RESUMO
Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.
Assuntos
Linfócitos B/imunologia , Ligante de CD40/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Linfócitos T Auxiliares-Indutores/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Ligante de CD40/imunologia , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/imunologia , Especificidade da Espécie , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
Measurement of the urinary albumin excretion rate (UAER) is essential for the early diagnosis and monitoring of diabetic nephropathy; immunonephelometry is a procedure used worldwide for routine screening of diabetic patients. Since we have met with occasional inconsistent values of UAER in serial urine collections, we searched for possible sources of analytic error. To assess the best working conditions of the instrument in use, the stability of urine samples during storage and the need for previous urine centrifugation, we assayed repeatedly the six automatically diluted points of the standard curve (55.6 to 1.7 mg/l), four control samples of human albumin in saline (100 to 1 mg/l) and 24-h urine collections from outpatient diabetic subjects. The last were also assayed with and without previous centrifugation, and both immediately after collection as well as after storage at -20 degrees C for 7, 42, 79, 97, 128 and 161 days. We concluded that: (1) pre-analytic centrifugation of urine samples in unnecessary; (2) the intra-assay coefficient of variation (CV) of the standard curve changed from 2.4% to 9.3% when moving from the highest to the lowest concentration; the inter-assay CV changed from 4.1% to 14.4%, respectively; (3) the intra-assay CV of the control samples (manually prepared) changed from 5.7% to 10.2% and the inter-assay CV from 7.7% to 22.9%; there was a constant and significant (P < 0.01) underestimation (from -9% to -30%) of the obtained values compared with the expected concentrations; (4) a progressive decrease in recovered albumin by multiple freezing and thawing of urine samples did occur, which became significant after 161 days of storage. In the BNA workbook (menu 7.1, assay protocols), a 7-day validity of the reference curve is reported. Moreover, to economize, pre-dilution cuvettes were often recycled in our hospital central laboratory. We observed that: the intra-assay CV for urine samples was 79.4% with recycled cuvettes and stored standard curve, 11.3% with new cuvettes and stored standard curve, 4.9% with both new cuvettes and newly performed standard curve; the inter-assay CV was 32.6%, 10.5% and 6.4%, respectively. These data emphasize, from the laboratory viewpoint, the need for both accurate calibration of BNA and use of native urines; in addition, they stress the importance of careful supervision of laboratory routine and interpreting analytic results in the clinical setting.