RESUMO
Human papillomavirus (HPV) 16 and 18 infections are related to many human cancers. Despite several preventive vaccines for high-risk (hr) HPVs, there is still an urgent need to develop therapeutic HPV vaccines for targeting pre-existing hrHPV infections and lesions. In this study, we developed a lipid nanoparticle (LNP)-formulated mRNA-based HPV therapeutic vaccine (mHTV)-03E2, simultaneously targeting the E2/E6/E7 of both HPV16 and HPV18. mHTV-03E2 dramatically induced antigen-specific cellular immune responses, leading to significant CD8+ T cell infiltration and cytotoxicity in TC-1 tumors derived from primary lung epithelial cells of C57BL/6 mice expressing HPV E6/E7 antigens, mediated significant tumor regression, and prolonged animal survival, in a dose-dependent manner. We further demonstrated significant T cell immunity against HPV16/18 E6/E7 antigens for up to 4 months post-vaccination in immunological and distant tumor rechallenging experiments, suggesting robust memory T cell immunity against relapse. Finally, mHTV-03E2 synergized with immune checkpoint blockade to inhibit tumor growth and extend animal survival, indicating the potential in combination therapy. We conclude that mHTV-03E2 is an excellent candidate therapeutic mRNA vaccine for treating malignancies caused by HPV16 or HPV18 infections.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Vacinas contra Papillomavirus , RNA Mensageiro , Animais , Camundongos , Vacinas contra Papillomavirus/imunologia , Humanos , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/prevenção & controle , Feminino , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Nanopartículas/química , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/genética , Camundongos Endogâmicos C57BL , Papillomavirus Humano 18/imunologia , Papillomavirus Humano 18/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfócitos T CD8-Positivos/imunologia , Proteínas Repressoras/imunologia , Proteínas Repressoras/genética , Proteínas de Ligação a DNA , LipossomosRESUMO
Tumor-infiltrating T cells often lose their effector function; however, the mechanisms are incompletely understood. We report that cholesterol in the tumor microenvironment induces CD8+ T cell expression of immune checkpoints and exhaustion. Tumor tissues enriched with cholesterol and cholesterol content in tumor-infiltrating CD8+ T cells were positively and progressively associated with upregulated T cell expression of PD-1, 2B4, TIM-3, and LAG-3. Adoptively transferred CD8+ T cells acquired cholesterol, expressed high levels of immune checkpoints, and became exhausted upon entering a tumor. Tumor culture supernatant or cholesterol induced immune checkpoint expression by increasing endoplasmic reticulum (ER) stress in CD8+ T cells. Consequently, the ER stress sensor XBP1 was activated and regulated PD-1 and 2B4 transcription. Inhibiting XBP1 or reducing cholesterol in CD8+ T cells effectively restored antitumor activity. This study reveals a mechanism underlying T cell exhaustion and suggests a new strategy for restoring T cell function by reducing cholesterol to enhance T cell-based immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Colesterol/sangue , Microambiente Tumoral/fisiologia , Animais , Western Blotting , Citometria de Fluxo , Humanos , Imunoprecipitação , Imunoterapia , Melanoma Experimental/sangue , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Plasmacytoid dendritic cells (pDCs) play a key role in antiviral responses by producing type-1 IFNs. However, recent studies showed that pDCs induce immune suppression and promote tumor growth in human ovarian cancer and myeloma. The molecular mechanisms underlying pDC acquisition of these properties are unknown. Here we show that human pDCs activated by CpG inhibited growth and induced apoptosis in myeloma cells via secreted IFN-α, but direct contact with myeloma cells converted pDCs into tumor-promoting cells by suppressing pDC IFN-α production. E-cadherin, expressed on both myeloma cells and pDCs, mediated these effects via a homophilic interaction - activation of E-cadherin signaling upregulated and activated TNFAIP3 to interact with TLR9, resulting in TLR9 ubiquitination and degradation, and inhibition of IFN-α production in pDCs. These findings were supported by an in vivo study in which pDC depletion induced tumor regression and better survival in the Vk*MYC myeloma mouse model. Furthermore, IFNAR1 expression level positively correlated to overall survival of patients with multiple myeloma (MM), and the IFN-α level in patient bone marrow was significantly lower than that in marrow of healthy individuals. This study reveals a novel mechanism underlying how MM tumors educate pDCs in their microenvironment and provides new targets for improving the treatment of MM.
Assuntos
Antígenos CD/imunologia , Caderinas/imunologia , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Tolerância Imunológica , Mieloma Múltiplo/imunologia , Proteínas de Neoplasias/imunologia , Animais , Antígenos CD/genética , Medula Óssea/imunologia , Medula Óssea/patologia , Caderinas/genética , Células Dendríticas/patologia , Feminino , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Oligodesoxirribonucleotídeos/farmacologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologiaRESUMO
CD8+ T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8+ or CD4+ T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Colesterol/farmacologia , Interleucina-9/biossíntese , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores X do Fígado/metabolismo , Camundongos Endogâmicos C57BL , Oxirredução , Oxisteróis/farmacologia , Sumoilação/efeitos dos fármacosRESUMO
We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.
Assuntos
Linfócitos B/imunologia , Interleucina-4/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Anticorpos/sangue , Autoanticorpos/sangue , Fator Ativador de Células B/sangue , Células Cultivadas , Técnicas de Cocultura , Centro Germinativo/imunologia , Imunoglobulina G/sangue , Interleucina-4/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/genética , Baço/citologia , Subpopulações de Linfócitos T/transplanteRESUMO
To prevent potentially damaging inflammatory responses, the eye actively promotes local immune tolerance via a variety of mechanisms. Owing to trauma, infection, or other ongoing autoimmunity, these mechanisms sometimes fail, and an autoimmune disorder may develop in the eye. In mice of the C57BL/10 (B10) background, autoimmune keratitis often develops spontaneously, particularly in the females. Its incidence is greatly elevated in the absence of γδ T cells, such that â¼80% of female B10.TCRδ(-/-) mice develop keratitis by 18 wk of age. In this article, we show that CD8(+) αß T cells are the drivers of this disease, because adoptive transfer of CD8(+), but not CD4(+), T cells to keratitis-resistant B10.TCRß/δ(-/-) hosts induced a high incidence of keratitis. This finding was unexpected because in other autoimmune diseases, more often CD4(+) αß T cells, or both CD4(+) and CD8(+) αß T cells, mediate the disease. Compared with wild-type B10 mice, B10.TCRδ(-/-) mice also show increased percentages of peripheral memory phenotype CD8(+) αß T cells, along with an elevated frequency of CD8(+) αß T cells biased to produce inflammatory cytokines. In addition, B10.TCRδ-/- mice have fewer peripheral CD4(+) CD25(+) Foxp3(+) αß regulatory T cells (Tregs), which express lower levels of receptors needed for Treg development and function. Together, these observations suggest that in B10 background mice, γδ T cells are required to generate adequate numbers of CD4(+) CD25(+) Foxp3(+) Tregs, and that in B10.TCRδ(-/-) mice a Treg deficiency allows dysregulated effector or memory CD8(+) αß T cells to infiltrate the cornea and provoke an autoimmune attack.
Assuntos
Doenças Autoimunes/imunologia , Córnea/imunologia , Ceratite/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead , Memória Imunológica , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-ß promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations.
Assuntos
Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Infecções por Picornaviridae/virologia , Trealose/farmacologia , Replicação Viral , Western Blotting , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Interferons/genética , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Sistema Respiratório/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/efeitos dos fármacos , Rhinovirus/fisiologiaRESUMO
OBJECTIVE: MUC18 or CD146, a transmembrane glycoprotein, is mainly expressed by endothelial cells and smooth muscle cells where it serves as a cell-cell adhesion molecule. We have found MUC18 up-regulation in airway epithelial cells of patients with asthma and chronic obstructive pulmonary disease (COPD). However, the function of MUC18 in airway epithelial cells remains unclear. In the present study, we tested the hypothesis that MUC18 exerts a pro-inflammatory function during stimulation with a viral mimic polyI:C or human rhinovirus infection. METHODS: Normal human primary airway epithelial cells were transduced with lentivirus encoding MUC18 cDNA to over-express MUC18 or with GFP (control), and treated with polyI:C or HRV for detection of pro-inflammatory cytokine IL-8 and anti-viral gene IFN-ß. Additionally, we performed cell culture of human lung epithelial cell line NCIH292 cells to determine the mechanisms of MUC18 function. RESULTS: We found that MUC18 over-expression promoted IL-8 production, while it inhibited IFN-ß expression following polyI:C stimulation or HRV infection. Increased phosphorylation of MUC18 serines was observed in MUC18 over-expressing cells. Reduction of MUC18 serine phosphorylation by inhibiting ERK activity was associated with less production of IL-8 following polyI:C stimulation. CONCLUSIONS: Our results for the first time demonstrate MUC18's pro-inflammatory and anti-viral function in human airway epithelial cells.
RESUMO
Regulatory T cells (Tregs) play a pivotal role in the maintenance of immunological self-tolerance. Deficiency or dysfunction of Tregs leads to severe autoimmune diseases. Although the forkhead/winged-helix family member FOXP3 is critical for Treg differentiation and function, the molecular basis for FOXP3 function remains unclear. In this study, we identified and characterized a human-specific FOXP3-interacting protein, referred to as FIK (FOXP3-interacting KRAB domain-containing protein). FIK is highly expressed in Tregs and acts as a bridging molecule to link FOXP3 with the chromatin-remodeling scaffold protein KAP1 (TIF-1ß/TRIM28). Disruption of the FOXP3-FIK-KAP1 complex in Tregs restored expression of FOXP3-target genes and abrogated the suppressor activity of the Tregs. These data demonstrate a critical role for FIK in regulating FOXP3 activity and Treg function.
Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Cromatina/imunologia , Cromatina/metabolismo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células Jurkat , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteína 28 com Motivo TripartidoRESUMO
Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic T lymphocytes (CTLs) and natural killer cells. However, whether it also plays a role in conventional CD4(+) T cell function remains unclear. Here we report that in perforin-deficient (PKO) mice, CD4(+) T cells are hyperproliferative in response to T cell receptor (TCR) stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD4(+) T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFAT1. Our results indicate that perforin plays a negative role in regulating CD4(+) T cell activation and immune response by affecting TCR-dependent Ca(2+) signaling.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio , Ativação Linfocitária , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos CD4/imunologia , Divisão Celular , Feminino , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Perforina/deficiência , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
FOXP3 is a forkhead family transcriptional repressor important for the development and function of CD4(+)CD25(+) regulatory T cells. In humans, FOXP3 is expressed as two isoforms, a full-length form and a smaller form lacking exon 2. These two isoforms are expressed in approximately equal amounts in circulating regulatory T cells, and are induced equally in freshly activated CD4(+)CD25(-) T cells. Herein, we show that FOXP3 interacts with retinoic acid receptor-related orphan receptor (ROR)alpha, and that this interaction inhibits transcriptional activation mediated by RORalpha. Full-length FOXP3, but not the isoform lacking exon 2, interacts with RORalpha, and the region of FOXP3 involved in the interaction is encoded by exon 2. Mutation of the LxxLL motif in FOXP3, located in exon 2, abolished interaction and repression by FOXP3. Additionally, the inhibition of RORalpha by FOXP3 does not require an intact forkhead domain, demonstrating a mode of FOXP3 function that is independent of DNA binding. Interestingly, expression of RORalpha in T cells leads to the expression of genes that define Th17 cells, and the expression of each of these gene was inhibited by coexpression of full-length, but not DeltaEx2, FOXP3. These data expand the possible targets of FOXP3-mediated repression and demonstrate functional differences between FOXP3 isoforms.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Linhagem Celular , Éxons/genética , Humanos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Extratos de Tecidos/metabolismo , Transativadores/genéticaRESUMO
Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-kappaB-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-kappaB binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-kappaB binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-kappaB binding site, which plays a pivotal role in controlling human granzyme B gene transcription.