David R. Webb

Transition stages of molecular drive in multiple-copy DNA families in Drosophila.

Multigene and non‐genic DNA families are in a state of turnover and hence are continually being replaced throughout a population by new variant repeats. To quantify such molecular processes, in the absence of selection, it is necessary to find and compare stages of transistion during the homogenization of at least two non‐genic families evolving in parallel in a closely related group of species. Detailed sequence analysis of patterns of variation, at each nucleotide position considered independently, amongst repeats of two tandem DNA families from seven related Drosophila species, reveals all stages of transition during the spread of randomly produced variant repeats. Variant repeats are found at different stages of homogenization and fixation in a population, irrespective of the loci, chromosomes or individuals from which they were cloned. Differences between the families in the relatively small number of variants at each transition stage and the greater number of fully homogenized and fixed variants between species of greater divergence indicate that the process of spread (molecular drive) is rapid relative to the mutation rate and occurs at seemingly different constant rates for each family. Occasional gene conversions, in addition to unequal exchanges, have contributed to family turnover. The significance of these results to the evolution of functional multigene families and divergence and conservation of sequences is discussed.

T‐cell subset‐specific expression of the IL‐4 gene is regulated by a silencer element and STAT6

During development of CD4+ T lymphocytes in the periphery, differential expression of cytokine genes, such as those of interleukin (IL)‐2 and IL‐4, occurs in distinct T‐cell subsets. IL‐4 is a cytokine produced by T‐helper 2 (Th2) cells, and the IL‐4 receptor (IL‐4R)‐mediated signaling pathway is thought to be required for commitment to the Th2 phenotype. However, the molecular basis for development of the Th subset‐specific production of IL‐4 remains unclear. We demonstrate here that the IL‐4 promoter is functional in Th1 and B cells which do not normally form IL‐4 transcripts as well as in IL‐4‐producing T cells. Based on studies of the effect of several different upstream and downstream regions of the IL‐4 gene on IL‐4 promoter activity, a Th1‐specific IL‐4 silencer element was identified in the 3′‐untranslated region. The silencer region contained a consensus sequence for a transcriptional factor that is normally regulated by the IL‐4 R signaling pathway, STAT6. Nuclear expression of STAT6 protein, which was shown to bind to the silencer region, was observed in Th2 cells but not in Th1 cells. Deletion of the STAT6‐binding site from the silencer region and inhibition of STAT6 function resulted in the appearance of silencing function even in Th2 cells. These results provide evidence that the silencer element, and the binding of STAT6 to this element, play a permissive role in determining the commitment into Th2 phenotype.

Saying the ‘S’ word in public

The mechanism by which self-reactive, peripheral lymphocytes are prevented from causing overt autoimmune disease is the subject of much debate. A recent meeting examined the evidence to support a role for T-cell mediated suppression in maintenance of peripheral tolerance.

Induction of T-cell dependent splenic prostaglandin F2α by T-cell dependent antigen

Summary The intravenous injection of mice with an immunogenic dose of sheep erythrocytes results in a nearly 100 fold increase in the level of prostaglandin F2α in the spleen within 2 min after injection. The increase in prostaglandin is dependent on the presence of thymus derived T cells since injection of sRBC into athymic mice results in only a limited (10 fold) increase in the level of prostaglandin F2α.

Transduction of the IFN-γ signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.

Inhibition of interleukin 1 (IL-1)-elicited leukocytosis and LPS-induced fever by soluble immune response suppresor (SIRS)

IL-1-induced leukocytosis was inhibited or blocked in a dose-dependent manner by SIRS, an antigen-nonspecific suppressive lymphokine, when administered intravenously or per os to CBA mice. Timing experiments showed that SIRS effectively inhibited the leukocytosis when administered within 30 minutes of the IL-1. An antipyrogenic activity of SIRS was observed in rabbits injected intravenously with LPS. SIRS, given intravenously in one or two doses, markedly reduced LPS-induced fever. SIRS (2000 units) was a more effective antipyretic agent than aspirin (3 mg/kg body weight, intramuscularly). The results suggest that SIRS may be a potential drug for use in IL-1-mediated disorders.

lnterleukin-2 Stimulates the Development of Anergy via the Activation of Nonspecific Suppressor T Cells

We have previously demonstrated that incubation of splenic lymphocytes from an unimmunized mouse with IL-2 IFN-α or γ resulted in the development of a population of nonspecific regulatory cells (Ts). These cells were shown to block the ability of lymphocytes to generate mixed-lymphocyte responses in vitro. In the studies reported here, we have investigated the cell populations involved in this phenomenon. The Ts cells develop over a period of 2 or more days in culture with IL-2. Antibody to the 55-kD chain of the IL-2 receptor blocks Ts generation while stimulating T-cell proliferation. Although NK activity develops in these cultures, the mechanism of suppression is not via a lytic mechanism. Generation of the Ts in culture in the presence of IL-2 requires adherent cells as well as CD8+ cells. In studies using Con-A as the stimulus, the generation of Ts clearly requires both CD4+ T cells as well as CD8+ T cells and adherent cells. The evidence suggests that the CD4+ T cells serve as a source of IL-2 that is necessary in the activation of the IL-2-responsive CD8+, nonspecific Ts.

Characterization of an Antigen-Specific Suppressor Factor Generated by Cell-Free Translation

Recent advances in hybridoma technology and biochemical methodology have enabled a more quantitative analysis of antigen-specific suppressor T-cell products. Using reverse phase and high performance liquid chromatography (HPLC), it has been possible to purify to apparent chemical homogeneity several of these suppressor factors, TsF, specific for the antigen, GAT (1,2). Although the availability of such purified material allows dissection of the TsF protein at the biochemical, serological and potentially functional levels, it does not permit any detailed analysis of the events involved in factor production or the molecular organization of factors at the DNA level.

The Relationship of T-Cell Receptors and Factors

It has been over ten years since the first description of antigen specific factors that act to regulate immune responses. Despite intensive investigations by numerous investigators a resolution of the central questions raised by the discovery of these factors has remained elusive. The major questions concern the molecular nature of the antigen-specific factors and their genetic relationship to the immunoglobulin super gene family and particularly to the recently described T-cell receptor; secondly, what is the molecular and genetic nature of the restricting element(s) that control the interaction of these antigen-specific factors and their target cells; and lastly why are antigen-specific (e.g. antigen-binding) factors necessary? The workshop convened to discuss these issues did not lead to their resolution, rather it served to underscore the urgent need for a more thorough molecular analysis of the T-cell factors and the restricting elements.