RESUMO
Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here we investigated whether recently proposed 'open' and 'closed' conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the 'closed' and 'open' conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of wildtype (WT) protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution dSTORM microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of 'closed' CD53 was higher and of 'open' CD81 lower than respective WT protein. In addition, knock-out cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, 'closed' CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19, and 'closed' CD81 interacted less with CD19 than WT CD81, but 'open' CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.
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Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular components that regulate DC cytokine secretion are poorly characterised. Galectin-9, a ß-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and sheds new light into the multiple roles galectins play in governing cell function.
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Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different receptors on the plasma membrane. Most tetraspanins are highly glycosylated at their large extracellular loop; however, little is known about the function of tetraspanin glycosylation in immune cells. In this study we investigated the effects of glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants of CD37 and CD53 and analyzed their localization, nanoscale plasma membrane organization, and partner protein interaction capacity. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas surface expression of CD53 was unaffected by its glycosylation. Single-molecule dSTORM microscopy revealed that the nanoscale organization of CD53 was not dependent on glycosylation. CD37 interaction with its partner proteins CD53 and CD20 was affected by glycosylation in a localization-dependent way, whereas its interaction with IL-6Rα was independent of glycosylation. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45, CD20, and, to a lesser extent CD37. Together, our data show that glycosylation affects the interaction capacity of immune-specific tetraspanins CD37 and CD53, which adds another layer of regulation to immune membrane organization.
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AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region.
Assuntos
Linfoma Folicular , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Rearranjo Gênico , Linfoma Folicular/patologia , Hibridização in Situ Fluorescente , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genéticaRESUMO
Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.
Assuntos
Antígenos de Neoplasias/genética , Privilégio Imunológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Tetraspaninas/genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/imunologia , Neoplasias do Sistema Nervoso Central/patologia , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Frequência do Gene , Inativação Gênica , Humanos , Privilégio Imunológico/genética , Linfoma Difuso de Grandes Células B/epidemiologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Mutação , Neoplasias Testiculares/genética , Neoplasias Testiculares/imunologia , Neoplasias Testiculares/patologia , Testículo/imunologia , Testículo/patologia , Tetraspaninas/química , Tetraspaninas/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologiaRESUMO
Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.
Assuntos
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Tetraspaninas/metabolismo , Actomiosina/metabolismo , Animais , Adesão Celular , Extensões da Superfície Celular/metabolismo , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Interleucina-6/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Ligação Proteica , Células RAW 264.7 , Tetraspaninas/deficiênciaRESUMO
Immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is characterized by IgA depositions in the kidney. Deficiency of CD37, a leukocyte-specific tetraspanin, leads to spontaneous development of renal pathology resembling IgAN. However, the underlying molecular mechanism has not been resolved. Here we found that CD37 expression on B cells of patients with IgAN was significantly decreased compared to B cells of healthy donors. Circulating interleukin (IL)-6 levels, but not tumor necrosis factor-α or IL-10, were elevated in Cd37-/- mice compared to wild-type mice after lipopolysaccharide treatment. Cd37-/- mice displayed increased glomerular neutrophil influx, immune complex deposition, and worse renal function. To evaluate the role of IL-6 in the pathogenesis of accelerated renal pathology in Cd37-/-mice, we generated Cd37xIl6 double-knockout mice. These double-knockout and Il6-/- mice displayed no glomerular IgA deposition and were protected from exacerbated renal failure following lipopolysaccharide treatment. Moreover, kidneys of Cd37-/- mice showed more mesangial proliferation, endothelial cell activation, podocyte activation, and segmental podocyte foot process effacement compared to the double-knockout mice, emphasizing that IL-6 mediates renal pathology in Cd37-/- mice. Thus, our study indicates that CD37 may protect against IgA nephropathy by inhibition of the IL-6 pathway.
Assuntos
Glomerulonefrite por IGA/metabolismo , Imunoglobulina A/metabolismo , Interleucina-6/metabolismo , Glomérulos Renais/metabolismo , Tetraspaninas/deficiência , Albuminúria/imunologia , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Antígenos CD/genética , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Modelos Animais de Doenças , Predisposição Genética para Doença , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/prevenção & controle , Humanos , Imunoglobulina A/imunologia , Interleucina-6/deficiência , Interleucina-6/genética , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Fenótipo , Podócitos/imunologia , Podócitos/metabolismo , Podócitos/patologia , Tetraspaninas/sangue , Tetraspaninas/genéticaRESUMO
CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in â¼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.
Assuntos
Linfócitos B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/patologia , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Análise Multivariada , Mutação/genética , NF-kappa B/metabolismo , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Análise de Sobrevida , Tetraspaninas/genética , Tetraspaninas/metabolismo , Resultado do Tratamento , Proteína Supressora de Tumor p53/genéticaRESUMO
Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.
Assuntos
Citoesqueleto de Actina/metabolismo , Antígenos CD/fisiologia , Movimento Celular/fisiologia , Quimiocina CCL21/metabolismo , Células Dendríticas/fisiologia , Matriz Extracelular/metabolismo , Semaforinas/fisiologia , Animais , Antígenos CD/genética , Adesão Celular , Movimento Celular/genética , Células Cultivadas , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Humanos , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Interferência de RNA , RNA Interferente Pequeno , Semaforinas/genéticaRESUMO
This study supports a new concept where the opposing functions of the tetraspanins CD37 and CD82 may coordinate changes in migration and Ag presentation during dendritic cell (DC) activation. We have previously published that CD37 is downregulated upon monocyte-derived DC activation, promotes migration of both skin and bone marrow-derived dendritic cells (BMDCs), and restrains Ag presentation in splenic and BMDCs. In this article, we show that CD82, the closest phylogenetic relative to CD37, appears to have opposing functions. CD82 is upregulated upon activation of BMDCs and monocyte-derived DCs, restrains migration of skin and BMDCs, supports MHC class II maturation, and promotes stable interactions between T cells and splenic DCs or BMDCs. The underlying mechanism involves the rearrangement of the cytoskeleton via a differential activation of small GTPases. Both CD37(-/-) and CD82(-/-) BMDCs lack cellular projections, but where CD37(-/-) BMDCs spread poorly on fibronectin, CD82(-/-) BMDCs are large and spread to a greater extent than wild-type BMDCs. At the molecular level, CD82 is a negative regulator of RhoA, whereas CD37 promotes activation of Rac-1; both tetraspanins negatively regulate Cdc42. Thus, this study identifies a key aspect of DC biology: an unactivated BMDC is CD37(hi)CD82(lo), resulting in a highly motile cell with a limited ability to activate naive T cells. By contrast, a late activated BMDC is CD37(lo)CD82(hi), and thus has modified its migratory, cytoskeletal, and Ag presentation machinery to become a cell superbly adapted to activating naive T cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Movimento Celular , Células Dendríticas/imunologia , Proteína Kangai-1/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Tetraspaninas/imunologia , Animais , Separação Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da PolimeraseRESUMO
To facilitate the myriad of different (signaling) processes that take place at the plasma membrane, cells depend on a high degree of membrane protein organization. Important mediators of this organization are tetraspanin proteins. Tetraspanins interact laterally among themselves and with partner proteins to control the spatial organization of membrane proteins in large networks called the tetraspanin web. The molecular interactions underlying the formation of the tetraspanin web were hitherto mainly described based on their resistance to different detergents, a classification which does not necessarily correlate with functionality in the living cell. To look at these interactions from a more physiological point of view, this review discusses tetraspanin interactions based on their function in the tetraspanin web: (1) intramolecular interactions supporting tetraspanin structure, (2) tetraspanin-tetraspanin interactions supporting web formation, (3) tetraspanin-partner interactions adding functional partners to the web and (4) cytosolic tetraspanin interactions regulating intracellular signaling. The recent publication of the first full-length tetraspanin crystal structure sheds new light on both the intra- and intermolecular tetraspanin interactions that shape the tetraspanin web. Furthermore, recent molecular dynamic modeling studies indicate that the binding strength between tetraspanins and between tetraspanins and their partners is the complex sum of both promiscuous and specific interactions. A deeper insight into this complex mixture of interactions is essential to our fundamental understanding of the tetraspanin web and its dynamics which constitute a basic building block of the cell surface.
Assuntos
Transdução de Sinais , Tetraspaninas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.
Assuntos
Actinas/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Citoesqueleto/imunologia , Neutrófilos/fisiologia , Tetraspaninas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígenos CD18/metabolismo , Adesão Celular , Movimento Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiotaxia/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Tetraspaninas/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.
Assuntos
Antígenos de Neoplasias/análise , Tecido Linfoide/química , Tecido Linfoide/metabolismo , Tetraspanina 25/análise , Tetraspaninas/análise , Antígenos de Neoplasias/biossíntese , Humanos , Imuno-Histoquímica , Tecido Linfoide/citologia , Microscopia Confocal , Baço/química , Baço/citologia , Baço/metabolismo , Tetraspanina 25/biossíntese , Tetraspaninas/biossínteseRESUMO
Previous studies on the role of the tetraspanin CD37 in cellular immunity appear contradictory. In vitro approaches indicate a negative regulatory role, whereas in vivo studies suggest that CD37 is necessary for optimal cellular responses. To resolve this discrepancy, we studied the adaptive cellular immune responses of CD37(-/-) mice to intradermal challenge with either tumors or model antigens and found that CD37 is essential for optimal cell-mediated immunity. We provide evidence that an increased susceptibility to tumors observed in CD37(-/-) mice coincides with a striking failure to induce antigen-specific IFN-γ-secreting T cells. We also show that CD37 ablation impairs several aspects of DC function including: in vivo migration from skin to draining lymph nodes; chemo-tactic migration; integrin-mediated adhesion under flow; the ability to spread and form actin protrusions and in vivo priming of adoptively transferred naïve T cells. In addition, multiphoton microscopy-based assessment of dermal DC migration demonstrated a reduced rate of migration and increased randomness of DC migration in CD37(-/-) mice. Together, these studies are consistent with a model in which the cellular defect that underlies poor cellular immune induction in CD37(-/-) mice is impaired DC migration.
Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Tetraspaninas/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Adesão Celular/imunologia , Proliferação de Células , Células Dendríticas/patologia , Feminino , Expressão Gênica , Interferon gama/biossíntese , Interferon gama/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Transplante de Neoplasias , Pele/imunologia , Pele/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/transplante , Tetraspaninas/deficiência , Tetraspaninas/genéticaRESUMO
Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.
Assuntos
Galectinas , Tetraspaninas , Humanos , Galectinas/análise , Galectinas/metabolismo , Tetraspaninas/análise , Tetraspaninas/química , Tetraspaninas/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Transdução de SinaisRESUMO
Fungal pattern-recognition receptors (F-PRRs), including C-type lectins, Toll-like receptors, scavenger receptors and Fc/complement receptors, are crucial for inducing anti-fungal immune responses by antigen-presenting cells. The recent identification of specific F-PRR interactions with tetraspanins has shed new light on the functioning of F-PRRs in the cell membrane and subsequent downstream signaling. Tetraspanins are small four-transmembrane proteins that can assemble immune receptors and signaling molecules into functional membrane microdomains. Here, we discuss the implications of this novel type of interaction between F-PRRs and tetraspanins in different subsets of antigen-presenting cells. We postulate that upon fungal binding tetraspanins modulate the function of F-PRRs by their recruitment into tetraspanin microdomains, leading to immune activation or tolerance.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Fungos/imunologia , Proteínas de Membrana/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Imunológicos , Fagócitos/imunologia , Fagócitos/metabolismo , Ligação Proteica/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologiaRESUMO
Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.
Assuntos
Melanoma , Linfócitos T , Humanos , Células Apresentadoras de Antígenos , Melanoma/terapia , Imunoterapia , Imunoterapia AdotivaRESUMO
Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.
RESUMO
Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.
Assuntos
Candidíase/genética , Códon sem Sentido , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Onicomicose/genética , Animais , Candida albicans/imunologia , Candidíase/imunologia , Candidíase Mucocutânea Crônica/genética , Candidíase Vulvovaginal/genética , Citocinas/biossíntese , Feminino , Predisposição Genética para Doença , Humanos , Lectinas Tipo C , Masculino , Mamíferos/genética , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , LinhagemRESUMO
The cooperative nature of tetraspanin-tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37(-/-) mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize "double knockout" mice (CD37(-/-)Tssc6(-/-)) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37(-/-)Tssc6(-/-) mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37(-/-)Tssc6(-/-) mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37(-/-)Tssc6(-/-) mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37(-/-) or Tssc6(-/-) mice, demonstrating a complementary role for these two molecules in cellular immunity.