RESUMO
OBJECTIVES: Microbial translocation and chronic immune activation were previously shown to be associated with impairment of T cell functions and disease progression during infection with human immunodeficiency virus type-1 (HIV-1); however, their impact on B cell function and number remains unknown. By measuring markers of immune activation and molecules involved in apoptosis regulation, we have evaluated the association between microbial translocation and loss of memory B cells in HIV-1-infected patients. METHODS: Markers of activation [the interleukin-21 receptor (IL-21R) and CD38] and apoptosis (Bim, Bcl-2 and annexin V) were measured in B cell subpopulations by multicolour flow cytometry. Levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS), measures of microbial translocation, were determined in plasma. Purified B cells were also exposed in vitro to Toll-like receptor (TLR) ligands. RESULTS: IL-21R expression was higher in cells from HIV-1-infected patients, compared with control subjects, with the highest levels in nontreated patients. An inverse correlation was observed between IL-21R expression and percentages of circulating resting memory (RM) B cells. IL-21R-positive memory B cells were also more susceptible to spontaneous apoptosis and displayed lower levels of Bcl-2. It is interesting that the levels of sCD14, which are increased during HIV-1 infection, were correlated with decreased percentages of RM B cells and high IL-21R expression. In the plasma of HIV-1-infected individuals, a correlation was found between sCD14 and LPS levels. TLR activation of B cells in vitro resulted in IL-21R up-regulation. CONCLUSIONS: Microbial translocation and the associated immune activation during HIV-1 infection may lead to high expression levels of the IL-21R activation marker in RM B cells, a feature associated with increased apoptosis and a reduced number of these cells in the circulation.
Assuntos
Linfócitos B/imunologia , Infecções por HIV/imunologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-21/metabolismo , Fatores Etários , Apoptose , Translocação Bacteriana/fisiologia , Biomarcadores , Estudos de Casos e Controles , HIV-1/imunologia , Humanos , SuéciaRESUMO
OBJECTIVES: Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. DESIGN: We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. RESULTS: We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. CONCLUSIONS: Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.
Assuntos
Infecções por HIV/imunologia , HIV-1 , Interleucina-1beta/imunologia , Interleucina-7/biossíntese , Linfócitos T/imunologia , Apoptose/imunologia , Células da Medula Óssea/imunologia , Citocinas/imunologia , Células Epiteliais/imunologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade nas Mucosas , Interferon gama/imunologia , Interleucina-7/genética , Modelos Imunológicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , Receptores de Quimiocinas/metabolismo , Células Estromais/imunologia , Células Tumorais Cultivadas , Receptor fas/metabolismoRESUMO
Defects in cell surface expression of major histocompatibility complex class I antigen molecules are common in tumour cells. We have previously described the generation of adaptive immunity to tumour cells deficient in the transporter associated with antigen processing molecule. In this study, we demonstrate enhanced in vivo protection against growth of beta(2)-microglobulin-deficient tumour cells in syngeneic C57Bl/6 mice, following vaccination with beta(2)-microglobulin-deficient dendritic cells. In vitro analysis suggested that vaccinated mice produced CD3+ cells, which could induce apoptosis in syngeneic beta(2)-microglobulin-deficient tumour and non-malignant cells. Further investigation of target cell recognition suggested that also tumour cells lacking expression of classical major histocompatibility complex class I heavy chains and functional transporter associated with antigen processing molecules were recognized by CD3+ effector cells from vaccinated mice. Histopathological examination of organs from vaccinated mice showed no significant vaccination-induced pathology. The present findings point to a new possible strategy to counteract the growth of major histocompatibility complex class I-deficient tumour cells.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/transplante , Neoplasias Experimentais/terapia , Microglobulina beta-2/deficiência , Animais , Apoptose/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Microglobulina beta-2/imunologiaRESUMO
The synthetic peptide comprising the 317-341 region of human influenza A virus (H1N1 subtype) hemagglutinin elicits peptide-specific antibody and helper T cell responses and confers protection against lethal virus infection. Molecular mapping of the 317-329 region, which encompasses the epitope recognized by peptide-specific T cells, revealed that the minimal size required for T cell activation was the 317-326 segment. The most likely peptide alignment, which placed 320Leu to pocket 1 of the I-E(d) peptide binding groove, was predicted by molecular mechanics calculations performed with the parental and with the Ala-substituted analogs. In line with the prediction data, the results of the peptide binding assay, where the relative binding efficiency to I-E(d) molecules expressed on the surface of antigen-presenting cells was monitored, identified the 320-326 core sequence interacting with the major histocompatibility class II peptide binding groove. Functional analysis of Ala-substituted variants by functional assays and by calculating the surface-accessible areas of the single peptidic amino acids in the I-E(d)-peptide complexes demonstrated that 324Pro is a primary contact residue for the T cell receptor. Our results show that this type of analysis offers a suitable tool for molecular mapping of helper T cell epitopes and thus provides valuable data for subunit vaccine design.