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1.
PLoS One ; 19(6): e0304985, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38843278

RESUMO

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.


Assuntos
Antígenos de Diferenciação , Antígeno CD47 , Imunoconjugados , Receptores Imunológicos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Animais , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Humanos , Camundongos , Imunoconjugados/farmacologia , Antígenos de Diferenciação/imunologia , Linhagem Celular Tumoral , Feminino , Trastuzumab/farmacologia , Inibidores da Topoisomerase I/farmacologia , Imunoterapia/métodos , Camundongos Endogâmicos BALB C
2.
Photodiagnosis Photodyn Ther ; 47: 104200, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38723757

RESUMO

BACKGROUND: Photodynamic therapy (PDT) is used for the treatment of centrally-located early lung cancers (CLELCs) and is recommended for tumors ≤ 1.0 cm in diameter. We previously reported that PDT using talaporfin sodium, second-generation photosensitizer, for tumors > 1.0 cm but ≤ 2.0 cm in diameter was able to achieve a therapeutic outcome comparable to that of tumors with a diameter of ≤ 1.0 cm. However, the effectiveness of PDT using talaporfin sodium for tumors > 2.0 cm in diameter remains unclear. We conducted a retrospective analysis of cases in which PDT was performed for flat-type CLELCs with tumor diameters of > 2.0 cm. METHODS: We retrospectively analyzed seven cases (eight lesions) with tumor diameters > 2.0 cm and no evidence of extracartilaginous invasion or lymph node metastasis. RESULTS: All the patients underwent multiple PDT sessions. The PDT treatment results over the study period were partial response in one case (14.3 %), stable disease (SD) in three cases (42.9 %), and progressive disease (PD) in three cases (42.9 %). At the time of writing this report, five of seven cases (71.4 %) are still undergoing treatment. The duration of SD-the time from the start of treatment until the criteria for PD were met (SD or better maintained)-ranged from 7 to 52 months (mean, 25.3 months). CONCLUSIONS: "Maintenance PDT" for CLELCs > 2.0 cm in diameter has the potential to inhibit tumor progression in the long term while maintaining quality of life, rather than simply aiming only for a quick radical cure.


Assuntos
Neoplasias Pulmonares , Fotoquimioterapia , Fármacos Fotossensibilizantes , Porfirinas , Humanos , Fotoquimioterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fármacos Fotossensibilizantes/uso terapêutico , Masculino , Idoso , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Porfirinas/uso terapêutico , Idoso de 80 Anos ou mais , Resultado do Tratamento
3.
PLoS One ; 17(7): e0268485, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35901072

RESUMO

Late-onset non-syndromic autosomal dominant hearing loss 9 (DFNA9) is a hearing impairment caused by mutations in the coagulation factor C homology gene (COCH). COCH encodes for cochlin, a major component of the cochlear extracellular matrix. Though biochemical and genetic studies have characterized the properties of wild-type and mutated cochlins derived from DFNA9, little is known about the underlying pathogenic mechanism. In this study, we established a cochlin reporter cell, which allowed us to monitor the interaction of cochlin with its ligand(s) by means of a ß-galactosidase assay. We found a class of highly sulfated glycosaminoglycans (GAGs), heparin, that were selectively bound to cochlin. The interaction was distinctly abrogated by N-desulfation, but not by 2-O- or 6-O-desulfation. The binding of cochlin to GAG was diminished by all of the point mutations found in DFNA9 patients. Through GAG composition analysis and immunostaining using mouse cochlin/immunoglobulin-Fc fusion protein, we identified moderately sulfated GAGs in mouse cochlea tissue; this implies that cochlin binds to such sulfated GAGs in the cochlea. Since GAGs play an important role in cell growth and survival as co-receptors of signal transduction mechanisms, the interaction of cochlin with GAGs in the extracellular matrix could aid the pathological research of autosomal dominant late-onset hearing loss in DFNA9.


Assuntos
Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Animais , Camundongos , Surdez/genética , Proteínas da Matriz Extracelular/metabolismo , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Heparina , Heparitina Sulfato , Sulfatos
4.
Carbohydr Polym ; 277: 118606, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34893207

RESUMO

Levan, a ß-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography-mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides.


Assuntos
Frutanos/farmacologia , Fatores Imunológicos/farmacologia , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/imunologia , Animais , Linhagem Celular , Citocinas/biossíntese , Erwinia/química , Frutanos/química , Humanos , Fatores Imunológicos/química , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/deficiência
6.
Commun Biol ; 2: 450, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815204

RESUMO

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aß1-42 - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

7.
Food Res Int ; 115: 10-15, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30599920

RESUMO

Lactulose-derived oligosaccharides (OsLu) are prebiotic galactooligosaccharides (GOS) beneficial for human health including immunomodulatory properties; however, the molecular mechanism is unclear. OsLu produced by enzymatic synthesis can be purified with Saccharomyces cerevisiae (OsLu-Sc). We show that this purification introduces yeast-derived proteins reactive to Dectin-2, an innate immune receptor for fungal polysaccharides. Using a cell-based bioassay, we tested the binding of OsLu and GOS samples to Dectin-2. While OsLu purified with active charcoal and commercial GOS failed to bind to Dectin-2, we found OsLu-Sc bound to this receptor. The carbohydrate-binding incompetent mutant of Dectin-2 failed to bind to OsLu-Sc. These data suggest that OsLu-Sc introduced carbohydrate ligands for Dectin-2. In accordance with this, proteomic analysis revealed OsLu-Sc contained S. cerevisiae-derived mannoproteins. Therefore, our data highlight the importance of the purification method for OsLu, which may positively affect the bioactivity of OsLu. Data are available via ProteomeXchange with identifier PXD010495.


Assuntos
Proteínas Fúngicas/metabolismo , Lactulose/química , Lectinas Tipo C/metabolismo , Oligossacarídeos/análise , Receptores Imunológicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Prebióticos , Proteômica
8.
Cell Chem Biol ; 26(1): 131-136.e4, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30393066

RESUMO

Macrophages (MØs) expressing the endocytic sialic acid-binding immunoglobulin-like lectin 1 (siglec-1, CD169, sialoadhesin) are known to be adept at antigen capture-primarily due to their strategic location within lymphatic tissues. Antigen concentrated in these cells can be harnessed to induce potent anti-tumor/anti-pathogen cytotoxic (CD8+) T cell responses. Here, we describe a chemical platform that exploits the CD169-mediated antigen capture pathway for biased priming of antigen-specific CD4+ or CD8+ T cells in vivo. In the absence of a toll-like receptor (TLR) agonist, antigen delivery through CD169 produced robust CD4+ T cell priming only. However, simultaneous treatment with targeted antigen and a TLR7 agonist induced CD8+ T cell priming, with concomitant suppression of the CD4+ T cell response. We exploited these observations to manipulate the activation ratio of CD4+/CD8+ T cells in the same animal. These findings represent a unique chemical strategy for targeting CD169+ macrophages to modulate antigen-specific T cell immunity.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Macrófagos/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética
9.
FASEB J ; 32(6): 3301-3320, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29401627

RESUMO

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein-carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly- N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule-grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.-Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.


Assuntos
Moléculas de Adesão Celular/química , Galectina 3/química , Lectinas Tipo C/química , Mucina-2/química , Receptores de Superfície Celular/química , Animais , Proteínas Sanguíneas , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Galectinas , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Espectrometria de Massas , Camundongos , Mucina-2/genética , Mucina-2/metabolismo , Domínios Proteicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade
10.
Front Immunol ; 8: 1397, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29118762

RESUMO

The human fungal microbiota known as mycobiota is increasingly recognized as a critical factor in human gut health and disease. Non-pathogenic commensal yeasts such as Saccharomyces cerevisiae promote homeostasis in the gut, whereas dysbiosis of the gut mycobiota is associated with inflammation. Glycan-binding receptors (lectins) are key host factors in host-mycobiota interaction in the gut. They are expressed on immune cells such as dendritic cells (DCs) and recognize fungal polysaccharides. This interaction is imperative to mount appropriate immune responses for immune homeostasis in the gut as well as clearance of fungal pathogens. Recent studies demonstrate that microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis (LAP) is involved in lectin-fungi interactions. Yet, the biological impact of LAP on the lectin function remains largely elusive. In this report, we demonstrate that in mouse LAP is linked to dendritic cell-associated lectin 2 (Dectin-2), a C-type lectin specific to fungal α-mannan polysaccharide. We found that mouse Dectin-2 recognizes commensal yeast S. cerevisiae and Kazachstania unispora. Mouse bone marrow-derived DCs (BMDCs) produced inflammatory cytokines TNFα and IL-1ß in response to the yeasts in a Dectin-2 and spleen tyrosine kinase (Syk)-dependent manner. We found that S. cerevisiae and K. unispora induced LAP in mouse BMDCs upon internalization. Furthermore, LC3 was activated by stimulation of BMDCs with the yeasts in a Dectin-2 and Syk-dependent manner. To address the biological impact of LAP on Dectin-2 yeast interaction, we established a knock-in mouse strain (Atg16L1E230, thereafter called E230), which BMDCs exhibit autophagy-active and LAP-negative phenotypes. When stimulated with yeasts, E230 BMDCs produced significantly less amounts of TNFα and IL-1ß. Taken together, we revealed a novel link between Dectin-2 and LAP that enables host immune cells to respond to mycobiota.

11.
Biochem Biophys Res Commun ; 494(3-4): 440-445, 2017 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-29042196

RESUMO

Dendritic cell inhibitory receptor 3 (DCIR3) is a member of dendritic immuno-receptor family, of which protein expression has been unknown. We established a specific monoclonal antibody against mouse DCIR3 and investigated the expression of DCIR3 on immune cells of various immune organs. We found that DCIR3 was expressed on monocytes, but not on eosinophils and neutrophils. We also found the existence of a dichotomy in the levels of the expression of DCIR3 on monocytes in bone marrow, blood and spleen. Further investigation of the expression of several cell surface markers on DCIR3High cells and DCIR3Low cells revealed that DCIR3High cells were Ly-6C- CD43High CD11c+ CD80+ NK1.1+ patrolling monocytes and that DCIR3Low cells were Ly-6C+ CD43Low CD11c- CD80- NK1.1- inflammatory monocytes. These results and our previous finding that DCIR4 is expressed at high level in patrolling monocytes and at a low level in inflammatory monocytes (Kameda et al., 2016) suggest that DCIR3 and DCIR4 are simultaneously expressed on monocytes. Indeed, DCIR4+ CD11b+ monocytes from various immune organs expressed DCIR3. We also found that DCIR1 was expressed on DCIR4Low inflammatory monocytes but not on DCIR4HIgh patrolling monocytes. The anti- DCIR3 antibody established in this study, together with the previously established anti-DCIR1 and anti-DCIR4 antibodies, would be a valuable tool to investigate biology and pathophysiology of monocytes.


Assuntos
Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/imunologia , Lectinas Tipo C/imunologia , Monócitos/imunologia , Receptores Imunológicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sangue/imunologia , Medula Óssea/imunologia , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/imunologia , Baço/imunologia
12.
Front Microbiol ; 8: 321, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28326063

RESUMO

The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB, upon interaction of L. reuteri ATCC PTA 6475 and ATCC 53608 strains with human monocyte-derived dendritic cells (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also indicated that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly, in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by atomic force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins.

13.
Biochem Biophys Res Commun ; 479(1): 1-4, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27524237

RESUMO

Intestinal microfold (M) cells are epithelial cells primarily present on Peyer's patches (PPs) in the small intestine. The ability of M cells to shuttle antigens into the PP for appropriate immune responses makes M cells a target for next-generation oral vaccine delivery. In this regard, discovery of M cell-specific receptors are of great interest, which could act as molecular tags for targeted delivery of cargo to M cells. Here, using a monoclonal antibody we generated to the Sialic acid-binding immunoglobulin-like lectin F (Siglec-F), we show that Siglec-F is expressed on mouse M cells in the small intestine. Immunohistochemical analysis of the PP tissue sections shows that Siglec-F is expressed on the surface of the M cell membrane exposed to the intestinal lumen. Anti-Siglec-F antibody injected into the mouse small intestine bound to M cells, demonstrating the potential to target M cells via Siglec-F.


Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Mucosa Intestinal/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Células CHO , Membrana Celular/imunologia , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Citometria de Fluxo , Células HEK293 , Humanos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Ligação Proteica/imunologia , Ratos Endogâmicos Lew , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
14.
J Biol Chem ; 291(34): 17629-38, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27358401

RESUMO

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-ß1,3]GalNAc-α1,3-GalNAc-ß1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.


Assuntos
Bactérias Gram-Negativas/imunologia , Lectinas Tipo C/imunologia , Células Mieloides/imunologia , Antígenos O/imunologia , Animais , Células HEK293 , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Lectinas Tipo C/genética , Masculino , Camundongos , Camundongos Knockout , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
15.
J Biol Chem ; 290(50): 30066-77, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26507663

RESUMO

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galß1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.


Assuntos
Linfócitos B/metabolismo , Centro Germinativo/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Sítios de Ligação , Humanos , Camundongos , Polissacarídeos/metabolismo
16.
Methods Mol Biol ; 1200: 337-51, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25117248

RESUMO

Animal lectins elicit biological functions through the interaction with glycan ligands. To clarify the functions of the lectins, both identification of their glycan ligand structure and assessment of impact of lectin-glycan interaction on the biological event are essential strategies. This chapter focuses on two of key useful methodologies for planning experiments based on the strategies. One is the detection of lectin-glycan interaction by the multivalent display of lectins and glycans. This methodology is a powerful means for identification of the glycan ligand structure and proteins and/or lipids carrying the glycan ligands for lectins. The other is the intervention of lectin-glycan interaction to assess the biological roles of lectins. Bioinformatics especially useful for animal lectins will be also described in this chapter. The concepts described in this chapter are versatile and applicable to a wide range of animal lectin research.


Assuntos
Biologia Computacional/métodos , Lectinas/metabolismo , Análise em Microsséries/métodos , Animais , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Ligantes , Polissacarídeos/metabolismo
17.
Methods Mol Biol ; 1200: 371-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25117251

RESUMO

Cell surface glycan remodeling is a useful method to modulate glycan-lectin interactions. In this chapter, a facile and reliable method to remodel mammalian cell surface N-glycans using inhibitors for N-glycan-processing enzymes is described. The method is widely applicable to many mammalian systems because those inhibitors work for the conserved glycosylation pathways among species.


Assuntos
Inibidores Enzimáticos/farmacologia , Polissacarídeos/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glicosilação/efeitos dos fármacos , Células HeLa , Humanos , Lectinas de Plantas/metabolismo , Coloração e Rotulagem , Fatores de Tempo
18.
J Immunol ; 193(4): 1560-6, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25000981

RESUMO

Lipids from mycobacteria can be presented to human T cells by group 1 CD1 Ag-presenting molecules (CD1a, CD1b, and CD1c). Group 1 CD1-restricted T cells are activated by lipid Ags presented by myeloid dendritic cells (DCs), after which they generate antibacterial effector functions, including IFN-γ secretion and cytolysis. Thus, mycobacterial lipids are being investigated as components of novel vaccines for mycobacterial infections. In this study we show that the mycobacterial lipid Ag C80 glucose-6-monomycolate can be delivered to human CD1b(+) DCs via targeted liposomal nanoparticles, leading to robust group 1 CD1-restricted activation of T cells. Targeting was achieved by decorating the liposomes with a high-affinity glycan ligand of sialic acid-binding Ig-like lectin (Siglec)-7, a siglec receptor expressed on DCs that mediates rapid endocytosis and transport of its cargo to lysosomes. An Ab to Siglec-7 completely blocked the binding of targeted liposomes to human monocyte-derived DCs (Mo-DCs), demonstrating their targeting specificity. Mo-DCs pulsed with targeted liposomes containing C80 glucose-6-monomycolate more potently activated a CD1b-restricted T cell line relative to Mo-DCs pulsed with free lipid Ag or antigenic liposomes without Siglec-7 ligand. These data suggest that the endocytic function of Siglec-7 can be exploited to deliver glycolipid Ags to their target cell and increase the efficiency of display to T cells.


Assuntos
Antígenos de Diferenciação Mielomonocítica/imunologia , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Glicolipídeos/imunologia , Lectinas/imunologia , Linfócitos T/imunologia , Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Antígenos CD1/imunologia , Linhagem Celular , Endocitose/imunologia , Glicolipídeos/administração & dosagem , Humanos , Interferon gama/metabolismo , Lipossomos/administração & dosagem , Lipossomos/imunologia , Ativação Linfocitária/imunologia , Lisossomos/imunologia , Mycobacterium/imunologia , Nanopartículas/administração & dosagem , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologia
19.
J Immunol ; 191(4): 1724-31, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23836061

RESUMO

Differentiation of self from nonself is indispensable for maintaining B cell tolerance in peripheral tissues. CD22 and Siglec-G (sialic acid-binding Ig-like lectin G) are two inhibitory coreceptors of the BCR that are implicated in maintenance of tolerance to self Ags. Enforced ligation of CD22 and the BCR by a nanoparticle displaying both Ag and CD22 ligands induces a tolerogenic circuit resulting in apoptosis of the Ag-reactive B cell. Whether Siglec-G also has this property has not been investigated in large part owing to the lack of a selective Siglec-G ligand. In this article, we report the development of a selective high-affinity ligand for Siglec-G and its application as a chemical tool to investigate the tolerogenic potential of Siglec-G. We find that liposomal nanoparticles decorated with Ag and Siglec-G ligand inhibit BCR signaling in both B1 and B2 B cells compared with liposomes displaying Ag alone. Not only is inhibition of B cell activation observed by ligating the BCR with Siglec-G, but robust tolerance toward T-independent and T-dependent Ags is also induced in mice. The ability of Siglec-G to inhibit B cell activation equally in both B1 and B2 subsets is consistent with our observation that Siglec-G is expressed at a relatively constant level throughout numerous B cell subsets. These results suggest that Siglec-G may contribute to maintenance of B cell tolerance toward self Ags in various B cell compartments.


Assuntos
Subpopulações de Linfócitos B/imunologia , Tolerância Imunológica/imunologia , Lectinas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sinalização do Cálcio , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Centro Germinativo/citologia , Memória Imunológica , Lectinas/biossíntese , Lectinas/genética , Ligantes , Lipossomos , Linfopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Especificidade de Órgãos , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
20.
Proc Natl Acad Sci U S A ; 110(19): 7826-31, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23610394

RESUMO

Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169(+) macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169(+) macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169(-/-) mice and is macrophage-dependent, demonstrating that targeting CD169(+) macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.


Assuntos
Lipídeos/imunologia , Macrófagos/metabolismo , Células T Matadoras Naturais/citologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Apresentação de Antígeno , Antígenos/imunologia , Linhagem Celular , Células Dendríticas/citologia , Endocitose , Glicolipídeos/imunologia , Humanos , Ligantes , Lipossomos/metabolismo , Fígado/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia
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