Escherichia coli hemolysin mutants with altered target cell specificity.

In order to understand the functional significance of HlyC-dependent acylation of the Escherichia coli hemolysin structural protein (HlyA), random as well as site-directed substitutions at the known regions of modification, i.e., those at lysine residues at amino acid positions 563 and 689 (HlyAK563 and HlyAK689, respectively), were isolated. Sixteen random hlyA mutations were identified on the basis of a screen for loss of immunoreactivity to the hemolysin-neutralizing D12 monoclonal antibody that reacts to only HlyC-activated HlyA. These substitutions occurred at the region from HlyAE684 to HlyAY696. A recombinant glutathione S-transferase-hemolysin gene fusion encoding glutathione S-transferase-HlyAS608-T725 residues reacts with monoclonal antibody when HlyC is coexpressed with the fusion protein. Therefore, at most only 12% of the total HlyA primary sequence is needed for HlyC-facilitated acylation at the HlyAK689 position, and this modification can occur in the absence of the proximal HlyAK563 acylation site. The cytolytic activities of these HlyA mutants against sheep erythrocytes and bovine and human lymphocyte cell lines (BL-3 and Raji cells, respectively) were analyzed. HlyAK563 and HlyAK689 substitutions displayed various degrees of loss of cytotoxicity that depended on the particular amino acid replacement. An HlyAK563C variant retained greater than 59 and 21% of its BL-3-lytic and erythrolytic activities, respectively, but was nearly inactive against Raji cells. An HlyA mutant with a K-to-E substitution at amino acid 689 (HlyAK689E) was essentially inactive against all three cell types, whereas an HlyAK689R substitution had a pattern of activity similar to that of the HlyAK563C mutant. Preceding the two in vitro acylated HlyA lysines are glycines that appear to be the only amino acids conserved in alignments of these regions among the RTX toxins. Remarkably, considering the retention of cytotoxic activity by some HlYAK689 mutants, each of three different substitutions at the HlyAG688 position was relatively inactive against all three cell types tested. This suggests that HlyAG688 plays a significant structural role in cytotoxic activity apart from its possible participation in an HlyC activation process which presumably requires recognition of pro-HlyA structures. The related RTX toxin, the Pasteurella haemolytica leukotoxin structural protein (LktA), can be activated in an E. coli recombinant background by HlyC. In amino acid sequence alignments, LktAK554 is equivalent to the HlyAK563 position but it has an asparagine (LktAN684) at the homologous HlyAK689 site. An LktAN684K substitution possesses wild-type leukotoxin activity against BL-3 cells and does not acquire hemolytic or Raji cell cytotoxic activity. Surprisingly, both LktAK554C and LktAK554T substitutions retain considerable BL-3 cytotoxicity (45 and 49%, respectively), indicating that there may be additional lysines within LktA that the HlyC activation mechanism is capable of acylating. Based on these results and a comparison of amino acid sequence alignments of 12 RTX toxins, a putative consensus structure of the RTX residues necessary for HlyC activation is hypothesized.     

Nonreciprocal complementation of the hlyC and lktC genes of the Escherichia coli hemolysin and Pasteurella haemolytica leukotoxin determinants.

The genetic organization of the Pasteurella haemolytica leukotoxin operon (lktCABD) is similar to that of the Escherichia coli hemolysin (hlyCABD). Their gene products share a sequence similarity of 66, 62, 90.5, and 75.6%, respectively. We investigated the role of the C proteins (LktC and HlyC) by performing reciprocal transcomplementation analyses in an E. coli recombinant background. In the absence of the C genes, neither LktA nor HlyA had their respective cytotoxic activities. When hlyC was provided in trans to lktA, the toxin that was produced had the same activity and target cell specificity as the wild-type leukotoxin; it was leukotoxic for bovine lymphoid cells but not human lymphoblast cells when it was evaluated by a 51Cr-release assay. We also detected a weak hemolytic activity for the active form of LktA against sheep erythrocytes. In contrast, an E. coli strain containing lktC with hlyA produced a form of HlyA which was neither hemolytic nor cytotoxic. A monoclonal antibody (D12) against HlyA which recognized an epitope specific to the active form of HlyA did not cross-react in immunoblots with LktA that was activated by either LktC or HlyC. We conclude that the mechanism for activation of leukotoxin and hemolysin by their respective C proteins (LktC and HlyC) is mechanistically similar but that the exact structural requirements involved in the process are different.     

Characterization of monoclonal antibodies against the Escherichia coli hemolysin.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.     

Biological effects of two genetically defined leukotoxin mutants of Mannheimia haemolytica

Mannheimia(Pasteurella)haemolytica serotype 1 is the primary causative agent responsible for bovine pneumonic mannheimiosis, also known as shipping fever in cattle. The bacterium produces a variety of virulence factors, foremost of which is the exotoxic leukotoxin. The leukotoxin is a calcium-dependent cytolysin that is a member of the RTX (repeats in toxin) family and exhibits a narrow cell-type and species specificity and has biological effects only on ruminant leukocytes and platelets. The genetic organization of the leukotoxin is comprised of four genes: lktC, lktA, lktB and lktD. The lktA structural gene encodes the protoxin (pro-LktA) and lktC encodes a transacylase that post-translationally modifies the inactive pro-LktA to a biologically active wild-type leukotoxin (LktA). The LktA has been implicated as the key factor that contributes to the pathogenesis of lung injury associated with the disease and considerable efforts have been employed in abrogating toxin function while retaining immunogenicity, with an eye towards design of attenuated vaccines. We hypothesized that the pro-LktA retains the ability to cause biological effects on target cells as has been reported in the case of the closely related RTX toxin alpha-hemolysin (HlyA). We also examined the biological effects of an amino-terminal truncation mutant leukotoxin DeltaLktA on target cells. Thus the objectives of our study were to investigate whether two different mutant leukotoxins, one a nonacylated pro-LktA, and the other lacking 344 amino acids at the N-terminal end of the LktA protein; DeltaLktA, are capable of (i). binding to the beta2-integrin leukotoxin receptor, (ii). inducing the elevation of second messenger intracellular calcium ([Ca(2+)](i)), and (iii). inducing inflammatory gene expression, reactive oxygen metabolites (ROMs) and cytolysis in target cells. Our results demonstrate that neither acylation nor the amino terminal 344 amino acids are required for LktA binding but are essential for LktA-induced [Ca(2+)](i) elevation, generation of ROM, generation of the inflammatory cytokine IL-8 and cytolysis in target cells.     

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.     

Assignment of human natural killer (NK)-like cells to the T cell lineage. Single allospecific T cell clones lyse specific or NK-sensitive target cells via distinct recognition structures

The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.     

The selection of somatic cell hybrids between human lymphoma cell lines

Somatic cell hybrids have been selected between three pairs of established human lymphoid cell lines producing pure lines of proliferating hybrid cells: Raji/Namalwa, Raji/Daudi, and Raji/BJAB. The hybrid cell lines have been characterized with respect to isozyme pattern, volume, and karyotype.Paper 1 of the series, Somatic cell hybrids between human lymphoma cell lines.     

Association of RTX toxins with erythrocytes.

A critical step in the target cell attack by RTX cytotoxins is their association with target cells. A binding assay was used to study the association of the Escherichia coli hemolysin protein (HlyA) with erythrocytes. Several parameters required for lysis by HlyA were tested for their effects on its initial association with erythrocytes. The results demonstrate that HlyA binding to target cells is independent of several structural components of the active toxin, including the N-terminal hydrophobic region, the glycine-rich repeat region, and the HlyC-dependent acylation of HlyA. Further, the association with erythrocytes was independent of Ca2+ concentration or temperature, while the lytic event is both Ca2+ dependent and temperature dependent. The association of two other RTX toxin proteins, the Pasteurella haemolytica leukotoxin (LktA) and the enterohemorrhagic E. coli toxin (EhxA), were also examined; these toxins bound to erythrocytes much less efficiently than did HlyA. The association of HlyA with erythrocytes occurred rapidly, within 12 s of incubation, and demonstrated no measurable dissociation. HlyA bound to erythrocytes with a maximum of approximately 2,000 molecules per cell. Competition between active HlyA and unacylated HlyA demonstrated no inhibition of binding by unacylated HlyA; rather, active HlyA appeared to displace unacylated HlyA on the cell surface. These data demonstrate that binding and lysis by HlyA are separable events and challenge the concept of nonspecific binding to the cell surface by RTX toxins.     

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.     

Cytolysis of actinomycin D-treated target cells by cell-free supernatants from human monocytes

The lytic mechanism of human peripheral blood monocytes was studied by using as targets actinomycin D-treated WEHI-164, an NK-insensitive murine fibrosarcoma cell line. Monocytes, but not lymphocytes, lysed WEHI-164 target cells pre-treated with actinomycin D within 6 h in 51Cr-release assays. Because cytolysis could not be inhibited competitively by unlabeled WEHI/D target cells, contact-independent mechanisms of cytolysis were investigated. Cell-free supernatants collected from monocytes cultured for 4-6 h at 37 degrees C lysed target cells as effectively as effector cell preparations of monocytes. Supernatants from lymphocytes cultured in parallel were not cytolytic. Cytolytic activity was not detected in supernatants from preparations of monocytes that were held on ice. However, monocytes produced cytolytic activity whether they were isolated by adherence or remained unseparated in suspensions of mononuclear cells. The cytolytic activity in cell-free supernatants (CFS) from monocytes was unaffected by incubation with protease inhibitors. CFS activity was destroyed by heat. Storage of CFS at 37 degrees, 22 degrees, 4 degrees, or -20 degrees C for 24 h decreased cytolytic activity; however, loss of cytotoxicity was minimized by storage at 4 degrees C. The cytolytic substance detected in 4-h CFS from monocytes appeared to be a protein(s) based on the sensitivity of the cytolytic activity to proteases. Cytolytic activity of CFS eluted from Sephacryl 200 in a single peak with an apparent molecular weight between 25,000 and 45,000 daltons.     

Effect of the host cell on the maintenance and replication of Epstein-Barr virus.

We have studied the role the host cell plays in controlling the expression of the Epstein-Barr virus (EBV) by using Burkitt somatic cell hybrids of human and mouse cells. Mouse/Burkitt somatic-cell hybrids were shown to contain a repressed EBV genome that was inducible with iododeoxyuridine. Electron microscopic examination of human/Burkitt hybrid cells (D98/HR-1 and D98/Raji) and Burkitt lymphoblastoid cells in which EBV was replicating showed an enhancement of virus replication concomitant with an enhancement of EBV-specific cytopathologic effect in D98/HR-1 and D98/Raji cells when compared to Burkitt lymphoblastoid cells. When the stability of the EBV genome in human/Burkitt hybrid cells was studied, it was found that the EBV genome in hybrids of the nonproducer Burkitt cells (Raji) was less stable over time than hybrid cells of producer cells (HR-1). The data obtained in this study support the concept that the cell in which the EBV genome resides plays a major role in the maintenance, expression, and replication of EBV.     

The Photobacterium damselae subsp. damselae hemolysins damselysin and HlyA are encoded within a new virulence plasmid

Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a marine bacterium that causes infections and fatal disease in a wide range of marine animals and in humans. Highly hemolytic strains produce damselysin (Dly), a cytolysin encoded by the dly gene that is lethal for mice and has hemolytic activity. We found that Dly is encoded in the highly hemolytic strain RM-71 within a 153,429-bp conjugative plasmid that we dubbed pPHDD1. In addition to Dly, pPHDD1 also encodes a homologue of the pore-forming toxin HlyA. We found a direct correlation between presence of pPHDD1 and a strong hemolytic phenotype in a collection of P. damselae subsp. damselae isolates. Hemolysis was strongly reduced in a double dly hlyA mutant, demonstrating the role of the two pPHDD1-encoded genes in hemolysis. Interestingly, although single hlyA and dly mutants showed different levels of hemolysis reduction depending on the erythrocyte source, hemolysis was not abolished in any of the single mutants, suggesting that the hemolytic phenotype is the result of the additive effect of Dly and HlyA. We found that pPHDD1-encoded dly and hlyA genes are necessary for full virulence for mice and fish. Our results suggest that pPHDD1 can be considered as a driving force for the emergence of a highly hemolytic lineage of P. damselae subsp. damselae.     

Genetic conservation of hlyA determinants and serological conservation of HlyA: basis for developing a broadly cross-reactive subunit Escherichia coli alpha-hemolysin vaccine.

The HlyA determinant among Escherichia coli isolates from patients with symptomatic urinary tract infection was compared in this report with a prototype HlyA encoded by pSF4000 by DNA-DNA hybridization tests with 20-base synthetic oligonucleotides and monoclonal antibody binding and neutralization assays. Hybridization results demonstrated that 349 (98%) of 357 definitive reactions among 54 hemolytic strains shared homology with seven DNA probes spanning many HlyA regions corresponding to residues (R) 41 to 47, 55 to 61, 248 to 254, 306 to 312, 336 to 343, 402 to 408, and 929 to 935. Genetic divergence was identified by lack of hybridization signals among 17 to 76% of the hemolytic strains within the distal portion of a predicted hydrophobic region corresponding to R491 to 319 and within a predicted hydrophilic region corresponding to R491 to 497 and R532 to 538. Serological studies demonstrated that 26 (81%) culture supernatants of 32 hemolytic strains were bound by all 12 monoclonal anti-HlyA antibodies. Among five of six remaining strains, the culture supernatants were bound by 3 to 11 monoclonal antibody preparations. There was only one hemolytic culture supernatant that failed to be bound by any monoclonal antibody, although the strain hybridized with nine hemolysin DNA probes. In addition, hemolytic activity of all 24 different culture supernatants tested was reduced by at least twofold by one monoclonal antibody specific for R2-161. These data extend and support previous views that the HlyA determinant is conserved among E. coli strains and suggest that a broadly cross-reactive HlyA subunit vaccine can be developed.     

Cytolytic T cell hybridomas. II. Interleukin 2-independent cytolytic variants

Interleukin 2 (IL2)-dependent cytolytic hybrids usually loose IL2 dependence and cytolytic activity concomittantly. Here we describe an exceptional IL2-independent variant which lost IL2 receptor but retained cytolytic activity. When the IL2-independent cytolytic hybrid cells were fused with IL2-dependent cytolytic clones which express IL2 receptors constitutively, IL2-dependent hybrids were obtained. This suggests that the expression of IL2 receptors can interfere with IL2-independent growth mechanisms.     

Leukemic cells arise from cloned cytotoxic lymphocytes during cell culture

In spite of many promising attempts to apply T cell clones to questions of in vitro and in vivo function of T cells it is still unclear to what extent continuous propagation of T lymphocytes in vitro effects their original properties. This study describes the appearance of malignant cells from long-term cultured C57BL/6 (B6) cytotoxic T lymphocytes (CTL). Four out of five T cell lines (CTLL.1,3,4,5) representing distinct stages of development of T effector cells in vitro were repeatedly cloned and all five CTLL were tested for various cellular parameters. It is shown that transformation of H-Y-specific CTLL into malignant cells in vitro was accompanied by alterations in growth characteristics, successive loss of specificity and cytolytic function and by quantitative changes in the expression of cell surface markers. Whereas growth of the H-Y-specific CTLL (CTLL.1) was dependent on antigen and concanavalin A (Con A) supernatant (Con ASN) the CTLL variants could be either maintained in Con ASN alone (CTLL.3) or in the absence of both antigen and lymphokine sources (CTLL.4,5). CTLL.1 was cytolytic for male B6 target cells and lysed P815 tumor targets in the presence but not the absence of lectin. In contrast, CTLL.3 lost its original specificity but lysed P815 cells in the absence or presence of lectin. CTLL.2 representing an intermediary stage showed cytolytic activity on both male B6 and P815 target cells. In contrast, CTLL.4 and CTLL.5 lost the ability to lyse any of the indicated target cells. Although all CTLL expressed the surface markers Thy-1, Lyt-2, Kb, Db and interleukin 2 receptor (IL 2 R), Thy-1 and Lyt-2 markers were drastically reduced and Kb/Db and IL 2 R structures significantly increased on CTLL.4 and CTLL.5 compared to CTLL.1,2,3. In addition, multiple karyotypic alterations including the appearance of metacentric chromosomes were observed in long-term cultured CTLL. Investigations on the expression of the alpha-, beta-, and gamma-chains of the T cell antigen receptor in CTLL.1-5 indicate that all three chains were expressed as mRNA irrespective of whether the lymphocytes expressed their original specificity and/or function. However, distinct beta variable chain genes were used by H-Y-specific CTLL and its long-term culture variants CTLL.2 and CTLL.3 suggesting that the expression of the new specificity was accompanied by the rearrangement of a new beta-chain gene in T effector cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin.

Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.     

The immune response of the mouse to lymphocytic choriomeningitis virus. V. High numbers of cytolytic T lymphocytes are generated in the spleen during acute infection

In the spleens of C57BL/6J (B6) and CBA/J (CBA) mice undergoing acute infection with lymphocytic choriomeningitis (LCM) virus, lymphocytes with the ability to develop in vitro into LCM virus-specific cytolytic clones were enumerated by use of the limiting dilution method. At intervals after virus inoculation, defined numbers of cells were cultivated with virus-infected syngeneic stimulator cells and T cell growth factor in multiple wells of microculture plates. After 7 days, individual cell cultures were tested for their ability to cause release of 51Cr from infected and uninfected syngeneic target cells. In cultures seeded with spleen cells from uninfected mice or from mice infected 3 days previously, no cytolytic activity was observed. On day 5, cells developing into LCM virus-specific cytolytic effector cells were detected. They rose in numbers, and on days 8 to 9 after infection, values of approximately 1/10 and 1/200 in B6 and CBA mice, respectively, were calculated. A low proportion of microcultures proved cytolytic also for noninfected syngeneic target cells, but the counts thus released were consistently much lower than the counts set free from infected targets, and no regular dose-response relationships existed between seeded cells and positive cultures. Determination of cell surface antigens of responder cells by negative and positive selection procedures disclosed that they were predominantly T lymphocytes and expressed Lyt-2 but not L3T4 surface markers. Lysis by the great majority of LCM virus-specific clones was restricted by products of the major histocompatibility gene complex (MHC), but a few lysed, in addition, allogeneic infected or uninfected targets; however, a consistent pattern of alloreactivity was not observed. Furthermore, cells of a proportion of the cultures also lysed uninfected YAC cells. Probably this natural killer-like activity was acquired by T lymphocytes during prolonged cultivation. We conclude that most spleen cells that during acute infection with LCM virus attained the ability to develop in vitro into LCM virus-specific cytolytic clones were derived from MHC-restricted Lyt-2+, L3T4- antigen-specific cytolytic T lymphocytes and their activated precursors.     

Vibrio cholerae hemolysin is apoptogenic to peritoneal B-1a cells but its oligomer shepherd the cells for IgA response

Vibrio cholerae hemolysin (HlyA) can exist as a monomer with hemolytic activity and an oligomer that agglutinates erythrocytes. Biochemical differences accompanying the change in state of aggregation led us to weigh possible differences between the two forms from mucosal immunoregulation perspective. HlyA oligomer-treated murine B-1a cells up-regulated TLR2 and involved the signaling molecules MyD88, TRAF6 and NF-kappaB. The cells subsequently expressed IgM and IgA. HlyA monomer treatment although resulted in TLR2 up-regulation, could not induce these effects. Apoptosis was detected in majority of the monomer-treated cells that involved caspase-9 and caspase-3. This study shows for the first time that two forms of the same protein could drive the host immune cell to two different outcomes, one of death and the other towards activation.     

Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.