Skip to content

Biomedical subjects

J E Hambor

Publications and source records attributed to J E Hambor.

7 recordsLinked to original sources

PubMed1995

Repetitive Alu elements form a cruciform structure that regulates the function of the human CD8 alpha T cell-specific enhancer.

We previously identified a T cell-specific enhancer in the last intron of the human CD8 alpha gene that is adjacent to a sequence element that significantly represses enhancer function. This negative regulatory region consists of a half-Alu sequence that has potential to base-pair with a downstream Alu element, which is part of the fully active enhancer, to form a cruciform structure. The activity of this half-Alu silencer sequence is position and orientation-dependent, suggesting that DNA structure plays an important role in its function. Using site-directed mutational analysis and P1 nuclease mapping, we directly demonstrate that formation of a cruciform structure is required for repression of enhancer function in transient transfection assays. Finally, a P1 nuclease-sensitive site is present in the endogenous CD8 alpha gene in T cell lines providing indirect evidence that the stem-loop may form in vivo. Taken together, these results suggest that Alu elements may contribute to the regulation of the CD8 alpha gene enhancer through the formation of secondary structure that disrupts enhancer function.

Base Sequence↗

PubMed1990

CD8 functions as an inhibitory ligand in mediating the immunoregulatory activity of CD8+ cells.

Antisense and sense transfection technologies were employed in cellular coculture systems to investigate the physiologic requirements for CD8-dependent immunoregulation. Our data indicate that cells bearing genetically engineered CD8 molecules incorporating a glycoinositolphospholipid membrane anchor, as well as fixed cells bearing natural CD8 molecules, retain specific, CD8-dependent immunoregulatory activity. These findings together support the novel concept that CD8, a molecule traditionally thought of as a receptor, can function as an inhibitory ligand. CD8-dependent inhibition was shown to induce nonresponsiveness, persisting for at least 24 h, in Ag-specific responders. Moreover, only cells undergoing primary, but not secondary, antigenic stimulation through their TCR were found to be susceptible to CD8-dependent inhibition. Both CD4+ and CD8+ responding T cells were inhibited by CD8+ modulatory cells. These functional analyses of inhibitory and responder cells in CD8-dependent inhibition lay the groundwork for developing artificial CD8-based immunomodulatory peptides and deciphering CD8's role in natural immunoregulation.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Regulation of allogeneic responses by expression of CD8 alpha chain on stimulator cells.

By antisense-mediated inhibition of CD8 expression in T cell clones and expression of CD8 in non-T cell lines, we have produced several sets of CD8+/CD8- paired cell lines. These cellular reagents have allowed us to assess the effect of CD8 surface expression on the immunogenic potential of stimulator cells. We found that the presence of CD8 on stimulator cells markedly decreased their capacity to stimulate proliferative responses or to induce the generation of cytotoxic activity. The CD8 alpha chain, in the absence of the beta chain, was sufficient to mediate this inhibitory effect. Our findings support the notion that CD8 functions as an immunoregulatory ligand and that auxiliary cell surface molecules on stimulator cells can have profound effects on the immunogenic capabilities of these cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

An immunoregulatory function for the CD8 molecule.

The molecular details of immunoregulatory phenomena associated with CD8+ T lymphocytes have not been clearly elucidated. We tested the hypothesis that the cell surface glycoprotein CD8 is itself essential in mediating the inhibitory effects associated with CD8+ T cells. For this purpose we utilized a T-cell clonal pair, consisting of a human CD8+ T-cell clone and a specific CD8- phenocopy of this clone obtained via antisense RNA mutagenesis, to modulate allogeneic responses in vitro. Our findings indicate that the expression of the CD8 molecule by the inhibitory cells is essential for down-regulation of both allogeneic proliferation and generation of cytotoxicity in mixed lymphocyte cultures. These results define an immunomodulatory function for the CD8 molecule and provide insights into the molecular basis of immunosuppression.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Functional consequences of anti-sense RNA-mediated inhibition of CD8 surface expression in a human T cell clone.

An experimental approach for defining the function of CD8 has been developed by linking anti-sense RNA mutagenesis and T cell cloning technologies. We have transfected an anti-sense CD8 episomal expression vector into a CD8+ nontransformed human T cell clone that is specific for the human class I alloantigen HLA-B35. Expression of CD8 on this T cell clone, JH.ARL.1, was selectively and efficiently inhibited. Stimulation of this CD8- variant with specific alloantigen resulted in a marked loss of a number of functional responses, including cytotoxicity, proliferation, IL-2 secretion, and IL-2-R expression. However, these same functional responses could be elicited with stimuli that do not require antigen recognition to activate the T cell (anti-CD3 mAbs, PHA). The results of our study support the hypothesis that CD8 is required for recognition of class I MHC alloantigens that results in activation of T cell functional responses.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone.

A methodology was developed for stable gene transfer into cloned nontransformed human T lymphocytes. Stable high-level gene expression was achieved in cloned human T cells by using a self-replicating Epstein-Barr virus (EBV) episomal replicon. A comparison of five eukaryotic promoters established that the Rous sarcoma virus 3' long terminal repeat (RSV 3' LTR) and the lymphopapilloma virus (LPV) 5' LTR are optimal for episome-based expression in T cells. Effective (greater than 95%), selective, and reversible anti-sense RNA-mediated gene inhibition of a model T-cell-associated molecule (CD8) was achieved in a cytotoxic human T-cell clone by using an EBV episome-based, RSV 3' LTR-driven expression system. The linking of anti-sense RNA mutagenesis and T-cell cloning technologies should contribute significantly to studies of human T-cell function.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1983

Impairment of macrophage migration inhibitory factor synthesis and macrophage migration in protein-malnourished mice.

Weanling CD2F1 mice were fed isocaloric diets that were protein sufficient (PS; containing 27% casein) or protein deficient (PD; containing 8% casein). Weight measurements demonstrated that the growth of PD mice was significantly impaired, thus indicating that the PD diet induced protein malnutrition. The cellular immune responsiveness of these mice was assessed from Day 21 to Day 49 of the diet using, as indicators, in vitro production of migration inhibitory factor (MIF) by splenic lymphocytes and MIF responsiveness of peritoneal macrophages. PD lymphocytes, when stimulated with the polyclonal activator concanavalin A, produced significantly less MIF than did PS lymphocytes. The amount of MIF produced by PD lymphocytes, however, increased throughout the study, possibly indicating delayed maturation of MIF synthetic capacity in PD mice. Normal CD2F1 mouse macrophages were used for these assays. MIF responsiveness of PD and PS macrophages was not significantly different when assayed using MIF produced by normal CD2F1 mouse lymphocytes. As compared to that of PS macrophages, the migratory ability of PD macrophages decreased progressively throughout the study. This impaired migratory ability did not interfere with MIF responsiveness of PD macrophages.

Animals↗