RESUMO
Exhausted immune responses to chronic diseases represent a major challenge to global health. We study CD4+ T cells in a mouse model with regulatable antigen presentation. When the cells are driven through the effector phase and are then exposed to different levels of persistent antigen, they lose their T helper 1 (Th1) functions, upregulate exhaustion markers, resemble naturally anergic cells, and modulate their MAPK, mTORC1, and Ca2+/calcineurin signaling pathways with increasing dose and time. They also become unable to help B cells and, at the highest dose, undergo apoptosis. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of T cell receptor (TCR) signals over a period of weeks. Upon antigen removal, the cells recover their functionality while losing exhaustion and anergy markers. Our data suggest an adjustable response of CD4+ T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease.
Assuntos
Antígenos/imunologia , Sinalização do Cálcio/imunologia , Anergia Clonal , Regulação da Expressão Gênica/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Células Th1/imunologia , Animais , Antígenos/genética , Linfócitos B/imunologia , Sinalização do Cálcio/genética , Feminino , Perfilação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLeA, as well as its defucosylated form sialyl LeC (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galß1-3GlcNAcß (LeC), thus resembling specificity of human natural cancer-associated anti-LeC antibodies. We propose that cancer-specific glycopeptide epitope includes Galß1-3GlcNAcß fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.
Assuntos
Anticorpos Monoclonais/imunologia , Antígeno CA-19-9/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Trissacarídeos/imunologia , Animais , Anticorpos Monoclonais/genética , Antígeno CA-19-9/genética , Epitopos/genética , Glicopeptídeos/genética , Glicopeptídeos/imunologia , Humanos , Camundongos , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/imunologia , Neoplasias/genética , Trissacarídeos/genéticaRESUMO
Aberrant glycosylation is one of the major hallmarks of cancer with altered gene expression signatures of sialyltransferases. ST3Gal-I, a sialyltransferase, is known to play a crucial role in sialylation of T antigen in bladder cancer and it has reported elevated expression in breast carcinogenesis with increased tumor progression stages. The aim of the current study is to develop new monoclonal antibodies (mAbs) against human ST3Gal-I and evaluate their diagnostic potential. We developed a repertoire of stable hybridoma cell lines producing high-affinity IgG antibodies against recombinant human ST3Gal-I, expressed in E. coli BL21-DE3 strain. In order to demonstrate the diagnostic value of the mAbs, various clones were employed for the immunohistochemistry analysis of ST3Gal-I expression in cancerous tissues. Antibodies generated by 7E51C83A10 clone demonstrated a strong and specific fluorescence staining in breast cancer tissue sections and did not exhibit significant background in fibroadenoma sections. In conclusion, the mAbs raised against recombinant ST3Gal-I recognize cellular ST3Gal-I and represent a promising diagnostic tool for the immunodetection of ST3Gal-I expressing cells. Specific-reactivity of clone 7E51C83A10 mAbs towards ST3Gal-I was also confirmed by immunoblotting. Therefore, our observations warrant evaluation of ST3Gal-I as a potential marker for cancer diagnosis at larger scale.