RESUMO
Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.
Assuntos
Antígenos CD1/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/imunologia , Glicoproteínas/metabolismo , Cadeias alfa de Integrinas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Fator de Crescimento Transformador beta1/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The generation and maintenance of CD8+ T cell memory is crucial to long-term host survival, yet the basic tenets of CD8+ T cell immunity are still being established. Recent work has led to the discovery of tissue-resident memory cells and refined our understanding of the transcriptional and epigenetic basis of CD8+ T cell differentiation and dysregulation. In parallel, the unprecedented clinical success of immunotherapy has galvanized an intense, global research effort to decipher and de-repress the anti-tumor response. However, the progress of immunotherapy is at a critical juncture, since the efficacy of immuno-oncology agents remains confined to a fraction of patients and often fails to provide durable benefit. Unlocking the potential of immunotherapy requires the design of strategies that both induce a potent effector response and reliably forge stable, functional memory T cell pools capable of protecting from recurrence or relapse. It is therefore essential that basic and emerging concepts of memory T cell biology are rapidly and faithfully transposed to advance therapeutic development in cancer immunotherapy. This review highlights seminal and recent reports in CD8+ T cell memory and tumor immunology, and evaluates recent data from solid cancer specimens in the context of the key paradigms from preclinical models. We elucidate the potential significance of circulating effector cells poised downstream of neoantigen recognition and upstream of T cell dysfunction and propose that cells in this immunological 'sweet spot' may be key anti-tumor effectors.
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Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunidade Celular , Memória Imunológica , Ativação Linfocitária/imunologia , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Homeostase , Humanos , Imunomodulação , Contagem de Linfócitos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Despite the different strategies used to treat ovarian cancer, around 70% of women/patients eventually fail to respond to the therapy. Cancer stem cells (CSCs) play a role in the treatment failure due to their chemoresistant properties. This capacity to resist chemotherapy allows CSCs to interact with different components of the tumor microenvironment, such as mesenchymal stem cells (MSCs), and thus contribute to tumorigenic processes. Although the participation of MSCs in tumor progression is well understood, it remains unclear how CSCs induce the pro-tumorigenic activity of MSCs in response to chemotherapy. Small extracellular vesicles, including exosomes, represent one possible way to modulate any type of cell. Therefore, in this study, we evaluate if small extracellular vesicle (sEV) derived from ovarian cancer spheroids (OCS), which are enriched in CSCs, can modify the activity of MSCs to a pro-tumorigenic phenotype. We show that sEV released by OCS in response to cisplatin induce an increase in the migration pattern of bone marrow MSCs (BM-MSCs) and the secretion interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA). Moreover, the factors secreted by BM-MSCs induce angiogenesis in endothelial cells and the migration of low-invasive ovarian cancer cells. These findings suggest that cisplatin could modulate the cargo of sEV released by CSCs, and these exosomes can further induce the pro-tumorigenic activity of MSCs.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Cisplatino/farmacologia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Expressão Gênica , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/patologia , Esferoides Celulares , Microambiente TumoralRESUMO
Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.
Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Inovirus/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.
Assuntos
Catalase/imunologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Estresse Oxidativo/fisiologia , Linfócitos T/imunologia , Western Blotting , Efeito Espectador/imunologia , Linhagem Celular , Separação Celular , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/imunologia , TransfecçãoRESUMO
Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells.
Assuntos
Inflamação Neurogênica/imunologia , Doença de Parkinson/imunologia , Receptores de Dopamina D3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Dopamina D3/genética , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/genética , Equilíbrio Th1-Th2RESUMO
Dendritic cells (DCs) are responsible for priming T cells and for promoting their differentiation from naive T cells into appropriate effector cells. Emerging evidence suggests that neurotransmitters can modulate T cell-mediated immunity. However, the involvement of specific neurotransmitters or receptors remains poorly understood. In this study, we analyzed the role of dopamine in the regulation of DC function. We found that DCs express dopamine receptors as well as the machinery necessary to synthesize, store, and degrade dopamine. Notably, the expression of D5R decreased upon LPS-induced DC maturation. Deficiency of D5R on the surface of DCs impaired LPS-induced IL-23 and IL-12 production and consequently attenuated the activation and proliferation of Ag-specific CD4(+) T cells. To determine the relevance of D5R expressed on DCs in vivo, we studied the role of this receptor in the modulation of a CD4(+) T cell-driven autoimmunity model. Importantly, D5R-deficient DCs prophylactically transferred into wild-type recipients were able to reduce the severity of experimental autoimmune encephalomyelitis. Furthermore, mice transferred with D5R-deficient DCs displayed a significant reduction in the percentage of Th17 cells infiltrating the CNS without differences in the percentage of Th1 cells compared with animals transferred with wild-type DCs. Our findings demonstrate that by contributing to CD4(+) T cell activation and differentiation to Th17 phenotype, D5R expressed on DCs is able to modulate the development of an autoimmune response in vivo.
Assuntos
Células Dendríticas/imunologia , Dopamina/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Receptores de Dopamina D5/fisiologia , Células Th17/imunologia , Transferência Adotiva , Animais , Comunicação Autócrina/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Dopamina/metabolismo , Dopamina/farmacologia , Encefalomielite Autoimune Experimental/terapia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Celular , Interleucina-17/biossíntese , Interleucina-17/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/biossíntese , Receptores de Dopamina D5/genética , Organismos Livres de Patógenos EspecíficosRESUMO
Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.
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In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.
Assuntos
Melanoma Experimental , Ribonucleases , Camundongos , Animais , Ribonucleases/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Imunidade Adaptativa , Ribonuclease Pancreático/metabolismo , Melanoma Experimental/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Células DendríticasRESUMO
Gallbladder cancer (GBC) is commonly diagnosed at late stages when conventional treatments achieve only modest clinical benefit. Therefore, effective treatments for advanced GBC are needed. In this context, the administration of T cells genetically engineered with chimeric antigen receptors (CAR) has shown remarkable results in hematological cancers and is being extensively studied for solid tumors. Interestingly, GBC tumors express canonical tumor-associated antigens, including the carcinoembryonic antigen (CEA). However, the potential of CEA as a relevant antigen in GBC to be targeted by CAR-T cell-based immunotherapy has not been addressed. Here we show that CEA was expressed in 88% of GBC tumors, with higher levels associated with advanced disease stages. CAR-T cells specifically recognized plate-bound CEA as evidenced by up-regulation of 4-1BB, CD69 and PD-1, and production of effector cytokines IFN-γ and TNF-α. In addition, CD8+ CAR-T cells up-regulated the cytotoxic molecules granzyme B and perforin. Interestingly, CAR-T cell activation occurred even in the presence of PD-L1. Consistent with these results, CAR-T cells efficiently recognized GBC cell lines expressing CEA and PD-L1, but not a CEA-negative cell line. Furthermore, CAR-T cells exhibited in vitro cytotoxicity and reduced in vivo tumor growth of GB-d1 cells. In summary, we demonstrate that CEA represents a relevant antigen for GBC that can be targeted by CAR-T cells at the preclinical level. This study warrants further development of the adoptive transfer of CEA-specific CAR-T cells as a potential immunotherapy for GBC.
Assuntos
Neoplasias da Vesícula Biliar , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Antígeno Carcinoembrionário/genética , Imunoterapia Adotiva/métodos , Antígeno B7-H1 , Neoplasias da Vesícula Biliar/terapia , Imunoterapia , Linfócitos TRESUMO
DNA vaccination is an attractive approach to induce antigen-specific cytotoxic CD8(+) T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be enhanced by codelivering gene-encoded adjuvants. Pattern recognition receptors (PRRs) that sense intracellular DNA could potentially be used to harness intrinsic immune-stimulating properties of plasmid DNA vaccines. Consequently, the cytosolic DNA sensor, DNA-dependent activator of interferon (IFN) regulatory factors (DAI), was used as a genetic adjuvant. In vivo electroporation (EP) of mice with a DAI-encoding plasmid (pDAI) promoted transcription of genes encoding type I IFNs, proinflammatory cytokines, and costimulatory molecules. Coimmunization with pDAI and antigen-encoding plasmids enhanced in vivo antigen-specific proliferation, and induction of effector and memory CTLs. Moreover, codelivery of pDAI effectively promoted CTL and CD4(+) Th1 responses to the TAA survivin. The DAI-enhanced CTL induction required nuclear factor κB (NF-κB) activation and type I IFN signaling, but did not involve the IFN regulatory factor 3 (IRF3). Codelivery of pDAI also increased CTL responses to the melanoma-associated antigen tyrosinase-related protein-2 (TRP2), enhanced tumor rejection and conferred long-term protection against B16 melanoma challenge. This study constitutes "proof-of-principle" validating the use of intracellular PRRs as genetic adjuvants to enhance DNA vaccine potency.
Assuntos
Adjuvantes Imunológicos/genética , Glicoproteínas , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA , Imunidade Adaptativa/genética , Animais , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Glicoproteínas/imunologia , Imunidade Inata/genética , Proteínas Inibidoras de Apoptose/imunologia , Oxirredutases Intramoleculares/imunologia , Ativação Linfocitária/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos/genética , Plasmídeos/imunologia , Proteínas de Ligação a RNA , Proteínas Repressoras/imunologia , Análise de Sobrevida , Survivina , Vacinas de DNA/imunologiaRESUMO
Dopamine has emerged as an important regulator of immunity. Recent evidence has shown that signalling through low-affinity dopamine receptors exerts anti-inflammatory effects, whilst stimulation of high-affinity dopamine receptors potentiates immunity in different models. However, the dopaminergic regulation of CD8+ T-cells in anti-tumour immunity remains poorly explored. Here, we studied the role of dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in the function of CD8+ T-cells and its consequences in the anti-tumour immune response. We observed that the deficiency of Drd3 (the gene encoding DRD3) in CD8+ T-cells limits their in vivo expansion, leading to an impaired anti-tumour response in a mouse melanoma model. Mechanistic analyses suggest that DRD3 stimulation favours the production of interleukin 2 (IL-2) and the surface expression of CD25, the α-chain IL-2 receptor, which are required for expansion and effector differentiation of CD8+ T-cells. Thus, our results provide genetic and pharmacologic evidence indicating that DRD3 favours the production of IL-2 by CD8+ T-cells, which is associated with higher expansion and acquisition of effector function of these cells, promoting a more potent anti-tumour response in a melanoma mouse model. These findings contribute to understanding how dopaminergic signalling affects the cellular immune response and represent an opportunity to improve melanoma therapy.
Assuntos
Melanoma , Linfócitos T Citotóxicos , Animais , Camundongos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Dopamina , Interleucina-2/metabolismo , Receptores Dopaminérgicos , Linfócitos T Citotóxicos/metabolismoRESUMO
DNA vaccines assisted by electroporation efficiently trigger antitumor cytotoxic CD8+ T cell responses in preclinical cancer models and hold potential for human use. They can be easily engineered to express either tumor-associated self-antigens, which are broadly expressed among tumor patients but also in healthy tissue, or tumor-specific neoantigens, which are uniquely expressed in tumors and differ among patients. Recently, it has been demonstrated that DNA vaccination generates both circulating and tissue-resident compartments of CD8+ T cells, which act concertedly against tumors. Here we describe the steps to obtain and test DNA vaccines against models of self-antigens and neoantigens in mice. It includes the evaluation of effector and memory CD8+ T cell responses, as well as assessing the antitumor potential in vivo using transplantable syngeneic tumor models.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Vacinas de DNA/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunização , Memória Imunológica , Imunofenotipagem , Camundongos , Neoplasias/imunologia , Neoplasias/terapia , Resultado do Tratamento , Vacinação , Vacinas de DNA/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ecto-5'-nucleotidase (CD73) is an enzyme present on the surface of tumor cells whose primary described function is the production of extracellular adenosine. Due to the immunosuppressive properties of adenosine, CD73 is being investigated as a target for new antitumor therapies. We and others have described that CD73 is present at the surface of different CD8+ T cell subsets. Nonetheless, there is limited information as to whether CD73 affects CD8+ T cell proliferation and survival. In this study, we assessed the impact of CD73 deficiency on CD8+ T cells by analyzing their proliferation and survival in antigenic and homeostatic conditions. Results obtained from adoptive transfer experiments demonstrate a paradoxical role of CD73. On one side, it favors the expression of interleukin-7 receptor α chain on CD8+ T cells and their homeostatic survival; on the other side, it reduces the survival of activated CD8+ T cells under antigenic stimulation. Also, upon in vitro antigenic stimulation, CD73 decreases the expression of interleukin-2 receptor α chain and the anti-apoptotic molecule Bcl-2, findings that may explain the reduced CD8+ T cell survival observed in this condition. These results indicate that CD73 has a dual effect on CD8+ T cells depending on whether they are subject to an antigenic or homeostatic stimulus, and thus, special attention should be given to these aspects when considering CD73 blockade in the design of novel antitumor therapies.
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BACKGROUND AND AIMS: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4ß7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. METHODS: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. RESULTS: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. CONCLUSIONS: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Inflamação/imunologia , Multimerização Proteica , Receptores CCR/metabolismo , Receptores de Dopamina D5/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Proliferação de Células , Colite/imunologia , Colite/patologia , Humanos , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Integrina beta1/metabolismo , Células Jurkat , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peptídeos/química , Fosforilação , Receptores CCR/deficiência , Receptores de Dopamina D5/deficiência , Transdução de Sinais , TropismoRESUMO
CD39 and CD73 are ectoenzymes that dephosphorylate ATP into its metabolites; ADP, AMP, and adenosine, and thus are considered instrumental in the development of immunosuppressive microenvironments. We have previously shown that within the CD8+ T cell population, naïve and memory cells express the CD73 ectonucleotidase, while terminally differentiated effector cells are devoid of this enzyme. This evidence suggests that adenosine might exert an autocrine effect on CD8+ T cells during T cell differentiation. To study the possible role of CD73 and adenosine during this process, we compared the expression of the adenosinergic signaling components, the phenotype, and the functional properties between CD73-deficient and WT CD8+ T cells. Upon activation, we observed an upregulation of CD73 expression in CD8+ T cells along with an upregulation of the adenosine A2A receptor. Interestingly, when we differentiated CD8+ T cells to Tc1 cells in vitro, we observed that these cells produce adenosine and that CD73-deficient cells present a higher cytotoxic potential evidenced by an increase in IFN-γ, TNF-α, and granzyme B production. Moreover, CD73-deficient cells presented a increased glucose uptake and higher mitochondrial respiration, indicating that this ectonucleotidase restrict the mitochondrial capacity in CD8+ T cells. In agreement, when adoptively transferred, antigen-specific CD73-deficient CD8+ T cells were more effective in reducing the tumor burden in B16.OVA melanoma-bearing mice and presented lower levels of exhaustion markers than wild type cells. All these data suggest an autocrine effect of CD73-mediated adenosine production, limiting differentiation and cytotoxic T cells' metabolic fitness.
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Survivin is an intracellular tumor-associated antigen that is broadly expressed in a large variety of tumors and also in tumor associated endothelial cells but mostly absent in differentiated tissues. Naked DNA vaccines targeting survivin have been shown to induce T cell as well as humoral immune responses in mice. However, the lack of epitope-specific CD8+ T cell detection and modest tumor protection observed highlight the need for further improvements to develop effective survivin DNA vaccination approaches. Here, the efficacy of a human survivin DNA vaccine delivered by intradermal electroporation (EP) was tested. The CD8+ T cell epitope surv(20-28) restricted to H-2 Db was identified based on in-silico epitope prediction algorithms and binding to MHC class I molecules. Intradermal DNA EP of mice with a human survivin encoding plasmid generated CD8+ cytotoxic T lymphocyte (CTL) responses cross-reactive with the mouse epitope surv(20-28), as determined by intracellular IFN-gamma staining, suggesting that self-tolerance has been broken. Survivin-specific CTLs displayed an activated effector phenotype as determined by CD44 and CD107 up-regulation. Vaccinated mice displayed specific cytotoxic activity against B16 and peptide-pulsed RMA-S cells in vitro as well as against surv(20-28) peptide-pulsed target cells in vivo. Importantly, intradermal EP with a survivin DNA vaccine suppressed angiogenesis in vivo and elicited protection against highly aggressive syngeneic B16 melanoma tumor challenge. We conclude that intradermal EP is an attractive method for delivering a survivin DNA vaccine that should be explored also in clinical studies.
Assuntos
Vacinas Anticâncer/administração & dosagem , Melanoma Experimental/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Animais , Vacinas Anticâncer/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Eletroporação , Humanos , Proteínas Inibidoras de Apoptose , Injeções Intradérmicas , Melanoma Experimental/patologia , Melanoma Experimental/prevenção & controle , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Survivina , Vacinas de DNA/imunologiaRESUMO
Caveolin-1 reportedly acts as a tumor suppressor and promotes events associated with tumor progression, including metastasis. The molecular mechanisms underlying such radical differences in function are not understood. Recently, we showed that caveolin-1 inhibits expression of the inhibitor of apoptosis protein survivin via a transcriptional mechanism involving the beta-catenin-Tcf/Lef pathway. Surprisingly, while caveolin-1 expression decreased survivin mRNA and protein levels in HT29(ATCC) human colon cancer cells, this was not the case in metastatic HT29(US) cells. Survivin down-regulation was paralleled by coimmunoprecipitation and colocalization of caveolin-1 with beta-catenin in HT29(ATCC) but not HT29(US) cells. Unlike HT29(ATCC) cells, HT29(US) cells expressed small amounts of E-cadherin that accumulated in intracellular patches rather than at the cell surface. Re-expression of E-cadherin in HT29(US) cells restored the ability of caveolin-1 to down-regulate beta-catenin-Tcf/Lef-dependent transcription and survivin expression, as seen in HT29(ATCC) cells. In addition, coimmunoprecipitation and colocalization between caveolin-1 and beta-catenin increased upon E-cadherin expression in HT29(US) cells. In human embryonic kidney HEK293T and HT29(US) cells, caveolin-1 and E-cadherin cooperated in suppressing beta-catenin-Tcf/Lef-dependent transcription as well as survivin expression. Finally, mouse melanoma B16-F10 cells, another metastatic cell model with low endogenous caveolin-1 and E-cadherin levels, were characterized. In these cells, caveolin-1-mediated down-regulation of survivin in the presence of E-cadherin coincided with increased apoptosis. Thus, the absence of E-cadherin severely compromises the ability of caveolin-1 to develop activities potentially relevant to its role as a tumor suppressor.
Assuntos
Caderinas/metabolismo , Caveolina 1/metabolismo , Regulação para Baixo/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Fatores de Transcrição TCF/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Clonais , Cães , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Imunoprecipitação , Proteínas Inibidoras de Apoptose , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Survivina , Transcrição GênicaRESUMO
Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.