Spontaneous Cytotoxicity Research Articles

Inhibition of natural killer (NK) activity by human colostral and serum IgA.

Natural killer (NK) cells are defined as effector cells with spontaneous cytotoxicity against tumor, virus-infected and undifferentiated normal cells. NK activity is closely associated with a subpopulation of lymphocytes, large granular lymphocytes (LGL), that contain azurophilic cytoplasmic granules. The NK cells show a characteristic organ distribution: they are numerous in peripheral blood and spleen, but relatively low numbers are found in lymph nodes, peritoneal cavity and bone marrow (1,2). Previous investigations have demonstrated that NK activity is influenced by various factors such as interferon, interleukin-2, hormones, suppressor cells, environmental factors and age. Recently, mucosal LGL have been isolated from the small intestines of humans, mice and rats (3–6). In the mouse and rat, mucosal LGL showed moderate levels of NK activity, while human mucosal LGL demonstrated low NK activity.KeywordsNatural KillerNatural Killer CellNatural Killer ActivityLarge Granular LymphocyteK562 Target CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Definite spontaneous cell-mediated cytotoxicity and HNK-1 cells in the human large intestine.

Spontaneous cell-mediated cytotoxicity (SCMC) and the marker of natural killer (NK) cells mediating SCMC of the human large intestine were studied. Lamina proprial lymphoid cells (LPL) were isolated by sequential dithiothreitol-EDTA-collagenase treatment of the gut specimen. SCMC was measured by the chromium release method. Target cells included P4788 in monolayer, a cell line derived from colon cancer, Chang cells in monolayer, and K562 in suspension. Target cells in monolayer including colon cancer cell line were chosen because they were thought to be more appropriate to assess SCMC for lymphoid cells in the solid organ. While lower compared to cytotoxicities (CT) by peripheral blood lymphoid cells (PBL), define CT were observed in LPL against all three targets. NK cells marker was studied both on LPL by an indirect fluorescent antibody method and on the gut tissue by indirect immunoperoxidase staining using anti HNK-1 monoclonal antibody which defines virtually all NK cells. HNK-1 positive (HNK-1 +) cells were identified in both methods. HNK-1 + cells were observed in the epithelium, lamina propria, and lymph follicle with or without germinal centers. These results clearly demonstrated the presence of SCMC and HNK-1 + cells in the human large bowel.

Cell-mediated cytotoxicity by natural killer and killer cells, lipid peroxidation and glutathione.

In the course of spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral lymphocytes as effector cells, no lipid peroxidation occurred as measured by the production of ethane and thiobarbituric acid-reactive material. Furthermore, impairment of major cellular defense systems of target cells (K562 cells for SCMC, Chang liver cells for ADCC), by decreasing their glutathione content, had no effect on either lipid peroxidation or the cytotoxic response. These findings indicate that peroxidative damage is not a mechanism of NK and K cell-mediated cytotoxicity.

Effect of short-term inhalation exposure to 1,3-butadiene on murine immune functions

Interest in 1,3-butadiene (BD) as a potential immunomodulator was prompted by reports of an increased incidence of neoplasia in humans exposed to BD during the manufacture of styrene-butadiene synthetic rubber, and by a recent study which demonstrated a high incidence of thymic lymphomas in B6C3F1 mice. B6C3F1 mice were exposed to 1250 ppm BD by inhalation 6 hr per day, 5 days per week, for 6 or 12 weeks. Immune function assays were selected to evaluate specific humoral and cell-mediated immunity and spontaneous cytotoxicity; lymphoid organ histopathology was also evaluated. A slight decrease in antibody plaque-forming cells (PFC) per spleen was observed in exposed mice, although PFC per 10 6 splenic lymphocytes was normal. Significant extramedullary hematopoiesis and erythroid hyperplasia was observed in spleens from exposed mice, and correlated with a twofold increase in thymidine incorporation in spontaneously proliferating splenocytes. No differences in proliferation to alloantigens were demonstrable between control and BD-exposed splenocytes. Mitogenesis by phytohemagglutinin, Concanavalin A, and lipo polysaccharide was suppressed in splenocytes from exposed mice, but may have been due to the cellular dilution effect of hematopoietic activity. Cytotoxic T-lymphocyte generation was suppressed after a 6-week exposure to BD, but was comparable to controls after 12 weeks of exposure. No differences in spontaneous cytotoxicity were observed between control and exposed mice. Overall, no persistent immunological defects were detectable after inhalation exposure to this tumorigenic agent.

Sensitivity of human fetal hemopoietic cells to NK cell-mediated lysis.

Hemopoietic cells from human fetal livers at 11 to 18 weeks of gestational age were studied as to whether they were sensitive to adult and autologous fetal NK cells. Cell line K562 was used as control target in the 51Cr release assay and in the cold target competition assay. Adult peripheral blood NK cells did not kill liver-derived hemopoietic cells of four fetuses tested. Neither did fetal liver cells, whether or not expressing spontaneous cytotoxicity against K562 cells, destroy autologous hemopoietic liver cells. Hemopoietic liver cells from six fetuses were studied in the cold target competition assay. Liver cells of two fetuses clearly competed with lysis of K562 cells by adult NK cells. Thymus cells from one 18 week-old fetus had a weak capacity to compete with lysis of K562 cells by adult cells, whereas cells from the spleen and liver of the same fetus did not. It is concluded that early fetal liver hemopoietic cells are not susceptible to lysis by NK cells but an interaction between fetal cells and NK effector cells results in a depressed NK cell activity against NK-sensitive target cells.

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin.

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.

Macrophage-like effector of spontaneous cytotoxicity from the shark

The effector of spontaneous cytotoxicity from shark peripheral blood has been shown to be a macrophage-like cell. Effector cells are isolated by centrifugation over Lymphocyte Separation Medium, adherence to glass, Percoll density gradient centrifugation and adherence to fibronectin sequentially. Effector cells are adherent to glass, sediment to densities of 1.048–1.052 g/ml and are adherent to fibronectin. The isolated effectors represent less than 1% of the peripheral blood leukocytes, and exhibit potent cytotoxic capability, both spontaneous and in the presence of phytohemagglutinin. In addition, the activity of these cells is resistant to 3000 rads gamma irradiation. Although nurse sharks have natural antibody to trinitrophenol, spontaneously cytotoxic cells are incapable of killing trinitrophenol modified targets indicating that natural antibody is not required for reactivity, and that natural antibody and spontaneous effectors do not have the same repertoire. However, cold target inhibition studies showed that these effector cells can recognize four of five human lymphomyeloid targets. It is concluded that the spontaneous, extracellular killing by the macrophage-like effector most closely resembles that of activated mammalian tumoricidal macrophages with the exception that they do not appear to require in vitro activation.

Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, gamma-interferon and interleukin-1.

Activated monocytes or macrophages can release soluble cytotoxic molecules capable of lysing tumour cells in vitro and thus represent an important component of the host defence mechanisms against malignancy. The recent availability of pure recombinant or natural human lymphokines and monokines and their respective polyclonal or monoclonal antibodies now makes it possible to dissect the interactions of these factors in the induction and performance of the cytotoxic event by the monocytes. Our studies indicate that pretreatment of monocytes with alpha-IFN or gamma-IFN, and also interleukin (IL)-1 or tumour necrosis factor (TNF) results in enhanced monocyte cytotoxicity. Although all these substances induce the production of IL-1 by monocytes, TNF mediates the enhanced cytotoxicity induced in monocytes by gamma-IFN, IL-1 and, in an autocrine manner, by TNF itself. Neither TNF, IL-1, gamma-IFN nor alpha-IFN mediate spontaneous monocyte cytotoxicity or that induced by alpha-IFN. Our studies thus reveal new interactions between the two monokines IL-1 and TNF and provide a dual role for TNF, as immunomodulator and mediator of monocyte cytotoxicity induced by certain specific lymphokine and monokine molecules.

Functional heterogeneity of murine macrophage precursor cells from spleen and bone marrow

We previously reported that highly purified bone marrow-derived macrophage precursors can exert strong spontaneous cytotoxicity against YAC-1 tumor cells, Candida albicans, and protozoa of the genus Leishmania. In the present paper, evidence is shown that macrophage precursors in normal untreated mice are not confined to the bone marrow compartment but can also be found in the spleen. These organ-associated cells, which have the same buoyant density as large granular lymphocytes, have been positively sorted by means of an indirect rosetting technique employing the macrophage-specific monoclonal antibodies F4/80 and M143. The rosetting fractions represented an extremely homogeneous population of macrophage precursors characterized by high candidacidal and natural killer activity and by a strong proliferative response to the macrophage-specific colony-stimulating factor CSF-1. Spleen- and bone marrow-derived macrophage precursors differed in their target selectivity. In addition, the mature macrophages derived in vitro from these two precursor populations displayed striking differences in their candidacidal activity. The implications of these findings in relationship to heterogeneity in the macrophage differentiation line are discussed.

Separation of human natural killer cell subpopulations differentially responsive to interferon potentiation.

Subsets of human natural killer (NK) cells were identified that differed in the capacity to be activated by interferon (IFN) or the IFN-inducer, polyriboinosinic:polyribocytidylic acid [poly(I):poly(C)]. These subsets, which represented effectors of both spontaneous and antibody-dependent cellular cytotoxicity, were physically separable on the basis of cell buoyant density changes induced by exposure of lymphocytes to hyperosmolar Ficoll-Hypaque solutions or by centrifugation of lymphocytes through hyperosmolar (350 mOs/kg) Percoll gradients. Hyperosmolar conditions per se altered neither cell viability, NK cell cytolytic activity, nor the capacity of NK cells in unseparated lymphocyte preparations to be activated by IFN. IFN-unresponsive NK cells, separated by centrifugation through a 350 mOs/kg Percoll layer of 1.069-1.070 g/cm3 specific density, constituted 20 +/- 4% of all active NK cells identified at the single cell level and, per active NK cell, killed comparably to unstimulated IFN-responsive NK cells in 51Cr release assays. Thus, the IFN-unresponsive phenotype was probably not attributable to NK cells that were in an activated state prior to IFN treatment. Surface marker analysis of active NK cells at the single cell level identified comparable proportions in each subfraction to be of the OKM1+, OKT8+, or OKT11+ phenotypes and few, if any, in either subfraction to be of the OKT3+ phenotype. The human IFN-unresponsive NK cell phenotype, in contrast to the corresponding phenotype in the mouse, was therefore not linked to expression of T-cell-associated membrane differentiation antigens.

T lymphocyte subsets implicated in cytotoxicity in autologous hepatocytes in chronic active hepatitis patients with active viral replication

We investigated inhibitory effect of various monoclonal antibodies on T-cell-mediated cytotoxicity against autologous hepatocytes in 24 patients with hepatitis B surface antigen/hepatitis B e antigen (HB sAg HB eAg) - positive chronic active hepatitis. A significant reduction of cytotoxicity index occurred after preincubation of T lymphocytes with anti-Leu 7 (killer-natural killer cells), D1 12 (la-positive cells), 5 9 (restricted helper/inducer cells), and MLR4 (“activated” and radiosensitive helper cells) monoclonal antibodies (MAb). Anti-Leu 2a (cytotoxic/suppressor cells) and anti-Leu 3a (helper/inducer cells) MAb did not affect cytotoxic activity. This finding supports the hypothesis that the T cytotoxic reaction in this in vitro system is probably due to two mechanisms: first, spontaneous cell membrane cytotoxicity sustained by anti-Leu-7-positive lymphocytes; and second, specific cytotoxicity mediated by activated Ia-positive cells. We also found that the presence of helper/inducer cells ( 5 9 positive) appears to be a prerequisite for the cytotoxic reaction.

Histamine H2-receptor-mediated regulation of human natural killer cell activity.

We have investigated effects of histamine on the spontaneous cytotoxic activity of human natural killer (NK) cells in vitro. Addition of histamine (10(-3) to 10(-7) M) to assay cultures of Percoll-fractionated mononuclear cells (MNC) and erythroleukemic K 562 target cells resulted in a strong enhancement of the cytotoxicity of low-density MNC, enriched for NK cell cytotoxicity (NKCC). No enhancing or suppressing effects of histamine could be detected after removal of monocytes/adherent cells from the effector cell suspensions. When unfractionated MNC were used as NK effectors, similar results were obtained, i.e., dose-dependent enhancement of NKCC by histamine in the presence of monocytes and lack of effect in nonadherent effector cells. Freshly isolated monocytes displayed low spontaneous cytotoxicity against K 562 targets and were not induced by histamine. The histamine-induced enhancement was mimicked by dimaprit, a specific histamine H2-receptor agonist, but not by N-methyldimaprit, a chemical control for H2-receptor agonist activity of dimaprit. Furthermore, the enhancement was completely antagonized by the specific histamine H2-receptor antagonists cimetidine and ranitidine. The effect of histamine could not be ascribed to endogenous interferon (IFN) production, since no IFN activity could be detected in histamine-treated MNC effectors. Also, the enhancing effects of histamine and human leukocyte IFN-alpha were clearly additive. On the basis of these findings, we suggest that histamine, via specific activation of H2 receptors, may be an important regulator of human NK cell activity.

Characterisation of the effector cells in antibody-dependent and spontaneous cell-mediated cytotoxicity in swine against target cells infected with transmissible gastroenteritis virus

When porcine peripheral blood leucocytes were fractionated, lymphocytes were the most active effectors in both antibody-dependent cell-mediated cytotoxicity (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC), although both polymorphs and macrophages showed some activity in ADCC. Adsorption of lymphocytes to antibody-sensitised or unsensitized PK-15-transmissible gastroenteritis (TGE) cells caused similar reductions in ADCC and SCMC effector activities. Over 60 per cent of the target-binding lymphocytes were non-specific esterase positive large lymphocytes, which did not form erythrocyte (E)-rosettes, and about 30 per cent were non-specific esterase positive medium sized lymphocytes, which formed low avidity E-rosettes. The remainder were non-specific esterase negative small lymphocytes, some of which formed high avidity E-rosettes. None of the eluted lymphocytes stained for surface immunoglobulin and all formed low avidity erythrocyte-antibody rosettes. Porcine killer and natural killer cells resembled in many respects those described in humans and rodents.

A subset of natural killer cells in peripheral blood displays a mature T cell phenotype.

Normal human PBMC were analyzed for the presence of cells expressing both T3 and NKH1 antigens, using direct two-color immunofluorescence. In six individuals, NKH1+T3+ cells were found to represent 2.5% of PBMC and 24% of the total number of NKH1+ cells. Purified NKH1+T3+ cells were shown to have the typical morphology of large granular lymphocytes (LGL). NKH1+T3+ cells also exhibited spontaneous cytotoxicity against K562 target cells and this lytic activity could be inhibited by anti-T3 mAb. Similar results were obtained with NKH1+T3+ cells cultured in vitro in lymphocyte-conditioned medium. Taken together, these results indicate that NKH1+T3+ cells represent a unique population of NK-active cells in normal peripheral blood. Although these cells exhibit LGL morphology and NK activity, this appears to be mediated through a functional T cell-like receptor for target antigen.

Concurrent enhancement of monocyte immunoregulatory properties and effector functions by recombinant interferon-γ

Interferon-γ is a critical factor in the activation of several mononuclear phagocyte effector and immunoregulatory properties. However, it remains uncertain if IFN-γ is capable of concurrent activation of both functions in the same cell population. Plastic adherent mononuclear cells (80–98% MN by cytochemical criteria) were cultivated in the absence or presence of recombinant interferon-γ (rIFN-γ, 0.1–100 U/ml) for 48 hr. MN surface DR antigen was assessed by flow cytometry (EPICS V) after staining with monoclonal antibodies OKIa1 or L243. Exposure to rIFN-γ (100 U/ml) increased MN surface DR antigen (mean fluorescence intensity) by 80 ± 20% ( P < 0.01) and 121 ± 52% ( P < 0.001), respectively, compared to untreated cells. The increase in DR antigen was maximal at 100 U/ml, dependent on protein and RNA synthesis and blocked by agents that increase cAMP levels. IL-1 activity was determined in the mouse thymocyte assay; rIFN-γ (100 U/ml) increased IL-1 activity in the supernatants of MN cultured in medium alone from 0.5 ± 0.2 to 7.8 ± 4.7 U/ml ( P < 0.05), and lipopolysaccharide-stimulated MN from 20.4 ± 19.1 to 71.7 ± 38.9 U/ml ( P < 0.05). Following rIFN-γ exposure, MN stimulation of the AMLR was increased at 6 days (29,269 ± 5224 vs 13,252 ± 4938 cpm, P < 0.01). Spontaneous cytotoxicity (SC) and antibody-dependent cell cytotoxicity (ADCC) were studied in a 51Cr release microculture assay using the human lymphoblastoid cell line CCRF-CEM as target. SC by MN increased linearly as a function of log[rIFN-γ] for effector: target (E:T) ratios of 5:1 ( r = 0.95, P < 0.01) and 10:1 ( r = 0.99, P < 0.01). ADCC by MN increased following rIFN-γ exposure (100 U/ml) at E:T ratios of 5:1 (22 ± 13 to 31 ± 4%, P < 0.025) and 10:1 (31 ± 4 to 38 ± 4%, P < 0.01). Thus, rIFN-γ not only activates MN effector function, but has concurrent stimulatory effects on multiple MN properties critical to immunoregulation.

The tissue distribution of spontaneous cell-mediated cytotoxicity effector lymphocytes in swine

Peripheral blood lymphocytes, intraepithelial lymphocytes from the small intestine and lymphocytes from the thymus, spleen, mesenteric lymph nodes and Peyer's patches from 5 young adult pigs were used as effector cells in a spontaneous cell-mediated cytotoxicity chromium release assay against PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. Both peripheral blood and intraepithelial lymphocytes caused marked specific chromium release, while the lymphocytes from the remaining tissues were inactive in spontaneous cell-mediated cytotoxicity.

Lack of cellular cytotoxicity by human mononuclear cells to Giardia.

Cytotoxicity of mononuclear leukocytes (MNL) to Giardia lamblia was compared by using two techniques. The first method assessed the viability of surviving Giardia directly by culturing and the second method measured release of incorporated [3H] thymidine. Cytotoxicity, as measured directly by culturing and visual assessment, showed that the numbers of surviving Giardia decreased over time whether cultured with or without MNL but that Giardia survived significantly better in the presence of MNL at 18 hr (21.8 +/- 8.3% with MNL compared with 3.4 +/- 1.5% without MNL). Release of [3H]thymidine increased whether Giardia were cultured with or without MNL and although there was a tendency for increased release with MNL, there was no significant difference. Dead labeled Giardia released significantly more label in the presence of MNL than without MNL, suggesting that MNL cause release of [3H]thymidine after phagocytosis. The thymidine release assay therefore does not measure spontaneous cytotoxicity of MNL to Giardia.

Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory bowel disease patients

We have studied the effects of sulfasalazine and its metabolites on cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from both control and inflammatory bowel disease (IBD) patients. Sulfasalazine and sulfapyridine, as well as hydrocortisone and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD peripheral blood cells. Sulfasalazine and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD intestinal mononuclear cells cultured for 72 h in media alone. In contrast, 5-aminosalicylate, indomethacin and benzylimidazole had no effect on cytotoxicity by any cell population. Lectin-induced, antibody-dependent and interleukin-2-induced cell-mediated cytotoxicity, as well as lymphokine-activated killing were not inhibited by the drugs: inhibitory effects in these assays were primarily upon the underlying spontaneous cell-mediated cytotoxicity. The inhibition induced by sulfapyridine and nordihydroguaiaretic acid could not be reversed by adding the lipoxygenase metabolites leukotrine B4 or 12-hydroxyeicosatetraenoic acid. These findings demonstrate that spontaneous cell-mediated cytotoxicity by control and IBD mononuclear cells can be inhibited by sulfasalazine.

Inhibition of cytotoxicity by sulfasalazine. II. Sulfasalazine and sulfapyridine inhibit different stages of the NK and NKCF lytic processes

Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibit spontaneous cytotoxicity mediated by human natural killer (NK) cells. The aim of this study was to determine which stage(s) of the NK cytotoxic reaction is inhibited by these compounds. Effector/target cell binding studies performed in parallel with cytoxicity assays using purified large granular lymphocytes indicated that inhibition is a post-binding event. The kinetic profile of inhibition in a calcium pulse assay showed that inhibition continues long after the effector cell triggering stage and that although sulfasalazine may have some inhibitory effect on the calcium-dependent events of the programming phase, sulfapyridine continues to inhibit during the calcium-independent or lethal hit phase of the cytotoxic sequence. The NK soluble cytotoxic factor (NKCF) assay was used as a measure of the lethal hit since the time course of this assay permits study of the various substages of this terminal event in the lytic sequence. Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibited NKCF-mediated target cell lysis. Different substages of the NKCF-induced lytic reaction were affected by these agents. Sulfasalazine appears to inhibit binding of NKCF to the target cell whereas sulfapyridine predominantly inhibits early post-binding events.

The role of interferon in spontaneous cell-mediated cytotoxicity in pigs

Specific release of 51Cr and the production of interferon (IFN) increased in parallel in a spontaneous cell-mediated cytotoxicity (SCMC) assay in which uninfected PK-15 cells or PK-15 cells persistently infected with transmissible gastroenteritis virus (PK-15-TGE cells) were used as targets, and peripheral blood lymphocytes (PBL) from a young adult pig were used as effector cells. Higher levels of both specific 51Cr release and IFN were obtained in the assays containing PK-15-TGE cells. Co-cultivation of PBL from newborn piglets with PK-15-TGE cells yielded similar levels of IFN to those produced by co-cultivation of adult PBL and PK-15-TGE cells, but lower levels of IFN were produced by co-cultivation with uninfected PK-15 cells. Pretreatment of adult PBL with IFN augmented their SCMC effector activity for both PK-15 and PK-15-TGE cells in the 51Cr release assay. Pretreatment of the PK-15-TGE target cells with IFN did not affect their release of 51Cr in the SCMC reaction, while IFN pretreatment of PK-15 targets protected them against SCMC. In a single cell cytotoxicity assay the effects of IFN pretreatment on the effector adult PBL and on the PK-15 and PK-15-TGE target cells were confirmed, and SCMC incompetent PBL from neonatal piglets were rendered cytotoxic by pretreatment with IFN. PBL from newborn piglets bound to either target cell with the same frequency as PBL from SCMC competent adult pigs, and IFN pretreatment of either effector or target cells had no effect on target-binding frequency.