A molecular map of T cell development.

Using a sensitive molecular marker for positive selection, the appearance of a particular functional TCR alpha chain sequence in cells from mice bearing a transgenic beta chain, we address several aspects of intrathymic T cell development. First, by examining specific TCR prior to and after maturation, we demonstrate how a restricted TCR repertoire is positively selected from a highly diverse immature TCR repertoire. Second, since this molecular marker is enriched in cells progressing toward the CD4 lineage and depleted in cells progressing toward the CD8 lineage, a map of the developmental pathway of alphabeta thymocytes can be inferred. Third, the first cells that show clear signs of positive intrathymic selection are identified.

The imprint of intrathymic self-peptides on the mature T cell receptor repertoire.

The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.

The orientation of a T cell receptor to its MHC class II:peptide ligands.

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.

Regional localization of the putative cell surface receptor for HTLV-I to human chromosome 17q23.2-17q25.3.

The gene for the cell surface receptor for HTLV-I, the etiologic agent of adult T-cell leukemia and HTLV-I-associated myelopathy, has been localized to distal human chromosome 17q. A panel of somatic cell hybrids containing fragments of human 17q as the only human genetic component was mapped with a set of 10 chromosome 17 probes and utilized to regionally localize the gene. When compared to the murine fibroblast fusion partner, L-M(TK-), and a hybrid cell line containing human chromosome 20, human 17q-containing hybrid cells bound high levels of both HTLV-I virions and the monoclonal antibody, Mab 34-23, which may be directed against the putative HTLV-I receptor. Additional experiments revealed that the human 17q-containing hybrids could also be more efficiently infected by cell-free HTLV-I virions than could the control cell lines. Western blot analyses of cell lysates showed that recombinant HTLV-I envelope gp46 protein and Mab 34-23 both bound to proteins of approximate MW 30 and 31 kDa which were found only in the hybrid cell lines which contained human chromosome 17q. The data suggest that the gene for the HTLV-I receptor is located on the distal region of human chromosome 17q demarcated by the tk-1 locus (17q23.2-17q25.3).

Variation in genomic Alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes.

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human: rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse: human hybrid cells that span the entire long arm of human chromosome 17.

An efficient technique for obtaining sequences flanking inserted retroviruses.

Genomic mapping studies frequently employ retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuLV-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome 17q hybrid cells.