RESUMO
Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.
Assuntos
Imunidade , Neoplasias/imunologia , Neoplasias/metabolismo , Obesidade/metabolismo , Microambiente Tumoral , Adiposidade , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Cinética , Linfócitos do Interstício Tumoral , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Análise de Componente Principal , Pró-Colágeno-Prolina Dioxigenase/metabolismo , ProteômicaRESUMO
Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.
Assuntos
Melanoma/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/administração & dosagem , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante de NeoplasiasRESUMO
Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)-mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell-mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.
Assuntos
Subpopulações de Linfócitos B/imunologia , Centro Germinativo/imunologia , Tolerância Imunológica , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Formação de Anticorpos , Células Cultivadas , Epigênese Genética , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa de Receptor de Interleucina-21/genética , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Imunoterapia/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Genômica , Humanos , Interferons/imunologia , Mutação com Perda de Função , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , NF-kappa B/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/deficiência , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Evasão Tumoral/genética , Resposta a Proteínas não Dobradas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.
Assuntos
Neoplasias da Mama , Imunoterapia , Neoplasias Mamárias Experimentais , Microambiente Tumoral/imunologia , Animais , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Transcriptoma/imunologiaRESUMO
PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunidade/imunologia , Imunoterapia/métodos , Receptor de Morte Celular Programada 1/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Humanos , Camundongos , Fosforilação , Transdução de SinaisRESUMO
Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type-specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity-promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell-specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Ferroptose/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológicoRESUMO
Oncogenic mutations in KRAS can be recognized by T cells on specific class I human leukocyte antigen (HLA-I) molecules, leading to tumor control. To date, the discovery of T cell targets from KRAS mutations has relied on occasional T cell responses in patient samples or the use of transgenic mice. To overcome these limitations, we have developed a systematic target discovery and validation pipeline. We evaluate the presentation of mutant KRAS peptides on individual HLA-I molecules using targeted mass spectrometry and identify 13 unpublished KRASG12C/D/R/V mutation/HLA-I pairs and nine previously described pairs. We assess immunogenicity, generating T cell responses to nearly all targets. Using cytotoxicity assays, we demonstrate that KRAS-specific T cells and T cell receptors specifically recognize endogenous KRAS mutations. The discovery and validation of T cell targets from KRAS mutations demonstrate the potential for this pipeline to aid the development of immunotherapies for important cancer targets.
Assuntos
Neoplasias Pulmonares , Linfócitos T , Camundongos , Animais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Mutação , Receptores de Antígenos de Linfócitos T/genética , Neoplasias Pulmonares/genética , Antígenos de Histocompatibilidade Classe I/genéticaRESUMO
Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.
Assuntos
Diabetes Mellitus Experimental/imunologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diabetes Mellitus Experimental/genética , Encefalomielite Autoimune Experimental/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes Neurológicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Receptor de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/genéticaRESUMO
Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Animais , Antígenos de Neoplasias , Epitopos , Humanos , Melanócitos , Melanoma/terapia , CamundongosRESUMO
It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1-deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.
Assuntos
Antígeno B7-H1/fisiologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Evasão Tumoral , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Ativação Linfocitária , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/fisiologia , Microambiente TumoralRESUMO
Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.
Assuntos
Autoimunidade/imunologia , Antígeno CTLA-4/genética , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Fatores Etários , Sequência de Aminoácidos , Animais , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Antígeno CTLA-4/imunologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Encefalomielite Autoimune Experimental/genética , Deleção de Genes , Interleucina-10/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Linfócitos T Reguladores/fisiologia , Tamoxifeno/farmacologiaRESUMO
BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (â¼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8(+) T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten(-/-) syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8(+) T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8(+) T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/imunologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Humanos , Imunocompetência , Indóis/administração & dosagem , Ipilimumab , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma/patologia , Melanoma/secundário , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Sulfonamidas/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Vemurafenib , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) ß chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10-14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones.
RESUMO
In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2- DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2-, and CD133-Sox2- (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133- cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2- spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2- cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function.
Assuntos
Proliferação de Células , Derme/citologia , Derme/metabolismo , Hidrogéis , Fatores de Transcrição SOXB1/metabolismo , Antígeno AC133 , Fosfatase Alcalina/metabolismo , Animais , Antígenos CD/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Glicoproteínas/metabolismo , Cabelo/anatomia & histologia , Folículo Piloso/crescimento & desenvolvimento , Integrina alfa1beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Peptídeos/metabolismoRESUMO
OBJECTIVES: To determine the expression of the p53 family member p73 in vestibular schwannoma (VS) and to determine the potential role of this tumor suppressor in regulating the proliferation of HEI193, a human papillomavirus E6-E7 immortalized VS cell line. METHODS: Immunohistochemical staining was used to investigate the expression of p73 in 34 cases of archived VS tissue, while Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of p73 in HEI193. After transfection of a full-length p73 plasmid (TAp73alpha), flow cytometry analysis was performed to determine the effect of p73 expression on cell cycle distribution, while annexin V-FITC (fluorescein isothiocyanate) analysis and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay were used to measure apoptosis. The effect of p73 expression on ionizing radiation-induced cell death was also investigated with annexin V staining, TUNEL assay, and flow cytometry analysis. RESULTS: Of the 34 vestibular schwannoma tissues examined, p73 was expressed in 14 (41%) but was not expressed in HEI193. Transfection of p73 alone resulted in increased apoptosis and necrosis, and G(1) accumulation with concomitant induction of p21. The presence of p73 also significantly increased early apoptosis (P = .046), late apoptosis (P < .001), and necrosis (P = .009) on exposure of the HEI193 cells to ionizing radiation. CONCLUSION: Forced expression of p73, perhaps by gene therapy, to induce apoptosis directly or to sensitize VS tumors to ionizing radiation may have relevant therapeutic applications.