Changes in cytosolic free calcium ([Ca2+]i) have been continuously imaged during the interaction of the H‐2Kb specific cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the H‐2Kb EL4.BU or the H‐2Kk RDM4 cell lines. Activation of the CTLs by EL4.BU raises [Ca2+]i to several hundred nanomolar in the CTL. Frequently [Ca2+]i is preferentially elevated in the region of the CTL furthest from the site of target contact. These responses require external Ca2+ suggesting that they are generated by the plasma membrane and not internal stores. Inappropriate targets such as RDM4 evoke no changes in [Ca2+]i. Activation of the BM 3.3 CTL is followed by increases of [Ca2+]i to several micromolar or higher in the EL4.BU targets. This massive increase can be mimicked by direct application of cytolytic granules isolated from rat natural killer cells. The increase in plasma membrane permeability is ion‐specific since external Mn2+ can also readily enter target cells that have been ‘hit’, as evidenced by the rapid selective quenching of fura‐2 in those targets. The flood of Ca2+ into the target cell is followed by a leakage of the trapped fura‐2. Since both processes continue after the CTL has disengaged, they provide a useful assay for the lethal hit. Furthermore, this technique can be used to follow complete cycles of CTL activation and lethal hit delivery, which under some circumstances can be as rapid as 6 min per cycle.
CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.
A B cell hybridoma (Désiré-1) was derived which secreted and expressed at its cell surface immunoglobulin (Ig) specific for the antigen-specific T cell receptor (Ti) of an H-2Kb-specific alloreactive cytotoxic T lymphocyte (CTL) clone (KB5-C20). It was found that the CTL clone could lyse hybridoma Désiré-1, whereas it could not lyse hybridoma which expressed surface Ig (sIg) binding to other cell surface structures of clone KB5-C20 such as the H-2Kk molecule. Blocking of CTL-target cell interactions using monoclonal antibodies (mAb) indicated that the CTL-target cell interaction was inhibited with appropriate anti-H-2 mAb and by anti-Lyt-2 mAb when CTL-H-2Kb interaction was involved but not when CTL-sIg interaction was involved. The two types of interactions were inhibited by anti-LFA-1 mAb. The involvement of the CTL-Ti structure was necessary to obtain a lytic interaction between CTL and target cells, but a major histocompatibility complex product on the target cells did not need to be involved. Comparison of CTL-target cell inhibition with cold target cells or with anti-clonotypic mAb indicated that the Ti-sIg cellular interaction was of much higher apparent affinity than the Ti-H-2Kb cellular interaction. These results further suggest potential regulatory effects of CTL-B cell cross-idiotypic interactions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.