RESUMO
The tumor microenvironment of glioblastoma IDH-wildtype is highly immune suppressive and is characterized by a strong component of myeloid-derived suppressor cells (MDSCs). To interfere with the immune suppressive functions of MDSCs, a comprehensive understanding on how MDSCs acquire their suppressive phenotype is essential. Previously, we and others have shown a distinct Sialic acid-binding immunoglobulin-like lectin (Siglec) receptor expression profile for MDSCs in glioblastoma. Siglec receptors can transmit inhibitory signals comparable to PD-1 and are suggested to act as glyco-immune checkpoints. Here, we investigated how glioma specific Siglec-sialic acid interactions influence myeloid immune suppressive functions. Co-culturing monocytes with glioblastoma cells induced CD163 expression on the monocytes. Upon desialylation of the glioblastoma cells, this induction of CD163 was hampered, and furthermore, the monocytes were now able to secrete higher amounts of IL-6 and TNFα compared to fully sialylated glioblastoma cells. Additionally, Siglec-specific triggering using anti-Siglec-7 or Siglec-9 antibodies displayed a decreased TNFα secretion by the monocytes, validating the role of the Siglec-Sialic axis in the co-culture experiments. Together, our results demonstrate that glioblastoma cells induce a myeloid immune-suppressive phenotype that could be partly rescued by lowering the glioblastoma-associated sialic acid levels. This manuscript supports further research of the Siglec-Sialic acid axis in the context of glioblastoma and its potential to improve clinical outcome.
RESUMO
BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Humanos , Animais , Camundongos , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/metabolismo , Microambiente TumoralRESUMO
Sialic acid sugar-carrying glycans, sialoglycans, are aberrantly expressed on many tumor cells and have emerged as potent regulatory molecules involved in creating a tumor-supportive microenvironment. Sialoglycans can be recognized by sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immunomodulatory receptors. Most mammalian Siglecs transmit inhibitory signals comparable with the immune checkpoint inhibitor programmed death protein 1 (PD-1), but some are activating. Recent studies have shown that tumor cells can exploit sialoglycan-Siglec interactions to modulate immune cell function, contributing to an immunosuppressive tumor microenvironment (TME). Interference with sialoglycan synthesis or sialoglycan-Siglec interactions might improve antitumor immunity. Many questions regarding specificity, signaling, and regulatory function of sialoglycan-Siglec interactions remain. We posit that sialoglycans and Siglecs present as potential glyco-immune 'checkpoints' for cancer immunotherapy.
Assuntos
Polissacarídeos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Microambiente Tumoral , Animais , Humanos , Imunidade , Imunoterapia , Neoplasias/terapia , Polissacarídeos/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Microambiente Tumoral/imunologiaRESUMO
Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid-Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.
Assuntos
Neoplasias Encefálicas/imunologia , Glioma/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Transcriptoma/imunologia , Adulto , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Feminino , Glioma/genética , Glioma/terapia , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.
Assuntos
Comunicação Celular/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Biomarcadores , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Neoplasias/patologia , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
OBJECTIVE: The prevalence of periodontitis is increased in patients with rheumatoid arthritis (RA), and the severity of periodontitis can affect the level of arthritis. Porphyromonas gingivalis is one of the main bacteria involved in periodontitis. Our aim was to determine if there are differences in the innate immune response against P gingivalis between healthy controls and RA patients. METHODS: Monocyte-derived dendritic cells (DCs) from healthy controls, RA patients, and patients with psoriatic arthritis (PsA) were stimulated with P gingivalis, a range of other bacteria, and Toll-like receptor agonists. Cytokine production was determined, and blocking studies were performed to determine which receptors were involved in differential recognition of P gingivalis. Effects on T cell cytokines were also determined in cultures of peripheral blood mononuclear cells (PBMCs). RESULTS: Upon stimulation with P gingivalis, RA patient DCs produced less tumor necrosis factor as compared to healthy control DCs, which was not observed in PsA patients or upon stimulation with other bacteria. In addition, P gingivalis-mediated activation of RA patient PBMCs showed a clear reduction of interferon-γ production. Among the various possible underlying mechanisms investigated, only blockade of CR3 abolished the difference between RA patients and healthy controls, suggesting the involvement of CR3 in this process. CONCLUSION: Immune cells from RA patients display a reduced response to P gingivalis, which has functional consequences for the immune response. This may result in prolonged survival of P gingivalis, possibly driving autoantibody formation and a self-perpetuating loop of chronic inflammation. The possible role of CR3 in this process warrants further investigation.
Assuntos
Artrite Reumatoide/imunologia , Infecções por Bacteroidaceae/imunologia , Células Dendríticas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Psoriásica/imunologia , Estudos de Casos e Controles , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Citocinas/imunologia , Feminino , Citometria de Fluxo , Humanos , Técnicas In Vitro , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Antígeno de Macrófago 1/imunologia , Masculino , Pessoa de Meia-Idade , Porphyromonas gingivalis , Linfócitos T/imunologia , Receptores Toll-Like/agonistas , Adulto JovemRESUMO
BACKGROUND: In rheumatoid arthritis (RA) macrophages play a major role in amplifying synovial inflammation. Important activating signals are those induced by Toll-like receptor (TLR) ligands and by activated T cells. The balance between activating and inhibitory Fc gamma receptors (FcγRs) on macrophages might be crucial in modulating these inflammatory responses. The purpose of this study was to determine FcγR expression on pro- and anti-inflammatory macrophages (gmMφ and mMφ, respectively) and identify functional consequences on immune complex uptake and macrophage activation. METHODS: Human monocytes were isolated and differentiated into gmMφ and mMφ. A full FcγR characterization of both macrophage subtypes was performed and uptake of fluorescent immune complexes (ICs) was determined. FcγRIIb isoforms were determined by qPCR. Macrophages were stimulated via different TLRs or cytokine activated T cells in the presence or absence of ICs and cytokine production was determined. Blocking studies were performed to look into the pathways involved. RESULTS: mMφ expressed high levels of the activating FcγRIIa and FcγRIII and low levels of the inhibitory FcγRIIb, while the FcγR balance on gmMφ was shifted towards the inhibitory FcγRIIb. This was accompanied by a clear increase in FcγRIIb1 mRNA expression in gmMφ. This resulted in higher IC uptake by mMφ compared to gmMφ. Furthermore, FcγR-mediated stimulation of gmMφ inhibited TLR2, 3, 4 and 7/8 mediated cytokine production via FcγRIIb and PI3K signaling. In addition, gmMφ but not mMφ produced TNFα upon co-culture with cytokine activated T cells, which was reduced by IC binding to FcγRIIb. The latter was dependent on PI3K signaling and COX2. CONCLUSIONS: FcγR expression patterns on gmMφ and mMφ are significantly different, which translates in clear functional differences further substantiating FcγRIIb as an interesting target for inflammation control in RA and other autoimmune/inflammatory diseases.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Inflamação/metabolismo , Macrófagos/imunologia , Receptores de IgG/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Interleucina-10/metabolismo , Ligantes , Ativação Linfocitária , Macrófagos/citologia , Macrófagos/metabolismo , Microscopia de Fluorescência , Monócitos/citologia , Monócitos/metabolismo , Fagocitose , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Transdução de Sinais , Linfócitos T/citologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4). In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.
Assuntos
Citocinas/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Complexo Antígeno-Anticorpo/farmacologia , Células Cultivadas , Citocinas/genética , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Imunoglobulina G/química , Imunoglobulina G/farmacologia , Interleucina-10/genética , Interleucina-23/metabolismo , Ligantes , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fenótipo , Temperatura , Receptores Toll-Like/química , Regulação para Cima , gama-Globulinas/farmacologiaRESUMO
OBJECTIVE: The pathogenesis of psoriatic arthritis (PsA) remains poorly understood. The underlying chronic inflammatory immune response is thought to be triggered by unknown environmental factors potentially arising from a defective immune function. We undertook this study to determine whether an impaired acute inflammatory response by dendritic cells (DCs) might compromise the clearance of bacteria and predispose to chronic inflammation. METHODS: We determined cytokine production by DCs from healthy controls and from patients with rheumatoid arthritis, PsA, and psoriasis in response to Mycobacterium tuberculosis, Mycobacterium avium paratuberculosis, and a range of other bacteria and Toll-like receptor (TLR) ligands. Phenotypic differences involved in cellular responses against (myco)bacteria were determined by quantitative polymerase chain reaction and flow cytometry. RESULTS: The secretion of proinflammatory cytokines by PsA DCs was impaired upon in vitro challenge with mycobacteria and TLR-2 ligands. This impairment was associated with elevated serum levels of C-reactive protein. The expression of TLR-2 and other receptors known to mediate mycobacterial recognition was unaltered. In contrast, the intracellular TLR inhibitors suppressor of cytokine signaling 3 and A20 were more highly expressed in DCs from PsA patients. PsA DCs further demonstrated up-regulated levels of ATG16L1, NADPH oxidase 2, and LL37, which are molecules implicated in the immune response against intracellular bacteria. CONCLUSION: Our findings indicate that DCs from PsA patients have a disordered immune response toward some species of (myco)bacteria. This might predispose to impaired immune responses to, and in turn impaired clearance of, these bacteria, setting the stage for the chronic inflammation of joints, entheses, skin, and the gut.
Assuntos
Artrite Psoriásica/imunologia , Citocinas/biossíntese , Células Dendríticas/metabolismo , Adulto , Artrite Psoriásica/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Células Cultivadas , Citocinas/imunologia , Humanos , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologiaRESUMO
TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-alpha but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.
Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Subpopulações de Linfócitos T/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Western Blotting , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interferon Tipo I/metabolismo , Interleucina-1/biossíntese , Interleucina-1/imunologia , Interleucina-12/biossíntese , Interleucina-12/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-6/biossíntese , Interleucina-6/imunologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismoRESUMO
Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcgammaRs and TLRs is accepted, their precise involvement remains to be elucidated. FcgammaRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcgammaRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcgammaRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcgammaRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcgammaRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcgammaRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcgammaRIIb in the induction of these phenomena. This TLR4-FcgammaRIIb interaction was shown to dependent on the PI3K and Akt pathway.