RESUMO
Antigen-specific T helper cells present in peripheral blood at very low frequencies are capable of rapid clonal expansion during antigenic challenge. The exquisite specificity of this response provides for activation and expansion of a very select cohort of T cells, a feature we have used to directly identify and quantify human epitope-specific T helper cells from peripheral blood. Soluble tetramerized class II MHC molecules, loaded with an immunodominant peptide from hemagglutinin (HA) and labeled with fluorescent dyes, were constructed and used to directly identify antigen-specific T cells from influenza-immune individuals. After 7 days of proliferation in response to stimulation by HA peptide or whole influenza vaccine, cells staining positive with the HA tetramer had undergone between 6 and 9 divisions and were CD3(+), CD4(+), CD25(+), and CD8(-), characteristic of activated T helper cells responding to antigen. The HA epitope-specific component of the complex response to whole influenza vaccine represented a major subset of proliferating T helper cells. Soluble class II tetramers allow a direct approach for the analysis of immunodominant antigenic specificities. The identification of antigen-specific T helper cells in the peripheral blood provides a means for tracking the immune response against infectious agents and in autoimmune disease. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vírus da Influenza A/imunologia , Fragmentos de Peptídeos/imunologia , Antígenos de Histocompatibilidade Classe II/química , Humanos , Ativação LinfocitáriaRESUMO
BACKGROUND: Initial doses of OKT3 are associated with a cytokine-induced acute clinical syndrome (ACS). This study assessed the safety of a recombinant human tumor necrosis factor receptor fusion protein (TNFR:Fc) given to minimize OKT3-ACS symptoms in renal allograft recipients undergoing induction therapy. METHODS: Sixteen patients were randomized into treatment or control groups. Treated patients received TNFR:Fc 1 hr before OKT3 on days 0 and 3. Patients were monitored after transplant for OKT3-ACS symptoms. Levels of cytokines, serum creatinine, and C-reactive protein were followed. RESULTS: Patients receiving TNFR:Fc had lower OKT3-ACS symptoms as measured by a scoring system. There was a higher incidence of infection in treated patients (10/12) compared to controls (1/4) in the 3 months after transplant, but the etiology of this difference was unclear. There were no significant differences in cytokine profiles. CONCLUSIONS: TNFR:Fc is well tolerated by renal transplant patients receiving OKT3 induction therapy and modestly decreases the symptoms associated with OKT3-ACS.
Assuntos
Fragmentos Fc das Imunoglobulinas/uso terapêutico , Transplante de Rim , Muromonab-CD3/efeitos adversos , Receptores do Fator de Necrose Tumoral/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Doença Aguda , Humanos , Interleucina-6/sangue , Transplante HomólogoAssuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/metabolismo , Gravidez em Diabéticas/imunologia , Adolescente , Adulto , Glicemia/metabolismo , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Feminino , Glutamato Descarboxilase/imunologia , Hemoglobinas Glicadas/análise , Humanos , Insulina/uso terapêutico , Isoenzimas/imunologia , Gravidez , Gravidez em Diabéticas/sangueRESUMO
Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.
Assuntos
Cálcio/metabolismo , Citometria de Fluxo/métodos , Compostos de Anilina , Benzofuranos , Citometria de Fluxo/estatística & dados numéricos , Corantes Fluorescentes , Humanos , Imidazóis , Técnicas In Vitro , Indóis , Líquido Intracelular/metabolismo , Linfócitos/metabolismo , Sensibilidade e Especificidade , XantenosRESUMO
Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Deleção Clonal , Antígeno HLA-DR4/imunologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Biopolímeros , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Epitopos/imunologia , Genótipo , Antígenos HLA-DR/imunologia , Antígeno HLA-DR4/química , Cadeias HLA-DRB1 , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Ligantes , Linfocinas/metabolismo , Substâncias Macromoleculares , Camundongos , Fragmentos de Peptídeos/imunologia , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância a Antígenos Próprios/imunologia , Transfecção , Microglobulina beta-2/deficiência , Microglobulina beta-2/genéticaRESUMO
Antigen-specific T cells acquire a distinctive phenotype during activation, with characteristic acquisition of surface markers and patterns of gene expression. Early after antigen stimulation, CD4(+) T lymphocytes increase their surface density of the CD4 marker, a trait which has been used to identify antigen-activated cells. The recent development of MHC tetramer technologies has greatly improved the ability to detect HLA class I-restricted T cells specific for known antigen epitopes. We have recently extended these studies to human class II-restricted CD4(+) T cell responses and now describe antigen-specific T cell responses from human peripheral blood in which elevated CD4 expression levels in human T cells following antigen stimulation identify the activated and proliferating subset of cells. The CD4(high) population is substantially enriched in epitope-specific cells identified by class II tetramer staining and almost all tetramer-positive cells are contained within the CD4(high) population. T cell clones derived from the tetramer-positive, CD4(high) population demonstrate antigen specificity and maintain tetramer staining, while the substantial number of CD4(high) cells which fail to stain with tetramer appear to proliferate as a result of bystander activation. Epitope-specific components of a polyclonal immune response are directly visualized and quantitated by tetramer detection, providing a direct measure of the heterogeneity of the human immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Antígenos CD4/análise , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular/genética , Divisão Celular/imunologia , Epitopos de Linfócito T/análise , Epitopos de Linfócito T/genética , Citometria de Fluxo , Humanos , Imunofenotipagem , Ativação Linfocitária/genética , Coloração e Rotulagem , Subpopulações de Linfócitos T/metabolismoRESUMO
T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.
Assuntos
Linfócitos T CD4-Positivos/química , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/análise , Epitopos Imunodominantes/análise , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Transformada , Células Clonais/química , Células Clonais/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Antígenos HLA-DR/análise , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/metabolismo , Cadeias alfa de HLA-DR , Cadeias HLA-DRB1 , Proteína Vmw65 do Vírus do Herpes Simples/análise , Proteína Vmw65 do Vírus do Herpes Simples/biossíntese , Proteína Vmw65 do Vírus do Herpes Simples/imunologia , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Herpesvirus Humano 2/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária , Substâncias Macromoleculares , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologiaRESUMO
Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.