Characterization and partial purification of a novel cytotoxic lymphokine (factor 2) produced by a human B cell line (Karpas 160).

A subclone (160b) of the human B cell (Karpas 160) was shown to produce a novel cytotoxic lymphokine [Factor 2 (F2)] in addition to tumour necrosis factor alpha (TNF-alpha) and beta (TNF-beta). F2 was found to have selective toxicity to numerous human tumour cell lines, particularly the erythroleukaemic cell line K562, whereas TNF-alpha/beta were not cytotoxic to these cells, even at relatively high concentrations. Our studies have shown that F2 activity in crude preparations is heterogeneous both in its molecular weight, isoelectric point (pI) and hydrophobicity, which depends not only on the source of F2 cytotoxicity but also on the conditions of methods for its production. Our studies also showed that F2 was separable from TNF by DE52, S-300 gel filtration and Rotofor isoelectric focusing. F2 was partially purified up to 1000-fold by two procedures. The major active form, as assessed by gel filtration was of mol. wt of 45-67 kDa. On SDS-PAGE, F2 activity was recovered mainly from two regions of the gels corresponding to 10-14 kDa and 60-70 kDa. Antibodies of human TNF-alpha, TNF-beta, IFN-alpha, IFN-gamma, and TGF-beta failed to prevent F2-mediated cytotoxicity to K562. F2 activity was not inhibited by mannose-6-PO4 or mouse mAb to rat granule content, both of which have been reported to block human natural killer cytotoxic factor. Our studies indicated that F2 is likely to be a distinct human cytokine with selective cytotoxic activity against tumour cells.     

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.     

Interleukin-1 and interleukin-2 production in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PMA stimulated the monocytes to exert cytolysis and suppressed NK cells by the generation of these highly toxic oxygen products. Hydrogen peroxide especially seemed to be the mediator in this oxygen-dependent monocyte-mediated cytotoxicity and NK-cell suppression.     

Limiting dilution analysis (LDA) of cells responding to recombinant interleukin-2 without previous stimulation: evidence that all responding cells are lymphokine-activated potent effectors

The relationship between peripheral blood mononucleated cells spontaneously bearing the IL-2 receptor (IL-2R) and cell cytotoxicity for the natural killer (NK)-sensitive K562 target cell line was investigated. For this purpose, three types of experiments were performed. (i) Positive selection of cells spontaneously bearing the IL-2R was carried out by culturing peripheral blood lymphocytes (PBL) in the sole presence of recombinant IL-2 (rIL-2). Cytotoxicity was assessed at Day 6 of the culture in a 4 hr cytotoxic assay. (ii) Negative selection was performed by complement mediated lysis using the B1.49.9 monoclonal antibody which is specific for the IL-2R. (iii) Limiting dilution analysis of non-adherent PBL was carried out in the presence of rIL-2 alone. The colonies obtained were divided and daughter colonies assayed for anti-K562 cytotoxicity in a 6 hr cytotoxic assay and for proliferation. The results show that: (i) a 6-day culture of human non-adherent PBL in the presence of rIL-2 alone leads to a sharp increase in anti-K562 cytotoxicity; (ii) depletion of B1.49.9 positive PBL strongly decreases cytotoxicity against K562 targets; (iii) limiting dilution analysis indicates that all colonies grown without activation in the presence of autologous serum and rIL-2 can mediate cytotoxicity against K562 targets, which is not the case when the starting population is activated. Thus, our data taken together strongly suggest that lymphocytes spontaneously bearing the IL-2R are directly involved in K562 lysis by fresh PBL (classical NK activity). Moreover, we demonstrate that all colonies able to proliferate without any activation, in the sole presence of rIL-2, are potent K562 killers (in this case, these cells correspond to the so-called lymphokine activated killers, LAK).     

Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediated cytotoxicity: use of cytoplasts and cells from chronic granulomatous disease patient

Human polymorphonuclear leukocytes (PMN) and granule-free cytoplasts were compared for their cytotoxic capacities against red blood cells (RBC) and K562 tumor cells. Phorbol myristate acetate (PMA) stimulated PMN to efficient lysis of RBC targets, while cytotoxicity against the tumor cell line K562 was moderate. Activated cytoplasts also lysed RBC targets but were not able to kill K562 tumor cells, even at high cell numbers. Suppression of the glutathione redox cycle of the K562 tumor targets markedly increased their susceptibility to lysis by PMA-activated PMN. Despite the enhanced susceptibility of antioxidant-depleted K562 tumor cells to oxygen radical-induced damage, PMA-stimulated cytoplasts did not kill these targets. Addition of exogenous myeloperoxidase or lactoferrin to cytoplasts devoid of granule did not improve the lysis of RBC and K562 tumor cells. Coating K562 targets with specific antibodies induced efficient PMN-mediated killing in comparison to PMA-stimulated lysis of non-coated targets. Cytoplasts, however, did not kill antibody-coated K562 tumor cells; this was not improved by glutathione depletion but showed some lysis of antibody-coated RBC. PMN from a patient with chronic granulomatous disease (CGD) showed normal antibody-dependent cell-mediated cytotoxicity (ADCC) against K562 tumor cells but were not able to lyse these targets after PMA stimulation. The analysis of target cell killing by cytoplasts and PMN from a CGD patient indicated that granular constituents are important mediators in the killing of nucleated target cells and that PMN-mediated ADCC does not require the release of reactive oxygen species. Differences in the susceptibility of target cells to oxygen-mediated lysis indicates that target cell antioxidant mechanisms play an important role in the outcome of the cytotoxic response.     

Two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate

Pulmonary alveolar macrophages (PAM) and peripheral blood monocytes (PBMO) of the miniature swine can be converted to cytolytically active effector cells by treating with phorbol myristate acetate (PMA) as determined by enhancement of cytotoxicity to various target cells. Kinetics of the PMA-activated PAM and PBMO in cytotoxicity show that the effective PMA concentration ranges from 10 to 1,000 ng/ml. Induction of cytotoxic macrophages and monocytes occurred as early as 30 min and to their maximum cytotoxicity after 1 hr exposure to PMA and the enhanced cytotoxic activity persisted up to 24 to 40 hr when PMA was removed by washing after 1 hr exposure, but prolonged exposure to PMA for more than 6 hr resulted in a drastic decrease of cytolytic activities suggesting the prolonged exposure to PMA causes macrophages and monocytes to become refractory to PMA stimulation. Target cells displayed varying degrees of cytotoxic sensitivity to the PMA-activated PAM and PBMO; PRBC, SRBC, and K562 were sensitive, WEHI-164 and U937 were relatively sensitive, and SB was very resistant to these activated effector cells. The mechanisms of PMA-induced cytotoxicity could largely be divided into two categories. One was the H2O2 mediated killing as shown by complete reduction of cytotoxicity after adding catalase in the assay. The other was the proteases mediated cytolysis, which could be blocked by protease inhibitors, Phenyl methyl sulfonyl fluoride (PMSF), and N- alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). H2O2 was the only mediator produced in large enough quantities from PBMO to kill target cells, whereas PAM could produce both mediators (H2O2 and proteases). PRBC, SRBC, and K562 appeared to be killed by H2O2 produced by PAM and PBMO. In contrast, U937 and WEHI-164 appeared to be killed by proteases in PAM mediated cytolysis but by H2O2 in PBMO-mediated cytolysis. These results suggest that the observed cytolytic mechanisms can be differed by type of target cells as well as the source of mononuclear phagocytes within the individual animal.     

Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines.

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.     

Stimulation of natural cytotoxic activity in mixed cell cultures and by culture supernatants containing interferon

We have previously demonstrated that lymphocyte preparations cultured for 5 days with a number of different mitomycin-C-treated lymphoid cell lines showed increased non-specific (NK-like) cytotoxic activity. We have now examined the activity induced in T- and B-cell-enriched responder populations, prepared by sheep red blood cell (SRBC) rosette separation, and found that increased cytotoxic activity was present mainly in the T-cell-enriched fraction.

To investigate whether soluble factors were involved in the mediation of enhanced cytotoxicity, supernatants from mixed cell cultures were used to pretreat freshly isolated effector cell populations before measurement of cytotoxicity against K562 cells. Effector cells pretreated with supernatants from mixed cell cultures incubated for 2–6 days showed increased cytotoxic activity, the greatest activity being obtained with supernatants from 4 and 5 day co-cultures. Increased activity was observed when effector cells were pretreated for 2, 4 and 18 hr with supernatants from mixed cell cultures, regardless of the nature of the stimulator cell (T- and B-cell-derived lines, K562 and normal allogeneic lymphocytes). Active supernatants were produced by T-cell-enriched, but not B-cell-enriched responder populations.

Supernatants from mixed cell cultures were also examined for the presence of interferon (IFN) and in general, those mediating large increases in cytotoxic activity also contained significant anti-viral activity. The IFN detected was not neutralized by antiserum to IFN-β but was partly neutralized by antiserum to IFN-α. pH 2 treatment also removed part of the activity while a combination of pH 2 and anti-α completely neutralized the activity suggesting the presence of IFN-γ and IFN-α. The candidacy of IFNs as mediators of enhanced cytotoxicity in this system is discussed.

     

Cytotoxicity of unstimulated and thrombin-activated platelets to human tumour cells.

Platelet cytotoxicity was examined in vitro using various tumour cell lines as target cells. Thrombin-activated platelets as well as unstimulated platelets exerted a cytotoxic effect on some tumour cell lines including K562, KU812, LU99A and KG1, but other tumour cell lines including U937, MIA PaCa-2 and MOLT-4 were completely insensitive to this effect. Electron microscopic examinations showed that unstimulated platelets adhered to target K562 cells but thrombin-activated platelets did not. Morphological changes of K562 cells induced by unstimulated and thrombin-activated platelets were indistinguishable. When platelets and K562 cells were co-cultured in the same vessel but were prevented from coming into direct cell-to-cell contact by means of a membrane barrier, cytotoxicity of unstimulated platelets was completely blocked but that of thrombin-activated platelets was still detectable. However, no cytotoxic activity to K562 cells was detected in the supernatants obtained after stimulation of platelets with either target cells or thrombin for 4 hr. Extracellular Ca2+ ion was not required for the platelet-mediated cytotoxicity. Esterase inhibitors SBTI and TPCK had no effect on the formation of platelet-target cell adhesion but inhibited the cytotoxicity of unstimulated platelets. In contrast, the inhibitors had no effect on the cytotoxic activity of thrombin-activated platelets. These results suggest that direct contact between platelets and target cells is essential for unstimulated platelets but not for thrombin-activated platelets to exert cytotoxicity and that some esterases play a role in the cytotoxic process of unstimulated platelets. They also provide evidence that some cytotoxic effectors are soluble and easily inactivated factors liberated by activated platelets. Our findings indicate that platelets may be one of the cytotoxic effector cells against certain neoplasia.     

Natural cytotoxic activity in human lungs

Disease-free surgical lung specimens from 13 patients with neoplastic or infectious diseases and from three subjects with non-neoplastic, non-infectious pathology were mechanically disaggregated. Natural cytotoxicity was tested against 51Cr-labelled K562 target cells. Unseparated lung cells had little cytotoxicity against K562 cells. Removal of plastic and nylon-wool-adherent cells resulted in cell preparations (morphologically 80% lymphoid) with increased cytolytic activity against K562 but cytotoxicity levels were considerably lower than those of blood lymphocytes tested in parallel. Similar results were obtained when phagocytic adherent cells were removed with carbonyl iron. The NK-resistant murine TU5 and human Raji lines were not affected by lung effector cells. In vitro exposure to partially purified fibroblast interferon enhanced the cytotoxicity of unseparated or non-adherent lung cells. Thus, unlike in mouse pulmonary tissue, low levels of natural cytotoxic activity are associated with the humans lung.     

Defective generation of killer cells against spontaneously Epstein-Barr virus (EBV)-transformed autologous B cells in a fatal EBV infection.

Killer cell activities were analysed in a 16-month-old boy with a sporadic form of fatal Epstein-Barr virus (EBV) infection, and compared with those in three patients with acute infectious mononucleosis (IM). We used spontaneously EBV-transformed autologous lymphoblastoid B cell lines (LCL) as target cells, because the results obtained with such targets can be expected to reflect most accurately the killer-versus-target reaction in vivo. The patient's fresh peripheral blood mononuclear cells (PBMC) had relatively high natural killer (NK) cell activity against K562 cells (128% of the control value), but they did not kill his autologous LCL. The patient's PBMC, unlike PBMC of acute IM, showed no cytotoxicity against Raji cells and autologous LCL after 5 days' culture in the presence of recombinant interleukin 2 (rIL-2), indicating defective generation of lymphokine-activated killer (LAK) cells. The patient's PBMC, unlike PBMC of acute IM, also could not induce cytotoxicity against autologous LCL when cocultured with mitomycin C-treated respective autologous LCL for 7 days. The addition of rIL-2 to the culture significantly restored their ability to generate cytotoxic T lymphocytes (CTL) against his LCL: the percent cytotoxicity value rose from 3.0% to 37.7%. With respect to this, the endogenous IL-2 production by the patient's PBMC was deficient. These results suggest that the defective EBV-selective CTL generation was due to deficient IL-2 production. The failure of the killer cells to eliminate EBV-infected cells seems to have been responsible for the patient's unusual course after primary EBV infection.     

Inhibition of T Cell Cytolytic Potential by Concanavalin A: A Result of Activation?

We investigated the effects of concanavalin A (Con A) on T cell-mediated lympholysis. Human cytotoxic T cell lines were generated from peripheral blood and these lines were shown to lyse Leetin-coated K562 target cells. Addition of soluble Con A to the assay resulted in a dosedependent inhibition of the cytolysis. Preincubation experiments demonstrated that this inhibitory effect was exerted at the effector cell level. F(ab')₂ fragments of WT32, a monoclonal extent. We furter showed that Con A strongly inhibited the cytolysis exerted by alloantigen-specific, major histocompatibility complex (MHC)-restricted cytotoxic T cell lines against their specific targets. On the other hand, con A had no clear inhibitory effect on the cytotoxicity of freshly isolated peripheral blood mononuclear cells against K562 target cells. We hypothesize that Con A-induced inhibition blood of cytotoxicity may be explained by a direct triggering of the lytic potential of activated T cells.     

双特异性抗体对LAK细胞增殖和细胞毒作用影响的体外研究

将抗ＣＤ３与抗ＨＢｓ的单克隆抗体经化学偶联得到双特异性抗体，观察该双特异性抗体对ＬＡＫ细胞增殖和增强细胞毒性的作用。结果显示双特异性抗体显著提高ＬＡＫ细胞与２．２．１５细胞结合率；促进淋巴细胞增殖。１２５Ｉ－ＵｄＲ释放试验检测发现双特异性抗体增强ＬＡＫ细胞对２．２．１５细胞的细胞毒性且与抗体浓度呈正相关。进一步对抗体的特异性研究表明，加入双特异性抗体后ＬＡＫ细胞对２．２．１５细胞毒性作用显著高于对照组。     

Natural cytotoxicity of human blood monocytes: production of monocyte cytotoxic factors (MCF) during interaction with tumor cells

Human blood monocytes obtained by EDTA-reversible adherence to autologous serum-coated plastic dishes expressed natural cytotoxicity against NK-sensitive K562 cells in a 4-h 51Cr release assay. These monocytes released soluble cytotoxic factors, termed monocyte cytotoxic factors (MCF), when cultured with target cells. In contrast, blood monocytes obtained by adherence to fetal calf serum-coated plastic surfaces failed to kill K562 cells and to produce MCF. Although some lysis could be detected at 18 h, optimal lysis of K562 cells by MCF was observed after 48 h incubation in a microcytotoxicity assay using trypan blue dye exclusion. The addition of actinomycin D to the cytotoxicity assay enhanced the sensitivity and then NCF activity was detectable in a 18-h Cr release assay. Neither supernatants produced by culture of monocytes alone nor lysates of monocytes were cytotoxic. In addition, cytochalasin A inhibited both direct cell-mediated lysis and generation of MCF. Optimal production of MCF occurred after 6-24 h of interaction with K562 cells, although significant activity was already present by 3 h. Treatment of monocytes with OKM1 monoclonal antibody plus complement abrogated both cell-mediated lysis and MCF generation, whereas Leu-11b plus complement were ineffective. These results indicate that human blood monocytes can release MCF during interaction with tumor cells and that this may be involved in the lytic mechanism of monocyte-mediated natural cytotoxicity.     

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.     

In vitro augmentation of human natural cytotoxic activity.

Stimulation of human blood lymphocyte preparations with mitomycin C-treated lymphoid cell lines produced increased levels of cytotoxicity against both NK-susceptible and NK-resistant target cell lines. The greatest effect was seen following stimulation by the B lymphocyte-derived lines, Bri8 and raji. K562 also stimulated high levels of activity while the T lymphocyte-derived lines, CCRF/CEM and MOLT 4, produced smaller increases activity was also found in PHA- and MLC-stimulated populations. Stimulation by lymphoid cell lines gave increased cytotoxic activity against all five cell lines when used as target cells and the pattern of target cell susceptibility was maintained, with K562, CCRF/CEM and MOLT 4 being more susceptible than Bri8 and Raji. No direct correlation was found between the level of cytotoxic activity and the level of 3H-thymidine uptake in stimulated effector cell populations. The B cell lines stimulated high levels of isotopic uptake, while the T cell lines gave no significant stimulation. Similarly, the level of 3H-thymidine incorporation following PHA and MLC stimulation showed no direct correlation with the level of cytotoxic activity. Stimulation of lymphocyte transformation did not appear to be necessary for the induction of cytotoxic activity, although the largest increases in cytotoxicity occurred in populations showing high isotope incorporation. No correlation was found between the target cell susceptibility of the different cell lines and their ability to stimulate cytotoxicity.     

Specificity of γδ Receptor-Bearing Cytotoxic T Lymphocytes Isolated from Human Peripheral Blood

T-cell receptor (TcR)-gamma delta-bearing lymphocytes were isolated from the peripheral blood of two healthy donors by immunomagnetic separation and subsequently cultured. The cell lines generated showed two distinct patterns of cytotoxicity. One TcR-gamma delta + cell line (HG.D) lysed K562 and U937 target cells, three TcR-gamma delta + cell lines lysed Daudi cells, and one TcR-gamma delta + cell line showed a shift from the former to the latter specificity during culture. Cold target inhibition experiments showed that the HG.D effector cells which were cytotoxic against U937 cells also lysed K562 cells. The cytotoxicity against Daudi cells was strongly inhibited by monoclonal antibodies (MoAb) against the CD3 complex, whereas the cytotoxicity of the HG.D cell line against K562 and U937 was unaffected by such antibodies. The cytotoxicity against Daudi cells was also strongly inhibited in the presence of anti-TcR-gamma delta MoAb. However, in two of the Daudi-specific cell lines, strong cytotoxicity against K562 cells was induced by anti-TcR-gamma delta MoAb. Anti-LFA-1 MoAb caused only a partial inhibition of cytotoxicity, while anti-CD2 and anti-TcR-alpha beta MoAb were found to have no effect. The results indicate that human gamma delta receptor-bearing T cells demonstrate a certain degree of target cell specificity, and that recognition of some target cells may be mediated through the TcR-gamma delta.     

Natural cytotoxicity in man: activity of lymph node and tumor-infiltrating lymphocytes.

Lymphocytes from blood, lymph node and tumor have been tested for cytotoxicity against the K562 cell line which is known to be highly sensitive to lysis by spontaneously reactive cells. Cytotoxicity was found in all 13 samples from healthy donors and in 17/32 cancer patients. By contrast, activity was determined in only 1/18 lymph node and 1/14 preparations of tumor-infiltrating lymphocytes. Lymph node cells were similarly nonreactive against 3 other cell lines known to be sensitive to natural cytotoxicity. Studies of the composition of the effector populations revealed no absolute deficit of a particular cell type although there were differences between them resulting from the different isolation procedures used. Enrichment of the lymph node population for non-T, non-B lymphocyte was ineffective in inducing cytotoxicity in previously nonreactive samples although this procedure uniformly increased the cytotoxic potential of blood lymphocytes. Tests with blood taken during operation showed that the lack of reactivity in these preparations was unlikely to be a result of the effects of anesthesia or surgery. The reason for the low cytotoxicity in the lymph node and tumor-infiltrating lymphocytes is as yet undefined.     

Natural Killer Cell Activity of Human Fetal Liver Cells after Allogeneic Stimulation

Cells isolated from the liver of human fetuses were confronted with mitomycin C-treated adult allogeneic cells. After this mixed leukocyte culture (MLC)-type reaction, the cytotoxic activity of fetal cells was tested against K562 cell line in a 4-h ⁵¹Cr release assay. Three of the seven fetuses tested (8 to 11 weeks of gestational age) expressed marginal cytotoxic activity before cultivation. Cells from one 8-week-old and one 9-week-old fetus were slightly more cytotoxic when cultured in the presence of allogeneic cells than when cultured in the medium only. Production of gamma interferon (IFN-γ) was not detected in these cultures. Cells of one 18-week-old fetus expressed strong cytotoxic activity against K562 cells after MLC. Thymocytes from the same fetus were not cytotoxic, either before or after MLC against K562 cells. The results indicate that the 'prethymic' human liver contains cytotoxic cells able to spontaneously kill natural killer (NK)-sensitive target cells. Generation of strong NK-like cytotoxicity from noncytotoxic precursor cells was observed only after the thymus becomes lymphoid, suggesting that thymus-processed cells may regulate the generation of NK-like cytotoxic activity. The results suggest a different ontogenity of spontaneous and MLC-induced NK-like cells in the human fetus.     

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity.

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.