoplasias de Cabeça e Pescoço , Humanos , Terapia Neoadjuvante , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/metabolismo , Imunoterapia , Fator de Crescimento Transformador beta/metabolismo , Adaptação Fisiológica , Linfócitos do Interstício TumoralRESUMO
Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.
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Neoplasias de Cabeça e Pescoço , Histona-Lisina N-Metiltransferase , Interferon Tipo I , Infecções por Papillomavirus , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Proteínas Estimuladoras de Ligação a CCAAT , Linfócitos T CD8-Positivos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Histona-Lisina N-Metiltransferase/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Ubiquitina-Proteína LigasesRESUMO
Background: Chordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma. Methods: Chordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments. Results: Chordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage. Conclusion: This is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing.
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Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS-rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS-rmIL-12-treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type-specific IL-12 receptor-KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12-induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS-IL-12.
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Carcinoma , Interleucina-12 , Animais , Linfócitos T CD8-Positivos , Interleucina-12/genética , Interleucina-12/metabolismo , Camundongos , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Linfócitos T ReguladoresRESUMO
TNFα is a key mediator of immune and radiotherapy-induced cytotoxicity, but many cancers, including head and neck squamous cell carcinomas (HNSCC), display TNF resistance due to activation of the canonical IKK-NF-κB/RELA pro-survival pathway. However, toxicities associated with direct targeting of the canonical pathway point to the need to identify mechanism(s) contributing to TNFα resistance and synthetic lethal targets to overcome such resistance in cancer cells. Here, RNAi screening for modulators of TNFα-NF-κB reporter activity and cell survival unexpectedly implicated the WEE1 and CDC2 G2-M checkpoint kinases. The IKKα/ß-RELA and WEE1-CDC2 signaling pathways are activated by TNFα and form a complex in cell lines derived from both human papillomavirus (-) and (+) subtypes of HNSCC. WEE1 inhibitor AZD1775 reduced IKK/RELA phosphorylation and the expression of NF-κB-dependent pro-survival proteins Cyclin D1 and BCL2. Combination of TNFα and AZD1775 enhanced caspase-mediated apoptosis in vitro, and combination treatment with radiotherapy and AZD1775 potentiated inhibition of HNSCC tumor xenograft growth in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insight into the interplay between NF-κB signaling and WEE1-mediated regulation of the G2-M cell-cycle checkpoint in HNSCC. IMPLICATIONS: Inhibiting WEE1 and IKK-RELA crosstalk could potentially enhance the effects of therapies mediated by TNFα with less systemic immune suppression and toxicity than observed with direct interruption of IKK-NF-κB/RELA signaling.
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Proteínas de Ciclo Celular , Neoplasias de Cabeça e Pescoço , Quinase I-kappa B , Proteínas Tirosina Quinases , Fator de Transcrição RelA , Apoptose , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfaRESUMO
BACKGROUNDHead and neck squamous cell carcinoma not associated with HPV (HPV-unrelated HNSCC) is associated with a high rate of recurrence and poor survival.METHODSWe conducted a clinical trial in 14 patients with newly diagnosed HPV-unrelated HNSCC to evaluate the safety and efficacy of neoadjuvant bintrafusp alfa, a bifunctional fusion protein that blocks programmed death ligand 1 (PD-L1) and neutralizes TGF-ß.RESULTSBintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses (PRs) were observed, and 12 of the 14 (86%) patients were alive and disease free at 1 year. Alterations in Treg infiltration and spatial distribution relative to proliferating CD8+ T cells indicated a reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not virus-specific responses, correlated with the development of a PR. Detection of neoepitope-specific responses and PRs in tumors was not correlated with genomic features or tumor antigenicity but was associated with reduced pretreatment myeloid cell tumor infiltration. These results indicate that dual PD-L1 and TGF-ß blockade can safely enhance tumor antigen-specific immunity and highlight the feasibility of multimechanism neoadjuvant immunotherapy for patients with HPV-unrelated HNSCC.CONCLUSIONOur studies provide insight into the ability of neoadjuvant immunotherapy to induce polyclonal neoadjuvant-specific T cell responses in tumors and suggest that features of the tumor microenvironment, such as myeloid cell infiltration, may be a major determinant of enhanced antitumor immunity following such treatment.TRIAL REGISTRATIONClinicalTrials.gov NCT04247282.FUNDINGThis work was funded by the Center for Cancer Research, the NCI, and the Intramural Research Program of the NIDCD, NIH. Bintrafusp alfa was provided by the health care business of Merck KGaA (Darmstadt, Germany), through a Cooperative Research and Development Agreement with the NCI. Additional funding was provided by ImmunityBio through a Cooperative Research and Development Agreement with the NIDCD.
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Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Antígenos de Neoplasias/uso terapêutico , Antígeno B7-H1 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Terapia Neoadjuvante , Infecções por Papillomavirus/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Fator de Crescimento Transformador beta , Microambiente TumoralRESUMO
BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types. METHODS: We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison. RESULTS: Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas. CONCLUSIONS: Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP. TRIAL REGISTRATION NUMBERS: NCT03707587 and NCT02859454.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Infecções por Papillomavirus/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Feminino , Humanos , Fatores Imunológicos/uso terapêutico , Camundongos , Células NIH 3T3 , Papiloma/tratamento farmacológico , Microambiente Tumoral/imunologiaRESUMO
Recurrent respiratory papillomatosis (RRP) is a debilitating neoplastic disorder of the upper aerodigestive tract caused by chronic infection with low-risk human papillomavirus types 6 or 11. Patients with severe RRP can require hundreds of lifetime surgeries to control their disease and pulmonary papillomatosis can be fatal. Here we report the comprehensive genomic and transcriptomic characterization of respiratory papillomas. We discovered and characterized distinct subtypes with transcriptional resemblance to either a basal or differentiated cell state that associate with disease aggressiveness and differ in key molecular, immune and APOBEC mutagenesis profiles. Through integrated comparison with high-risk HPV-associated head and neck squamous cell carcinoma, our analysis revealed divergent molecular and immune papilloma subtypes that form independent of underlying genomic alterations. Cumulatively our results support the development of dysregulated cellular proliferation and suppressed anti-viral immunity through distinct programs of squamous cell differentiation and associated expression of low-risk HPV genes. These analyses provide insight into the pathogenesis of respiratory papillomas and provide a foundation for the development of therapeutic strategies.
Assuntos
Genoma , Papillomavirus Humano 11/genética , Papillomavirus Humano 6/genética , Infecções por Papillomavirus/virologia , Infecções Respiratórias/virologia , Transcriptoma , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: As heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control. METHODS: Here, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1). RESULTS: In engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-γ) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo. CONCLUSIONS: These results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-γ production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.
Assuntos
Edição de Genes , Neoplasias de Cabeça e Pescoço/terapia , Imunoterapia Adotiva , Células Matadoras Naturais/transplante , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos Quiméricos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Linfócitos T/transplante , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Bases de Dados Genéticas , Antígenos HLA/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos Quiméricos/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules. METHODS: NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice. RESULTS: We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types. CONCLUSION: These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.