Faculty Bibliography

The majority of human T cells express an antigen receptor consisting of a disulphide-linked heterodimer (Ti) of relative molecular mass 80,000-90,000 (Mr 80-90K) which is noncovalently associated with a set of at least three proteins of Mr 20-28K termed CD3 (Leu4, T3). Whereas both chains of Ti, an acidic alpha-chain of Mr 48-54K and a more basic beta-chain of Mr 40-44K, contain variable and constant region domains, the component peptides of CD3 are invariant. Several laboratories have more recently reported the expression of CD3 in association with a novel protein. On the surface of long-term T-cell lines and one thymocyte clone this novel structure consists of a 40K protein noncovalently linked to a 55 or 62K protein identified as the protein product of the Ti gamma-chain gene, a T-cell specific gene which like the Ti alpha- and Ti beta-chain genes undergoes rearrangement of variable (V) and joining (J) region gene segments. On the human T-cell leukaemic line PEER we have detected only a single 55K glycoprotein associated with CD3. We here demonstrate that an anti-Ti gamma-peptide antiserum reacts with the 55K CD3-associated protein on PEER. Most previously described human Ti gamma-chain complementary DNA clones encode the products of non-functional rearrangements. One of the Ti gamma cDNAs isolated from PEER, however, represents a functional rearrangement reported for the first time in a cell which expresses a Ti gamma-chain protein product on the cell surface. Interestingly, a 48-base-pair (bp) sequence in the constant (C) region domain of this functional Ti gamma-chain cDNA is triplicated in PEER and duplicated in other cDNAs isolated from PEER and other cell lines

The T-cell surface glycoprotein, CD4, is expressed predominantly on helper T cells and is thought to play a major role in cell-cell interactions. Monoclonal antibodies against CD4 have been shown to block numerous T-cell functions; moreover, recent results suggest that the CD4 molecule may be involved in transmembrane signal transduction. The human CD4 glycoprotein has also been shown to form at least part of the receptor for the AIDS virus, HIV-1. Elucidation of the functions of CD4 will be facilitated by the ability to manipulate the protein by genetic means. Because the mouse system is well suited for a variety of functional studies, we have isolated, sequenced and expressed cDNA clones encoding the murine CD4 (L3T4) glycoprotein. Comparison of the mouse and human CD4 sequences reveals striking evolutionary conservation of the cytoplasmic domain, suggesting that this region is essential for CD4 function. In addition, both the human and mouse CD4 gene contain a large intron in the coding region of the V-like domain. As no other members of the immunoglobulin gene superfamily have been shown to contain similarly placed introns, this finding may have important implications regarding the evolution of this gene family in particular and of introns in general

The surface glycoproteins T4 and T8 define functionally distinct populations of T lymphocytes. We have obtained cDNA and genomic clones encoding the T4 molecule and used these as probes to determine the chromosomal location of this gene. Genomic blotting experiments, along with in situ hybridization analyses, indicate that the T4 gene resides on the short arm of human chromosome 12, at region p12-pter. Thus, the T4 gene is not linked to any known member of the immunoglobulin gene family, including its counterpart gene, T8, which resides on human chromosome 2 immediately distal to the immunoglobulin kappa locus

Taken together, our studies suggest a mechanism of AIDS virus infection that initially involves the specific association of the AIDS virus with T4 molecules on the cell surface. This association does not require additional T-cell-specific molecules and can be demonstrated on both B lymphocytes and epithelial cell lines. The T4-AIDS virus complex is likely to be internalized in endosomes via receptor-mediated endocytosis. The virus can then fuse with the vacuolar membrane, releasing the viral nucleocapsid into the cytoplasm to undergo uncoating. Viral replication does not appear to require the environment of a T lymphocyte because active infection is also observed in human T4+ B lymphocytes and epithelial cell lines. Moreover, the T4 gene is expressed in the brain as well as in lymphocytes, providing an explanation for the dual neurotropic and lymphotropic character of the virus. In this manner, a T-lymphocyte surface protein thought to be important in mediating effector cell-target cell interactions has been exploited by a human lymphotropic virus to target the AIDS virus specifically to populations of T4+ cells

The surface glycoproteins T4 and T8 define different functional subsets of T lymphocytes and may act as recognition molecules mediating appropriate interactions between the T cell and its target. Previously we employed gene transfer and subtractive hybridization to isolate a T8 cDNA; now we have isolated and sequenced a cDNA clone encoding the T4 molecule. The deduced protein sequence reveals that T4 is an integral membrane protein that shares significant amino acid and structural homologies with members of the immunoglobulin supergene family. The overall structure of T4 consists of an N-terminal variable (V)-like domain, a joining (J)-like region, a third extracellular domain, a membrane-spanning region homologous to class II MHC beta-chains, and a highly charged cytoplasmic domain. Comparison of the protein sequences deduced from the T4 and T8 cDNAs reveals structural similarities consistent with their postulated role as recognition molecules, as well as differences suggesting that the two proteins recognize different structures on the target cell

The T cell surface glycoproteins T4 and T8 are thought to mediate efficient cell-cell interactions in the immune system and in this way may be responsible for the appropriate targeting of subpopulations of T cells. We have used gene transfer combined with subtractive hybridization to isolate both cDNA and functional genomic clones encoding the T8 protein. The sequence of the cDNA reveals that T8 is a transmembrane protein with an N-terminal domain which shares significant homology to immunoglobulin variable region light chains. This immunoglobulin-like structure is likely to be important in the function of T8 during differentiation and in the course of the immune response

The study of immune phenomena dependent on the major histocompatibility complex (MHC) would be greatly simplified by the use of MHC antigen-containing liposomes in various functional systems. Towards this end, we have constructed unilamellar phosphatidylcholine liposomes containing H-2 and Ia antigens. These molecules were not simply trapped within the aqueous compartment of the liposome as assessed by their accessibility to papain digestion. They were shown to be integrally inserted in the liposome bilayer because they could not be dissociated from the liposome with high salt and EDTA concentrations but could be solubilized by detergent. A sensitive radioimmunoassay showed that the Ia molecules were antigenically active in the liposome environment. Both Ia and H-2 antigens could be immunoprecipitated from detergent-solubilized liposomes. By comparing liposome-associated Ia activity in the presence and absence of detergent and by showing accessibility of the Ia antigens to papain, it was concluded that the majority of Ia antigens were exposed on the external surface of the liposome. These results suggest that the orientation of MHC antigens in liposomes closely parallels their natural orientation in the cell membrane