- Email: [email protected]
Internalization and recyling of the T-cell receptor for antigen
174
Abstracts
INTERNALIZATION AND RECYCLING OF THE T-CELL RECEPTOR FOR ANTIGEN. Michael S. Krangel; Dana-Farber Cancer Institute, Boston, MA A variety of receptors are specifically involved in a process of receptor mediated endocytosis, whereby they enter the cell via clathrin coated vesicles, pass through an endosomal compartment, and recycle back to the cell surface. This process has typically been followed by monitoring the fate of bound ligand. In order to examine the dynamics of various lymphocyte surface glycoproteins for which a high affinity, natural ligand is not available, a novel assay was devised. This involves labeling viable cells by cell surface iodination on ice, warming cells for variable lengths of time, and assessing the subsequent internalization of iodinated molecules by assaying their protection from digestion when intact cells are treated with neuraminidase. Results are assayed as neuraminidase dependent mobility shifts of immunoprecipitated material in SDS-PAGE. This approach has been used to assess the dynamics of the T-cell receptor (TCR) on the tumor cell line HPB-MLT. The results indicate the spontaneous internalization and recycling of the TCR-T3 complex on this cell line. lodinated TCR molecules are rapidly internalized upon warming the cells. External and internal pools equilibrate within 1 hr, with roughly 15% of TCR molecules intracellular. Internalized molecules can be directly demonstrated to return to the cell surface. Anti-T3 MoAb rapidly reduce the levels of surface TCR by perturbing this dynamic equilibrium. MoAb induced internalization is more rapid than the basal level; the equilibrium intracellular pool is three times normal, but internalized molecules still recycle to the cell surface. Internalization and recycling could also be demonstrated for the transferrin receptor. However, no evidence for the internalization ofT1 molecules could be obtained. Thus the TCR is specifically endocytosed in these cells. The spontaneous endocytosis and recycling of TCR molecules may provide a means of rapidly regulating the levels of surface receptors in response to activation of T cells. The comparative dynamics of other lymphocyte surface glycoproteins will be determined as well. POLYMORPHISMS OF THE HUMAN T-CELL RECEPTOR ALPHA GENE. Edward J. Ball, Lori Dombrausky, Marie Hoover, J. Donald Capra, and Peter Stastny; Departments of Internal Medicine and Microbiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas. T X The T-cell receptor for antigen (Ti) is a heterodimeric glycoprotein of 90 kd. It is encoded by two nonlinked genes; Ti-alpha and Ti-beta. Both of these genes have been shown to be polymorphic with respect to restriction endonuclease recognition sites. We have characterized a new RFLP of the Ti-alpha gene identified by the enzyme Taql. Two allelic Taql patterns were found: one a 7.8-kb fragment and the second a pair of fragments of 5.8 and 2.2 kb. The 7.8-kb fragment was found in 41/70 (58.6%) and the 5.8/2.2 kb pair in 59/70 (84.3%) unrelated individuals. Hybridization with fragments of the Ti-alpha gene clone localized the Taql and the previously described BglII polymorphic sites to separate exon regions of the gene. In 100 haplotypes examined with both enzymes, there was no evidence of strong linkage disequilibrium between the two markers. Thus, the two enzymes defined four distinct alleles of the Ti-alpha gene: T7.8, B3.2 (29%); T7.8, B2.9 (6%); T5.8/2.2, B3.2 (60%); T5.8/2.2, B2.9 (5%). This has allowed fully informative genotyping for Ti-alpha in a number of families. The use of multiple independent RFLP markers of the Ti-genes will allow one
Abstracts
INTERNALIZATION AND RECYCLING OF THE T-CELL RECEPTOR FOR ANTIGEN. Michael S. Krangel; Dana-Farber Cancer Institute, Boston, MA A variety of receptors are specifically involved in a process of receptor mediated endocytosis, whereby they enter the cell via clathrin coated vesicles, pass through an endosomal compartment, and recycle back to the cell surface. This process has typically been followed by monitoring the fate of bound ligand. In order to examine the dynamics of various lymphocyte surface glycoproteins for which a high affinity, natural ligand is not available, a novel assay was devised. This involves labeling viable cells by cell surface iodination on ice, warming cells for variable lengths of time, and assessing the subsequent internalization of iodinated molecules by assaying their protection from digestion when intact cells are treated with neuraminidase. Results are assayed as neuraminidase dependent mobility shifts of immunoprecipitated material in SDS-PAGE. This approach has been used to assess the dynamics of the T-cell receptor (TCR) on the tumor cell line HPB-MLT. The results indicate the spontaneous internalization and recycling of the TCR-T3 complex on this cell line. lodinated TCR molecules are rapidly internalized upon warming the cells. External and internal pools equilibrate within 1 hr, with roughly 15% of TCR molecules intracellular. Internalized molecules can be directly demonstrated to return to the cell surface. Anti-T3 MoAb rapidly reduce the levels of surface TCR by perturbing this dynamic equilibrium. MoAb induced internalization is more rapid than the basal level; the equilibrium intracellular pool is three times normal, but internalized molecules still recycle to the cell surface. Internalization and recycling could also be demonstrated for the transferrin receptor. However, no evidence for the internalization ofT1 molecules could be obtained. Thus the TCR is specifically endocytosed in these cells. The spontaneous endocytosis and recycling of TCR molecules may provide a means of rapidly regulating the levels of surface receptors in response to activation of T cells. The comparative dynamics of other lymphocyte surface glycoproteins will be determined as well. POLYMORPHISMS OF THE HUMAN T-CELL RECEPTOR ALPHA GENE. Edward J. Ball, Lori Dombrausky, Marie Hoover, J. Donald Capra, and Peter Stastny; Departments of Internal Medicine and Microbiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas. T X The T-cell receptor for antigen (Ti) is a heterodimeric glycoprotein of 90 kd. It is encoded by two nonlinked genes; Ti-alpha and Ti-beta. Both of these genes have been shown to be polymorphic with respect to restriction endonuclease recognition sites. We have characterized a new RFLP of the Ti-alpha gene identified by the enzyme Taql. Two allelic Taql patterns were found: one a 7.8-kb fragment and the second a pair of fragments of 5.8 and 2.2 kb. The 7.8-kb fragment was found in 41/70 (58.6%) and the 5.8/2.2 kb pair in 59/70 (84.3%) unrelated individuals. Hybridization with fragments of the Ti-alpha gene clone localized the Taql and the previously described BglII polymorphic sites to separate exon regions of the gene. In 100 haplotypes examined with both enzymes, there was no evidence of strong linkage disequilibrium between the two markers. Thus, the two enzymes defined four distinct alleles of the Ti-alpha gene: T7.8, B3.2 (29%); T7.8, B2.9 (6%); T5.8/2.2, B3.2 (60%); T5.8/2.2, B2.9 (5%). This has allowed fully informative genotyping for Ti-alpha in a number of families. The use of multiple independent RFLP markers of the Ti-genes will allow one