Antibody- and interferon-dependent killer cells are part of the NK cell receptor positive subpopulation of human peripheral blood cells.

Cytotoxicity by human non-adherent peripheral blood lymphocytes was analysed after effector cell activation with either interferon (IF) or by target cell specific IgG antibodies (T-IgG). Four different cell lines were used as target cells that differed in susceptibility to natural killer cell (NK) activity; a highly susceptible T cell line (Molt-4), a medium susceptible B lymphoma line (Daudi), a resistant B cell line established by Epstein-Barr virus transformation (LCL-LS) and a resistant mouse mastocytoma line (P815). Three different parameters influencing killing were investigated; lytic potential, target cell binding and efficiency of the lytic phase from which the absolute number of effector cells and their recycling capacity could be estimated. It was found that, when using human target cell lines, IF and T-IgG influenced the system in a similar way by activating the lytic phase and the effector cell recycling but not the early binding phase. With the NK resistant mouse mastocytoma cell line P815 a comparatively small target binding population was found which, however, increased markedly with T-IgG treatment. Taken together, the results indicate that the effector population responsible for antibody-induced killing belong to a subpopulation of cells that have the ability to spontaneously conjugate to the present target cells by virtue of naturally occuring undefined cell surface receptors designated NKR (NK receptor) and that the role of T-IgG in the present system is similar to that of IF. In contrast, if target cells are used that do not express binding structures for NKR receptors, T-IgG may also fulfill a receptor function through Fc receptors for IgG.     

Surface markers and cytotoxic activity of blood natural killer cells studied at the single cell level in Hodgkin''s disease.

Purified peripheral blood lymphocytes (PBL) from nine untreated patients with Hodgkin's disease (HD), two HD patients in complete remission and 17 healthy donors were studied for natural killer (NK) cell activity against the K-562 cell line using a single cell cytotoxic assay, which allowed enumeration of effector cells and characterization of their surface membrane phenotypes after staining with monoclonal antibodies. The frequency of NK cells was significantly lower in HD patients than in controls (mean % +/- s.d., 1.9 +/- 0.9 and 2.8 +/- 1.2, respectively), while the fraction of target binding cells was similar in the two groups. The fraction of cytotoxic lymphocytes increased after pre-treatment of PBL with 500 iu leucocyte interferon in all tested control donors (n = 12) and the two patients in remission but only in four of seven untreated patients. No relation between the impaired NK cell frequency and age, tumour histology and clinical stage could be revealed. Subtyping of the target cell binding NK cells by monoclonal antibodies disclosed a marked heterogeneity of effector cells. NK effector cells reactive with M1 and anti-Ia antibodies were enriched while T3+ and T4+ NK lymphocytes tended to be reduced as compared to PBL. There was no difference between patients and healthy donors with regard to the surface antigen patterns of NK cells. Interferon treatment did not alter significantly the phenotypic characteristics of cytotoxic lymphocytes in patients and controls. It is concluded that the impairment of NK cell activity in HD is partly attributed to a lower frequency of cytotoxic effector cells among a normal number of target binding cells. The defect could not be attributed to a selective defect of effector cell subsets.     

Virus-Dependent Cellular Cytotoxicity in Vitro

When human peripheral blood lymphocytes were incubated with 51Cr-labelled tissue culture cells (T24 bladder carcinoma cells or Chang liver cells), their natural cytotoxicity (NK) usually stopped after 8 h of incubation. The 51Cr release induced by lymphocytes treated with small amounts of live or ultraviolet-inactivated mumps virus was strongly enhanced and lasted longer. When the lymphocytes were fractionated by Percoll gradient centrifugation, the highest NK activity was found in the low-density fraction enriched in large granular lymphocytes, whereas that of the T-cell-enriched high-density fractions was low. In contrast, the virus-dependent cellular cytotoxic (VDCC) activity was more evenly distributed between these fractions. However, there was a difference between the target cells in that the T24 cells were more susceptible to the cytotoxicity of lymphocytes in the high-density fractions than the Chang cells. Studies of Percoll fractions in the single-cell agarose assay showed that virus treatment increased the proportion of both target binding cells and killer cells in all fractions. Moreover, in the high-density fractions the increase in the number of killer cells was greater than that in binding cells, suggesting that the enhanced target cell killing induced by the virions reflected both increased binding and effector cell activation. Surface marker analysis of unfractionated lymphocytes indicated that the number of T3+ effector cells was greater than that of the HNK-1+ effector cells, regardless of whether the lymphocytes were treated with virus or not. However, for both NK and VDCC, the T3 to HNK-1 distribution ratio on the effector cells was 5-8:1 for T24 and 2:1 for Chang. Taken together, the results indicate that both NK and VDCC effector cells are phenotypically heterogeneous and that the target cells may play an active role in the recruitment of those effector cells that are most efficient in that system. The enhancement of lymphocyte cytotoxicity primarily reflects effector cell recruitment.     

The use of VEP13 monoclonal antibody for definition of natural killer cells: spontaneous killer cells directed against fresh human leukaemia cells carry the VEP13 antigen.

VEP13, an IgM monoclonal antibody (MoAb), produced against human large granular lymphocytes, is able to deplete natural killer (NK) cell activity in complement-dependent lysis. Here we report that VEP13 also reacts with the majority of interferon (IFN) activated NK cells. By contrast cytotoxic activity of unstimulated monocytes and cytotoxic T cells directed against allogeneic lymphocytes were unaffected by VEP13 plus complement treatment. Thus among the major types of cytotoxic cells VEP13 selectively reacts with NK cells and hence can be employed to identify these cells. We therefore used VEP13 in complement-dependent lysis and FACS separation to analyse NK cells involved in enhanced killing of fresh leukaemia cells. Spontaneous cell-mediated lysis of human leukaemia cells was enhanced in two ways: (a) effector cells were pre-treated with beta-IFN and (b) leukaemia cells were pre-treated with a pulse of actinomycin D. In complement-dependent lysis VEP13 removed all NK cell activity of IFN activated PBM against untreated and against ActD pre-treated leukaemia cells. FACS separation of VEP13 positive cells further supported this finding, in that all activity of IFN activated NK cells against actinomycin D pre-treated targets was found in the VEP13 positive fraction. Thus enhanced killing of fresh human leukaemia cells appears to be mediated VEP13 positive NK cells which are distinct from cytotoxic T cells and cytotoxic monocytes.     

Decreased NK killing in patients with multiple sclerosis: An analysis on the level of the single effector cell in peripheral blood and cerebrospinal fluid in relation to the activity of the disease

Natural killer cell activity has earlier been shown to be depressed in patients with multiple sclerosis (MS) (Benczur et al., 1980). In the present study, this defect was more clearly characterized in different stages of the disease. By using a single-cell cytotoxicity assay in agarose (Grimm & Bonavida, 1979), in combination with the conventional ⁵¹Cr-release, the number of target-binding cells (TBCs) and the fraction of active killer cells therein could be compared with the radioisotope release in the different patient groups. It was found that patients with active and chronic MS showed lower natural killer (NK) activity in the ⁵¹Cr-release assay as compared with age and sex-matched controls, in contrast to stable MS patients who were comparable with their control group. The single cell cytotoxicity assay demonstrated that acute MS patients had a decreased number of TBCs in peripheral blood and that they also had a decreased percentage of active NK cells in their TBC fractions. Patients with chronic MS were normal in the single-cell cytotoxicity assay. When cells present in CSF were analysed in acute and chronic MS, few cells were found with target binding capacity and only in two instances out of 13 could any cytotoxicity at all be detected. Patients with other neurological diseases (OND) were found to have detectable NK activity in CSF in six cases out of ten in the single-cell assay. OND patients as a group also had higher peripheral NK activity in the ⁵¹Cr-release assay as compared with the control group. When peripheral and CSF cells from MS patients and OND patients were treated with interferon, no increase in TBCs or fraction of killer cells in TBCs was found. In the ⁵¹Cr-release assay, comparable increases in cytotoxicity were found in all groups. One possible explanation for the stage-related NK suppression seen in the present investigation may be a decreased interferon production combined with immune-complex induced, macrophage-produced prostaglandins which are known to influence the human NK system in a comparable way in vitro. Additionally, immune-complexes may directly interact with human NK cells as the majority of these are known to express receptors for the Fc portion of IgG.     

Umbilical-Cord-Blood-Derived Suppressor Cells of the Human Natural Killer Cell Activity Are Inhibited by Interferon

The natural killer (NK) activity of umbilical-cord-derived lymphocytes was studied. The general level of activity was lower than with adult lymphocytes against K-562 cells and fetal fibroblasts. The activity could be boosted by interferon pretreatment of effector cells, but not to the same extent as with adult lymphocytes. Umbilical cord lymphocytes were fractionated with Percoll density gradient centrifugation, and the suppressive activity of different fractions was tested on highly enriched adult buffy-coat-derived NK cells. Allogeneic adult NK cell activity could be inhibited in 9 of 20 cases tested with small and medium-sized T lymphocytes (Percoll fractions 4–5) from the umbilical cord. The suppressive capacity was further enriched in fractions forming rosettes (RFC) with antibody-coated human erythrocytes (EA). Such EA-RFC of Percoll fractions 4–5 from umbilical cord exerted a strong suppressive activity in each case tested. Pretreatment of EA-RFC with interferon regularly abolished the suppressive effect. We conclude that there are Fc-receptor-positive small/medium-sized T lymphocytes in the umbilical cord blood which can efficiently suppress the cytotoxic activity of NK cells and that the suppressive activity can be abolished by interferon pretreatment of the suppressor cells.     

Human fetal thymus and bone marrow contain target cells for natural killer cells

Previous studies in the mouse natural killer (NK) system have indicated that NK cells may be involved in lysing normal, primary hematopoietic tissues. In the present report, this was analyzed in the human NK system using fetal bone marrow (BM) cells and thymocytes as well as adult BM cells from healthy donors as target cells in a conventional 6 to 12-h ⁵¹Cr-release assay. Adult BM cells showed low but significant levels of sensitivity, which could be increased by using interferon (IFN)-activated peripheral blood leukocytes (PBL) as effector cells. BM cells from 16 to 19-week-old fetuses consistently showed higher sensitivity for lysis than adult BM, and also fetal thymocytes proved very sensitive for lysis, in contrast to what has previously been reported for adult thymocytes. When used as effector cells against K562 targets, adult BM showed a clear lytic activity which could be further activated with IFN. In contrast, fetal BM was totally NK-inactive also after IFN activation. Among healthy adult donors, autologous BM was lysed as efficiently as allogeneic BM, and when different NK cell donors were used, the same classification of these as NK high or low reactive cells was seen regardless of the source of BM targets. IFN could augment lysis in autologous as well as in allogeneic effector BM target combinations. IFN could also protect adult BM cells from NK lysis, but no protection was seen with fetal BM cells. The highest NK activity against BM targets was seen among nylon-nonadherent, E rosette-negative PBL and, therefore, the effector cell seemed to be of the same nature as that active against continuous cell lines. These results show that in the human NK system, NK cells can lyse normal BM cells and thymocytes. The higher sensitivity expressed by fetal BM cells and thymocytes as compared to adult cells may suggest an increased frequency of a particular primitive NK-sensitive cell type in these tissues, a finding which is in line with results seen in the mouse NK system.     

Differential effects of ifosfamide on dendritic cell-mediated stimulation of T cell interleukin-2 production, natural killer cell cytotoxicity and interferon-gamma production

Ifosfamide is a DNA-alkylating agent used frequently in chemotherapy of human malignancies. Ifosfamide and its major decomposition products deplete intracellular glutathione (GSH). Glutathione is the major intracellular thiol reductant that protects cells against oxidative injury. Ifosfamide depletion of intracellular GSH in human dendritic cells (DC), T cells and natural killer (NK) cells impairs their functional activity which can be restored by reconstituting GSH. Here we assessed the effect of ifosfamide on DC-mediated stimulation of NK cell proliferation via T cells and on direct DC stimulation of NK cell cytotoxicity and interferon (IFN)-gamma production. Indirect DC stimulation of NK cell proliferation via T cells and T cell-derived interleukin (IL)-2 were reduced by ifosfamide treatment of DC and reconstitution of GSH in DC restored both responses. When DC and NK cells were treated with ifosfamide, DC could overcome the negative effect of ifosfamide on NK cytotoxic function whereas NK cell IFN-gamma production was less efficiently restored. The ability of IL-2 activated NK cells to kill autologous immature DC or to induce DC maturation was reduced moderately by treatment of both cell types with ifosfamide. Overall, our results suggest that DC may stimulate anti-tumour effector cells in patients even if they had received treatment with chemotherapeutic agents such as ifosfamide.     

Restoration of anti-interleukin-1 depressed natural killer activity by human recombinant interferon alpha or gamma, human recombinant interleukin-2 and indomethacin

The lymphokines interleukin-2 and interferon alpha or gamma are synthesized and secreted by activated mononuclear cells (MC) and play a critical role in the proliferative expansion of T-lymphocyte effector cells during the immune response. The pretreatment of human peripheral blood mononuclear cells (PBMC) with antibody IgG against human interleukin-1 (IL-1) from normal rabbit serum, inhibited their natural killer (NK) activity against both myeloid (K562) and lymphoid (MOLT-4) cell lines. Percent specific lysis of tumor cells decreased by increasing the antibody anti-IL-1 dose in an almost linear fashion. When the effector cells were pretreated with human recombinant interleukin-2, human interferon alpha or gamma and indomethacin alone or in combination, the inhibitory effect of antibody against human IL-1 was almost totally reversed.     

Enhancement of Human Spontaneous Cell-Mediated Cytotoxicity by a Monoclonal Antibody against the Large Sialoglycoprotein (CD 43) on Peripheral Blood Lymphocytes

A murine monoclonal antibody, MoAb B1B6 (IgG1 chi), which recognizes the large sialoglycoprotein (LSGP) on human peripheral blood lymphocytes (PBL) effectively enhanced the spontaneous cytotoxicity of these cells against the natural killer (NK)-sensitive target cells K562 and Molt-4. Whereas preincubation of the lymphocytes with MoAb B1B6 resulted in increased cytotoxicity, preincubation of the target cells had no effect, indicating that the MoAb amplified cytotoxicity at the effector cell level. Kinetic analysis of the data revealed no differences between the control and the MoAb-treated lymphocytes with regard to Vmax, usually considered to reflect the overall lytic potential of the cells. The slopes of the saturation curves, however, differed significantly for the two cell populations, indicating a substantial increment in the activity of the MoAb-treated cells. When studied at the single cell level and with K562 as targets, treatment of PBL with the MoAb resulted in the recruitment of new effector lymphocytes from the pool of non-binding cells. In contrast, when Molt-4 cells were employed as targets, no additional effector cells were recruited. These results indicate that the enhanced cytotoxicity induced by MoAb B1B6 is the result of either recruitment of new effector lymphocytes or of an increased recycling capacity of preexisting effector cells. Together with previous observations, these findings support the conclusion that LSGP belongs to the set of surface molecules which regulate human lymphocyte activation.     

Production of interferon and tumour necrosis factor by cloned human natural cytotoxic lymphocytes and T cells.

Cell lines and clones, derived from natural killer (NK) cell-enriched (B73.1+) peripheral blood lymphocytes (PBL) from several human donors, that expressed distinct surface phenotypes and were cytolytically active against K562 target cells were tested for their capacity to produce interferon (IFN) and tumour necrosis factor (TNF), IFN and TNF were measured firstly in biological assays and secondly in specific immunoassays for alpha-IFN, gamma-IFN and tumour necrosis factor (TNF alpha). It was found that the majority of NK-derived lines and clones were highly cytotoxic towards K562, but generally produced relatively low or undetectable levels of gamma-IFN and TNF alpha following stimulation with phytohaemagglutinin. No alpha-IFN was detected in supernatants from these cells. In comparison, cell lines and clones, derived from T lymphocyte (B73.1-) enriched PBL from the same donors were poorly cytotoxic towards K562, but generally produced higher levels of gamma-IFN and TNF than NK-derived cells. Thus, neither gamma-IFN nor TNF production were shown to correlate well with the capacity of NK-derived or T cell clones to effect cytotoxic action towards K562 in vitro. These results suggest that the co-production of gamma-IFN and TNF is not indicative of cytotoxic potential.     

Natural killer activity of human blood lymphocytes

Natural killer (NK) activity is an operational designation. It implies the in vitro cytotoxicities registered in short-term tests exerted by lymphocytes derived from donors with no known immunization history against the particular target. The strength of the effect exerted by unmanipulated blood lymphocytes shows an individual variation. Short-term in vitro treatment with interferon elevates the lytic potential of lymphocytes. Owing to the heterogeneity of the cytotoxic blood lymphocytes with regard of cell surface properties it is not possible to separate all active cells and inactive cells in clean populations. A considerable enrichment of active cells can be achieved if nylon wool non-adherent, large, granular Fc gamma receptor positive SRBC receptor negative--or low-avidity SRBC receptor positive--OKM1-reactive cells are separated. Negative cells are concentrated in the Fc receptor and OKM1-negative high-avidity SRBC receptor positive high cell density subset. The activity of lymphocytes in the former category is potentiated by interferon and the latter acquire the lytic function if PHA is added to the assay system. Freshly separated, non-cultured tumor cells are not or weakly sensitive to the effect of unmanipulated lymphocytes. However, when the lymphocytes are treated with interferon prior to the assay a lytic potential can be induced even against these in allogeneic effector target combinations. Cytotoxic cells which acquired the function after in vivo and/or in vitro immunization are designated as 'cytotoxic T-lymphocytes' (CTL), and were shown to act on the basis of antigen recognition. The expression of known T-markers on at least a fraction of the active cells and the recognition of alloantigens in NK systems suggest that the distinction between CTL and NK cells is not as sharp as initially suggested.     

A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance

In cancer patients, NK cell inhibitory receptors (IR) are expressed on a fraction of melanoma-specific lymphocytes with a unique reactivity for tumor antigens derived from normal, nonmutated genes (differentiation antigens). It is presently not known whether expression of these receptors is induced during an immune response against melanoma cells or whether these receptors can be found on T cells harboring a self specificity for such differentiation antigens in healthy donors. By analyzing short-term cultures of CD8+ T cells primed in vitro with melanocyte differentiation antigens, we found expression of NK cell receptors on a small but consistent fraction of CD8+ T cells derived from healthy donors. Both long and short forms of NK cell receptors were expressed. Interestingly, only long forms were functional and inhibited effector functions (cytotoxicity and IFN-gamma production) of these CD8+ T cells. Short forms were devoid of any detectable activating function. The presence of T cells specific for differentiation antigens and expressing NK cell receptors, with an exclusive inhibitory function, in healthy donors strengthens the notion that IR may serve to control T cell tolerance to some peripheral antigens.     

CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis

Natural killer (NK) cells are innate immune cells that are enriched in the liver, but the processes underlying NK cell trafficking to the liver and cellular activation within the liver of patients with T cell-mediated liver diseases remain poorly defined. Concanavalin A (Con A) hepatitis is a murine model mimicking many aspects of human T cell-mediated liver diseases. Here we demonstrate that severe hepatitis in CCR5-deficient (KO) mice is associated with increased hepatic NK cell recruitment driven by enhanced hepatic production of CCL5 acting via CCR1 and by enhanced hepatic NK cell activation relative to that observed in wild-type mice after Con A administration. Furthermore, NK cell depletion ameliorated severe hepatitis in CCR5 KO mice but did not alter hepatitis in wild-type mice after Con A treatment. We propose that in the setting of CCR5 deficiency NK cells assume a profound effector role in Con A hepatitis via enhanced CCL5-CCR1 driven hepatic recruitment in addition to augmented cytokine-driven NK cell activation to produce interferon-gamma. These results highlight the potential profound impact of altered chemokine receptor expression on the innate immune response in the setting of T cell-mediated hepatitis.     

Enrichment by counterpart centrifugal elutriation of human lymphocytes cytotoxic to human tumour cells.

The enriched fractions of cytotoxic cells responsible for natural killer (NK) activity against both human sarcoma and neuroblastoma (LA-N2) cell lines were readily obtained by countercurrent centrifugal elutriation (CCE). The NK cells were obtained in the larger lymphocyte fractions (fraction 6 +/- 1), having a mean cell volume of 180 u3. The cytotoxic-enriched fraction contained 51% large lymphocytes having cytoplasmic granules. On the other hand, monocytes were purified to greater than 90% and isolated in another fraction (final fraction) and these cells had the lowest NK activity against both human tumour cell lines. However, compared with the lymphocyte fractions, small and large monocytes displayed greater antibody-dependent cellular cytotoxicity (ADCC) activity against human B erythrocytes. These results indicate that NK found to have activity against both tumour cells lines were larger lymphocytes, not small monocytes. Thus, countercurrent centrifugal elutriation (CCE) can provide a sensitive method to obtain enriched fractions of large lymphocytes contained tumoricidal activity against human sarcoma and neuroblastoma cell lines.     

Hyperprolactinemia inhibits natural killer (NK) cell functionin vivo and its bromocriptine treatment not only corrects it but makes it more efficient

We studied NK cell function in eight patients with pathological hyperprolactinemia by measuring⁵¹Cr release by K562 cells exposed to their mononuclear cells and found it decreased compared to normal controls (P<0.01). Bromocriptine (BrC) treatment corrected NK function but also made it more efficient at 12:1 than at 25:1 or 50:1 effector:target ratios (ANOVA;P=0.01). The study of NK cell function in agarose revealed that its decrease in hyperprolactinemia is due to their low active binding to target cells, active killing, and recycling capacity. BrC tended to correct them but also increased recycling capacity to levels higher than those of controls (P<0.05). Sequential studies in three hyperprolactinemic patients before and after BrC showed correction of NK function within 1 week but its increased efficiency at the 12:1 effector:target ratio required 8 weeks. We conclude that hyperprolactinemia decreases NK cell function. BrC corrects this by decreasing prolactin levels but also makes NK function more efficient by increasing the capacity of NK cells to recycle after killing.     

Enhancement of NK, but not K cell activity by different interferons

Two different interferons derived either from a human lymphoblastoid cell line (Namalva) or from human fibroblasts were tested for their ability to modulate natural killer (NK) or killer (K) cell activity. The lymphoblastoid interferon was purified by ion-exchange chromatography on SP-Sephadex C-25 and gel filtration on Sephadex G-100, the fibroblast-derived interferon was purified by chromatography on porous glass beads. Evidence is presented that NK cell activity is enhanced by both of these interferons being active to a similar extent. When tissue culture cells are employed as targets for measurement of K cell activity, the augmentation of cytotoxicity by interferons has to be attributed to the inherent NK cell activity. With the use of the autologous hapten-coated target cells and of affinity chromatography purified antibodies, the cytotoxicity is displayed solely by K cells and this activity is not enhanced by either interferons tested.     

Generation of Lymphokine-Activated Killer Cells by Adherent LGL Phenotype Cells and Non-Adherent T Lymphocytes

In this work we separated human blood lymphocytes (PBL) in two populations (A-LAK and NALAK cells) by the adherence plastic method. A maximum adherence of cells was obtained after 2 days of PBL incubation in LAK medium containing 500 U/ml rIL-2. The A-LAK cells had LGL phenotype but 40% of them had a macrophage phenotype marker and less than 20% weakly expressed a T-cell marker. This population, when reincubated in culture, produced an increasing titre of interferon. At the same time, a significant NK activity against K562 target cells was measured just after enrichment; these enriched adherent cells also developed an increased LAK activity against DAUDI cell lines, ninefold more at 6 days than when assayed just after enrichment. In contrast, 75% of the NA-LAK enriched cells expressed T-cell marker; these produced two- to threefold less interferon than A-LAK cells at all time-points. The NA-LAK lymphocytes enhanced principally LAK activity measured by 70% lysis against DAUDI target cells tested at 6 days of culture. Further studies are in progress to determine the nature of the effector cells that mediated LAK activity.     

Modulation of human natural killer cytotoxicity by influenza virus and its subunit protein.

The influence of intact influenza virus and purified detergent solubilized haemagglutinin (HA) subunits from these viruses on human natural killer (NK) cell activity was examined. Effector cells incubated with whole influenza virus for 18 hr initiated the production of alpha interferon which was associated with the enhancement of NK cell activity. In contrast, purified influenza virus HA suppressed NK activity in a dose-dependent manner, when added at the onset of the cytotoxicity assay, or when used to pre-treated effector cells prior to assay for cytotoxicity against K562 target cells. Effector cells exposed to influenza HA for 90 min, washed and re-incubated in fresh medium for up to 18 hr, failed to regain their cytotoxicity. Suppression of NK cell cytotoxicity could not be ascribed to direct toxicity of HA preparations or residual detergent and preservative in these preparations. The augmented cytotoxicity of activated human effector cells was also susceptible to suppression by virus HA, and pretreatment of human PBL effector cells with HA for 90 min, prior to exposure to human alpha interferon caused NK effector cells to become refractive to the enhancing effects of HIFN. That direct interaction between influenza virus HA and effector cells was a requirement for suppression of activity was shown in experiments using Bromelain-released influenza HA, which would not be expected to bind to cells and which failed to suppress NK cell activity.     

Mechanism of action of a Db-specific helper clone and factor in cytotoxic responses to alloantigens.

The evidence that NK cells can recognize and kill allogeneic lymphocytes has hitherto been based mainly on experiments in intact animals. Here we report results from an in vitro assay, showing allogeneic lymphocyte cytotoxicity in cell suspensions enriched for NK activity against tumour cells by Percoll gradient centrifugation of nylon-wool non-adherent cells. The addition of phytohaemagglutinin (PHA) to the NK-target cell cultures greatly enhanced the cytotoxic response against K562 and allogeneic, but not syngeneic, lymphocytes. The effector cells of ALC are present in the spleen of both euthymic and athymic nude rats, and to a lesser extent in the blood. ALC is augmented by interferon pretreatment of the effector cells, and by depleting the effector cell suspensions of all T cells and helper T cells with the monoclonal antibody MRC Ox19 and W3/25, respectively. Conversely, the activity was nearly abolished by depleting the cell suspensions of MRC Ox8+ cells reacting with rat cytotoxic T cells and NK cells. Furthermore, removal of residual B cells (Ox12+ cells) from the effector cells or attempts to block any putative antibody-dependent cellular cytotoxic mechanism in vitro with the monoclonal antibody Ox12 did not inhibit the NK activity against allogeneic lymphocytes nor against tumour cells. ALC in vitro did not discriminate between T and B or large and small lymphocyte targets. These characteristics of the ALC effector cells substantiate that they are present within the thymus-independent population of cells with NK activity, and are dependent on neither B cells nor immunoglobulin for their recognition and destruction of the target.