Pierre A. Henkart

Lymphocyte-mediated cytotoxicity : two pathways and multiple effector molecules

Pierre A. Henkart Experimental Immunology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 Important progress has been made over the last several years in our understanding of the mechanism of lympho- cyte cytotoxicity. Experiments published in recent months have built on this to provide definitive answers to basic questions, as I will review here. The Granule Exocytosis Pathway: Effects of CytolysinlPerforin Deletion Until recently, the granule exocytosis pathway had been the only molecularly defined cytotoxicity pathway, and is diagrammed in the right portion of Figure 1. This basic process and the considerable evidence supporting it have been reviewed elsewhere (Podack et al., 1991; Henkart et al., 1994). The functional cytotoxic mediator delivered by the killer lymphocyte in this pathway has traditionally been thought to be the membrane-damaging agent cytolysin or perforin. This interesting protein is found uniquely in cytotoxic T lymphocyte (CTL) granules, and is a potent lytic agent after its release into the physiological ionic environment. The pores it forms in the target cell have been shown to allow passage of large proteins, as well as ions, and it was proposed that this damage was lethal to the target cell. Although avery considerable body of evidence had been developed to support the granule exocytosis model, per- sistent questions arose regarding its in vivo relevance (Berke, 1991). The issue came down to whether the rela- tively small level of granule components in in vivo CTL, detectable in some studies but not others, could account for the cytotoxic function, and no definitive answer emerged. The recent development of knockout mice in which the gene for cytolysinlperforin is disrupted either in the germ- line or in B and T lymphocytes selectively has allowed these in vivo questions to be addressed in a particularly clear way. The report of Kagi et al. (1994a), one of four labs that have developed such mice, is a beautiful example of the success of the gene disruption appproach, since problems with indirect effects of the knockout and redun- dant molecular pathways seem not to be significant. Thus, CD8’ T cells were found in normal levels in lymphoid or- gansof the knockout mice, and became activated normally in response to LCMV infection. However, specific in vitro cytotoxic activity was negligible when spleen cells from virus- infected knockout mice were compared with infected con- trol mice. Alloreactive CTL originating from primary in vitro cultures as well as in vivo immunizations likewise were severely crippled in terms of their ability to lyse the appro- priate allospecific fibroblasts, and to a considerable ex- tent, allogeneic tumor cells as well. NK activity also ap-

Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A

The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-cytolysin and RBL-cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%-60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.

Target Cell Lysis by CTL Granule Exocytosis Is Independent of ICE/Ced-3 Family Proteases

Activation of ICE/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas-Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas-Fas ligand pathway. The peptide caspase inhibitors also blocked drug-induced apoptotic cell death in tumor cells. In contrast, the caspase inhibitors blocked CTL granule exocytosis-induced target apoptotic nuclear damage, but did not inhibit target lysis. These results are consistent with recent demonstrations that granzyme B can activate caspases leading to apoptotic nuclear damage, but show that target cell lysis by CTL granule exocytosis occurs by a caspase-independent pathway.

Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B–specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B–specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

IL-15 mimics T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8+ memory T cells

Generation of CD8+ memory T cells requires antigenic stimulation through T cell receptor (TCR); however, maintenance of CD8+ memory T cells seems to be mediated by cytokines, such as IL-15, in a TCR-independent manner. Compared with the TCR-induced activation, less is known about the mechanisms of IL-15 action. We report here a comparative and kinetic analysis of the responses of memory phenotype CD8+ T cells to IL-15 or TCR (anti-CD3) stimulation in vitro. These two stimuli induce highly similar responses in memory phenotype CD8+ T cells as measured by cellular proliferation, gene expression changes, synthesis of effector molecules (IFNγ, tumor necrosis factor β, granzyme B, and perforin), and induction of cytotoxicity. From 189 genes/expressed sequence tags (ESTs) whose expression changed in CD8+ memory T cells after IL-15 and anti-CD3 stimulation identified by cDNA microarray analysis, 77% of the genes/ESTs exhibit a highly similar pattern of expression between IL-15 and anti-CD3-treated cells, and only 16% and 7% of the genes/ESTs are differentially expressed in response to IL-15 and anti-CD3 treatments, respectively. These results show that IL-15 and anti-CD3 stimulation induced remarkably similar gene expression and effector function. Thus, IL-15 acts not only as a crucial growth factor but also as an antigen-independent activator of effector functions for CD8+ memory T cells.

Inducible Nonlymphoid Expression of Fas Ligand Is Responsible for Superantigen-Induced Peripheral Deletion of T Cells

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)-induced peripheral deletion of Vbeta8+ T cells. We found that peripheral deletion was defective in radiation chimeras with non-functional tissue FasL, regardless of the FasL status of the bone marrow-derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

Regulation of Cyclin D1 by Calpain Protease

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Perforin and the granule exocytosis cytotoxicity pathway.

Perforin defects have been identified in humans with familial hematophagocytic lymphohistiocytosis. The pathology of these patients has dramatically illustrated an under-appreciated role for perforin in the regulation of T-cell responses in vivo, and experimental studies are shedding light on the mechanisms involved. The detailed molecular mechanisms of perforins mandatory role in the cytotoxic T lymphocyte (CTL)-mediated granule exocytosis death pathway and of granzyme entry into target cells remain unclear. In model systems measuring apoptosis by granzyme B and sublytic perforin, pore formation is undetectable during granzyme entry. Selfprotection of cytotoxic lymphocytes after degranulation can be explained by surface expression of the granule protease cathepsin B, as shown by suicidal degranulation in the presence of specific inhibitors.

Human CD8+ T Cells Store RANTES in a Unique Secretory Compartment and Release It Rapidly after TcR Stimulation

The chemokine RANTES is secreted rapidly after activation of human CD8+ T cells, with a cycloheximide-resistant burst during the first hour. This pattern was observed in purified memory and effector phenotype CD8+ cells from blood as well as in blasts. In contrast, secretion of other chemokines and interferon-gamma by these cells was sensitive to cycloheximide and detectable only after a lag. Immunofluorescence microscopy of CD8+ memory and effector cells and blasts showed RANTES present in intracellular vesicles that do not significantly colocalize with cytotoxic granule markers or other markers of defined cytoplasmic compartments. Immunoelectron microscopy confirmed that RANTES is stored in small vesicles distinct from the lysosomal secretory granules. RANTES+ vesicles polarize rapidly in response to TcR engagement and are more rapidly depleted from the cytoplasm. These results show that CD8+ T cells have two distinct TcR-regulated secretory compartments characterized by different mobilization kinetics, effector molecules, and biological function.

Antigen-stimulated apoptotic T-cell death in HIV infection is selective for CD4+ T cells, modulated by cytokines and effected by lymphotoxin

Objective To characterize the mechanism of in vitro antigen-induced apoptotic T-cell death in the peripheral blood mononuclear cells (PBMC) of HIV-1-infected individuals. Design and methods PBMC from HIV-1-infected and uninfected individuals were unstimulated or stimulated with HIV-1 envelope synthetic peptides (Env) or influenza A virus to determine the extent of antigen-stimulated apoptotic T-cell death, whether this death was limited to the CD4+ subset, and the effects of cytokines on T-cell death. Death was assessed by apoptotic nuclear morphology after 7 days of culture by fluorescence microscopy using a DNA-specific dye. Transwell cultures and supernatant transfers were utilized to test whether a soluble factor produced by HIV-positive PBMC induced death of HIV-negative T cells. Exogenous cytokines [interleukin (IL)-12, interferon (IFN)-γ, IL-4 and IL-10, as well as antibodies against endogenously produced cytokines (IL-4, IL-10, IL-12, and lymphotoxin) were tested for their ability to modulate death. Results Antigenic stimulation induced death in PBMC from HIV-positive donors, but not in PBMC from HIV-negative donors. Antigen-stimulated death was seen in CD4+ but not CD8+ T-cell subset from the HIV-positive patients. Apoptotic death was blocked by IL-12, IFN-γ, anti-IL-4, anti-IL-10, and anti-lymphotoxin, but not by anti-IL-12. Transwell and supernatant transfer experiments indicated that antigen-stimulated HIV-positive PBMC produced a factor that killed T-cell blasts. The factor was inhibited by anti-lymphotoxin, but not by anti-IL-10. Conclusions Stimulation of HIV-positive PBMC with CD4-dependent antigens results in selective death of CD4+ T cells that is modulated by cytokines. Our results suggest that apoptotic death is not limited to HIV-infected or HIV-specific T cells, but occurs in bystander cells. Lymphotoxin is a mediator of antigen-stimulated T-cell death in this in vitro model.