Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Evidence of selective processing of immunodominant epitopes in virally infected cells

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.     

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (10⁵ cells mL^-1) at densities of 10⁷, 10⁵, and 10³ PA14 cells mL^-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10?days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30?days; total MCs: 91.0%, 86.9%, and 61.6% at 30?days, respectively). However, the activity of PA14 diminished when its density decreased to less than 10⁶ cells mL^-1. At three initial algal densities (10⁶, 10⁵, and 10³ cells mL^-1), PA14 at a density of 10⁵ cells mL^-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30?days, respectively). Host range assays showed that at lower initial PA14 inoculation (10⁵ cells mL^-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (10⁷ cells mL^-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 10⁵ Pseudomonas aeruginosa PA14 cells mL^-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.     

Production of lymphokines affecting tumor cells by T-T hybridomas

T-Cell hybridomas were constructed by fusing BW5147, an AKR lymphoma, with concanavalin A-stimulated murine splenic lymphocytes. The hybrids which were formed were studied for their ability to produce a lymphokine which inhibits tumor cell migration (TMIF) as well as macrophage migration (MIF) using in vitro assays. Clones were identified which affected tumor cell motility without exerting similar effects on murine macrophages, although the opposite effect was not observed. Although noncoordinate production of these factors cannot be unequivocally established, these results demonstrate that clones can be constructed that preferentially secrete TMIF. In these experiments, we also tested the supernatants for another lymphokine effect on tumor cells; namely, the ability to inhibit tumor cell binding to endothelial monolayers. A number of clones were identified that lacked TMIF activity, but could inhibit the tumor cell-endothelial interaction, suggesting the possibility that these effects may be due to separate mediators.     

Immune responses to weakly immunogenic virally induced tumors. III. Genetically unrestricted cytolysis of allogeneic tumor target cells.

Splenocytes from A mice injected with YAC-1 or RBL5 could generate, after in vitro culture with or without stimulation, a genetically nonrestricted cytotoxic response against the allogenic tumor RBL5. YAC-1 tumor is an in vitro carried tumor induced in A mice (H-2a) by Moloney virus. RBL5 tumor is a Rauscher virus-induced tumor of C57BL/6 mice (H-2b). These tumors cross-react serologically. The effector cells that were generated after the in vitro cultivation recognized tumor-associated antigens on the target cells. H-2 alloantigens were not recognized by the effector cells. The effector cells that killed RBL5 tumor in a genetically nonrestricted manner were identified as T cells. The in vivo carried tumor YAC, in contrast to the in vitro carried tumor YAC-1, could not induce anti-RBL5 reactive cells in A mice. Instead, YAC tumor induced suppressor cells in A mice, which could abrogate the anti-RBL5 cytotoxic response of RBL5-primed splenocytes, but not that of YAC-1 primed splenocytes.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage

The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells.     

Echinococcus multilocularis: in vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells

Hybrid cells were produced from Echinococcus multilocularis metacestode germinal cells and murine tumor cells. Small colonies were formed which, while ceasing to grow after a few generations, remained viable for at least 10 weeks. These hybridoma cells secrete antigen(s) reacting in indirect immunofluorescence and ELISA specifically with sera from patients suffering from an E. multilocularis infection. The antigen(s) appear suitable for the differential diagnosis of E. multilocularis and E. granulosus. Thus, hybridoma cells may produce helminth antigens.     

Effect of migillin on protein synthesis in intact and virally infected TKCE and FL cells]

The effect of migillin was studied with respect to protein synthesis in the cells of TKCE (transplantable line of cells of the kidneys of cow embryos) and FL--intact and infected with viruses of aphtous fever A22, strain 550 and poliomyelitis of type III, strain Saukett respectively. Simultaneously the effect of migillin on sensitivity of the cells to the above viruses was tested. The synthesis of protein was determined by incorporation of C14-glycine into the acid insoluble fraction of the cells. It was found that protein synthesis significantly increased under the effect of migillin in both the intact cells and the cells infected with the viruses. The viruses of aphtous fever and poliomyelitis inhibited the synthesis of proteins. The antibiotic increased the activity of dehydrogenases and respiration of HEp-2 cells. Migillin markedly suppressed reproduction of the poliomyelitis virus, the effect on the virus of aphtous fever was lower in the cell culture. In guinea pigs the antiviral effect of the antibiotic resulted in prolongation of the incubation period and retardation of the aphtous fever process generalization. The results of the experiments showed that migillin stimulated the activity of dehydrogenases, respiration, protein synthesis in the cell cultures and possessed antiviral activity in vitro and in vivo.     

Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm

A bacteriophage specific for Aggregatibacter actinomycetemcomitans serotype b, able to kill the bacterium within a biofilm, was isolated. Random mutagenesis of this phage rendered a bacteriophage able to kill 99% of the bacteria within a biofilm. This is the first report of a biocontrol experiment against A. actinomycetemcomitans.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

Effect of selenium on virally induced and transplantable tumor models

Se is effective in inhibiting both virally induced and transplantable tumors. Continued intake of Se at quantities greater than those required to optimize growth and glutathione peroxidase (EC 1.11.1.9) activities appear to be needed to achieve maximal tumor inhibition. Differences in the sensitivity to Se of various tumor cell lines are evident. The efficacy of Se depends on the form and mode of administration of this trace element. Total tumor mass also appears to affect the efficacy of Se. Evidence now suggests that selenodiglutathione or some other intermediate in Se metabolism is responsible for the antitumorigenic properties of this trace element.     

In vivo polarization of IFN-gamma at Kupfer and non-Kupfer immunological synapses during the clearance of virally infected brain cells

Kupfer-type immunological synapses are thought to mediate intercellular communication between antiviral T cells and virally infected target Ag-presenting brain cells in vivo during an antiviral brain immune response. This hypothesis predicts that formation of Kupfer-type immunological synapses is necessary for polarized distribution of effector molecules, and their directed secretion toward the target cells. However, no studies have been published testing the hypothesis that cytokines can only form polarized clusters at Kupfer-type immunological synapses. Here, we show that IFN-gamma and granzyme-B cluster in a polarized fashion at contacts between T cells and infected astrocytes in vivo. In some cases these clusters were found in Kupfer-type immunological synapses between T cells and infected astrocytes, but we also detected polarized IFN-gamma at synaptic immunological contacts which did not form Kupfer-type immunological synaptic junctions, i.e., in the absence of polarization of TCR or LFA-1. This indicates that TCR signaling, which leads to the production, polarization, and eventual directed secretion of effector molecules such as IFN-gamma, occurs following the formation of both Kupfer-type and non-Kupfer type immunological synaptic junctions between T cells and virally infected target astrocytes in vivo.     

P. Falciparum infected erythrocytes are capable of endocytosis

Summary P. falciparum, an intraerythrocytic parasite, obtains nourishment primarily through phagocytosis of the host cytosol but also through the incorporation of extracellular small molecules which enter through the parasitized red cell's membrane via pores. Normal mature erythrocytes are incapable of endocytosis. Several lines of evidence suggest that extracellular large molecules may be taken up when the mature red cell is parasitized byP. falciparum, but direct evidence has been lacking. We now report the use of ferritin, an electron dense protein, to demonstrate endocytosis inP. falciparum infected red cells. Parasitized red cells incubated with ferritin internalize that macromolecule as demonstrated by electron microscopy. While normal red cells incubated with ferritin took up none of the tracer molecule, parasitized red cells internalized substantial amounts. In addition both ferritin and apoferritin inhibited the growth ofP. falciparum in a dose dependent fashion, again indicating endocytosis of a macromolecule. These data indicate thatP. falciparum can somehow stimulate the mature erythrocyte to engage in endocytosis. We also note that both infected and non-infected red cells in a culture in whichP. falciparum is growing become abnormally sticky for ferritin. Moreover, parasitized red cells bind I¹²⁵-transferrin while non-parasitized erythrocytes do not. These observations suggest that a soluble parasite product alters the red cell membrane in a non-global manner, causing selective effects in relation to different proteins.     

Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells

Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.