Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.