RESUMO
Having discovered that the dendritic cell (DC)-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4(+) T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4(-/-) naive T cells during EAE induction into Rag2(-/-) mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti-DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4(-/-) mice, DC-HIL(-/-) mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL(-/-) mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL(+) cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.
Assuntos
Autoimunidade/imunologia , Proteínas do Olho/imunologia , Glicoproteínas de Membrana/imunologia , Células Mieloides/imunologia , Sindecana-4/imunologia , Transferência Adotiva , Animais , Autoimunidade/genética , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Citometria de Fluxo , Interferon gama/imunologia , Interferon gama/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/transplante , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Sindecana-4/genética , Sindecana-4/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Acute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo-reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen-presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan-4 (SD-4) on effector T cells mediates the inhibitory function of DC-HIL on APC, we proposed that SD-4 negatively regulates the T-cell response to allo-stimulation in acute GVHD, using SD-4 knockout mice. Although not different from their wild-type counterparts in responsiveness to anti-CD3 stimulation, SD-4(-/-) T cells lost the capacity to mediate the inhibitory function of DC-HIL and were hyper-reactive to allogeneic APC. Moreover, infusion of SD-4(-/-) T cells into sub-lethally γ-irradiated allogeneic mice worsened mortality, with hyper-proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD-4 deletion had no deleterious effect on T-cell-suppressive activity compared with SD-4(+/+) regulatory T cells. We conclude that SD-4, as the T-cell ligand of DC-HIL, is a potent inhibitor of allo-reactive T cells responsible for GVHD and a potentially useful target for treating this disease.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Sindecana-4/imunologia , Linfócitos T Reguladores/imunologia , Doença Aguda , Animais , Proteínas do Olho , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Imunológicos/agonistas , Receptores Imunológicos/genética , Sindecana-4/genética , Linfócitos T Reguladores/patologia , Transplante HomólogoRESUMO
Because syndecan-4 (SD-4) on effector and memory T cells inhibits T-cell activation by binding dendritic cell-associated heparan sulfate proteoglycan-integrin ligand (DC-HIL) on antigen presenting cells and because malignant cells of the cutaneous T-cell lymphoma (CTCL) subset, Sézary syndrome (SS), exhibit memory T-cell phenotype, we posited SS cells to express SD-4. Indeed, malignant T cells from patients with SS and from CTCL cell lines constitutively expressed SD-4 at high levels, in contrast to T cells from healthy volunteers and patients with other inflammatory skin diseases and to non-CTCL cell lines that did not. SS cells also bound to DC-HIL at a level higher than normal T cells activated in vitro, resulting in their inhibited proliferation to anti-CD3 antibody. SD-4 on SS cells also trapped transforming growth factor-ß1 to their cell surface, enhancing their ability to inhibit activation of syngeneic and allogeneic normal T cells. All of these inhibitory properties were dependent on overexpression of distinct heparan sulfate (HS) moieties by SD-4 on SS cells. Finally, we showed toxin-conjugated DC-HIL to abrogate the ability of SS cells to proliferate in vitro. These findings indicate that SD-4 bearing distinct HS moieties plays a pathogenic role in SS and may be targeted for treatment.
Assuntos
Heparitina Sulfato/fisiologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Síndrome de Sézary/imunologia , Sindecana-4/genética , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/metabolismo , Idoso , Idoso de 80 Anos ou mais , Membrana Celular/metabolismo , Feminino , Heparitina Sulfato/química , Heparitina Sulfato/imunologia , Heparitina Sulfato/metabolismo , Humanos , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Masculino , Glicoproteínas de Membrana/fisiologia , Pessoa de Meia-Idade , Ligação Proteica/imunologia , Ligação Proteica/fisiologia , Transporte Proteico , Receptores Imunológicos/imunologia , Síndrome de Sézary/genética , Síndrome de Sézary/metabolismo , Síndrome de Sézary/patologia , Sindecana-4/química , Sindecana-4/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof(Flox/Flox) (Mof (F/F)) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof(F/F)/Lck-Cre(+)) display a marked reduction in thymus size compared with Mof(F/F)/Lck-Cre(-) mice. In contrast, the spleen size of Mof(F/F)/Lck-Cre(+) mice was increased compared with control Mof(F/F)/Lck-Cre(-) mice. The thymus of Mof(F/F)/Lck-Cre(+) mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4(+)CD8(+) double-positive T-cell levels were reduced, whereas the immature CD4(-)CD8(-) double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44(-)CD25(+)) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof(F/F)/Lck-Cre(+) and Mof(F/F)/Lck-Cre(-) mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof(F/F)/Lck-Cre(+) mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof(F/F)/Lck-Cre(+) mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof(F/F)/Lck-Cre(-) mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation.
Assuntos
Diferenciação Celular/genética , Deleção de Genes , Instabilidade Genômica , Histona Acetiltransferases/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Camundongos , Camundongos Knockout , Micronúcleos com Defeito Cromossômico , Tamanho do Órgão , Tolerância a Radiação/genética , Baço/metabolismo , Baço/patologia , Linfócitos T/imunologia , Timo/metabolismo , Timo/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Most coinhibitory receptors regulate T-cell responses through an ITIM that recruits protein tyrosine phosphatases (PTPs) to mediate inhibitory function. Because syndecan-4 (SD-4), the coinhibitor for DC-associated heparan sulfate proteoglycan integrin ligand (DC-HIL), lacks such an ITIM, we posited that SD-4 links with a PTP in an ITIM-independent manner. We show that SD-4 associates constitutively with the intracellular protein syntenin but not with the receptor-like PTP CD148 on human CD4(+) T cells. Binding to DC-HIL allowed SD-4 to assemble with CD148 through the help of syntenin as a bridge, and this process upregulated the PTP activity of CD148, which is required for SD-4 to mediate DC-HIL's inhibitory function. Using a mouse model, we found SD-4 to be located away from the immunological synapse formed between T cells and APCs during activation of T cells. These findings indicate that SD-4 is unique among known T-cell coinhibitors, in employing CD148 to inhibit T-cell activation at a site distal from the synapse.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Sindecana-4/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ativação Enzimática/genética , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Hibridomas , Sinapses Imunológicas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/imunologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Sindecana-4/genética , Sindecana-4/imunologia , Sinteninas/metabolismo , Transgenes/genéticaRESUMO
Because syndecan-4 (SD-4) is expressed by some (but not all) T cells following activation and serves as the exclusive ligand of dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL), we envisioned the DC-HIL/SD-4 pathway to be a therapeutic target for conditions mediated by selectively activated T cells. We conjugated soluble DC-HIL receptor with the toxin saporin (SAP; DC-HIL-SAP) and showed it to bind activated (but not resting) T cells and become internalized by and deplete SD-4(+) T cells. In hapten-sensitized mice, DC-HIL-SAP injected i.v. prior to hapten challenge led to markedly suppressed contact hypersensitivity responses that lasted 3 wk and were restricted to the hapten to which the mice were originally sensitized. Such suppression was not observed when DC-HIL-SAP was applied during sensitization. Moreover, the same infusion of DC-HIL-SAP produced almost complete disappearance of SD-4(+) cells in haptenated skin and a 40% reduction of such cells within draining lymph nodes. Our results provide a strong rationale for exploring use of toxin-conjugated DC-HIL to treat activated T cell-driven disease in humans.
Assuntos
Dermatite de Contato/prevenção & controle , Imunoterapia/métodos , Imunotoxinas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Sindecana-4/imunologia , Linfócitos T/imunologia , Animais , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Dermatite de Contato/imunologia , Proteínas do Olho , Feminino , Imunofluorescência , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Saporinas , Pele/efeitos dos fármacos , Pele/imunologia , Sindecana-4/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Previously, we discovered antigen-presenting cells to express DC-HIL receptor and to secrete its soluble form (soluble DC-HIL [sDC-HIL]), both of which bind to syndecan-4 on T cells and endothelial cells (ECs), with the former binding attenuating T-cell function and the latter binding promoting angiogenesis. In this study, we examined the effects of sDC-HIL binding to EC on T-cell extravasation using an allergic contact dermatitis model in mice. The hapten oxazolone applied to ear skin in sensitized mice upregulated cutaneous expression of sDC-HIL, which downregulated the allergic reaction by reducing transendothelial migration of T cells but not other immune cells (neutrophils and mast cells). Moreover, intravenously infused sDC-HIL bound to EC in blood vessels of oxazolone-challenged skin in a scattered and patchy pattern, and intravital microscopic analysis revealed that blood-circulating T cells firmly adhere to DC-HIL-treated endothelia. This regulatory property of sDC-HIL requires syndecan-4 expression by both EC and T cells. Our findings indicate that the DC-HIL/syndecan-4 pathway mediates a cross-talk between T cells and ECs, regulating the cutaneous immune response by preventing extravasation of activated T cells into inflamed skin.
Assuntos
Ativação Linfocitária , Sindecana-4 , Animais , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Oxazolona , Pele , Sindecana-4/metabolismo , Linfócitos TRESUMO
(1) We hypothesized that adding concurrent stereotactic ablative radiotherapy (SAbR) would increase the time to progression in patients with metastatic castrate-resistant prostate cancer (mCRPCA) treated with sipuleucel-T. (2) Patients with a history of prostate cancer (PC), radiographic evidence of metastatic disease, and rising prostate-specific antigen (PSA) > 0.2 ng/dL on castrate testosterone levels were enrolled in this single-arm phase II clinical trial and treated with sipuleucel-T and SAbR. The primary endpoint was time to progression (TTP). Cellular and humoral responses were measured using ELISpot and Luminex multiplex assays, respectively. (3) Twenty patients with mCRPC were enrolled and treated with SAbR to 1−3 sites. Treatment was well tolerated with 51, 8, and 4 treatment-related grade 1, 2, and 3 toxicities, respectively, and no grade 4 or 5 adverse events. At a median follow-up of 15.5 months, the median TTP was 11.2 weeks (95% CI; 6.8−14.0 weeks). Median OS was 76.8 weeks (95% CI; 41.6−130.8 weeks). This regimen induced both humoral and cellular immune responses. Baseline M-MDSC levels were elevated in mCRPC patients compared to healthy donors (p = 0.004) and a decline in M-MDSC was associated with biochemical response (p = 0.044). Responders had lower baseline uric acid levels (p = 0.05). No clear correlation with radiographic response was observed. (4) While the regimen was safe, the PC-antigen-specific immune response induced by SAbR did not yield a synergistic clinical benefit for patients treated with sipuleucel-T compared to the historically reported outcomes.
RESUMO
The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-kappaB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-kappaB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-kappaB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-kappaB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-kappaB, indicating that Hyphae-induced NF-kappaB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-kappaB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-kappaB activation, they regulate different signaling events in which CARD9 mediates IkappaBalpha kinase ubiquitination, whereas Syk regulates IkappaBalpha kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-kappaB activation in response to C. albicans hyphae challenging.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Candida albicans/imunologia , Hifas/imunologia , Quinase I-kappa B/metabolismo , Lectinas Tipo C/metabolismo , NF-kappa B/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Quinase SykRESUMO
APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1beta, and TNF-alpha. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Candida albicans/imunologia , Dermatomicoses/imunologia , Glicoproteínas de Membrana/imunologia , Microsporum/imunologia , Receptores Imunológicos/imunologia , Sindecana-4/metabolismo , Trichophyton/imunologia , Animais , Células Apresentadoras de Antígenos/microbiologia , Bactérias/imunologia , Bactérias/metabolismo , Dermatomicoses/metabolismo , Proteínas do Olho , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Sindecana-4/imunologia , Linfócitos T/imunologia , Linfócitos T/microbiologia , Tirosina/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
T-cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC-HIL on APC to syndecan-4 (SD-4) on activated T cells potently inhibits T-cell activation. In humans, we now show that DC-HIL also binds to SD-4 on activated T cells through recognition of its heparinase-sensitive saccharide moiety. DC-HIL blocks anti-CD3-induced T-cell responses, reducing secretion of pro-inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC-HIL phosphorylates SD-4's intracellular tyrosine and serine residues. Anti-SD-4 Ab mimics the ability of DC-HIL to attenuate anti-CD3 response more potently than Ab directed against other inhibitory receptors (CTLA-4 or programmed cell death-1). Among leukocytes, DC-HIL is expressed highest by CD14(+) monocytes and this expression can be upregulated markedly by TGF-beta. Among APC, DC-HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC-HIL expression on CD14(+) monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF-beta reduced this capacity, whereas knocking down the DC-HIL gene augmented it. Our findings indicate that the DC-HIL/SD-4 pathway can be manipulated to treat T-cell-driven disorders in humans.
Assuntos
Citocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Sindecana-4/metabolismo , Subpopulações de Linfócitos T/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Fosforilação/imunologia , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Sindecana-4/imunologia , Subpopulações de Linfócitos T/metabolismo , Transfecção , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: Immune checkpoint inhibitors (ICI) benefit only a minority of treated patients with cancer. Identification of biomarkers distinguishing responders and nonresponders will improve management of patients with cancer. Because the DC-HIL checkpoint differs from the PD1 pathway in expression and inhibitory mechanisms, we examined whether DC-HIL expression regulates ICI responsiveness. EXPERIMENTAL DESIGN: Plasma samples were collected from patients with advanced non-small cell lung carcinoma (NSCLC) (n = 76) at baseline and/or follow-up after ICI monotherapy. Blood-soluble DC-HIL (sDC-HIL) was determined and analyzed for correlation with the early tumor response. To study the mechanisms, we measured effect of anti-DC-HIL versus anti-PDL1 mAb on growth of mouse tumor cells in experimentally metastatic lung. Influence of DC-HIL to anti-PDL1 treatment was assessed by changes in tumor response after deletion of host-DC-HIL gene, injection of DC-HIL-expressing myeloid-derived suppressor cells (MDSC), or induction of sDC-HIL expression. RESULTS: Nonresponders expressed significantly higher levels of baseline sDC-HIL levels than responders. Among patients (n = 28) for fluctuation with time, nonresponders (14/15 cases) showed increasing or persistently elevated levels. Responders (12/13) had decreasing or persistently low levels. Among various tumors, B16 melanoma exhibited resistance to anti-PDL1 but responded to anti-DC-HIL mAb. Using B16 melanoma and LL2 lung cancer, we showed that deletion of host-derived DC-HIL expression converted the resistant tumor to one responsive to anti-PDL1 mAb. The responsive state was reversed by infusion of DC-HIL+MDSC or induction of sDC-HIL expression. CONCLUSIONS: sDC-HIL in the blood and probably DC-HIL receptor expressed by MDSC play an important role in regulating response to ICI in advanced NSCLC.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/imunologia , Células Supressoras Mieloides/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapiaRESUMO
The discovery of several transmembrane receptors expressed by antigen presenting cells, including those that detect and interact with specific sugar moieties on the surface of microbes, has improved our understanding of how immunity against infection is generated. This knowledge, in turn, prompted us to review such interactions with emphasis on C-type lectin receptors and a focus on the roles of dectin-1 and dectin-2 in anti-fungal immunity.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Polissacarídeos/imunologia , Animais , Antígenos CD/imunologia , Colectinas/imunologia , Humanos , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Receptores de Superfície Celular/imunologia , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vetores Genéticos/imunologia , Lentivirus/imunologia , Vacinas Virais/imunologia , Animais , Antígenos de Neoplasias/genética , Células Cultivadas , Epitopos/imunologia , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Lectinas Tipo C/genética , Lentivirus/genética , Linfonodos/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Baço/imunologia , Vacinas Virais/genéticaRESUMO
Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel-17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal-sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary-cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by alpha-melanocyte-stimulating hormone (MSH) (but not beta-MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN-gamma and TNF-alpha. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD-dependent manner. These results indicate that Gpnmb is a melanosome-associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.
Assuntos
Proteínas do Olho/metabolismo , Queratinócitos/citologia , Melanócitos/citologia , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligopeptídeos/metabolismo , Neoplasias Cutâneas/patologia , Motivos de Aminoácidos , Animais , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Olho/efeitos da radiação , Humanos , Integrinas/metabolismo , Queratinócitos/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Glicoproteínas de Membrana/efeitos da radiação , Camundongos , Neoplasias Cutâneas/metabolismo , Raios UltravioletaRESUMO
PURPOSE: Blocking the function of myeloid-derived suppressor cells (MDSC) is an attractive approach for cancer immunotherapy. Having shown DC-HIL/GPNMB to be the T-cell-inhibitory receptor mediating the suppressor function of MDSCs, we evaluated the potential of anti-DC-HIL mAb as an MDSC-targeting cancer treatment. EXPERIMENTAL DESIGN: Patients with metastatic cancer (n = 198) were analyzed by flow cytometry for DC-HIL or PDL1 expression on blood CD14+HLA-DRno/lo MDSCs. Their suppressor function was assessed by in vitro coculture with autologous T cells, and the ability of anti-DC-HIL or anti-PDL1 mAb to reverse such function was determined. Tumor expression of these receptors was examined histologically, and the antitumor activity of the mAb was evaluated by attenuated growth of colon cancers in mice. RESULTS: Patients with metastatic cancer had high blood levels of DC-HIL+ MDSCs compared with healthy controls. Anti-DC-HIL mAb reversed the in vitro function in â¼80% of cancer patients tested, particularly for colon cancer. Despite very low expression on blood MDSCs, anti-PDL1 mAb was as effective as anti-DC-HIL mAb in reversing MDSC function, a paradoxical phenomenon we found to be due to upregulated expression of PDL1 by T-cell-derived IFNγ in cocultures. DC-HIL is not expressed by colorectal cancer cells but by CD14+ cells infiltrating the tumor. Finally, anti-DC-HIL mAb attenuated growth of preestablished colon tumors by reducing MDSCs and increasing IFNγ-secreting T cells in the tumor microenvironment, with similar outcomes to anti-PDL1 mAb. CONCLUSIONS: Blocking DC-HIL function is a potentially useful treatment for at least colorectal cancer with high blood levels of DC-HIL+ MDSCs.See related commentary by Colombo, p. 453.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Imunofenotipagem , Interferon gama , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Células Supressoras Mieloides/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Linfócitos T/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Soluble factors from the primary tumor induce recruitment of bone marrow-derived progenitors to form tumor-supportive microenvironments or pre-metastatic niches in distal organs before metastasis. How tumor-secreted factors condition the sites for tumor progression remains ambiguous. B16 melanoma produces the secreted form of T cell-inhibitory DC-HIL (sDC-HIL) that travels to distal organs and potentiates the metastatic capacity of tumor cells. We studied the molecular mechanisms and found that sDC-HIL binds to select endothelial cells that co-localize with the sites where bone marrow-derived progenitors and tumor cells migrate. sDC-HIL-bound endothelial cells exist at a similar frequency in mice with or without tumors, and they are strongly associated with survival of intravenously injected tumor cells in the lung. sDC-HIL binding conferred T-cell suppressor function on the ECs and awakened the angiogenic property by inducing vascular endothelial growth factor expression, resulting in enhanced transendothelial migration of bone marrow-derived progenitors and tumor cells, but not for T cells. This selectivity is achieved by the T-cell binding of sDC-HIL, which prevents formation of the leading edges required for chemotaxis. Finally, inducing tumor expression of sDC-HIL significantly reduced tumor-infiltrated T cells. Therefore, the highly metastatic attribute of B16 melanoma can be explained by the endothelial gatekeeper function of sDC-HIL that limits lymphocyte transmigration to pre-metastatic niches.
Assuntos
Células Endoteliais/fisiologia , Proteínas do Olho/metabolismo , Melanoma/imunologia , Glicoproteínas de Membrana/metabolismo , Neoplasias Cutâneas/imunologia , Linfócitos T/imunologia , Animais , Humanos , Ativação Linfocitária , Melanoma/patologia , Melanoma Experimental , Camundongos , Metástase Neoplásica , Neoplasias Experimentais , Neoplasias Cutâneas/patologia , Migração Transendotelial e Transepitelial , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neurofibromatosis type 1 (NF1) is an autosomal genetic disorder. Patients with NF1 are associated with mono-allelic loss of the tumor suppressor gene NF1 in their germline, which predisposes them to develop a wide array of benign lesions. Intriguingly, recent sequencing efforts revealed that the NF1 gene is frequently mutated in multiple malignant tumors not typically associated with NF1 patients, suggesting that NF1 heterozygosity is refractory to at least some cancer types. In two orthogonal mouse models representing NF1- and non-NF1-related tumors, we discover that an Nf1+/- microenvironment accelerates the formation of benign tumors but impairs further progression to malignancy. Analysis of benign and malignant tumors commonly associated with NF1 patients, as well as those with high NF1 gene mutation frequency, reveals an antagonistic role for NF1 heterozygosity in tumor initiation and malignant transformation and helps to reconciliate the role of the NF1 gene in both NF1 and non-NF1 patient contexts.
Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neurofibromina 1/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Heterozigoto , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Neurofibroma/patologia , Neurofibromina 1/metabolismo , Fenótipo , Neoplasias Cutâneas/patologia , Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Dendritic cells (DC) are far more potent to activate T cells than other antigen presenting cells (e.g., macrophages) and distributed to many organs where DC develop to functionally and phenotypically distinctive subsets. To isolate DC-differentially expressed genes, we used a subtractive cDNA cloning (XS52 DC minus J774 macrophages), resulting in the identification of d2 isoform of vacuolar (V) H+-ATPase subunit d. Unlike the ubiquitously expressed isoform (d1), d2 mRNA manifested expression restricted to particular subsets of DC (e.g., skin- and bone marrow-derived DC) among leukocytes and encoded two transcripts (1.6 and 3.0 kb) that differed in the length of the 3'-untranslated region. The d2 protein displayed association with membranes and the localization in lysosomes and antigen-containing endosomes. Interestingly, XS52 DC expressed seven-fold higher V-ATPase proton-pump activity than J774 macrophages and distinguished from the macrophage by high levels of isoforms a1 and a2 expression among V-ATPase subunits. These results indicated that d2 is a new marker for DC and it may, co-operatively with subunit a isoforms, regulate V-ATPase activity.
Assuntos
Células Dendríticas/enzimologia , Células Dendríticas/imunologia , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Apresentação de Antígeno , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Células Dendríticas/classificação , Endossomos/enzimologia , Endossomos/imunologia , Expressão Gênica , Técnicas In Vitro , Leucócitos/classificação , Leucócitos/enzimologia , Leucócitos/imunologia , Ativação Linfocitária , Macrófagos/enzimologia , Macrófagos/imunologia , Membranas/enzimologia , Camundongos , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologiaRESUMO
Psoriasis vulgaris is an inflammatory skin disease caused by hyperactivated T cells regulated by positive and negative mechanisms; although the former have been much studied, the latter have not. We studied the regulatory mechanism mediated by myeloid-derived suppressor cells (MDSCs) and showed that MDSCs expanded in melanoma patients express dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand, a critical mediator of T-cell suppressor function. We examined expansion of DC-HIL(+) MDSCs in psoriasis and characterized their functional properties. Frequency of DC-HIL(+) monocytic MDSCs (CD14(+)HLA-DR(no/low)) in blood and skin was markedly increased in psoriatic patients versus healthy control subjects, but there was no statistically significant relationship with disease severity (based on Psoriasis Area and Severity Index score). Blood DC-HIL(+) MDSC levels in untreated patients were significantly higher than in treated patients. Compared with melanoma-derived MDSCs, psoriatic MDSCs exhibited significantly reduced suppressor function and were less dependent on DC-HIL, but they were capable of inhibiting proliferation and IFN-γ and IL-17 responses of autologous T cells. Psoriatic MDSCs were functionally diverse among patients in their ability to suppress allogeneic T cells and in the use of either IL-17/arginase I or IFN-γ/inducible nitric oxide synthase axis as suppressor mechanisms. Thus, DC-HIL(+) MDSCs are expanded in psoriasis patients, and their mechanistic heterogeneity and relative functional deficiency may contribute to the development of psoriasis.