Biomedical subjects

D L Wiest

Publications and source records attributed to D L Wiest.

32 records · Page 2Linked to original sources

PubMed1995

The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes.

Immature thymocytes express clonotype-independent CD3 complexes that, when engaged by anti-CD3 antibodies, can signal CD4-CD8- thymocytes to differentiate into CD4+CD8+ cells. Clonotype-independent CD3 complexes consist of CD3 components associated with an unknown 90 kDa surface protein. We now report the surprising finding that this 90 kDa surface protein is the molecular chaperone calnexin, an integral membrane protein previously thought to reside only in the endoplasmic reticulum (ER). We found that calnexin-CD3 complexes escaping to the cell surface utilize interchain associations distinct from those utilized by calnexin-CD3 complexes remaining within the ER. Specifically, we demonstrate that carbohydrate-mediated luminal domain interactions that are necessary for formation of most internal calnexin-CD3 complexes destined to be expressed on the cell surface, and we provide evidence that cytoplasmic domain interactions between calnexin and CD3 epsilon chains mask calnexin's ER retention signal, permitting calnexin and associated proteins to escape ER retention. Thus, the present study demonstrates that partial T cell antigen receptor complexes can escape the ER of immature thymocytes in association with their molecular chaperone to be expressed at low levels on the cell surface where they may function as a signaling complex to regulate thymocyte maturation.

Amino Acid Sequence↗

PubMed1995

Developmental regulation of alpha beta T cell antigen receptor assembly in immature CD4+CD8+ thymocytes.

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic alpha beta chains associated with invariant CD3- gamma delta epsilon components and sigma chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of alpha beta TCR complexes on their surfaces expressed by mature CD4+ and CD8+ T cells. Recent evidence demonstrates that low surface TCR density on CD4+CD8+ thymocytes results from the limited survival of a single TCR component within the ER, the TCR alpha chain, which as a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCR alpha proteins in immature CD4+CD8+ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of alpha beta TCR complexes in CD4+CD8+ thymocytes and comment on the functional significance of TCR alpha instability during T cell development.

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PubMed1994

Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post-translational limitations on receptor assembly.

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PubMed1994

Developmentally regulated expression of CD3 components independent of clonotypic T cell antigen receptor complexes on immature thymocytes.

CD3 signal transducing proteins are thought to be expressed on the surface of T cells only as part of clonotypic T cell receptor (TCR) complexes. Contrary to this paradigm, the present study describes surface expression of CD3 proteins independently of clonotypic TCR complexes, but only on immature thymocytes. Such novel clonotype-independent CD3 (CIC) complexes are composed primarily of CD3 gamma epsilon and secondarily of CD3 delta epsilon heterodimers that are independent of one another and are expressed on the cell surface in association with an unknown 90-100 kD protein termed CD3-associated protein (CD3AP). CIC complexes are expressed in normal mice on early thymocytes through the CD4+CD8+ stage of development, but not on mature peripheral T cells. Furthermore, CIC complexes are expressed by both TCR- severe combined immunodeficiency (SCID) thymocytes and thymoma cell lines, in the absence of any clonotypic chains. The isolation and biochemical characterization of surface CIC complexes provides a structural basis for the signaling effects of anti-CD3 epsilon antibody treatment in early thymocyte development.

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PubMed1994

Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ thymocytes into mature T cells.

Immature precursor cells are induced in the thymus to express clonotypic T-cell antigen receptors (TCRs) and to differentiate into mature T cells. Perhaps the least understood event which occurs during intrathymic development is the positive selection of immature CD4+CD8+ thymocytes for differentiation into mature CD4+ and CD8+ T cells based on the TCR specificity individual thymocytes express. TCR expression by CD4+CD8+ thymocytes is quantitatively regulated by CD4-mediated activation of p56lck protein-tyrosine kinase whose activity can in turn be regulated by the membrane-bound protein-tyrosine-phosphatase CD45. Here we show that antibody engagement of CD45 external domains enhances Lck tyrosine kinase activity in CD4+CD8+ thymocytes, inhibits TCR expression, and inhibits differentiation of immature CD4+CD8+ thymocytes into mature T cells. Thus, engagement of the external domains of CD45 tyrosine phosphatase can regulate the ability of immature CD4+CD8+ thymocytes to undergo positive selection, suggesting an important regulatory role for intrathymic ligands that are capable of engaging CD45 within the thymus.

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PubMed1993

Decreased signaling competence as a result of receptor overexpression: overexpression of CD4 reduces its ability to activate p56lck tyrosine kinase and to regulate T-cell antigen receptor expression in immature CD4+CD8+ thymocytes.

Thymic selection of the developing T-cell repertoire occurs in immature CD4+CD8+ thymocytes, with the fate of individual thymocytes determined by the specificity of T-cell antigen receptor they express. However, T-cell antigen receptor expression in immature CD4+CD8+ thymocytes is actively down-regulated in CD4+CD8+ thymocytes by CD4-mediated tyrosine kinase signals that are generated in the thymus as a result of CD4 engagement by intrathymic ligands. In the present study we have examined the effect of CD4 overexpression in CD4+CD8+ thymocytes on activation of CD4-associated p56lck tyrosine kinase and regulation of T-cell antigen receptor expression. Augmented CD4 expression in CD4+CD8+ thymocytes did not result in commensurate increases in associated p56lck molecules, so that CD4 expression was quantitatively disproportionate to that of its associated signaling molecule p56lck. Interestingly, we found that CD4 overexpression significantly interfered with the ability of CD4 crosslinking to activate associated p56lck molecules and significantly interfered with the ability of CD4 to regulate T-cell antigen receptor expression. Thus, this study provides an example in which receptor overexpression leads to decreased receptor signaling competence.

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PubMed1993

Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56lck tyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules.

Signals transduced through the T cell antigen receptor (TCR) are modulated by the src family tyrosine kinase p56lck (lck), which associates in mature T cells with the coreceptor molecules CD4 and CD8. Here we describe a novel function of lck in immature CD4+CD8+ thymocytes, that of regulating TCR expression. Activation of lck in immature CD4+CD8+ thymocytes by intrathymic engagement of CD4 maintains low TCR expression by causing most TCR components to be retained and degraded within the endoplasmic reticulum. Importantly, activation of lck in immature CD4+CD8+ thymocytes results from engagement of surface CD4 molecules, but not surface CD8 molecules, despite the nearly fourfold greater surface expression of CD8 than CD4. The competence of CD4 to activate lck in CD4+CD8+ thymocytes relates to the fact that a relatively large fraction of surface CD4 molecules (25-50%) are associated with intracellular lck molecules, whereas only 2% of surface CD8 molecules are associated with lck. The amount of lck associated with CD4 in CD4+CD8+ thymocytes is diminished by chronic CD4 engagement in the thymus, as activated lck molecules subsequently dissociate from CD4. Indeed, the amount of lck associated with CD4 in CD4+CD8+ thymocytes is markedly increased in major histocompatibility complex (MHC) class II- mice that lack the intrathymic ligand for CD4 and in which surface CD4 molecules are consequently not engaged. Thus, the present study demonstrates that (a) activation of lck in CD4+CD8+ thymocytes regulates distribution and expression of TCR components; (b) unlike CD4 molecules, CD8 molecules on CD4+CD8+ thymocytes cannot efficiently activate lck despite their significantly greater surface expression; and (c) the amount of lck associated with CD4 in the CD4+CD8+ thymocytes is inversely related to the extent of CD4 engagement by MHC class II molecules in the thymus.

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PubMed1993

Subcellular localization of T-cell receptor complexes containing tyrosine-phosphorylated zeta proteins in immature CD4+CD8+ thymocytes.

The T-cell antigen receptor (TCR) is a complex of at least six different proteins (alpha, beta, gamma, delta, epsilon, and zeta) that is assembled in the endoplasmic reticulum (ER) and transported to the cell surface. Unlike mature T cells, most immature CD4+CD8+ thymocytes retain within the ER and degrade greater than 90% of some of the TCR components they synthesize, resulting in low surface expression of TCR complexes. The few surface TCR complexes that most immature CD4+CD8+ thymocytes do express are only marginally capable of transducing signals mobilizing intracellular calcium. The inverse relationship with TCR expression and function suggested that phosphorylated zeta (P-zeta) molecules might function in CD4+CD8+ thymocytes either as an ER retention signal for newly synthesized TCR complexes or as a negative regulatory modification of TCR complexes present on the cell surface. The present study sought to evaluate these two possibilities by determining the subcellular location of TCR complexes containing P-zeta chains. We found that, unlike unmodified zeta chains, all P-zeta chains in CD4+CD8+ thymocytes existed in assembled TCR complexes and that all TCR complexes containing P-zeta molecules had undergone carbohydrate processing events indicative of transit through the Golgi apparatus. These results demonstrate that P-zeta chains are exclusively associated with mature TCR complexes, excluding the possibility that P-zeta serves as an ER retention signal in immature thymocytes. Although we could not directly determine the representation of P-zeta chains among surface TCR complexes, we found that 60-70% of surface TCR complexes on immature CD4+CD8+ thymocytes were associated with tyrosine-phosphorylated protein(s) and that this percentage was inversely correlated with their signaling competence. These results support the concept that tyrosine phosphorylation serves as a negative regulatory modification of certain TCR-associated proteins.

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PubMed1993

Structurally distinct T cell receptor complexes on developmentally distinct T cell populations in severe combined immunodeficiency mice expressing a TCR beta transgene.

T cell differentiation and TCR expression were assessed in severe combined immunodeficiency (SCID) mice possessing an already rearranged TCRV beta 8 transgene. Unlike nontransgenic SCID mice, TCRV beta 8-transgenic SCID mice contained a broad spectrum of T cell populations, including both immature thymocytes and mature T cells. In TCRV beta 8-transgenic SCID mice, immature CD4-CD8- and CD4+CD8+ thymocytes expressed surface TCR complexes structurally distinct from those expressed by mature single-positive T cells. Immature CD4+CD8+ thymocytes expressed surface TCR beta chains without a clonotypic TCR partner chain and largely without associated CD3 components. In contrast, mature single-positive T cells expressed fully assembled surface TCR complexes containing disulfide-linked heterodimers consisting of transgenic TCR beta chains and endogenous TCR alpha chains. The surface TCR complexes on mature T cells were associated with CD3 components and were competent to transduce TCR-mediated proliferative signals. Thus, T cells at different stages of development in TCRV beta 8-transgenic SCID mice express structurally distinct surface TCR complexes, demonstrating that the developmental stage achieved by T cells in these mice is related to the structure of the surface TCR complexes they express. Indeed, the present results indicate that successful differentiation into single-positive T cells requires surface expression of fully assembled TCR complexes.

Animals↗

PubMed1992

Activation-induced ubiquitination of the T cell antigen receptor.

The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.

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PubMed1990

Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately.

The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins.

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PubMed1989

Expression of intracisternal A-type particles is increased when a B-cell lymphoma differentiates into an immunoglobulin-secreting cell.

The murine B-cell lymphoma CH12, like many other murine cells, expresses intracisternal A-type particles (IAPs). When CH12 cells are treated with lipopolysaccharide, the cells differentiate and secrete immunoglobulin M. The expression of IAP genes was also increased, in parallel with the increased expression of immunoglobulin genes. The amount of IAP mRNA increased within 48 h of lipopolysaccharide treatment and reached a level fivefold higher than that in unactivated CH12 cells. The two major IAP transcripts (7.2 and 5.4 kilobases) were induced to similar extents. The increased level of mRNA was reflected in higher levels of the major IAP structural protein p70, whose abundance increased 3.6-fold. The number of IAP particles per cell also increased upon activation of CH12, from 67 in nonsecreting CH12 to 290 in secreting cells. All of the IAPs were confined to the cisternae of the endoplasmic reticulum (ER), regardless of the differentiation state of the cell. Accompanying the induction of p70 was the induction of the related IAP polypeptide p102. A third viral polypeptide, p120, was also made in CH12; its abundance was almost unchanged. Localization of the IAP proteins on ultrathin frozen sections showed that most were assembled into particles in the ER. However, there were small pools of unassembled proteins both in the ER and on the plasma membrane. p70 and p120 could be detected, by iodination, on the surfaces of both secreting and nonsecreting CH12 cells. Unlike p70 and p120, p102 did not seem to be membrane associated. Taken together, these observations show that IAP expression is regulated developmentally in B lymphocytes. Also, these observations point to possible interactions between IAP genes and other cellular genes.

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PubMed1987

The expression of membrane and secreted immunoglobulin during the in vitro differentiation of the murine B cell lymphoma CH12.

The expression of membrane and secreted IgM was analyzed during mitogen-induced differentiation of the murine B cell lymphoma CH12. To characterize the Ig genes used by CH12, the nucleotide sequences of the variable gene segments (V mu and V kappa) were determined. The expressed V mu gene segment belongs to the VHII NPb-related family. The D (FL16.1a) and J (JH2) segments are the same as those used by the NP-specific hybridoma B1-8. The V kappa used by CH12 is almost identical to those used by the oxazolone-specific hybridomas NQ5.89.4 and NQ7.7.1. Treatment with lipopolysaccharide (LPS) induces up to 80% of CH12 cells to secrete IgM within 48 hr of culture. The steady state levels of secreted mu (mu s) and kappa mRNA increase four to fivefold over this period in cells stimulated with LPS compared with unstimulated cells. The kinetics are similar for both mRNA and parallel the increase in IgM secretion. EL-4 supernatants induce comparable changes in m mu s and kappa transcript levels. The simultaneous increase in m mu s and kappa transcripts suggests that coordinate control of RNA levels is used to increase the synthesis of secretory IgM during differentiation. The level of mRNA encoding the membrane form of mu (mu m) remains constant in stimulated cells and increases slightly in unstimulated cells. While the net rates of synthesis of membrane-bound mu-chains remain similar during LPS stimulation, the level of surface IgM on secreting cells is reduced three to fivefold. These observations suggest that the level of surface IgM expression during differentiation of CH12 is controlled largely by post-translational mechanisms. Our results demonstrate that the CH12 cell line regulates the expression of membrane and secreted IgM differently during its differentiation. The changes in IgM expression in CH12 parallel those occurring in normal B cells after mitogen or antigen challenge. Thus, the in vitro differentiation of CH12 is a good model for the analysis of late stages of B cell development.

Amino Acid Sequence↗

PubMed1986

Characterization of a colony-stimulating factor produced by the human monocytic leukemia cell line, THP-1.

The human monocytic leukemia cell line, THP-1, acquires macrophage-like characteristics following exposure to mezerein. Serum-free medium conditioned by mezerein-activated cells was observed to contain colony-stimulating factor (CSF) activity in assays with murine bone marrow cultures. Isoelectrofocusing revealed that CSF activity displayed charge heterogeneity and migrated in a pl range of 4.4-5.3. Treatment with neuraminidase did not affect biological activity but did reduce charge heterogeneity. Reisofocusing of neuraminidase-treated CSF revealed a peak of activity at pl 4.9. The active component was shown to be an acidic sialoglycoprotein, resistant to proteolytic cleavage but completely inactivated by 2-mercaptoethanol. This CSF has been purified from THP-1-conditioned serum-free medium by preparative isoelectrofocusing, gel filtration through Sephacryl S-200, ion exchange chromatography on DEAE-Sephacel, neuraminidase treatment, and tris-glycinate polyacrylamide gel electrophoresis (PAGE). Elution from SDS-PAGE revealed a single peak of activity corresponding to an apparent molecular weight of 70,000 daltons. Preliminary characterization of the bone marrow cells in colonies showed that THP-1 cells produced macrophage-specific CSF.

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