T-cell-mediated lysis of B cells induced by a CD19xCD3 bispecific single-chain antibody is perforin dependent and death receptor independent

A recently developed bispecific antibody construct, directed against CD19 and CD3 (bscCD19xCD3), induces T-cell-mediated lysis of allogeneic and autologous B cells in a specific and highly efficient manner. Since knowledge of the molecular mechanisms underlying this lysis is limited, a study on bscCD19xCD3-activated T-cell-effector pathways was performed. BscCD19xCD3-induced lysis of target B-cell lines Nalm-6, Daudi, and Raji and of autologous primary B cells is caused by the perforin-dependent granule-exocytosis pathway but not by the death ligands FasL, TRAIL, or TNF-. When activated by bscCD19xCD3 and Raji cells, T cells express FasL mRNA, but incubation of Raji cells with cell-free supernatants from cytotoxicity experiments caused an upregulation of c-Flip_l, possibly accounting for the cells insensitivity toward death-receptor-mediated lysis. In addition to granule exocytosis, Raji cells are lysed by at least one mechanism independent of perforin, which requires transport through the T cells Golgi apparatus.This investigation was supported by the Deutsche Forschungsgemeinschaft, Klinische Forschergruppe, grant no. KFO 105/1.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

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.     

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.     

Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Human perforin employs different avenues to damage membranes

Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro.     

Cytotoxicity responses to selected ESAT-6 and CFP-10 peptides in tuberculosis

Cytotoxicity responses were studied for the ESAT-6 peptides Esp1, Esp6, Esp7, Esp8, and CFP-10 peptides, Cfp6, Cfp7, Cfp8, Cfp9 (synthetic 20-mer peptides) and the recombinant ESAT-6, CFP-10 proteins. Cytolytic molecules perforin, granzymes A and B, granulysin responses in healthy household contacts (HHC) and pulmonary tuberculosis patients (PTB), were studied by intracellular flow cytometry. Functional cytotoxicity was studied in both the groups for the peptides Esp6 and Cfp8 by an enzyme (lactate dehydrogenase) based assay. The results revealed that cytolytic molecule positive CD4+ and CD8+ T cells were increased in HHC in response to Esp1, Esp6, Cfp8 and Cfp9 immunogenic peptides compared to PTB. Functional cytotoxicity results showed higher cytotoxicity (not statistically significant) to be exhibited by the peptide Esp6 than Cfp8 in the HHC.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.