Showing papers by "Mark M. Davis published in 1989"

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.

Abstract: In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.

Phenotypic differences between αβ versus β T-cell receptor transgenic mice undergoing negative selection

Abstract: T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.

Model for the interaction of T-cell receptors with peptide/MHC complexes.

Abstract: The immune response against a viral infection is mediated by two different types of cells known as B and T lymphocytes. The receptor on the B cell is the well-characterized antibody molecule, which exists in a membrane-bound form and in a secreted form involved in the initiation of complement-mediated killing and the inactivation of viral particles by direct binding. The recognition molecule on T cells is the membrane-bound T-cell antigen receptor, which has specificity for a combination of foreign antigen with a molecule of the major histocompatibility complex (MHC), as first demonstrated by Zinkernagel and Doherty (1974). MHC proteins exist in two closely related forms called class I and class II MHC molecules, both of which are cell-surface glycoproteins that are highly polymorphic in the human population. In general, class II MHC molecules are involved in interactions with T-helper cells, which cooperate with B cells to make antibody.

The XLR sequence family: dispersion on the X and Y chromosomes of a large set of closely related sequences, most of which are pseudogenes

Abstract: The XLR sequence family encodes RNA transcripts specific to late-stage T and B cells and their neoplasms. Only one apparently functional mRNA has been identified thus far and this encodes a novel 25 kDa nuclear protein. In this report, we find that the XLR gene family is composed of 50-75 copies per haploid genome which localize to at least two different portions of the mouse X chromosome. Neither of these locations are near the xid mutation that earlier work had correlated with XLR. In addition, some members of this family are also on the Y chromosome. Another surprising finding is that while the fourteen genomic clones examined to date have the same exon-intron structure and are closely related with respect to sequence conservation (90%), all appear (in most cases by multiple criteria) to be non-functional, raising the possibility that all but one of the members of this large semi-dispersed family are pseudogenes.

A Model for T Cell Receptor and MHC/Peptide Interaction

Abstract: For some time it has been known that T cell recognition of antigen occurs in an MHC restricted fashion (1–3). Much recent evidence suggests that the antigens ‘seen’ by T cell receptors (TcR) are fragments (presumably derived by intracellular processing) bound to MHC molecules at a single site (4–10) By contrast, the immunoglobulin (Ig) B cell receptor can bind to native antigen alone. Structurally and genetically however, both immunoglobulins and T cell receptors seem very similar. Both are derived from the relatively random juxtaposition of different coding segments (V, D and J) of DNA to produce proteins that differ in their N-terminal domains (V-domains), but are the same elsewhere (C domains) (11–13). Ig V region domains from the heavy and light chain polypeptides (VH and VL) pair to form the ligand binding region (14). By analogy, it seems likely that the binding site for antigen and MHC is formed by pairs of TcR V-domains (either α:β or γ: δ). In the Ig variable regions, sequence diversity is concentrated in three distinct ‘hypervariable regions’(15, 16). These amino acids form the principal points of contact with antigens and are thus referred to as complementarity determining regions (CDR’s) (17, 18).

The XLR gene product defines a novel set of proteins stabilized in the nucleus by zinc ions.

Abstract: The major product of the XLR (X-chromosomal, lymphocyte-regulated) locus is found to be a 30-kD nuclear protein with a relatively short (t1/2 approximately equal to 2 h) half-life. Together with its stage- and tissue-specific pattern of expression, this suggests a role for this protein in the regulation of differentiation in T and B lymphocytes. Interestingly, the XLR protein almost completely leaches out of the nucleus after lysis of cells in low salt buffer, but is stabilized in that location by metal cations, particularly Zn++. This stabilization is reversible by chelating agents (o-phenanthroline, EDTA) which also release a number of other polypeptides in addition to XLR. These results suggest that XLR represents a novel class of nuclear proteins, and that cations such as zinc may play a role in the localization of these proteins in the nucleus.

TCR recognition and selection in vivo.

Abstract: Much has been accomplished in identifying the molecules and genes responsible for T-cell recognition. We are now familiar with two distinct heterodimers, αβ and γδ, and we know that the former (at least) confers on a T cell the ability to recognize antigens complexed with specific molecules of the major histocompatibility complex (MHC) (Dembic et al. 1986; Saito and Germain 1987). Because of the recent solution of an MHC class I structure (Bjorkman et al. 1987a,b), its apparent generalization to class II molecules (Brown et al. 1988), as well as the similarity of T-cell receptor (TCR) primary sequences to immunoglobulins (Igs), we can guess a great deal about how they might interact (Chothia et al. 1988, Claverie et al. 1989; Davis and Bjorkman 1988; see also Bjorkman and Davis, this volume).

A Possible Basis for Major Histocompatibility Complex-Restricted T-Cell Recognition

Abstract: Four distinct T-cell antigen-receptor gene loci have now been identified and partly characterized: ɑ, β, y and δ. All of these loci can rearrange in an immunoglobulin- like fashion and express polypeptides that contribute to either ɑ:β or y:δ T-cell receptor-CD3 complexes. Surprisingly, the T-cell receptor (TCR) δ coding regions are located entirely, or almost entirely, within the TCR ɑ locus and share at least some of the V region gene segments, thus at least partly linking the two different types of receptor heterodimers. Analysis of potential T-cell receptor diversity, particularly that of the δ chain, indicates a striking concentration of somatic polymorphism in the V-J junctional region of the two heterodimers, four to six orders of magnitude higher than similar calculations for immunoglobulin light- and heavy-chain combinations. In contrast, the number of possible V region combinations in T-cell receptors is one hundredth to one thousandth that of immunoglobulins. TCR ɑ:β heterodimers are known to recognize many possible fragments of antigens embedded in the peptide- binding clefts of a relatively small number of major histocompatibility complex (MHC) molecules. Thus it is attractive to speculate that the V-J junctional portions of both types of T-cell receptor contact peptide antigens, whereas the remaining diversity regions contact the MHC. This contention is supported by molecular modelling studies and has interesting implications for the evolution of antigen-receptor genes.

Selection of an αβ T Cell Antigen Receptor In Vivo and Engineering a Solulizable Form

Abstract: Much progress has now been made in terms of identifying and characterizing the molecules important in T cell recognition, both receptors and ligands. Many issues remain unresolved, however, among these are: how and on what basis T cell receptors are selected in the thymus? and the precise nature of the T cell receptor-antigen-MHC recognition event. Towards addressing these issues we have developed two types of experimental systems. The first involves mice transgenic for specific T cell receptor genes crossed onto different MHC backgrounds. With suitable serological probes we can follow the expression of both α and β chains and the progress (or lack thereof) of T cells bearing these receptors in the animal. Together with the work of von Boehmer and colleagues and Loh and colleagues (this volume) the results of these studies give us some very clear ideas about the reality and efficiency of both positive and negative selection in the thymus. In addition, these transgenic systems should make possible the biochemical characterization of these phenomenon in the near future.