RESUMO
Two monoclonal antibodies with specificity for rat gammadelta T cell receptor (TCR) were generated. One, called V65, reacts with all CD3+ alphabeta TCR- rat Tcells and thus recognizes a constant determinant of the rat gammadelta TCR (Kühnlein et al., Journal of Immunology 1994, 153: 979). The other, called V45, reacts with approximately 80% of gammadelta T cells in peripheral lymphoid organs. In rat epidermis, V65 but not V45 detects a dense network of the dendritic epidermal Tcells (DETC). Analysis of epidermal RNA by polymerase chain reaction (PCR) indicated that Vgamma3 and Vdelta1 are the predominant, if not exclusive TCR V transcripts present at this site. Sequence analysis of cDNA clones obtained by reverse transcription-PCR with Vgamma3- and Vdelta1-specific primers revealed that the variable domains of rat DETC gamma and delta chains are very homologous to those described in mice (92% and 95% identity at the protein level). The complete conservation between the two species of the amino acid sequences at the V-(D)-J transitions of this monomorphic receptor indicates that the interaction of the DETC TCR with its as yet unknown ligand must be of central importance for DETC function.
Assuntos
Sequência Conservada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epiderme/imunologia , Epiderme/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Sequência de Bases , Clonagem Molecular , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/isolamento & purificação , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The rat Tcra V gene locus is only poorly characterized, although rats are widely used in a variety of T-cell-mediated experimental animal models. Recently, we described the first monoclonal antibody, G99, directed against a rat Tcra V4 segment. We examined cDNA transcripts of G99-positively sorted T cells and show that the monoclonal antibody G99 most likely recognizes at least two members of the Tcra V4 family. Moreover, we analyzed the genomic repertoire of this VA family and report 15 novel Tcra V4 DNA sequences. Based on sequence and Southern blot analysis, the Tcra V4 family could be divided into four subgroups, which were also detected in mice. These findings corroborate previous findings of a similar genetic organization of the Tcra V loci in both species.
Assuntos
Família Multigênica/genética , Ratos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Camundongos/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Using a panel of newly developed mAb to two rat TCR V alpha and four TCR V beta segments, TCR V usage in CD4 and CD8 T cells of eight RT1 congenic strains sharing the LEW background was analyzed by flow cytometry. While no striking effects on V beta 8.2 and 8.5 usage were observed, a 3- to 4-fold over-representation of V beta 10 in the CD4 as compared with the CD8 subset in all strains suggested a preference of V beta 10 for MHC class II products. The degree of 'overselection' was mapped to the RT1.B/D region. In addition, an allele-specific overselection of V alpha 4+ CD4 T cells was mapped to RT1.B/Du and of V beta 16+ CD8 T cells to RT1.Au. Finally, a dramatic overselection of V alpha 8+ CD8 T cells by RT1f (14% in RT1f versus 1-2% in other haplotypes) provides the most striking case yet for an intrinsic affinity of a TCR V segment for an MHC product. V alpha 8+ CD8 T cells are not only overselected by RT1f in the thymus, but also during the alloreactive response of peripheral CD8 T cells to RT1f. The implications of these findings for the contribution of TCR V segments to TCR-MHC interactions in repertoire selection and alloreactivity are discussed.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timo/imunologia , Animais , Anticorpos Monoclonais , Diferenciação Celular , Citometria de Fluxo , Isoantígenos/imunologia , Ratos , Ratos Endogâmicos Lew , Ratos Mutantes , Timo/citologiaRESUMO
The in vitro response of unprimed rat T cells to retroviral and bacterial superantigens (SAg) was analyzed with TCR V beta 8.2-, 8.5-, 10-, and 16-specific mAbs. Specific stimulation of V beta 8.2 and 8.5 CD4 cells was observed in the response to Mls1a, the retroviral SAg encoded by integrated provirus Mtv-7 (Mtv-7 SAg), which was presented by mouse B cells or mouse fibroblasts transfected with DR1 genes and the Mtv-7 SAg. Additionally, a strong response of V beta 16 CD4 cells to an as yet unidentified mouse SAg was found. Only some of the bacterial SAg known to stimulate mouse and human T cells also activated rat lymph node cells. SEA, SEE, and TSST-1 stimulated rat T cells well; SEB, SEC1, and SED did not. This defect was apparently a result of weak binding to rat MHC class II molecules because presentation by human MHC class II molecules restored T cell activation. Under these conditions, SEB stimulated V beta 8.2+ and 8.5+ CD4 and CD8 cells from Lewis rats. A comparison of several rat strains revealed an unresponsiveness to SEB or Mtv-7 SAg for V beta 8.2 cells from F344 and DA rats. Determination of the nucleotide sequences of the Tcrb-V8.2 of these strains revealed differences between SAg-responsive and SAg-unresponsive Tcrb-V8.2 in seven amino acids, four of them located in the putative SAg contact site. The significance of these findings for the evolution of TCR-SAg interactions is discussed.
