Lymphocyte-mediated Cytolysis Research Articles

Cooperation between chemotherapy and immune checkpoint blockade to enhance anti-tumour T cell immunity in oesophageal adenocarcinoma

Response rates to immune checkpoint blockade (ICB) remain low in oesophageal adenocarcinoma (OAC). Combining ICB with immunostimulatory chemotherapies to boost response rates is an attractive approach for converting ‘cold’ tumours into ‘hot’ tumours. This study profiled immune checkpoint (IC) expression on circulating and tumour-infiltrating T cells in OAC patients and correlated these findings with clinical characteristics. The effect of first-line chemotherapy regimens (FLOT and CROSS) on anti-tumour T cell immunity was assessed to help guide design of ICB and chemotherapy combinations in the first-line setting. The ability of ICB to enhance lymphocyte-mediated cytolysis of OAC cells in the absence and presence of post-FLOT and post-CROSS chemotherapy tumour cell secretome was assessed by a CCK-8 assay. Expression of ICs on T cells positively correlated with higher grade tumours and a subsequent poor response to neoadjuvant treatment. First-line chemotherapy regimens substantially altered IC expression profiles of T cells increasing PD-1, A2aR, KLRG-1, PD-L1, PD-L2 and CD160 and decreasing TIM-3 and LAG-3. In addition, pro-inflammatory T cell cytokine profiles were enhanced by first-line chemotherapy regimens. T cell activation status was significantly altered; both chemotherapy regimens upregulated co-stimulatory markers ICOS and CD69 yet downregulated co-stimulatory marker CD27. However, ICB attenuated chemotherapy-induced downregulation of CD27 on T cells and promoted differentiation of effector memory T cells into a terminally differentiated state. Importantly, dual nivolumab-ipilimumab treatment increased lymphocyte-mediated cytolysis of OAC cells, an effect further enhanced in the presence of post-FLOT tumour cell secretome. These findings justify a rationale to administer ICBs concurrently with first-line chemotherapies.

Exogenously delivered heat shock protein 70 displaces its endogenous analogue and sensitizes cancer cells to lymphocytes-mediated cytotoxicity.

Hsp70 chaperone is known to stimulate anti-tumour immunity in a variety of cancer models. Here we demonstrated that the addition of purified recombinant Hsp70 to the culture medium facilitated cancer cell cytolysis by lymphocytes. Importantly, exogenous Hsp70 triggered secretion of the intracellular Hsp70 to a cell surface and extracellular milieu, which played a role in cytolysis because down-regulation of the endogenous Hsp70 reduced both its presence at the cell surface and the lymphocyte-mediated cytolysis. Inhibitors that target both the ATPase and the peptide-binding domains of Hsp70 molecule potently decreased its anti-tumor effect. Using a variety of cell transport markers and inhibitors, we showed that the exchange of exogenous and intracellular Hsp70 is supported by classical and non-classical transport pathways, with a particular role of lipid rafts in the chaperone's intracellular transport. In conclusion, exogenous Hsp70 can eject endogenous Hsp70, thus exerting anticancer activity.

Granzyme K Directly Processes Bid to Release Cytochrome c and Endonuclease G Leading to Mitochondria-dependent Cell Death

Granule-mediated cytolysis is the major pathway for killer lymphocytes to kill pathogens and tumor cells. Little is known about how granzyme K functions in killer lymphocyte-mediated cytolysis. We previously showed that human GzmK triggers rapid cell death independently of caspase activation with single-stranded DNA nicks, similar to GzmA. In this study we found that GzmK can induce rapid reactive oxygen species generation and collapse of mitochondrial inner membrane potential (DeltaPsim). Blockade of reactive oxygen species production by antioxidant N-acetylcysteine or superoxide scavenger Tiron inhibits GzmK-induced cell death. Moreover GzmK targets mitochondria by cleaving Bid to generate its active form tBid, which disrupts the outer mitochondrial membrane leading to the release of cytochrome c and endonuclease G. Thus, we showed herein that GzmK-induced caspase-independent death occurs through Bid-dependent mitochondrial damage that is different from GzmA.

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at −1085 and −1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4–DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.

Middle ear mucosa changes following exposure to Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (1 microgram/20 microliters) was instilled into the middle ear cavity in the rat. Morphological changes of the mucosa were analyzed after various intervals using light (LM) and transmission electron microscopy (TEM). An inflammatory cell reaction was seen in which there was a predominance of polymorphonuclear leukocytes (PMNs) and mononuclear cells that were mostly lymphocytes and monocytes/macrophages. Epithelial cells often showed signs of metaplasia and hyperplasia, followed by degradation through necrosis and/or apoptosis, resulting in denudation of the submucosal layer. A characteristic feature was the physical interaction between intraepithelial lymphocytes and epithelial cells. Lymphocytes adhering to the cell coat of epithelial cells showed signs of directed secretion that seemed to end in necrosis or apoptosis of the target cell. These changes occurred simultaneously with phagocytosis of cells and cell debris by PMNs and macrophages. Ultrastructural analysis suggested that intraepithelial, lymphocyte-mediated cytolysis and apoptosis may be important events in degradation of the epithelium following exposure to Pseudomonas sp. exotoxin. These findings indicate that denuding of the lamina propria may facilitate the penetration of toxin into the labyrinth, thus explaining subsequent inner ear damage.

Human Perforin: Rapid Enrichment by Immobilized Metal Affinity Chromatography (IMAC) for Whole Cell Cytotoxicity Assays

Perforin, a potent pore-forming protein, plays an important role in lymphocyte-mediated cytolysis causing necrosis or, when combined with the granzymes, apoptosis. The studies on perforin, although already extensive, have been hampered by the limited amount of material available from killer lymphocytes. Using a cell line that expresses high levels of human perforin, we describe a straightforward purification scheme that allows isolation of the lytic protein in approximately 8 hours. Perforin is enriched from the YT-INDY cell line by cavitation followed by differential centrifugation and ion metal affinity chromatography. In addition to demonstrating the lytic activity of human perforin toward various cell lines, we show that the conditions of the cytotoxicity assay influence both the kinetics and magnitude of perforin-mediated cytotoxicity.

Lymphocyte-mediated cytolysis and disease.

Lymphocytes are equipped to eradicate noxious agents (microbes, cancer cells, and grafts) that disturb the body's equilibrium, but when their cellular activity is excessive, the results are harmful. The list of abnormalities known to be caused by excessive lymphocyte activity is extensive. We review two distinct pathways that account for most of the cellular injury induced by lymphocytes, with an emphasis on their clinical implications. The Molecular Basis of Lymphocyte-Mediated Cytolysis Unlike humoral immune responses, which are mediated through antibodies and complement and can be transferred in serum to unimmunized subjects, cellular immune responses require the direct participation of effector . . .

Perforin and lymphocyte-mediated cytolysis.

We have discussed in the previous sections the recent progress made toward elucidating the regulatory mechanism of perforin gene transcription and the domain structure of the perforin molecule. It appears that the expression of perforin is, at least partially, controlled at the transcription level through the interaction between killer cell-specific cis- and trans- acting factors. One of such cognate pairs, NF-P motif (an EBS-homologous motif) and NF-P2 (a killer cell-specific DNA-binding protein), has been described. The regulatory mechanism of gene transcription, however, is likely to involve multiple factors which act in a coordinated fashion to bring about the most efficient expression of perforin limited strictly to activated killer lymphocytes. Through studies using synthetic peptides and recombinant perforins, it has been suggested that the N-terminal region of the perforin molecule is an important, though not the only, domain responsible for the lytic activity. Further studies are warranted to elucidate the role(s) of other potential amphiphilic structures located in the central portion of the perforin molecule in the overall pore-forming activity. The molecular basis underlying the resistance of killer lymphocytes to perforin-mediated lysis still remains an open question. Preliminary results, however, suggest that the surface protein(s) restricted to killer cells may account for their self-protection against perforin. Based on recent studies using perforin-deficient mice, the involvement of perforin in lymphocyte-mediated cytolysis both in vivo and in vitro has been confirmed. Two functional roles, a direct (lytic) and an indirect (endocytosis enhancer; conduit), both of which may contribute critically to the cell-killing event can be attributed to perforin. The fact that lymphocytes may also employ perforin-independent killing mechanism(s), e.g. Fas-dependent pathway, is beyond the scope of this review. There is, nevertheless, no doubt that these alternative cytolytic mechanisms may also play important roles in immune effector and/or regulatory responses associated with killer lymphocytes. Obviously, we are still a long way from concluding on the functional relevance of each individual cytolytic mechanism seen in different physiopathological situations. Suffice it to say, however, that a wealth of information on lymphocyte-mediated killing has already emerged through the multidisciplinary efforts conducted in our and other laboratories that promise to further dissect this complicated event in the years to come.

Morphologic and functional characterization of perforin-deficient lymphokine-activated killer cells.

Mice deficient in perforin, a key mediator of lymphocyte-mediated cytolysis, have recently been generated using the gene knockout technique. CTL and NK cells derived from these mice have been shown to be defective in the granule-dependent cytolytic pathway. To investigate whether the granule-formation process has been altered in these perforin-deficient cytotoxic cells, rendering them defective in using the other granule mediators, we have examined in the present study the morphologic and functional characteristics of perforin-deficient LAK cells. Perforin-deficient LAK cells, similar to wild-type LAK cells, were shown to contain a large number of granules in their cytoplasm. By electron microscopy, the morphology of the granules present in these two cell populations appeared indistinguishable. The complete depletion of perforin in LAK cells derived from perforin gene-knockout mice was further confirmed by immunoelectron microscopy using anti-perforin antiserum. The expression of other cytolytic mediators, present either within the granules (granzymes A and B) or elsewhere (Fas ligand), appeared to be unperturbed, as investigated using the reverse transcription-PCR technique. Like the CTL and NK cells isolated from perforin-deficient mice, perforin-deficient LAK cells could lyse only target cells that express high levels of Fas molecule. Furthermore, these perforin-deficient LAK cells, similar to wild-type LAK cells and a CTL clone, were resistant to perforin-mediated cytolysis.

CD43 diminishes susceptibility to T lymphocyte-mediated cytolysis.

CD43 is a major membrane sialoglycoprotein expressed by cells of hematopoietic origin. One property of CD43 is its ability to interfere with heterotypic and homotypic cellular adhesion. To determine whether CD43 expression can affect cell functions requiring intercellular adhesion, we compared a CD43-positive human T cell line (CEM) and its CD43-negative counterpart derived by gene targeting for susceptibility to cell-mediated lysis. CD43-negative CEM cells were more susceptible than CD43-positive cells to lysis by allospecific T cell lines derived from several donors. Induction of CD43 expression on transfected HeLa cells also imparted resistance to lectin-mediated lysis by a CD8+ T cell clone. The effect of CD43 expression on reducing susceptibility to lysis was more pronounced in short-term cytotoxicity assays and tended to disappear as the time of contact between the effector cell and its target increased. The enhanced susceptibility of CD43-negative cells to lysis was not associated with increased expression of adhesion molecules known to mediate antigen-independent cellular adhesion. Sialic acid residues on CD43 contributed to the CD43 protective effect. These results suggest that either diminished CD43 expression or incomplete sialylation may render hematopoietic cells more susceptible to T lymphocyte-mediated cytolysis.

The natural killer cell serine protease gene Lmet1 maps to mouse chromosome 10

Cytotoxic lymphocytes play a key role in immune responses against viruses and tumors. Lymphocyte-mediated cytolysis by both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is often associated with the formation of membrane lesions on target cells caused by exocytosis of cytoplasmic granule serine proteases and a pore-forming protein, perforin. A variety of granzymes have been found to reside within the cytoplasmic granules of cytotoxic lymphocytes, but unlike perforin, isolated serine proteases are not intrinsically lytic. However, a role for serine proteases in cellular cytotoxicity has been supported by the ability of protease inhibitors to completely abrogate lymphocyte cytotoxicity, and the demonstration that serine proteases can initiate DNA fragmentation in target cells transfected or pretreated with a sublytic concentration of perforin. Granzymes cloned in human, mouse, and rat encode four granzyme activities and all are expressed in either T cells, their thymic precursors, and/or NK cells. In particular, a rat granzyme that cleaves after methionine residues, but not phenylalanine residues and its human equivalent, human Met-ase 1, are unique granzymes with restricted expression in CD3-NK cells. 24 refs., 2 figs.

Characterization of Recombinant Mouse Perforin Expressed in Insect Cells Using the Baculovirus System

Perforin, a potent pore-forming protein, plays an important role in killer lymphocyte-mediated cytolysis. The studies on perforin, although already extensive, have been hampered by the limited amount of perforin naturally available from killer lymphocytes. In the present study, a full-length mouse perforin was expressed in insect cells using recombinant baculovirus. Recombinant perforin appeared to be functional in terms of lysing erythrocytes and nucleated target cells. These results suggest that the recombinant DNA approach developed in the present study may offer a new way for preparing sufficient amounts of engineered perforin for use in the studies aimed at dissecting the functional domains of the perforin molecule.

Perforin and its role in T lymphocyte-mediated cytolysis.

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.

Role of Perforin in Lymphocyte-Mediated Cytolysis

Publisher Summary This chapter discusses structure and expression of perforin. Perforin is a primary candidate as mediator of the cellular cytotoxicity exhibited by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The granule exocytosis model of lymphocyte-mediated cytolysis, wherein perforin is expected to play a central role, has been proposed on the basis of a great deal of circumstantial evidence. The central role of perforin and universality of the granule exocytosis model have been challenged based on evidence that some CTLs apparently lacking perforin expression are found and that some CTLs and lymphokine-activated killer cells lysed certain target cells without apparent granule exocytosis. The physiological relevance of the perforin-dependent and -independent pathways of lymphocyte-mediated cytolysis, mainly based on recent observations is discussed. The propriety of the perforin/granule exocytosis model has been challenged by extensive studies using various T cell clones, including classical CD8+ CTLs and recently characterized CD4+ helper/killer T cells. Perforin expression in primary CTLs in peritoneal exudate lymphocytes (PELS) after intraperitoneal immunization with allogeneic spleen cells is examined.

Perforin: structure, function, and regulation.

The mechanism of lymphocyte-mediated cytolysis is controversial (see recent reviews Bleackley et al. 1988; Brunet et al. 1988; Clark et al. 1988; Mueller et al. 1988; Müller-Eberhard 1988; Munger et al. 1988; Nagler-Anderson et al. 1988; Sitkovsky 1988; Young et al. 1988; Podack et al. 1988; Jenne and Tschopp 1988a, b; Bleackley 1988; Hershberger et al. 1988; Stevens et al. 1988; Tschopp and Nabholz 1990; Podack et al. 1991). The main contentions concern the question whether secretory functions of the killer cell, as proposed by the vectorial granule secretion model of cytotoxicity (Podack 1985; Henkart 1985), are required for target cell lysis and whether secretory models of cytotoxicity account for most of the cytotoxic activity. Other open questions are related to the role of DNA degradation (apoptosis, programmed cell death) in target cell lysis (Russel et al. 1982; Russel 1983; Duke et al. 1983; Lovell and Martz 1987) and how this process is mediated molecularly. No molecular tools are available at the present time to assess the importance, or even existence, of the putative secretion-independent pathway of lymphocyte-mediated cytolysis. Similarly, since the mechanism of DNA degradation is not understood, it is difficult to quantitate its role in cytolysis.

Efficacy of cyclophosphamide in toxic epidermal necrolysis: Clinical and pathophysiologic aspects

In this article we describe the immunocytochemical and electron microscopic findings in five patients with toxic epidermal necrolysis. They indicate the occurrence of necrotic keratinocytes with nuclear disintegration associated with apposed dendritic cells with the nuclear chromatin configuration of T lymphocytes. These findings, including the presence of blebbing of the keratinocytes and membrane defects associated with cytoplasmic processes from these apposed lymphoid cells, fit known electron microscopic criteria that suggest the involvement of T lymphocyte-mediated cytolysis of drug-altered target keratinocytes in toxic epidermal necrolysis. The effector cell appears to be a dendritic subset, with the phenotypic characteristics (CD3+, CD4-, CD8+, CD2+, DR+) of a T cell subset. There is some evidence that tumor necrosis factor alpha, secreted by activated macrophages, may play a role in necrolysis of the epidermis. The dramatic response of our patients to cyclophosphamide, which is known to inhibit cell-mediated cytotoxicity by inhibiting both the recognition and lethal hit stages, together with the rapid regrowth of the epidermis within 4 days to a week in patients who received adequate dosage of the drug, supports the preceding concepts.

The cytolytic T lymphocyte and its mode of action.

While the binding step of cytolytic T lymphocyte (CTL) target cell interaction resulting in conjugate formation is a well-characterized event, there seems to be more than one mechanism whereby lymphocytes kill the target. In recent years, infliction of complement (C)-like "holes" (I.D. 10-20 nm) on the target cell membrane, believed to be produced by the Ca2+-dependent lytic protein(s) perforin/cytolysin of secretory lytic granule origin has been proposed to be the mechanism of lymphocytotoxicity. More recent evidence, however, suggests that Ca2+-dependent exocytosis of lytic granules (where detectable) is not involved in lymphocyte-mediated cytolysis. Furthermore, neither formation of C-like "holes" in targets exposed to CTL, nor higher-than-background levels of lytic granules, perforin or BLT-esterases, have been detected in highly potent, peritoneal exudate CTL (PEL) derived directly from the animal or in cytocidal PEL-hybridomas. Hence exocytosis of perforin and formation of the above pores may apply to certain effector cells, particularly those grown in vitro in IL-2, but not to in vivo primed CTL such as PEL. On the other hand, work from this laboratory with Ca2+ probes has shown that lysis induced by CTL such as PEL-not involving lytic granules, perforin or formation of the above "holes"-is preceded by a marked prelytic elevation of cytosolic Ca2+ in the target. CTL-induced target cell membrane perturbation--a direct result of receptor-mediated effector-to-target interaction or through a membrane-bound or secreted effector component(s)--may be responsible for triggering the prelytic influx of Ca2+ from external sources, or its mobilization from internal stores in the target. We propose that CTL-induced, persistent elevation of cytosolic Ca2+, above a critical level, rather than formation of 10-20 nm pores, is responsible for the catastrophic prelytic events observed in the target, such as bleb formation, metabolic exhaustion and DNA degradation, ultimately leading to lysis.

3-Deazaadenosine 5′-triphosphate: A novel metabolite of 3-deazaadenosine in mouse leukocytes

Evidence has been obtained for the metabolic formation of small amounts (1–2% of the ATP pool) of 3-deazaadenosine 5′-triphosphate (c 3ATP) from 3-deazaadenosine (c 3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c 3Ado. Moreover, in the presence of MgCl 2, NaCI and IMP, purified rat liver 5′-nucleotidase catalyzed the phosphorylation of c 3Ado to 3-deazaadenosine 5′-monophosphate (c 3AMP). Two lines of evidence suggest that the metabolic formation of c 3ATP is not involved in the inhibition of leukocyte function caused by c 3Ado. First, the inhibitory action of c 3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c 3ATP remained constant in lymphocytes and macrophages after removal of c 3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c 3ATP were formed from both c 3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c 3ATP metabolite is of relevance for the mechanism of action of c 3Ado in mouse leukocytes.

Lymphocyte‐Mediated Cytolysis

Lymphocyte‐Mediated Cytolysis

Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms.

Cholera toxin (CT), but not pertussis toxin (PT), treatment of cloned murine CTL inhibited target cell lysis in a dose-dependent fashion. The effects of CT were mimicked by forskolin and cyclic adenosine monophosphate (cAMP) analogues. Inhibition of cytotoxicity by CT and cAMP analogs was mediated in part by attenuation of conjugate formation. Additionally, both CT and cAMP analogs blocked the increase in intracellular Ca2+ induced by stimulation of the TCR complex by mAbs. These findings indicate that cAMP inhibits the activity of CTL by two distinct mechanisms and suggests a role for this second messenger in CTL-mediated cytolysis.