Calcium influx and signaling in cytotoxic T-lymphocyte lytic granule exocytosis

Summary:  Cytotoxic T lymphocytes (CTLs) kill targets by releasing cytotoxic agents from lytic granules. Killing is a multi-step process. The CTL adheres to a target, allowing its T-cell receptors to recognize antigen. This triggers a signal transduction cascade that leads to the polarization of the microtubule cytoskeleton and granules towards the target, followed by exocytosis that occurs specifically at the site of contact. As with cytokine production by helper T cells (Th cells), target cell killing is absolutely dependent on Ca²⁺ influx, which is involved in regulating both reorientation and release. Current evidence suggests that Ca²⁺ influx in CTLs, as in Th cells, occurs via depletion-activated channels. The molecules that couple increases in Ca²⁺ to reorientation are unknown. The Ca²⁺/calmodulin-dependent phosphatase calcineurin, which plays a critical role in cytokine production by Th cells, is also involved in lytic granule exocytosis, although the relevant substrates remain to be identified and calcineurin activation is only one Ca²⁺ -dependent step involved. There are thus striking similarities and important differences between Ca²⁺ signals in Th cells and CTLs, illustrating how cells can use similar signal transduction pathways to generate different functional outcomes.     

MHC-specific cytotoxic T lymphocyte killing of dissociated sympathetic neuronal cultures.

Experiments were conducted to determine whether neurons in culture can serve as targets for immunologic attack mediated by major histocompatibility complex (MHC)-specific cytotoxic T lymphocytes (CTLs) which recognize Class I antigens. Allogeneic C3H/He primary neuronal cultures were quickly destroyed after CTL addition, while syngeneic C57BL/6J neurons were not lysed. Alterations in the distribution of chromatin were the first ultrastructural changes that occurred, followed by loss of nuclear morphology, cytosolic changes, and eventually fragmentation of both the nucleus and cytosol. With Campenot chambers, it was possible to separate the membrane and nuclear lesions. CTLs exposed to neurites, but separated from the cell body by the chamber barrier, caused degeneration of neurites but did not cause lysis and cell death. Neuronal lysis mediated by antibody and complement appeared to be distinct from CTL-mediated lysis. These experiments demonstrate that neurons in culture are targets for MHC-specific CTLs, and therefore probably express functional levels of Class I antigens. The signal for killing by CTLs is not retrogradely transported from the neurite to the cell body, and morphologic events following CTL-neuron interaction resemble those that occur in dividing tumor target cell populations.     

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

Midline 1 (MID1) is a microtubule‐associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss‐of‐function mutations in MID1 lead to the X‐linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1^?/? CTLs is impaired. Transfection of MID1 into MID1^?/? CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.     

The actin cytoskeleton and cytotoxic T lymphocytes: evidence for multiple roles that could affect granule exocytosis-dependent target cell killing

One important mechanism cytotoxic T lymphocytes (CTLs) use to kill virus-infected, transplanted or tumour targets is exocytosis of granules that contain cytotoxic agents such as perforin and granzymes. Granule exocytosis-dependent target cell killing is a complex process, involving initial T-cell receptor (TCR)-dependent signalling that includes Ca²⁺ influx and activation of protein kinase C, shape changes that serve to bind the CTL to the target and, finally, exocytosis of lytic granules at the site of contact with the target cell. Although there is reason to propose that multiple steps in the lytic process could involve the actin cytoskeleton of CTLs, few studies have examined this issue, and those that have do not allow the specific step(s) involved to be determined. We have used the potent membrane-permeant actin cytoskeleton-modifying drugs jasplakinolide and latrunculin A to investigate the actin dependence of defined processes that are expected to be important for granule exocytosis-dependent killing. Our results, obtained using TALL-104 human leukaemic CTLs as a model system, are consistent with the idea that a functional actin cytoskeleton is required for TCR/CD3-dependent signalling, for activation of store-dependent Ca²⁺ influx and for CTL shape changes. When cells were stimulated with solid-phase anti-CD3 antibodies, treatment with either jasplakinolide or latrunculin A abolished granule exocytosis. However, when cells were stimulated in a manner that bypasses TCR/CD3-dependent signalling, granule exocytosis was not significantly altered, suggesting that the actin cytoskeleton does not function as a barrier to exocytosis.     

Protein kinase C activity is required for cytotoxic T cell lytic granule exocytosis, but the theta isoform does not play a preferential role

CTLs kill virus-infected, tumor, and transplanted targets via secretion of lytic agents including perforin and granzymes. Knowledge of the signals controlling this important process remains vague. We have tested the idea that protein kinase C (PKC)theta, a member of the novel PKC (nPKC) family, which has been shown to play a preferential role in critical Th cell functions, plays a similar, preferential role in CTL lytic granule exocytosis using T acute lymphoblastic leukemia-104 (TALL-104) human leukemic CTLs as a model. We provide evidence consistent with the idea that PKC activity is important for the degranulation step of lytic granule exocytosis, as opposed to upstream events. In contrast with previous work, our results with pharmacological agents suggest that conventional PKCs (cPKCs) and nPKCs may participate. Our results suggest that stimulation with soluble agents that bypass the TCR and trigger granule exocytosis activates PKCalpha and PKCtheta, which can both accumulate at the site of contact with a target cell, although PKCtheta did so more often. Finally, using a novel assay that detects granule exocytosis specifically in transfected, viable cells, we find that overexpression of constitutively active mutants of PKCalpha or PKCtheta can synergize with increases in intracellular [Ca(2+)] to promote granule exocytosis. Taken together, our results lend support for the idea that PKCtheta does not play a preferential role in CTL granule exocytosis.     

Alloreactive cytotoxic T cells induce DNA fragmentation in peritoneal macrophages: evidence for target cell killing by cytotoxic T cells in vivo

This report addresses the question whether cytotoxic T cells can cause target cell death in vivo by examining target cell DNA fragmentation. The results show that alloreactive cytotoxic T cells induced significant DNA fragmentation in peritoneal macrophages in vivo and that the DNA fragment was a multiple of 180 +/- 30 bp. Furthermore, the effector cells which caused this characteristic DNA fragmentation were CD8+ T cells. These results demonstrate that cytotoxic T cells can cause target cell death in vivo.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells

We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock‐out (MID1^?/?) mice have a 25–30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild‐type (WT) mice. We wondered why the MID1 gene knock‐out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1^?/? CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1^?/? CTL upregulated MID2 two–twenty‐fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1^?/? CTL completely rescued exocytosis of lytic granules in MID1^?/? CTL, and vice versa, knock‐down of MID2 inhibited exocytosis of lytic granules in both WT and MID1^?/? CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL.     

A role for endobrevin/VAMP8 in CTL lytic granule exocytosis

CTL clear virus‐infected cells and tumorigenic cells by releasing potent cytotoxic enzymes stored in preformed lytic granules. The exocytosis process includes polarization of lytic granules toward the immunological synapse, tethering of lytic granules to the plasma membrane and finally fusion of lytic granules with the plasma membrane to release cytotoxic enzymes. Although much is known about the molecular machineries necessary for the earlier steps in lytic granule exocytosis, the molecular machinery governing the final step in the fusion process has not been identified. Here, we show using control and VAMP8 KO mice that VAMP8 is localized to the CTL lytic granules. While the immunological synapse and granule polarization appears normal in both VAMP8 KO and control CTL, CTL‐mediated killing was reduced for the Vamp8^–/– CTL. Analysis of lytic enzyme secretion demonstrated that granzyme A and granzyme B secretion is significantly compromised in VAMP8^–/– CTL, while the levels of the lytic enzymes in the cells are unaffected. Our results clearly show that VAMP8 is one of the v‐SNARE that regulate the lytic ability of CTL by influencing the ability of the lytic granules to fuse with the plasma membrane and release its contents.     

Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility

We investigated the functional role of the cAMP pathway in human cytotoxic T lymphocyte (CTL)-target interaction. Pharmacological increase of intracellular cAMP concentration ([camp]_i) inhibits killing, especially at low effectorto-target ratios, suggesting an inhibitory effect on CTL recycling. We show that this inhibitory effect is primarily at the level of conjugate formation. Pharmacological increase in [cAMP]_i, as well as treatment with cytochalasin D, results in a “rounding up” of the CTL and inhibition of the dramatic changes in shape that occur when a CTL forms a conjugate, even with an irrelevant target. In addition, pharmacological increase in [camp]_i affects the cytoskeleton of the CTL since it induces a decrease of filamentous actin, as detected by flow cytometry on phalloidin-stained CTL, and a stabilization of microtubules, as detected by increased resistance to the disrupting action of nocodazole. In mature CTL (but not in their immature precursors), T cell receptor triggering by specific targets results in a measurable increase in cAMP levels and strongly synergizes with adenylyl cyclase activators such as prostaglandin E2, cholera toxin and forskolin. We suggest that T cell receptor triggering may induce accumulation of cAMP that interferes with cytoskeleton function and, thus, terminates CTL secretion and adhesion. These effects of cAMP are rapidly reversible and may regulate CTL recycling.     

Induction of foot-and-mouth disease virus-specific cytotoxic T cell killing by vaccination

Foot-and-mouth disease (FMD) continues to be a significant threat to the health and economic value of livestock species. This acute infection is caused by the highly contagious FMD virus (FMDV), which infects cloven-hoofed animals, including large and small ruminants and swine. Current vaccine strategies are all directed toward the induction of neutralizing antibody responses. However, the role of cytotoxic T lymphocytes (CTLs) has not received a great deal of attention, in part because of the technical difficulties associated with establishing a reliable assay of cell killing for this highly cytopathic virus. Here, we have used recombinant human adenovirus vectors as a means of delivering FMDV antigens in a T cell-directed vaccine in pigs. We tested the hypothesis that impaired processing of the FMDV capsid would enhance cytolytic activity, presumably by targeting all proteins for degradation and effectively increasing the class I major histocompatibility complex (MHC)/FMDV peptide concentration for stimulation of a CTL response. We compared such a T cell-targeting vaccine with the parental vaccine, previously shown to effectively induce a neutralizing antibody response. Our results show induction of FMDV-specific CD8(+) CTL killing of MHC-matched target cells in an antigen-specific manner. Further, we confirm these results by MHC tetramer staining. This work presents the first demonstration of FMDV-specific CTL killing and confirmation by MHC tetramer staining in response to vaccination against FMDV.     

Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones.

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.     

Role of endogenous peptide in human alloreactive cytotoxic T cell responses.

The T x B hybrid 174 x CEM.T2 (T2) has been shown to be defective in the processing of proteins for presentation by MHC class I molecules. It continues, however, to express significant quantities of HLA-A2.1, suggesting that this class I molecule is expressed either in a largely peptide-free form or in association with a small subset of peptides. In this paper T2 was used in conjunction with limiting dilution analysis to provide a direct estimate of the fraction of alloreactive cytotoxic T lymphocytes (CTLs) that were dependent upon the presence of peptides for their recognition of HLA-A2.1. Alloreactive cytotoxic T cell lines generated by stimulation with HLA-A2.1 expressing peripheral blood lymphocytes recognized T2 poorly. Split-well analysis of 240 clonal limiting dilution cultures demonstrated that this reflected the existence of two subpopulations. An average 85% of HLA-A2.1 specific CTLs recognized HLA-A2.1 on normal cells but not on T2. The remainder recognized HLA-A2.1 on both T2 and normal targets. CTL lines with the latter specificity could be generated by using T2 as a stimulator cell. Using target cells that either expressed a lower density of HLA-A2.1 or that expressed HLA-A2 molecules that had been mutated to affect CD8 binding, no significant differences in avidity between T2-reactive and T2-unreactive CTLs were seen. Thus the failure of the majority of alloreactive CTLs to recognize T2 is not a consequence of the lower level of HLA-A2.1 surface expression on this cell, but is instead due to the absence of appropriate epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel cytotoxic T lymphocyte activation assay. Optimized conditions for antigen receptor triggered granule enzyme secretion

A method is described for the quantitative studies of cytotoxic T lymphocyte (CTL) activation. This functional assay is based on the measurements of secreted granule-associated enzymatic activity (BLT esterase (BLT-E) ) after incubation of CTL with activating stimuli. Immobilized mAb against CTL's antigen receptor (anti-TcR mAb), concanavalin A or a combination of PMA and ionophore A23187, were able to trigger the secretion of enzyme in the absence of target cells. Soluble anti-TcR mAb alone did not activate CTL, but using their conjugate with immobilized rabbit anti-mouse Ig antibody (RAMIg) TcR-mediated secretion of BLT-E was detected. Use of non-ionic detergents Nonidet P-40 or Triton X-100 (0.0125-0.2%) did not affect measurements of BLT-E activity. The efficiency of CTL exocytosis triggering by anti-TcR mAb which were immobilized on the surface of different plasticware is compared and conditions for studies of small and large numbers of CTL are described. The intensity of CTL response varies markedly with changes in buffer system, culture medium, additions of proteins. The optimal conditions for TcR complex triggered activation of murine CTL are described. Intensity of secretion can be easily manipulated by changing the surface density of immobilized anti-TcR mAb, thereby providing the possibility to screen inhibiting or activating agents (drugs or mAb) at selected sub-optimal levels of CTL activation. The potential for the use of described assay in screening of hybridoma supernatants for the presence of activating or inhibitory mAb against CTL's surface proteins is discussed. Since BLT-E secretion reflects exocytosis of granules from CTL, the conditions described here could be used for the detection of secretion of other markers of granules in future modifications of granule exocytosis assay.     

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. III. Antigen-specific T helper cells release nonspecific mediator(s) able to help induction of H-2-restricted cytotoxic T lymphocyte responses across cell-impermeable membranes

T helper cell induction and the specificity of T cell-mediated help as generated during alloreactive and H-2-restricted, virus- or hapten-specific cytotoxic T lymphocyte (CTL) responses have been compared. With the use of a double-chamber culture system, it was possible to dissect and separately analyze the induction phase of T helper cells from the T helper cell effector function. The data obtained revealed that during alloreactive as well as H-2-restricted T cell responses, antigen-specific T helper cells are induced. Upon specific restimulation of T helper cells, helper cell function is mediated across a cell-impermeable membrane via soluble products in an apparently nonspecific and nonrestricted manner. The data suggest that similar rules govern T-T cell interactions in alloreactive and H-2-restricted CTL responses.     

Role of Toll-like receptors in costimulating cytotoxic T cell responses

Stimulation of Toll-like receptors (TLR) by pathogen-derived compounds leads to activation of APC, facilitating the induction of protective immunity. This phenomenon is the basis of most adjuvant formulations currently in development. Here, we tested the ability of TLR2, 3, 4, 5, 7 and 9 signaling to enhance CTL responses upon vaccination with virus-like particles. Stimulation of TLR2 and 4 failed to increase CTL responses, whereas ligands for TLR3, 5 and 7 exhibited moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased CTL responses, indicating that ligands for TLR9 are likely to be the most promising candidates for the development of novel adjuvant formulations for stimulating CTL responses.     

Kinetic analysis of cloned cytotoxic T lymphocyte reactivity against normal and leukaemic target cells

The kinetics of unrestricted killing of normal and leukaemic lymphocyte target cells by a Qa-1b-specific murine cytotoxic T lymphocyte (CTL) clone were evaluated in a manner analogous to enzyme kinetic assays in which the effector and target cells corresponded to the enzyme and substrate, respectively. In order to apply the enzyme-substrate analogy to clonal cytolytic reactions, it was first established that the lytic reactions exhibited initial steady-state velocity of lysis at the effector and target cell concentrations used. The lytic reaction maintained linearity for velocity of lysis during the first 90 min of incubation, then plateaued. Vmax (the maximal rate of target cell lysis achieved by a given effector population) and Km (the target cell number resulting in 1/2Vmax) values were determined over a wide range of target and effector cell concentrations. Both parameters were found to be directly proportional to the number of effector cells. At a given concentration of cloned CTL, the lytic parameters of Vmax and Km were not significantly different for normal or leukaemic target cells that express Qa-1b. Additional kinetic parameters for lysis of normal and leukaemic target cells by a cloned CTL were also compared. The lytic efficiency of the CTL clone (i.e. maximal killing rate with an infinite number of targets) and the intrinsic affinity between effector and target cells were the same with either normal or leukaemic targets. However, the maximal lysis of target cells at an infinite number of effectors was significantly less for normal compared with leukaemic targets. This suggests that the normal target cells were more heterogeneous in their expression of the target (Qa-1b) antigen. Enzyme-like kinetic analysis of cell-mediated lysis reactions can be useful for comparing the relative affinities of effector and target cells obtained from various sources.     

Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.     

The role of monovalent cations in the interaction between the cytotoxic T lymphocyte and its target

Evidence is provided for the participation of monovalent cations in both the binding and lytic phases of the lytic cycle. There appears to be one sodium requirement in the cytotoxic T lymphocyte-target binding event (effective dose₅₀ ～ 35 mM) and possibly another sodium requirement in the delivery of the lethal hit (ED₅₀ <7 mM). Potassium can replace sodium in the binding event but antagonizes the lethal hit phase (inhibitory dose₅₀ ～ 110 mM). Lithium antagonizes the binding event (ID₅₀ ～ 15 mM).