RESUMO
OBJECTIVE: To explore the immune pathogenesis of aplastic anemia (AA) and the therapeutic effects of Shengxue Mixture (SM) through the gene expressions of subfamilies of T-cell receptor variable region beta (TCR Vbeta) using immunologic and molecular biologic technology. METHODS: Gene expressions of TCR Vbeta sub-families in peripheral blood mononuclear cells from 20 AA patients were detected before and after treatment with SM using RT-PCR and gene scanning method. RESULTS: TCR Vbeta gene repertoire of the 24 subfamily genes deviated in AA patients, and the oligoclonal gene expressions increased obviously compared with those in healthy people (P < 0.01), including Vbeta2, 5, 6, 15, 16, 22, and 23 were found in 30%-50% AA patients, and Vbeta8, 21 were in more than 50% patients. These oligoclonal genes reduced significantly after treatment with SM compared with those before treatment (P < 0.05). CONCLUSION: Multiple TCR Vbeta subfamilies of clonal proliferation participate in the pathogenesis of AA. SM can rectify the deviation of TCR Vbeta gene repertoire, reduce the abnormal clonal proliferation of T cells, thus to alleviate the immune injury to hematopoietic tissue, and thus to benefit the recovery of hematopoiesis of bone marrow.
Assuntos
Anemia Aplástica/imunologia , Medicamentos de Ervas Chinesas/farmacologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Adolescente , Adulto , Idoso , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/genética , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Fitoterapia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle disease in ruminant livestock. In this study, mice were fed a selenium-deficient diet for three generations. Female mice from the third depleted generation of these mice were given water containing either no added selenium or 0.1 or 1.0 ppm selenium as sodium selenate; DNA microarrays were used to compare gene expression in the muscle from mice fed the selenium diets to that from mice remaining on the depleted diet. The most prominent expression increases were observed with Ptger2 (a prostaglandin E receptor), Tcrb-V13 (a T-cell receptor beta), Tcf-7 (a T-cell transcription factor), and Lck (lymphocyte protein tyrosine kinase), and the major consistent decrease was Vav2, an oncogene in mice consuming the selenium containing diets.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Selênio/administração & dosagem , Selênio/deficiência , Animais , DNA/análise , DNA/metabolismo , Suplementos Nutricionais , Feminino , Fator 1-alfa Nuclear de Hepatócito , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/biossíntese , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-vav/biossíntese , Proteínas Proto-Oncogênicas c-vav/genética , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Prostaglandina E/biossíntese , Receptores de Prostaglandina E/genética , Selênio/farmacologia , Fator 1 de Transcrição de Linfócitos T/biossíntese , Fator 1 de Transcrição de Linfócitos T/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The nervous system and the immune system share several functional molecules involved in various cell-cell interaction events. In this study, we used in situ hybridization to identify immune molecules that are expressed by a restricted population of neurons in the mouse brain and found that mRNA for the beta subunit of T-cell receptor (TCRbeta) was predominantly and strongly localized to neurons in deep layers of the cerebral neocortex and weakly expressed in the thalamus. Developmentally, TCRbeta mRNA expression started at embryonic day 15 in the thalamic nuclei and at postnatal day 1 in the cerebral neocortex. The level of TCRbeta mRNA in the neocortex subsequently increased until postnatal day 21, and it remained high in the adult. Detailed analysis revealed that only the Cbeta2 segment of TCRbeta, not the Cbeta1 or Vbeta segments, was expressed by the brain neurons. By the 5' rapid amplification of cDNA ends method, we determined a brain-specific transcription start site in the Jbeta2 region locus, not in the Vbeta region locus. Furthermore, we confirmed that the aberrant transcription around the Jbeta2 region took place only in neurons and lymphocytes in transgenic mice. These results demonstrate that the transcriptional machinery for unrearranged TCRbeta expression is shared by the nervous and immune systems and raise a possibility of gene rearrangement in neurons under certain circumstances.
Assuntos
Encéfalo/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transcrição Gênica/imunologia , Processamento Alternativo/genética , Processamento Alternativo/imunologia , Animais , Sequência de Bases/genética , Encéfalo/citologia , Encéfalo/metabolismo , Comunicação Celular/imunologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência Molecular , Neocórtex/citologia , Neocórtex/imunologia , Neocórtex/metabolismo , Vias Neurais/citologia , Vias Neurais/imunologia , Vias Neurais/metabolismo , Neuroimunomodulação/genética , Neuroimunomodulação/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Tálamo/citologia , Tálamo/imunologia , Tálamo/metabolismo , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Gênica/genéticaRESUMO
Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4(+)CD8(-)TCR alphabeta(+) Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.
Assuntos
Alérgenos/imunologia , Artemisia/imunologia , Epitopos Imunodominantes/imunologia , Proteínas de Plantas/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Alérgenos/análise , Alérgenos/metabolismo , Reações Antígeno-Anticorpo , Antígenos de Plantas , Linhagem Celular , Células Clonais , Epitopos de Linfócito T/análise , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Antígenos HLA-DQ/análise , Antígenos HLA-DR/análise , Teste de Histocompatibilidade , Humanos , Immunoblotting , Epitopos Imunodominantes/análise , Epitopos Imunodominantes/metabolismo , Imunoglobulina E/sangue , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Pólen/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T/químicaRESUMO
OBJECTIVE: To explore the therapeutic effect of Hejie Decoction (HJD) in treating chronic hepatitis B and its relationship with T-cell receptor V beta 7 (TCRV beta 7) gene expression. METHODS: Forty-five patients of chronic hepatitis B were randomly divided into two groups. The 30 patients in the treated group were treated by HJD, and the 15 patients in the control group were treated by conventional western medicine. The therapeutic effect and changes of TCRV beta 7 gene expression after treatment were observed. RESULTS: After 6 months treatment, the ALT level in the two groups were obviously decreased (P < 0.01). No significant difference was shown in comparison of the total effective rate between the two groups but it did show in comparison of markedly effective rate between them. TCRV beta 7 expression was detected in 5 patients of the treated group, and HBV-DNA and HBeAg in the 5 patients were all negatively converted. While in the control group, no one had TCRV beta 7 expression detected, either no one with negative conversion of HBV-DNA and HBeAg. TCRV beta 7 could not be detected in all the patients whose HBV-DNA and HBeAg hasn't negatively converted, though their liver function could be normalized. CONCLUSION: HJD might have the effect of regulation on TCRV beta 7 expression, it possibly is the important way for HBV replication inhibition and virucidal action of HJD.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Hepatite B Crônica/tratamento farmacológico , Fitoterapia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Adolescente , Adulto , Idoso , Feminino , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
OBJECTIVE: To explore the correlation of clinical effect and TCRV beta 7 expression of He Jie Decoction (HJD) on chronic hepatitis B patients. METHODS: 45 patients of chronic hepatitis B were divided randomly into two groups. 30 patients in the treated group were treated by HJD, and 15 patients in the control group were treated by usual western medicine. The TCRV beta 7 level were detected before and after being treated. RESULTS: After six months treatment, the ALT level of two groups were obviously decreased (P < 0.01), TCRV beta 7 expression were detected out in 6 patients of the treated group, and HBV-DNA and HBeAg of the 6 patients were negative conversion. The TCRV beta 7 expression couldn't be detected out in the control group, and HBV-DNA and HBeAg of the control group weren't negative conversion. There was no significant difference between the clinical total effective rate of the two groups (P > 0.05), but the apparent rate of the treated group was significantly higher than that of the control group (P < 0.01). CONCLUSION: HJD might have the effect on improving the TCRV beta 7 expression of chronic hepatitis B patients, which might be the ways of HJD inhibiting and killing hepatitis B virus.
Assuntos
Antivirais/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Plantas Medicinais , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Adolescente , Adulto , Idoso , Bupleurum/química , DNA Viral/sangue , Medicamentos de Ervas Chinesas/isolamento & purificação , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/metabolismo , Humanos , Pessoa de Meia-Idade , Pinellia/química , Plantas Medicinais/química , Scutellaria baicalensis/químicaRESUMO
The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes, and the majority of CD4(-)CD8(+) and CD4(-)CD8(-) [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP(+) cells was detected in non-T lineage subsets, including mature and immature B cells, CD5(+) B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP(+) cells detected were found in the CD44(+)CD25(-) DN thymocyte subpopulation. The developmental potential of GFP(-) and GFP(+) cells in the CD44(+)CD25(-) DN fraction was examined using in vitro culture systems. The generation of substantial numbers of alphabeta and gammadelta T cells as well as NK cells was demonstrated from both GFP(-) and GFP(+) cells. However, no development of B cells or dendritic cells was detected from GFP(+) CD44(+)CD25(-) DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44(+)CD25(-) DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/biossíntese , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Regiões Promotoras Genéticas/imunologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/enzimologia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Células Dendríticas/citologia , Regulação da Expressão Gênica/imunologia , Proteínas de Fluorescência Verde , Receptores de Hialuronatos/biossíntese , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Receptores de Interleucina-2/biossíntese , Cifozoários , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Timo/crescimento & desenvolvimento , Timo/imunologiaRESUMO
Some TCR variable regions are preferentially expressed in CD4+ or CD8+ T cells, reflecting a predilection for interacting with MHC class II or class I molecules. The molecular basis for MHC class bias has been studied previously, in particular for V alpha 3 family members, pointing to a dominant role for two amino acid positions in complementary-determining regions (CDRs) 1 and 2. We have evaluated the generality of these findings by examining the MHC class bias of V alpha 2 family members, an attractive system because it shows more variability within the CDR1 and -2, exhibits variation in the framework regions, and includes a member for which the crystal structure has been determined. We find that preferential recognition of MHC class I or II molecules does not always depend on residues at the same positions of CDR1 and -2; rules for one family may be reversed in another. Instead, there are multiple influences exerted by various CDR1/2 positions as well as the CDR3s of both the TCR alpha- and TCR beta-chains.
Assuntos
Substituição de Aminoácidos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Substituição de Aminoácidos/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Família Multigênica/imunologia , Ovalbumina/genética , Ovalbumina/metabolismo , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Cognate interaction between TCRs and MHC class II molecules plays an important role in initiating the allergen-specific immune response. Therefore, we analyzed the TCR distribution of human PBLs of 56 atopic and nonatopic (NA) individuals, including 4 monozygotic twin pairs, from two extended and four nuclear families. The expression of 23 V beta and 3 V alpha elements was analyzed. The blood samples of symptomatic birch pollen-sensitized individuals that were taken < or = 6 wk after the birch pollen season (n = 8) showed a significantly higher frequency of V beta 16.1+ and V beta 20.1+ T cells compared with the blood samples of birch pollen-sensitized individuals that were obtained out of allergen season (n = 10) or from NA individuals (p < 0.0005 and p < 0.0001, respectively). Allergen-specific lymphocyte proliferation was detected in the allergic individuals, and the distribution of V beta 16.1+ and V beta 20.1+ T cells returned to normal levels after the pollen season. The frequency of these V beta-expressing T cells correlated with the levels of allergen-specific IgE Abs. In addition, cat-sensitized individuals (n = 8) showed a significantly higher frequency of V beta 17.1-expressing T cells than did NA individuals (p < 0.005). Our results indicate restricted TCR-V beta gene usage in cat and birch pollen allergies; we suggest that both genetic and environmental factors contribute to TCR-V beta gene expression and to the development of a specific T cell response.
Assuntos
Gatos/imunologia , Hipersensibilidade/imunologia , Pólen/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Humanos , Hipersensibilidade/genética , Masculino , Pessoa de Meia-Idade , Linhagem , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Rinite Alérgica Sazonal/genética , Rinite Alérgica Sazonal/imunologia , Árvores , Gêmeos MonozigóticosRESUMO
In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr-C alpha region on Tcr-alpha,beta/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr-alpha,beta/CD3-gamma epsilon,delta epsilon complexes and zeta 2 homodimers. The Tcr-C alpha region around phe195 seems together with the same region in the Tcr-C beta region to constitute an interaction site for zeta 2 homodimers. The presence of phe195 on both Tcr-C alpha and Tcr-C beta causes high avidity interaction with zeta 2 homodimers, whereas his195 in both Tcr-C gamma and Tcr-C delta results in an apparently lower avidity interaction with zeta 2 homodimers. It is suggested that the phe195 region (on beta-strand F) and eventually adjacent aromatic amino acid residues on beta-strand B region may play an important role in Tcr-alpha,beta/CD3 membrane expression, in Tcr-alpha,beta/CD3 competition with Tcr-gamma,delta/CD3 complexes for zeta 2 homodimers and in the control of formation of 'mixed' Tcr heterodimers.
Assuntos
Complexo CD3/biossíntese , Fenilalanina/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Linhagem Celular , Membrana Celular/metabolismo , Sequência Conservada , DNA Complementar , Humanos , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Homologia de Sequência de Aminoácidos , Ovinos , Linfócitos T/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Surface expression of the T cell antigen receptor (TCR) in mature T cells requires the association of a variable heterodimer (alpha.beta or gamma.delta) with six invariant CD3 polypeptides (gamma, delta, epsilon-epsilon, zeta-zeta, or zeta-eta). We described here that deletion of the cytoplasmic tail polypeptide sequence (Lys-Lys-Lys-Asn-Ser) of TCR beta-chain (beta CT) results in expression of the truncated beta-chain on the surface of a mature T cell hybridoma line, in the absence of TCR-alpha, as a glycophosphatidylinositol (GPI)-anchored monomeric polypeptide. The GPI-anchored TCR-beta CT is not associated with CD3-epsilon and is incapable of conventional signal transduction. Association with TCR-alpha prevents beta CT from GPI-linkage formation. The alpha beta CT heterodimer binds the CD3 polypeptides, and the resultant TCR alpha beta CT/CD3 complex is capable of signal transduction. Our data show that a signal sequence for GPI-linkage formation is present in TCR-beta, and this alternative membrane anchoring mechanism can be utilized by beta-chain polypeptide lacking the CT sequence. We conclude therefore that in the absence of TCR-alpha expression, the beta-chain CT sequence plays an essential function in hindering GPI-linkage formation, thereby preventing escape of incompletely assembled TCR beta-chain to the cell surface of mature T cells.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Deleção de Sequência , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Sequência de Bases , Membrana Celular/imunologia , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Imunofluorescência , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Transdução de Sinais/imunologia , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , TransfecçãoRESUMO
A spontaneous TCR cell surface variant (3P11) of the Jurkat T cell line is described and characterized. 3P11 was selected by incubation of Jurkat cells with anti-TCR mAb followed by passage through Ig anti-Ig columns and cloning. 3P11 contained mRNA for both Ti alpha and Ti beta and CD3 gamma, delta, epsilon and zeta. Biochemical analyses demonstrated that all of the TCR components were produced in 3P11 cells. The Ti alpha beta/CD3 gamma delta epsilon zeta complex was assembled in the endoplasmic reticulum but the zeta did not associate with this complex. Epitopes recognized by the Ti beta chain specific mAb beta F1 and JOVI as well as anti-V beta 8 were affected in the 3P11 Ti beta chain indicating that the 3P11 Ti beta chain was mutated. Transfection of a wild-type Ti beta cDNA into 3P11 cells reconstituted TCR expression. Sequence analyses of the 3P11 Ti beta chain demonstrated a guanine to adenine change in the second nucleotide of the triplet coding for cysteine191 resulting in a cysteine to tyrosine exchange. Cysteine191 is the C-terminal cysteine involved in the intrachain disulfide bond in the C domain of the Ti beta chain; thus, the 3P11 Ti beta chain did not contain this disulfide bond. Transfection of a site-directed Ti beta chain containing the 3P11 mutation into a Ti beta negative variant of the Jurkat cell line resulted in a TCR phenotype identical with 3P11 demonstrating that the mutation identified in the 3P11 Ti beta chain was the sole cause for the 3P11 defect.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , DNA Complementar/química , Epitopos/análise , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/química , Relação Estrutura-Atividade , TransfecçãoRESUMO
Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease induced in animals by immunization with retinal proteins (or synthetic fragments derived from them) in adjuvant, and it is considered a model of human autoimmune diseases of the eye. To study the T cell clonotypes that may be involved in EAU, we analyzed the T cell repertoire of three related T cell lines: the pathogenic line LR16, specific to the major uveitogenic epitope of IRBP; its pathogenic subline J; and its nonpathogenic subline A. We examined the expression of the genes coding for the variable regions of the 20 known Lewis rat T cell antigen receptor (TCR) V beta families. The nonpathogenic subline was found to contain mostly T cells expressing V beta 5, V beta 8.2, and V beta 19 while the pathogenic subline consisted mainly of cells expressing V beta 8.3 TCRs. Genomic Southern blot analysis of DNA from the pathogenic subline showed that V beta 8.3-expressing T cells were the dominant clonotype, and DNA sequence analyses of V beta 8.3 cDNAs revealed that two V beta 8.3 TCRs were expressed in the pathogenic subline. One of the V beta 8.3 cDNAs encoded a variable region gene segment identical to previously reported rat V beta 8.3 TCR while the other differed by two amino acids in the second complementarity determining region (CDR2). Taken together with previous data showing overrepresentation of V beta 8-expression in T cell lines that induce EAU, but not in nonuveitogenic T cell lines, our results suggest that V beta 8.3-expressing T cells represent a pathogenic clonotype in IRBP-induced EAU.