Studies on the induction and expression of T-cell-mediated immunity. XIII. Membrane-associated antigens of cytotoxic T lymphocytes involved in cytotoxicity

An xenogeneic rat anti-mouse T-cell serum, designated RAT*, has been shown to block the cytolytic activity of cytotoxic T lymphocytes (CTL) at a postbinding step. RAT* serum or the IgG fraction was extensively absorbed with the target cell, P815, a DBA mastocytoma, and used with or without further absorption to immunoprecipitate specific molecules from radiolabeled membrane extracts of CTL derived from either in vivo-allosensitized mice or from cytotoxic clones maintained in in vitro cultures. Cell surface sialic acid residues were labeled by oxidation with sodium periodate (NaIO4) and reduction with tritiated sodium borohydride ([3H]NaBH4). Alternatively, cell surface proteins were labeled with 125I by lactoperoxidase-catalyzed iodination. Nonidet P-40 (NP-40)-solubilized radiolabeled membranes were then immunoprecipitated with RAT* serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Three membrane-associated molecules of 95,000, 140,000 and 180,000 Mr were found by such analysis. The sensitivity of these three molecules to trypsinization and their susceptibility to labeling with [3H]NaBH4 suggested that they are glycoproteins. Moreover, when RAT* serum or the IgG fraction was absorbed with various cell types, its ability to immunoprecipitate the three molecules correlated with its ability to block cytolysis. Adsorption of RAT* serum with CTL, but not with nonimmune thymocytes, significantly reduced the ability of RAT* serum to inhibit cytotoxicity and to immunoprecipitate the 95k, 140k, and 180k molecules. Thus, these findings suggest that one or more of these cell surface molecules of CTL may be involved in the cytolytic process.     

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: Some implications

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by ⁵¹chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15–30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing >70% large granular lymphocytes (LGL)) was the most rapidly lytic population by ⁵¹chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 °C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.     

Display of lead-binding proteins on Escherichia coli surface for lead bioremediation

Cell surface display of heavy metal-binding proteins has been used to enhance the adsorption capacity of heavy metals and the engineered microbial cells can be potentially used for the bioremediation of heavy metals. In this study, the proteins PbrR, PbrR691, and PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli BL21 cells, with the N-domain of ice-nucleation protein as the anchor protein to achieve specific adsorption of lead ions (Pb²⁺) and bioremediation of lead in the soil. The localization of fusion proteins was confirmed by western blot analysis. We investigated the effects of fusion pattern, expression level, heavy metal concentration, and the presence of other heavy metal ions on the adsorption of Pb²⁺ by these engineered bacteria, and the optimal linker peptide (flexible linker) and inducer concentration (0.5 mM) were obtained. The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb²⁺. The maximum Pb²⁺ adsorption capacity of strains displaying the three proteins (PbrR, PbrR691, and PbrD) were 942.1-, 754.3-, and 864.8-μmol/g cell dry weight, respectively, which was the highest reported to date. The engineered E. coli bacteria were also applied to Pb²⁺-contaminated soil and the detoxification effects were observed via the seed germination test and the growth of Nicotiana benthamiana in comparison with the control BL21, which provides the proof-of-concept for in situ remediations of Pb²⁺-contaminated water or soil.