Biomedical subjects

M K Saizawa

Publications and source records attributed to M K Saizawa.

4 recordsLinked to original sources

PubMed1997

Changes in arrangement and in conformation of molecular components of peripheral T-cell antigen receptor complex after ligand binding: analyses by co-precipitation profiles.

Amounts of co-precipitating CD3 components by anti-T-cell receptor (TCR)V beta or anti-CD4/8 monoclonal antibodies were compared between non-stimulated and stimulated splenic T cells. The amounts of co-precipitating CD3 delta, epsilon and gamma chains with TCR alpha beta and with CD4/8 were not significantly changed after TCR ligation. The apparent amount of CD3 zeta chain co-precipitated with TCR alpha beta increased up to threefold, while the actual amount of co-precipitating CD3 zeta with TCR alpha beta and the total amount of specifically precipitated CD3 zeta are not changed after cross linking of TCR. The apparent amount of CD3 zeta chain co-precipitated with CD4/8 also increased. Unlike co-precipitation with TCR alpha beta, the actual amount of CD3 zeta co-precipitated with CD4/8 increased significantly. This observation suggests a conformational change as well as the relocation of CD3 zeta molecules within the TCR complex after the signal delivery. After TCR ligation, CD3 zeta chains relocate to the vicinity of either CD4 or CD8 molecules. In addition, when cross linking and binding signals are compared, CD3 chains undergo two distinct phases of conformational change. The responses, while the later conformational change caused by the cross linking of TCR does not induce but enhances the proliferative response.

Animals↗

PubMed1993

Epitope-specific differential modulations within the T-cell antigen receptor complex.

The feeding of cloned helper T cells by irradiated spleen cells results in the modulation of T-cell antigen receptors (TCRs) as detected by FACS. When two groups of anti-TCR antibodies--anti-idiotypic and anti-V beta--are compared, time-dependent patterns of modulation of TCR are drastically different. Whereas up to 80% of the idiotypic determinants disappear within 2 days after stimulation, almost no change is observed for the determinants recognized by V beta-specific antibodies within the same period. Sequential immunoprecipitations clearly showed that anti-idiotypic and anti-V beta antibodies recognize the same population of TCR. Moreover, surface labelling followed by immunoprecipitation of TCR did not show rapid and drastic changes in the amount of specifically precipitated material. The modulation is not significantly affected by the addition of specific antigen, but is specific for the clone's MHC restriction element. Functionally, the modulation of TCR epitopes affects the ability of anti-TCR antibody but not of the MHC/antigen complex to stimulate T cells. We conclude that the observed modulation of fluorescence intensity in the TCR complex after MHC recognition (1) is epitope specific, (2) is independent of the presence of nominal antigen and (3) does not affect the efficiency of antigen-specific stimulation.

Animals↗

PubMed1992

Autoreactivity of low but not of high CD4 variants of an antigen-specific, I-A-restricted mouse T cell clone.

Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.

Animals↗

PubMed1992

Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes.

The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.

Animals↗