Mechanisms of cellular cytotoxic innate resistance in tilapia (Oreochromis nilotica)

Mechanisms of innate cytotoxic immunity in tilapia (O. nilotica) were measured by characterization of the activity, distribution and functions of nonspecific cytotoxic cells (NCC). Active cytotoxic cells were obtained from anterior kidney. spleen and peripheral blood whereas nonlytic but anti-NCC monoclonal antibody 5C6 positive cells were obtained from tilapia liver. Thymocytes were not cytotoxic and were mab 5C6+. Unfractionated anterior kidney cells were 6% mab 5C6+ and had very low cytotoxicity of HL-60 target cells. Percoll (45.5%) purified NCC were 44% mab 5C6+ and had 35% HL-60 cytotoxicity (160:1 E:T ratio). Transformed mouse and human target cells were tested for sensitivity to NCC lysis. HL-60, U937, K562, IM-9 and NC-37 human targets were lysed by NCC. YAC-1 targets were insensitive to lysis. The killing of HL-60 targets by tilapia NCC was inhibited by mab 5C6. Experiments to determine optimal conditions for the cytotoxicity assay revealed that tilapia required 15-20h for optimum lysis of targets. Incubation at 37 C produced the highest cytotoxicity. The proliferative competence of Percoll purified anterior kidney cells was determined. A significant increase in in vitro uptake of tritiated thymidine by anterior kidney cells occurred following stimulation by mab 5C6, Con-A, PMA and calcium ionophore A23187. Purified spleen cells also produced significant increased uptake of tritiated thymidine following in vitro activation with PMA and mab 5C6, but not Con-A. These studies indicated that NCC may provide innate cytotoxic immunity similar to that provided by the NCC of catfish.     

Partial Amino Acid Sequence of a Novel Protozoan Parasite Antigen that Inhibits Non-Specific Cytotoxic Cell Activity

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC-37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e. g. NC-37, YAC-1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46–50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25–40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM-9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited NCC lysis of a variety of human and murine transformed targets (e. g. HL-60, MOLT-4, DAUDI, NC-37, U-937, YAC-1, EL-4). Inhibition was specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequencc analysis. Direct N-terminal analysis yielded a 12 residue sequence. Additional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology to known sequences (Swiss-Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.     

Inhibition of SK cell activity in frogs by certain drugs and sugars

We present similarities between mammalian natural killer (NK) cells and (anuran amphibian) frog spontaneous killer (SK) cells. A cytotoxic assay utilizing allogeneic erythrocytes as target cells was used and lysis assessed by measuring release of hemoglobin. SK effector cells, just as mammalian NK cells, are not sensitive to cycloheximide nor most simple sugars (50 mM glucose, glucose-6-phosphate, galactose, fucose, mannose). However, SK activity is inhibited by chloroquine, colchicine and mannose-6-phosphate. When SK cells were co-incubated with mammalian tumor cells, they were able to lyse only the NK-sensitive target YAC-1, but not other mammalian tumor cell targets including K562, Molt-4, Raji, P815 and EL4. Lysis of YAC-1 cells was also inhibited by colchicine and chloroquine. These results allow speculation on the evolution of cell mediated cytotoxicity since natural cytotoxic cells are present in ectothermic vertebrates.     

Natural Killer Cells and Cytotoxic T Cells Induce DNA Fragmentation in Both Human and Murine Target Cells in Vitro

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC-1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC-1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.     

Natural antitumor defense system of the murine liver

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1(4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Corynebacerium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver macrophages from untreated mice do not have tumoricidal activity per se but can "activate" NK cells to kill tumor cells normally resistant to NK cells.     

N-α-Benzyloxycarbonyl-l-Lysine Thiobenzyl Ester (BLT) Serine Esterase in Human Cytolytic Effector Cells and Cell Line Targets

The granules of in vitro primed cytotoxic mouse T cells and cytotoxic cell lines have been shown to contain high levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase. The enzyme activity has been suggested to be associated with the cytotoxic capacity of killer cells. We investigated human leucocytes and found that neutrophils, monocytes, cytotoxic T lymphocytes (CTL), natural killer (NK) cells [large granular lymphocytes (LGL)], and interleukin 2 activated killer (LAK) cells, which all display efficient cytotoxic capacity, show only marginal BLT esterase activity. The low BLT esterase activity in human lymphocytes increases about twofold when cells are stimulated in vitro with interleukin 2 (IL-2), phytohaemagglutinin (PHA), or cultured in mixed lymphocyte culture (MLC). Mouse T lymphocytes have about 20 times more BLT esterase activity than human T lymphocytes. The BLT activity in mouse T cells also increases about twofold in MLC. The human leukaemia cell lines (K562, U937, MOLT-4, Jurkat) and the mouse mastocytoma line (P815), which are frequently used as target cells, contain more BLT esterase activity than human resting or activated lymphocytes. We did not find a direct correlation between the cytotoxic capacity and the BLT esterase activity of killer cells.     

Mapping of the Epitope Recognized by Non-Specific Cytotoxic Cells: Determination of the Fine Specificity Using Synthetic Peptides

NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N-terminal, C-terminal and internal NKTag (deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20-mer peptide equivalent to amino acids (aa) nos. 55–74 specifically inhibited NCC lysis of human EBV transformed target cells (IM-9). Inhibitory effects were nonreversible and concentration dependent; and 30 min pre-incubation produced optimum inhibition. The inhibitory 20-mer peptide was truncated into 17, 14, 10, 9 and 6-mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6-mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68–74 (ARG-ASN-LEU-THR-PHE-ILE-LEU-)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14-mer peptide composed of aa nos 61–74 inhibited lysis of HL-60, IM-9, DAUDI, YAC-1, U937 and NC-37 target cells. Flanking peptides (aa nos 35–54 and 75–94) were negative. Biotinylated aa nos 61–74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9-mer obliterated the inhibitory effects. The 17-mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM-9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7–9 amino acids in the N-terminal region of NKTag.     

Phagocytosis of candida albicans by lymphatic tumour cells in vitro

Experiments were carried out to investigate whether different lymphatic tumour cell lines have similar kinetic characteristics of phagocytosis of microorganisms. Six tumour cell lines were used. These were a human T-cell line (CEM), a mouse T-cell line (YAC-1), a human B-cell line (LAZ), and a human erythroleukemic tumour cells (K562), whereas 2 cell lines of professional phagocytosis were used as controls, a human macrophage cell line (THP1) and a mouse macrophage cell line (P388D1). Tumour cells were mixed with candida albicans at a ratio of 10:1 of candida to tumour cells and the percentage of tumour cells that had attached/phagocytosed candida was determined. After 4 h coculture with candida, tumour cells not of T-cell origin (LAZ and K562) showed moderate level of phagocytosis (28%), whereas tumour cells of T-cell origin (CEM and YAC-1) demonstrated low levels of phagocytosis (15%) as compared to macrophage cell lines (THP1 and P388D1) that showed maximum phagocytosis (64-78%). Acid phosphatase (AcPase) activity was increased by 33% during coculture of YAC-1 cells and yeast cells. In conclusion, the results suggest that lymphatic tumour cells of nonphagocytic origin acquire phagocytic properties during the course of malignancy, and digestion of phagocytosed yeast cells maybe related with AcPase activity, as well as that of other lysosomal enzymes. This phenomenon may represent one mechanism by which tumour cells downregulate immune surveillance.     

Nonspecific cytotoxic cells of rainbow trout (Oncorhynchus mykiss) kill YAC-1 targets by both necrotic and apoptic mechanisms

Nonspecific cytotoxic cells (NCC) have been identified in a number of fish species and are thought to be evolutionary progenitors of mammalian natural killer cells. We show here that trout NCC are functionally similar to cytotoxic cells of higher vertebrates in that they mediate cytotoxicity through both mechanisms of apoptosis and necrosis. To demonstrate that trout NCC inflict apoptic and necrotic lesions in tumor target cells, DNA fragmentation and 51chromium release assays were conducted using leukocytes isolated from peripheral blood, spleen, and anterior kidney. At effector-target ratios of 25:1, 50:1, 100:1, and 200:1, the release of thymidine-labeled DNA fragments and the release of 51chromium from YAC-1 target cells paralleled one another. Percent chromium release and DNA fragmentation increased when effector:target incubation times were extended from 4 to 18 h. As evidenced in agarose gels, the pattern of fragmentation induced by trout effector cells was identical to that produced by BALB/c NK cells. Similar to human and murine NK cells, trout NCC were maximally inhibited by 50 mM mannose-6-phosphate. Morphologic characteristics of rainbow trout NCC were examined using light and electron microscopy. Photomicrographs of effector:target cell mixtures after a 1 h incubation show NCC binding to target YAC-1 cells. Transmission electron micrographs of the conjugates revealed that the cells responsible for killing are small (4.2-4.5 microns), agranular mononuclear leukocytes.     

Target Cell Lysis by Natural Killer Cells is Influenced by β2-Microglobulin Expression

Natural killer (NK) cells form part of the vertebrate defence against viruses and tumours, but show only limited specificity. The molecule(s) recognized by NK cells on target cells are at present unknown. Major histocompatibility complex (MHC) class I antigen concentration on target cells is inversely correlated with NK cell lysis. Here we show that MHC class I-unassociated beta 2-microglobulin (beta 2-m) expression is involved in NK cell-target cell interaction. Two human MHC class I negative cell lines, Daudi and K562, are differentially susceptible to NK cell lysis. Daudi cells are beta 2-m-negative and resistant to NK lysis, K562 are beta 2-m-positive and highly susceptible to lysis by NK cells. Interferon (IFN) treatment augments beta 2-m expression and NK lysis of K562, but not in Daudi cells. NK cell lysis of K562, but not YAC-1 cells, can be inhibited by monoclonal anti-human beta 2-m antibody. Furthermore, susceptibility of mouse embryo fibroblasts (MEF) to NK lysis can be increased by infection with recombinant vaccinia virus expressing the human beta 2-m gene.     

Use of a P815-derived line with an amplified adenosine deaminase gene: an improved target for cellular cytotoxicity

We describe a cytotoxic T lymphocyte-mediated cytotoxicity assay in which the release of a cytoplasmic enzyme, adenosine deaminase (ADA), instead of the widely used radioactive chromium is a measure of target lysis. In this enzyme-release assay the target is a mastocytoma P815-derived cell line, noted P815 ADA++, isolated by applying a selection procedure devised to specifically amplify the ADA gene. Gene amplification in P815 ADA++ was indeed demonstrated. Routine measurement of ADA activity from numerous supernatants is performed using a specific and sensitive colorimetric assay. The use of 96-well microtiter plates as well as of an automatic Multiscan spectrophotometer makes this measurement rapid and convenient. We show that this ADA-release assay is significantly more sensitive than the classical chromium-release test because of its consistently lower (5 to 10-fold) spontaneous release in 4 h, short-term cytotoxicity experiments. We also found that it is especially suited for the rapid detection, by visual screening, of rare, active killer clones among large, heterogeneous cytotoxic T lymphocyte populations. The assay could easily be adapted to other tumor targets (EL4, YAC-1, K562) of common use in studies involving immune lysis; indeed, the procedure of amplifying the ADA gene used in the isolation of the P815 ADA++ hyperactive line may be generally applied to these targets.     

Enumeration andCharacterization of Human Killer and Natural Killer Cells by a Modified Single-Cell Assay

Human natural killer (NK) and killer (K) cells were assayed in a modified single-cell cytotoxicity assay using poly-L-lysine-coated cover slips. When human Chang liver cells were used as targets, 20% of the lymphocytes formed conjugates and 2% were active NK cells. When anti-Chang antibodies were present, the proportion of target-binding cells (TBC) increased to 30% and that of the cytotoxic effector cells (comprising NK + K) to 6%. With the mouse mastocytoma cells (P815), which are not susceptible to NK, similar proportions of lymphocytes formed conjugates, and 6-9% were active as K cells. By an in situ rosetting assay a significant fraction of the TBC and cytotoxic effector cells bound either C3b or C3bi in both systems, with a certain predominance of C3bi-binding cells among the K cells. However, by indirect immunofluorescence, significantly more OKT3+ cells than OKM1+ cells were TBC or cytotoxic in the Chang cell system, whereas the OKT3+/OKM1+ ratios for both TBC and cytotoxic cells were 1:1 in the mouse mastocytoma system. The results indicate that TBC, NK and K cells are heterogeneous with respect to surface marker expression and that effector cells of different phenotypes predominate in different target systems.     

Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors

Exposure of tilapia (Oreochromis niloticus) to water temperatures of 10-15 degrees C for 3-5 min produces physiological stress responses characterized by immediate phenotypic and immunological changes. In the present study, this general stress response was utilized as a model system to study innate immunity mediated by soluble factors and cytotoxic cells. Acute innate cytotoxic responses of nonspecific cytotoxic cells (NCC) in the peripheral blood (PBL), anterior kidney (AK) and spleen (SPL) were measured. Following temperature stress, the levels of NCC activity depended on the presence of soluble factors and on the cell compartments from which the NCC were obtained. NCC from PBL of stressed tilapia had 30x or greater cytotoxic activity compared to nonstressed PBLs from controls. NCC activity from the AK and SPL of stressed tilapia was lower than controls. Flow cytometric analysis of NCC in each tissue showed that increased cytotoxicity was not produced by increased numbers of NCC. To determine the mechanism of amplification of cytotoxicity, NCC from nonstressed tilapia were passively treated with serum from temperature stressed tilapia. Serum containing the "stress activated serum factor" (SASF) passively increased naive NCC cytotoxicity (from PBL) 3-4 fold. The cytotoxic cell response was inhibited by addition of anti-NCC monoclonal antibody 5C6. These data indicated that NCC are (at least one of) the target cells for SASF. SASF required only 15 min pre-incubation with naive NCC to activate cytotoxicity. Activation was nonreversible and concentration dependent. Pretreatment of NCC with SASF reduced the assay time required to amplify target cell cytotoxicity from 12-24 h to 6 h. SASF amplification of NCC cytotoxicity was not restricted by different histological types of target cells. Determination of select physical/chemical properties of SASF revealed: complete heat inactivation of cytotoxicity amplification following 55 degrees C and 65 degrees C pretreatment; SASF was thermostable at room temperature to 45 degrees C for 15 min; and freeze-thaw treatment reduced but did not completely remove amplification activity. The molecular weight range of SASF activity was identified in a 50-100 kDa fraction obtained by differential dialysis. SASF appears to be a protein sensitive to trypsin digestion.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). I. Optimum requirements for target cell lysis

Nonspecific cytotoxic cells (NCC) obtained from the head (anterior) kidney of fish (Ictalurus punctatus) lyse human transformed B-cell targets. Lysis depended on direct cell-cell contact. Fish size, age, environmental holding temperatures, and lytic reaction conditions such as osmolality and optimum effector:target cell ratios were optimized. Experiments to characterize optimum kinetics demonstrated highly efficient killing after two hours incubation. This rapid cytolysis was further studied by determining NCC activity against appropriately labeled target cells after 30, 60, 90 and 120 minutes of cocultivation. At 160:1 (E:T) greater than 40% of the 5 hour percent specific release value was produced after 30 minutes. After 90 minutes, more than 90% of total percent specific release was observed. At least one mechanism of regulation of NCC killing was described. In the presence of normal (homologous or heterologous) catfish serum (CFS), essentially no NCC activity was observed. This suppression was reversible by preincubation in 10% fetal bovine serum (FBS). NCC "activation" by preincubation in 10% FBS was time-dependent (at least four hours was required to generate significant lysis). NCC activation could be reversed by treating potentially lytic cells with supernatants containing dissociated CFS. In addition, reversible activation could be demonstrated by treating potentially lytic effector cells with CFS to produce suppression. Regulation occurred at the effector cell level because treated target cells did not suppress NCC activity. These data demonstrate a population of nonspecific effector cytolytic cells that potentially represent a phylogenetic precursor to mammalian natural killer cells.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). VI. Flow cytometric analysis

Nonspecific cytotoxic cells (NCC) from channel catfish (Ictalurus punctatus) were enriched using flow cytometry (EPICS V, 753). Parameters of forward angle and 90 degrees light scatter were utilized to sort cytolytically functional populations of NCC. Fullbright (GR. II) 9.75 microns diameter fluorescent polystyrene microspheres were used for optical alignment. Cells obtained from the anterior kidney, spleen and peripheral blood were analyzed by one and/or two parameters of forward angle light scatter (FALS) and log integral 90 degrees light scatter (L90 degrees LS). Populations identified from FALS or L90 degrees LS histograms were sorted and cells were tested for cytotoxicity against NC-37 target cells. Anterior kidney contained at least four different sized populations of NCC. Spleen cells were sorted by FALS and four different populations of NCC detected. Peripheral blood contained little NCC activity. Enrichment of NCC activity from anterior kidney tissue was compared using Percoll density gradient separation and flow cytometry. Three to four fold higher cytotoxic activity was obtained from sorted cells compared to the population obtained from 45.5% Percoll. Additional experiments to determine the enrichment of sorted NCC were conducted by analyzing the number of enriched NCC required to kill an individual NC-37 target cell. Approximately 1.5-2.0 NCC could lyse a chromium-51 labelled target cell. These studies demonstrated that NCC could be enriched by size and granularity discrimination using flow cytometry. Effector:target cell ratios of 10:1 and lower produced significant lysis when sorted cells were analyzed.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Regulatory Role of CD8 in Major Histocompatibility Complex-Unrestricted Tumoricidal Activity of Mouse T Cells Activated with Anti-CD3 Monoclonal Antibody

Antigen-nonspecific CD8⁺ cytotoxic T cells induced with anti-CD3 monoclonal antibody (mAb) are able to kill tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion. However, the role of CD8 in the MHC-independent tumoricidal activity of anti-CD3-activated killer T (AK-T) cells has not been investigated. Here we show that anti-CD8α mAb inhibits, in a dose-dependent fashion, lysis of P815 and YAC-1 tumor cells by mouse AK-T cells. The inhibition of MHC-unrestricted cytotoxicity by anti-CD8α mAb cannot be attributed to interference with an adhesion-like function of CD8 towards class I MHC molecules on the target cells because anti-CD8α mAb (i) had equal inhibitory effects on the cytolysis of tumor target cells regardless of their relative level of class I MHC molecule expression and (ii) did not interfere with the formation of conjugates between AK-T cells and class I MHC-bearing P815 tumor cells. However, anti-CD8α mAb abrogated AK-T cell granule exocytosis in the presence of P815 tumor cells, indicating a regulatory role for CD8 in the signal transduction events which result in lysis of the tumor target cell. Immunoblot analysis of the post-nuclear fraction of lysates from AK-T cells exposed to P815 tumor cells in the presence of anti-CD8α mAb revealed reduced phosphorylation of tyrosine residues on a protein with an Mr of -62 kDa. Taken together, these data suggest that CD8 is able to affect the tumoricidal activity of MHC-unrestricted AK-T cells independent of class I MHC molecules on the target cell.     

Natural Killer Cells may be the Only Cells in Normal Mouse Lymphoid Cell Populations Endowed with Cytolytic Ability for Antibody-Coated Tumour Target Cells

Mouse normal lymphoid cells were analysed as to their ability to perform in three cytolytic systems: Ability to act as 'natural killer', NK, cells against a NK sensitive tumour target, YAC; as effector cells against IgG-coated 815 cells, or to function as effector cells against IgG-coated CRBC. NK activity and ADCC against the IgG-coated P815 cells were found to vary in parallel as affected by age, organ distribution and genotype of the effector cells. On the other hand, ADCC against CRBC was largely carried out by effector cells distinct from those functioning as NK cells or in ADCC against P815. Temperature pretreatment schedules at 37 degrees C showed both NK cells and ADCC ability against P815 to be highly sensitive on contrast to ADCC against CRBC. Likewise, inoculation of Corynebacterium parvum intraperitoneally will lead to reduction in ADCC ability against CRBC but increase in ADCC against P815 and NK activity. Blocking experiments using 'cold' inhibitor cells in the cytolytic assays indicated that NK cells and effector cells against IgG-coated P815 cells are the very same cells. We thus conclude that NK cells in the mouse also have the ability to express K cell activity against IgG-coated tumour target cells. In fact, our data suggest that the NK cells may be the only cell type in the mouse equipped with cytolytic potential for antibody-coated murine nucleated cells     

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity.