RESUMO
Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order-disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order-disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.
Assuntos
Anticorpos Bloqueadores/metabolismo , Molécula 1 de Adesão Celular/metabolismo , Epitopos/imunologia , Imunoglobulinas/imunologia , Animais , Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , CoelhosRESUMO
Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.
Assuntos
Molécula 1 de Adesão Celular/genética , Sistema Livre de Células/química , Domínios de Imunoglobulina/genética , Imunoglobulinas/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Sítios de Ligação , Molécula 1 de Adesão Celular/imunologia , Molécula 1 de Adesão Celular/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hibridomas/química , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Due to their increasing rates of morbidity and mortality, childhood malignancies are considered a global health priority, with acute lymphoblastic leukemias (ALLs) showing the highest incidence worldwide. Control of malignant clone emergence and the subsequent normal-leukemic hematopoietic cell out-competition require antitumor monitoring mechanisms. Investigation of cancer surveillance innate cells may be critical to understand the mechanisms contributing in either disease progression or relapse, and to promote displacement of leukemic hematopoiesis by the normal counterpart. We report here that NK cell production is less and low hematopoietic progenitor numbers contribute to this defect. By investigating the expression of the activation molecule class I restricted T-cell associated molecule (CRTAM) along the hematopoietic lineage differentiation pathway, we have identified lymphoid precursor populations coexpressing CD34, CD56/CD3/CD19, and CRTAM as the earliest developmental stage where activation may take place in specialized niches that display the ligand nectin-like-2. Of note, bone marrow (BM) from patients with ALL revealed high contents of preactivated CD56high NK cells expressing CRTAM and endowed with an exhaustion-like phenotype and the functional capability of producing IL-10 and TGF-ß in vitro. Our findings suggest, for the first time, that the tumor microenvironment in ALL directly contribute to exhaustion of NK cell functions by the CRTAM/Necl-2 interaction, and that the potential regulatory role of exhausted-like NK cells may favor malignant progression at the expense of anti-tumor responses. Phenotypic and functional identity of this unique suppressor-like NK cell population within the leukemic BM would be of special interest for the pathobiology of ALL and development of targeting strategies.
Assuntos
Medula Óssea/imunologia , Molécula 1 de Adesão Celular/genética , Proteínas da Matriz Extracelular/genética , Células Matadoras Naturais/imunologia , Chaperonas Moleculares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Microambiente Tumoral/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Medula Óssea/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Molécula 1 de Adesão Celular/imunologia , Diferenciação Celular , Criança , Técnicas de Cocultura , Citotoxicidade Imunológica , Proteínas da Matriz Extracelular/imunologia , Regulação da Expressão Gênica , Humanos , Vigilância Imunológica , Imunofenotipagem , Interleucina-10/genética , Interleucina-10/imunologia , Células K562 , Células Matadoras Naturais/patologia , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/patologia , Chaperonas Moleculares/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Microambiente Tumoral/genéticaRESUMO
Class I-restricted T cell-associated molecule (CRTAM) is an activation marker expressed on the cell surface of activated invariant natural killer T (iNKT) cells, CD8+ T cells, and a small subset of CD4+ T cells. CRTAM has also been associated with a proinflammatory profile in murine CD4+ T cells. However, CRTAM has not been thoroughly explored in human cells. This work focused on evaluating CRTAM expression in human iNKT lymphocytes after activation with α-galactosylceramide, its widely used specific glycolipid antigen. We also analyzed the involvement of costimulatory molecules in CRTAM expression and whether CRTAM expression is associated with a specific effector cytokine profile. We found that the signal produced by invariant T cell receptor (iTCR) engagement with α-galactosylceramide is sufficient to trigger CRTAM expression on human iNKT cells after 18 h of stimulation. Moreover, we observed a clear association between CRTAM expression and IFN-γ production in iNKT cells from healthy subjects and patients with type 1 diabetes. However, blocking the engagement of costimulatory molecules, such as CD40, CD80, and CD86, did not modify CRTAM expression. These results indicate that CRTAM may also play a role in triggering the production of IFN-γ in human iNKT cells and that CRTAM could be used as a marker to identify these inflammatory cells.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Imunoglobulinas/metabolismo , Interferon gama/biossíntese , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Biomarcadores , Estudos de Casos e Controles , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Humanos , Mediadores da Inflamação/metabolismoRESUMO
T cell activation leads to the induction of genes that are required for appropriate immune responses. This includes CRTAM (Class-I MHC-restricted T cell associated molecule), a protein that plays a key role in T cell development, proliferation, and generating cell polarity during activation. We previously characterized the CRTAM promoter and described how AP-1 family members are important for inducing CRTAM expression upon antigenic activation. Here, we show that CRTAM is a molecular target for ZEB1 (zinc finger E-box-binding protein), a homeodomain/Zn finger transcription factor. Overexpression of ZEB1 repressed CRTAM promoter activity, as well as endogenous CRTAM levels in human T cells. ZEB1-mediated transcriptional repression was abolished when E-box-like elements in the CRTAM promoter are mutated. In summary, ZEB1 functions as a transcriptional repressor for the CRTAM gene in both non-stimulated and stimulated T cells, thereby modulating adaptive immune responses.