RESUMO
Lymphatic endothelial cells (LECs) express MHC class II (MHC-II) upon IFN-γ stimulation, yet recent evidence suggests that LECs cannot activate naive or memory CD4+ T cells. In this article, we show that IFN-γ-activated human dermal LECs can robustly reactivate allogeneic human memory CD4+ T cells (hCD4+ TMs), but only when TGF-ß signaling is inhibited. We found that in addition to upregulating MHC-II, IFN-γ also induces LECs to upregulate glycoprotein A repetitions predominant, which anchors latent TGF-ß to the membrane and potentially inhibits T cell activation. Indeed, hCD4+ TM proliferation was substantially increased when LEC-CD4+ TM cultures were treated with a TGF-ß receptor type 1 inhibitor or when glycoprotein A repetitions predominant expression was silenced in LECs. Reactivated hCD4+ TMs were characterized by their proliferation, CD25 expression, and cytokine secretion. CD4+ TM reactivation was dependent on LEC expression of MHC-II, confirming direct TCR engagement. Although CD80 and CD86 were not detected on LECs, the costimulatory molecules OX40L and ICOSL were upregulated upon cytokine stimulation; however, blocking these did not affect CD4+ TM reactivation by LECs. Finally, we found that human dermal LECs also supported the maintenance of Foxp3-expressing hCD4+ TMs independently of IFN-γ-induced MHC-II. Together, these results demonstrate a role for LECs in directly modulating CD4+ TM reactivation under inflammatory conditions and point to LEC-expressed TGF-ß as a negative regulator of this activation.
Assuntos
Linfócitos T CD4-Positivos , Antígenos de Histocompatibilidade Classe II , Humanos , Antígenos de Histocompatibilidade Classe II/metabolismo , Células Endoteliais , Antígenos CD4 , Citocinas , Moléculas de Adesão Celular , Interferon gama , Fator de Crescimento Transformador betaRESUMO
Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Complexo Antígeno-Anticorpo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Humanos , Proteínas Virais/genéticaRESUMO
In allergic airway inflammation, VEGFR-3-mediated lymphangiogenesis occurs in humans and mouse models, yet its immunological roles, particularly in adaptive immunity, are poorly understood. Here, we explored how pro-lymphangiogenic signaling affects the allergic response to house dust mite (HDM). In the acute inflammatory phase, the lungs of mice treated with blocking antibodies against VEGFR-3 (mF4-31C1) displayed less inflammation overall, with dramatically reduced innate and T-cell numbers and reduced inflammatory chemokine levels. However, when inflammation was allowed to resolve and memory recall was induced 2 months later, mice treated with mF4-31C1 as well as VEGF-C/-D knockout models showed exacerbated type 2 memory response to HDM, with increased Th2 cells, eosinophils, type 2 chemokines, and pathological inflammation scores. This was associated with lower CCL21 and decreased TRegs in the lymph nodes. Together, our data imply that VEGFR-3 activation in allergic airways helps to both initiate the acute inflammatory response and regulate the adaptive (memory) response, possibly in part by shifting the TReg/Th2 balance. This introduces new immunomodulatory roles for pro-lymphangiogenic VEGFR-3 signaling in allergic airway inflammation and suggests that airway lymphatics may be a novel target for treating allergic responses.
Assuntos
Memória Imunológica , Linfangiogênese , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Alérgenos , Animais , Biomarcadores , Suscetibilidade a Doenças , Imunofenotipagem , Linfangiogênese/genética , Camundongos , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/patologia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours, CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD-IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD-IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.
Assuntos
Colágeno/metabolismo , Inflamação/patologia , Interleucina-12/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Imunidade Inata/efeitos dos fármacos , Interleucina-12/farmacologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Indução de Remissão , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Although a clinical breakthrough for cancer treatment, it remains that a minority of patients respond to checkpoint inhibitor (CPI) immunotherapy. The composition of tumor-infiltrating immune cells has been identified as a key factor influencing CPI therapy success. Thus, enhancing tumor immune cell infiltration is a critical challenge. A lack of the chemokine CCL4 within the tumor microenvironment leads to the absence of CD103+ dendritic cells (DCs), a crucial cell population influencing CPI responsiveness. Here, we use a tumor stroma-targeting approach to deliver CCL4; by generating a fusion protein of CCL4 and the collagen-binding domain (CBD) of von Willebrand factor, we show that CBD fusion enhances CCL4 tumor localization. Intravenous CBD-CCL4 administration recruits CD103+ DCs and CD8+ T cells and improves the antitumor effect of CPI immunotherapy in multiple tumor models, including poor responders to CPI. Thus, CBD-CCL4 holds clinical translational potential by enhancing efficacy of CPI immunotherapy.
Assuntos
Antígenos CD/imunologia , Quimiocina CCL4/genética , Imunoterapia , Cadeias alfa de Integrinas/imunologia , Melanoma Experimental/imunologia , Melanoma/imunologia , Animais , Antígenos CD/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Quimiocina CCL4/imunologia , Quimiocina CCL4/farmacologia , Colágeno/genética , Colágeno/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Cadeias alfa de Integrinas/genética , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/genética , Melanoma/terapia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Ligação Proteica/genética , Microambiente Tumoral/efeitos dos fármacos , Fator de von Willebrand/genéticaRESUMO
Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137+ and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.
Assuntos
Exsudatos e Transudatos/metabolismo , Linfa/metabolismo , Vasos Linfáticos/metabolismo , Melanoma/sangue , Melanoma/patologia , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/patologia , Animais , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Citocinas/análise , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Excisão de Linfonodo , Metástase Linfática , Melanoma/secundário , Melanoma/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/análise , Proteômica/métodos , Proteínas S100/análise , Neoplasias Cutâneas/secundário , Neoplasias Cutâneas/cirurgiaRESUMO
Cancer immunotherapy with immune checkpoint inhibitors (CPIs) and interleukin-2 (IL-2) has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death ligand 1 antibody (αPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both αCTLA4 + αPD-L1 combination therapy and IL-2, for example, eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells. In an orthotopic breast cancer model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically administered tumor drugs with high translational promise.
Assuntos
Anticorpos Antineoplásicos/imunologia , Colágeno/metabolismo , Citocinas/imunologia , Imunoterapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Citotoxicidade Imunológica , Modelos Animais de Doenças , Humanos , Imunidade , Injeções Intravenosas , Interleucina-2/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos , Resultado do TratamentoRESUMO
CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144) to enhance anti-CD40's effects when administered locally. Peritumoral injection of PlGF-2123-144-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2123-144-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered BrafV600E ßCatSTA melanoma model that does not respond to checkpoint inhibitors, PlGF-2123-144-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy.Implications: Extracellular matrix-binding peptide conjugation to agonistic anti-CD40 antibody enhances antitumor efficacy and reduces treatment-related adverse events. Mol Cancer Ther; 17(11); 2399-411. ©2018 AACR.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Afinidade de Anticorpos , Antígenos CD40/agonistas , Matriz Extracelular/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Antígenos CD40/imunologia , Linhagem Celular Tumoral , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Humanos , Imunidade , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Nus , Peptídeos/química , Fator de Crescimento Placentário/metabolismo , Linfócitos T/imunologia , beta Catenina/metabolismoRESUMO
Lymphatic vessels are the primary route of communication from peripheral tissues to the immune system; as such, they represent an important component of local immunity. In addition to their transport functions, new immunomodulatory roles for lymphatic vessels and lymphatic endothelial cells have come to light in recent years, demonstrating that lymphatic vessels help shape immune responses in a variety of ways: promoting tolerance to self-antigens, archiving antigen for later presentation, dampening effector immune responses, and resolving inflammation, among others. In addition to these new biological insights, the growing field of immunoengineering has begun to explore therapeutic approaches to utilize or exploit the lymphatic system for immunotherapy.
Assuntos
Imunomodulação , Imunoterapia , Vasos Linfáticos/imunologia , Animais , Autoantígenos/imunologia , Encéfalo/imunologia , Humanos , Inflamação/imunologia , Inflamação/terapia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfangiogênese , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)-super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2123-144 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2123-144-anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2123-144-anti-PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2123-144-Abs increased tumor-infiltrating activated CD8+ and CD4+ T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.
Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Matriz Extracelular/metabolismo , Imunoterapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Anticorpos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4 , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Engenharia Genética , Heparina/metabolismo , Imunidade , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Fator de Crescimento Placentário/química , Resultado do TratamentoRESUMO
In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naïve T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.
Assuntos
Imunoterapia , Linfangiogênese , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Linfócitos T/patologia , Animais , Proliferação de Células , Quimiocina CCL21/metabolismo , Intervalo Livre de Doença , Epitopos/imunologia , Humanos , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica , Receptores CCR7/metabolismo , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8(+) T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide-MHCII complexes from DCs and induce CD4(+) T cell dysfunction by presenting transferred complexes to naive CD4(+) T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin.