RESUMO
The binding of immunogenic peptides to DR molecules is influenced by residues that point into the peptide-binding groove. The T-cell response toward a peptide complexed to an MHC molecule depends on the presence of a sufficient number of T cells reactive with peptide-MHC complex on the surface of APCs. From 96 overlapping HIV peptides, we have selected 11 that show a significant binding to either DR1, DR103, or both. These two DR molecules are identical except for three amino acids at positions 67, 70, and 71 on the beta chain. Peptide-specific T-cell lines and clones were generated with cells from nonimmunized donors homozygous for DR1 or DR103 by using either individual peptides or peptide pools for the in vitro priming. Three of the peptides induced T-cell-specific proliferative response in both individuals, and these peptides were not among those with highest affinity. Most of the peptides induced strong responses against autologous APCs. This might reflect cross-reactivity between HIV and self-peptides. Definition of peptides that both show promiscuous binding to DR and elicit a strong T-cell response is important for design of efficient synthetic vaccines.
Assuntos
Antígenos HIV/imunologia , Antígenos HLA-DR/metabolismo , Linfócitos T/imunologia , Vacinas contra a AIDS/imunologia , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , Antígeno HLA-DR1/metabolismo , Humanos , Ativação Linfocitária/imunologiaRESUMO
Daudi Burkitt's lymphoma cells activate Vgamma9/Vdelta2 T cells through TCR ligation by an unknown antigen. This activity is for a large part revealed by their lack of HLA class I antigen expression, allowing their escape from KIR downregulation. We characterize here a culture variant of the Burkitt's lymphoma line Raji, RJ-A3, which is able to promote as efficiently as Daudi cells the outgrowth of Vgamma9/Vdelta2 T cells in cocultures in spite of unchanged HLA class Ia/Ib antigen expression. RJ-A3 is resistant to lysis by most Vgamma9/Vdelta2 lines and clones, even those lacking CD9-4/NKG2 and p58, p70 p140 KIR molecules. However, one Vgamma9/Vdelta2 line which can efficiently kill RJ-A3 do so in a TCR-dependent manner since killing is modulated by anti-TCR antibodies. The CDR3 sequences of the T cell clones amplified with Daudi and RJ-A3 reveal that some clones can be expanded with both lines while others are expanded preferentially with one or the other but not both. This indicates differences in the antigenic determinants of the two Burkitt's lines. The occurrence of this Raji variant line demonstrates that the stimulatory phenotype for Vgamma9/Vdelta2 cells can be acquired by some tumors independently of the loss of class I antigens and comforts the hypothesis of an anti-tumoral function for the Vgamma9/Vdelta2 T cell population.
Assuntos
Linfoma de Burkitt/imunologia , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/imunologia , Linfoma de Burkitt/virologia , Células Clonais , Técnicas de Cocultura , Citotoxicidade Imunológica , Expressão Gênica , Antígenos HLA-A , Antígenos HLA-B , Herpesvirus Humano 4/isolamento & purificação , Humanos , Região Variável de Imunoglobulina , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T Citotóxicos , Células Tumorais CultivadasRESUMO
In adult vessels the proliferation rate of differentiated endothelial cells is very low. In response to several environmental stimuli the expression of so-called 'angiogenic factors' is upregulated and the messenger RNAs are actively translated in secreted factors which induce the proliferation of endothelial cells; the digestion of their basement membrane then allows their migration and differentiation. Considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis. Vascular endothelial growth factor turned out to represent the major inducer of angiogenesis. Optional splicing of its pre-messenger RNA generates various isoforms which differ not only by their storage in the extracellular matrix but also by their signaling pathways.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Matriz Extracelular/fisiologia , Linfocinas/fisiologia , Neovascularização Fisiológica/fisiologia , Adulto , Animais , Embrião de Mamíferos/irrigação sanguínea , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Regulação da Expressão Gênica , Humanos , Linfocinas/biossíntese , Linfocinas/genética , Neovascularização Patológica/fisiopatologia , Fator de Crescimento Placentário , Proteínas da Gravidez/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fator B de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The binding ability of 23 overlapping peptides, all derived from the CB11 fragment of CII, was tested on several HLA-DR molecules associated or not with disease susceptibility. These experiments were performed on a variety of cells expressing different HLA-DR molecules, using both indirect and direct binding assays. The CII (256-271) fragment was shown to bind to a restricted population among which the HLA-DR molecules associated with susceptibility to rheumatoid arthritis. The results also clearly indicate that the binding specificity of CII (256-271), among the DR4 molecules, is controlled by the nature of the HLA-DR molecule beta-chain residues 71 and 74, residues previously shown by X-ray crystallography to be involved in the HLA-DR/peptide interaction. The human CII (256-271) peptide is thus likely to play a role in the disease process.
Assuntos
Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Colágeno/metabolismo , Antígenos HLA-DR/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Artrite Reumatoide/genética , Doenças Autoimunes/genética , Sítios de Ligação , Ligação Competitiva , Colágeno/imunologia , Predisposição Genética para Doença , Antígenos HLA-DR/genética , Humanos , Epitopos Imunodominantes/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Two closely-related molecules, DR(alpha,beta1*0101) and DR(alpha,beta1*0103), whose beta chains only differ by three amino acids at positions 67, 70, and 71, and six intermediate molecules obtained by site-directed mutagenesis were used to ascertain the respective roles of the three polymorphic residues. Substitutions at positions 70 (D-->Q), 71 (E-->R) and 67 (I or L-->F) strongly affected HA 306-318-specific T-cell recognition. The consequences of the substitution of residue 67 by a phenylalanine depended on the modified HLA-DR molecule. Although this substitution completely inhibited peptide-specific DR1-restricted T-cell recognition, its manifestations on the DR103-restricted T-cell response were variable (abolishing proliferation of some cell lines and not others), no matter what the peptide presented was (HA 306-319 or HIV P25 peptides). We also observed that inhibition of the proliferation of an alloreactive anti-DR103 T-cell clone, caused by a substitution at position 70, was completely cancelled by substitution of residue 67 by a phenylalanine. The observations based on functional experiments, thus, suggest that residue 67 plays an important role in determining conformation of the peptide presented to the T cells. Molecular modeling was used to predict changes induced by amino acid substitutions and highly supports functional data. Substitution of residue 67 by a phenylalanine could have repercussions on the structure of HLA-DR molecule/peptide complexes and affect T-cell recognition.
Assuntos
Apresentação de Antígeno , Antígeno HLA-DR1/imunologia , Peptídeos/imunologia , Linfócitos T/imunologia , Adulto , Animais , Sítios de Ligação , Divisão Celular , Linhagem Celular Transformada , Sobrevivência Celular , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Células Clonais , Glutamina/genética , Glutamina/imunologia , Antígeno HLA-DR1/genética , Humanos , Isoleucina/genética , Isoleucina/imunologia , Leucina/genética , Leucina/imunologia , Camundongos , Modelos Moleculares , Fenilalanina/genética , Fenilalanina/imunologia , Conformação Proteica , Relação Estrutura-Atividade , Linfócitos T/citologiaRESUMO
A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306-318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306-318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Based usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells.
Assuntos
Alelos , Variação Genética , Antígenos HLA-DR/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Cadeias HLA-DRB1 , Humanos , Interleucina-2/biossíntese , Peptídeos/imunologiaRESUMO
Despite all the structural and functional data that have been accumulated regarding major histocompatibility complex (MHC) class II molecules during recent years, the relative contribution of putative T cell receptor (TcR)-contacting residues and peptide-binding MHC polymorphisms to MHC-restricted and allospecific T cell responses remains a point of contention. Some authors emphasize the importance of direct interaction between the allospecific TcR and polymorphic MHC residues whereas other emphasize the role of naturally processed MHC-bound peptides. We have previously described a new HLA-DRB1 allele: DR BON (DRB1*0103). This gene differs from DRB1*0101 by six base pairs clustered in the third variable region of the second exon leading to three amino acid changes at positions 67, 70 and 71 of the beta chain of the HLA-DR molecule. To define the respective role of these residues in allorecognition, we have performed site-directed mutagenesis on the DRB1*0103 allele to create six mutants which are intermediary between the DR BON and the DR1 alleles. These mutant cDNA were expressed in mouse fibroblasts and the transfectants with the highest expression of class II molecules were used as stimulators for a panel of ten anti-DR BON and five anti-DR1 alloreactive T cell clones. We demonstrate that the residue at the peptide-binding position 71 is of paramount importance in the alloresponse of these clones. In addition some clones were sensitive to amino acid substitution at the TcR-contacting position 70, while substitution at position 67 affects very few clones. The dominance of residue 71 was also observed with an influenza hemagglutinin-specific HLA-DR BON-restricted T cell line.