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.     

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.     

Generation of Inositol Phosphates during Triggering of Cytotoxicity in Human Natural Killer and Lymphokine-Activated Killer Cells

We investigated early molecular mechanisms involved in the triggering of cytolytic responses in natural killer (NK) and lymphokine-activated (LAK) cells. When NK or LAK cells were conjugated to the sensitive target cells K562, an increased formation of both inositol monophosphate (IP1) and inositol trisphosphate (IP3) was detected. Target cells like Raji or Jok-1, which form conjugates with NK cells but are insensitive to NK lysis, did not elicit IP1 formation. Treatment of NK cells with interleukin 2 increased the basal turnover of inositol phosphates and enhanced the phosphatidyl inositol breakdown upon confrontation with sensitive targets. These finding indicate that hydrolysis of phosphatidyl inositols is associated with the signal which triggers the cytolytic response in NK and LAK cells. These events therefore constitute an early marker of the cytolytic activation.     

Protective Effect (s) of rIL-2 Modulation I. The Effects of Chemotherapeutic/Cytotoxic Agents on Human Natural Killer Cells and Lymphokine-Activated Killer Cells.

This study investigated whether in vitro immunotherapy with rIL-2 which augments human natural cytotoxicity and generation of lymphokine-activated killer cells diminished or inhibits the severity of therapeutically induced human in vitro NK immunosuppression. We demonstrate that rIL-2 induces a rapid and potent enhancement of cytolytic killing and that pretreatment of effecter cells for one hour with rIL-2 yields effecter cells which are more resistant to drug-induced immunosuppression. Additionally, we demonstrate cells pretreated for 24 hours with rIL-2 were less sensitive to drug inhibitory effects than rIL-2 non-treated or one hour pretreated effecter cells. Our data suggest prophylactic treatment with IL-2 for drug induced immunosuppression is feasible.     

Kinetic Analysis of Lymphokine-Activated Killer (LAK) Cells: Lytic Parameters and Determination of LAK Cell Frequency

Kinetic analysis was used to define lytic events in murine lymphokine-activated killer (LAK) cell-mediated tumour cell lysis. The maximum rate of target cell lysis (Vmax) and Km (target cell number resulting in 1/2 Vmax) were determined. Single cell lytic assays demonstrated that only LAK effector cells bound to target cells (i.e. non-lytic, bystander lymphocytes did not influence the determination of kinetic parameters) in contrast to natural killer (NK) cell lysis. This finding allowed for LAK cell frequency determinations where Km approximates the concentration of lytic LAK effector cells within a given number of lymphocytes. Frequencies determined in this manner were not significantly different from those obtained using the more cumbersome single cell lytic assay. Furthermore, frequencies determined for the same lymphocyte population against four different NK-resistant tumour targets, that varied in their sensitivity to LAK cell lysis, were not significantly different. In addition, LAK cell lytic programming of target cells was found to be the rate limiting lytic event. This study provides a means of determining reliable estimates of LAK cell frequencies within a lymphocyte population, which will be useful in studies evaluating LAK cytolytic mechanisms and the effects of drugs, biological response modifiers, or disease states on LAK cell lytic activity.     

Protective Effect (s) of rIL-2 Modulation I. The Effects of Chemotherapeutic/Cytotoxic Agents on Human Natural Killer Cells and Lymphokine-Activated Killer Cells.

Abstract

This study investigated whether in vitro immunotherapy with rIL-2 which augments human natural cytotoxicity and generation of lymphokine-activated killer cells diminished or inhibits the severity of therapeutically induced human in vitro NK immunosuppression. We demonstrate that rIL-2 induces a rapid and potent enhancement of cytolytic killing and that pretreatment of effecter cells for one hour with rIL-2 yields effecter cells which are more resistant to drug-induced immunosuppression. Additionally, we demonstrate cells pretreated for 24 hours with rIL-2 were less sensitive to drug inhibitory effects than rIL-2 non-treated or one hour pretreated effecter cells. Our data suggest prophylactic treatment with IL-2 for drug induced immunosuppression is feasible.     

The Reactivtiy of Leukaemic Cells to HL-A Typing Sera

Despite the atypical reactions of leukaemic cells in the microcytotoxicity test, it is possible to discern three broad catagories of behaviour. Acute lymphatic leukaemias usually give clear cut results, whereas the antigenic profile in chronic lymphatic leukaemia is obscured by many seemingly 'extra'antigens. Individuals showing apparent changes of antigens are usually those with acute myeloid leukaemia. The chronic lymphatic leukaemia group was further characterised by always giving positive results with antiserum Reid. The cases of lymphosarcoma studied were divided evenly into those in which the cells behaved in a manner similar to acute lymphatic leukaemia giving unequivocal results, and those similar to chronic lymphatic leukaemia having 'extra'antigens.     

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Abstract

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. the combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. the coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10^?6 10^?5, 10^?4 and 10^?3 dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. the lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. the significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.     

Lymphocyte Subpopulations in Man: Characterization of Human Killer Cells against Allogeneic Targets Sensitized with HLA Antibodies

Human killer cells mediating antibody-dependent cytotoxicity against allogeneic lymphoblasts presensitized with HLA antibodies have been studied by rosette fractionation experiments. Enriched and/or depleted cell suspensions have been tested in dose-response studies. Two different populations can act as killer cells. The major cytotoxic capacity is retained among T cells with high-avidity Fc receptors, whereas a minor cytotoxic capacity was found among non-T cells with high-avidity Fc receptors. These two populations have different dose-response curves, indicating different effector mechanisms.     

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. the combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. the coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10^-6 10^-5, 10^-4 and 10^-3 dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. the lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. the significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.     

Lymphokine-Activated Killer (LAK)-Mediated Lysis of Sequentially Isolated Ovarian Cancer Cell Lines

PROBLEM: Understanding immunologic eradication of cancer is hampered by failure to explore tumor evolution. Cell surface molecules may alter with therapy and disease progression. These alterations can translate into variable susceptibility to immune-mediated cell lysis. METHOD OF STUDY: Cell lines from a patient with ovarian carcinoma isolated at surgical debulking (UL-3A), during chemotherapy (UL-3B), and after progression (UL-3C) were used to study changes in cell lysis by natural killer (NK) and lymphokine-activated killer (LAK) cells. The role of adhesion molecules ICAM-1, LFA-3, and glycoproteins in the demonstrated differential killing was also examined. RESULTS: An inverse relationship between attachment and lysis was demonstrated. UL-3C, the most sensitive to lysis (50%), attached the least lymphocytes (40%), whereas UL-3A, the least sensitive (33%), attached the most lymphocytes (71%). A correlation with ICAM-1 and LFA-3 expression was not demonstrated. CONCLUSION: Ovarian cancer cells evolve throughout the disease course, and this may manifest as differential sensitivity to immune-mediated cell lysis.     

Role of K+ Ion Channels in Lymphokine-Activated Killer (LAK) Cell Lytic Function

Cells of the immune system possess K⁺ ion channels which have been implicated in various cellular functions including activation, differentiation and cytolytic function. To define the role of K⁺ ion channels in the lytic function of lymphokine-activated killer (LAK) cells, we investigated the effects of K⁺ channel blockers on their cytolytic activity. Results show that when LAK cell mediated cytolysis of AKIL-20 tumor cexis was carried out in the presence of: a) the K⁺ channel blocker, 4-aminopyridine (4-AP); b) the monoamine, serotonin (5-hydroxytryptamine; 5-HT); c) the serotonin agonist, quipazine; d) or the Ca⁺⁺ dependent K⁺ Channel blocker, quinidine, the cytolytic activity of the LAK cells was inhibited in a dose-dependent manner. Preincubation of LAK effector cells also inhibited lysis in a dose-dependent manner, whereas preincubation of the AKIL-20 tumor target cells produced no inhibitory effects. This study demonstrates that K⁺ ion channels are involved in the LAK cell cytolytic process and that compounds, including neuroendocrine products, which modulate K⁺ ion channel function are capable of modulating the lytic activity of these effector cells.     

Kinetic Analysis of K+ Ion Channel Functio in Lymphokine-Activated Killer (Lak) Cells'

Abstract

K⁺ ion channels of lymphocytes have been implicated in cellular differentiation, activation and cytolytic functions. We previously demonstrated that K⁺ channel blockers modulate lytic activity of CTLs and LAK cells. In the present study, we define and quantitate the inhibitory effects of ion channel blockers on the lytic process using kinetic analysis of lysis. The K⁺ channel blocker, 4-aminopyridine, the neuroendocrine monoamine, serotonin, its agonist, quipazine, and the Ca⁺⁺ dependent K⁺ channel blocker, quinidine were found to non-competitively inhibit the lytic process in a dose-dependent manner. These compounds inhibit lytic activity by causing a decrease in the maximum velocity (Vmax) by which LAK cells lyse tumor targets. These ion channel blockers did not alter effector or target cell viability or the binding of LAK cells to tumor cells. The inhibitory effects occurred at the effector cell level, since pre incubation of LAK effector cells resulted in a dose-dependent decrease in Vmax which was related to a slower rate of target cell lytic programming (k₂) by the LAK effector cells. Modulation of LAK cell lytic function occurs at a post-binding step, perhaps in the generation or release of the lytic signal.     

Inhibition of Human Natural Killer Cell and Lymphokine-Activated Killer Cell Cytotoxicity and Differentiation by Vitamin D3

Recent data suggest that vitamin D3 may be capable of immunoregulation after it is converted to an active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The effect of vitamin D3 and 1,25(OH)2D3 on human natural killer (NK) cells and their activation by interferon (IFN) and interleukin 2 (IL-2) was investigated. Vitamin D3 and 1,25(OH)2D3 inhibited NK cytotoxicity in a dose-dependent manner. Pretreatment of non-adherent (NA) cells at 37 degrees C for 18 h with the vitamins also led to inhibition of NK activity. Both the inhibition of NK lysis and pretreatment of NA cells were dependent on the concentrations of fetal calf serum (FCS) in the medium. The inhibition of NK activity was less effective in the presence of 10% FCS than with 1% FCS. Vitamin D3 inhibited both IFN and IL-2 activation of NK activity. However, increasing doses of IL-2 were able to abrogate the inhibition caused by vitamin D3. Vitamin D3 was able to inhibit NK activity of phytohaemagglutinin and IL-2-activated cells, and also inhibit the proliferation and lymphokine-activated killer activity induced by IL-2. NA cells pretreated with vitamin D3 did not respond well to IL-2. NA cells pretreated with low doses of IL-2 were sensitive to inhibition by vitamin D3 while those pretreated with high doses of IL-2 were not. The data presented suggest that vitamin D3 and 1,25(OH)2D3 inhibit NK activity and LAK cellular differentiation.     

苯乙酸钠增加乳腺癌细胞对LAK细胞杀伤敏感性的研究

本文分析了分化诱导剂苯乙酸钠处理乳腺癌细胞后,其对LAK细胞杀伤敏感性的变化。结果发现苯乙酸钠处理乳腺癌细胞MCF-7和MDA-453后,两者对LAK细胞杀伤的敏感性均增加。同时,苯乙酸钠能够增加乳腺癌细胞表面ICAM-1的表达以及LAK细胞和肿瘤细胞之间的粘附活性。因此,苯乙酸钠增加乳腺癌对LAK细胞杀伤的敏感性可能在于增强效靶细胞之间的识别以及结合。     

Transmission of Toxoplasma gondii from Infected Dendritic Cells to Natural Killer Cells

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. In the present study, we observed that a large number of natural killer (NK) cells were infected by T. gondii early after intraperitoneal inoculation of parasites into C57BL/6 mice. Interestingly, one mechanism of NK cell infection involved NK cell-mediated targeting of infected dendritic cells (DC). Perforin-dependent killing of infected DC led to active egress of infectious parasites that rapidly infected adjacent effector NK cells. Infected NK cells were not efficiently targeted by other NK cells. These results suggest that rapid transfer of T. gondii from infected DC to effector NK cells may contribute to the parasite''s sequestration and shielding from immune recognition shortly after infection.Toxoplasma gondii causes chronic infections in up to one-third of the human population and in animals (22, 31). In healthy individuals, primary T. gondii infection causes relatively mild symptoms, whereas in the immunocompromised patient or in the developing fetus, life-threatening manifestations lead to severe neurological and ocular damage (11, 28, 37). Following oral infection, T. gondii parasites typically pass across restrictive biological barriers and rapidly disseminate (13). In this process, T. gondii actively infects a great variety of cell types, including epithelial cells and blood leukocytes (12, 21). In infected cells, the parasites establish nonfusigenic parasitophorous vacuoles, where they can replicate (27, 32, 38).Natural killer (NK) cells and dendritic cells (DC) are two important cell types of the innate immune system. DC-NK cell interactions are important not only in host defense but also for the development of adaptive immune responses (5, 9). The activation of DC by pathogens leads to cytokine secretion, which activates NK cells, which in turn, via cytokines or by direct cell-cell contact, may determine the adaptive immune responses that follow (9, 29). DC are sensitive to NK cell-mediated lysis in vitro and can be eliminated by NK cells in vivo (4, 6, 17, 19, 33, 43). Viral or bacterial infection of DC can reduce their sensitivity to NK cell-mediated lysis by increasing the expression of classical and nonclassical major histocompatibility complex class I molecules on the cell surface (14, 35, 43).DC and NK cells play critical roles in innate immunity during acute Toxoplasma infection, being early sources of interleukin-12 (IL-12) and gamma interferon (IFN-γ), respectively (16, 20, 24, 34, 40). It has recently been suggested that infected DC, and possibly other leukocytes, can act as Trojan horses, potentiating the dissemination of the parasite from the point of infection to distal parts (8, 26). In the early phase of infection with T. gondii, NK cell recruitment to the site of infection is mediated by CCR5-binding chemokines (24). IFN-γ production by NK cells, induced by IL-12 from infected DC or macrophages, has been suggested to be the primary contribution of NK cells to the host defense against T. gondii (18, 25, 39). It can also drive cytotoxic CD8⁺ T-cell immunity to T. gondii even in the absence of CD4⁺ T cells (7). NK cells can also kill T. gondii-infected target cells (42), and perforin has been demonstrated to be important in protecting mice in the chronic stage of infection (10). In the present study, we investigated NK cell interactions with T. gondii-infected DC and, surprisingly, demonstrated how this interaction leads to T. gondii infection of NK cells.     

A Supportive Role of Neurol Cell Adhesion Molecule (NCAM) in Adhesion Between Leukaemic Blasts and Cytotoxic Lymphocytes

The expression and function of neural cell adhesion molecule (NCAM, CD56, Leu19) on leukaemic blasts was investigated. The expression of NCAM was frequent (64%) in 14 studied cases of acute myeloid leukaemia (AML) but not in chronic lymphocytic leukaemia (CLL: 1/3 cases positive) or immunocytoma (IC: no positivity). No correlation with the expression of other AM (intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1). VLA beta chain) or with AML type, according to FAB classification, was observed. Blocking of NCAM with anti-Leu 19 MoAb on AML targets resulted in a significant decrease of their susceptibility to LAK killing and in inhibition of conjugate formation. In the case of B prolymphocytic leukaemia (B-PLL) which did not express ICAM-1 or LFA-1 but was NCAM+, a complete resistance to LAK activity and lack of conjugate formation was observed. Blocking of NCAM on LAK effectors did not decrease their cytotoxic activity. Our results suggest that NCAM, in the presence of other AM, may have a supportive role in adhesion of leukaemic targets to LAK effectors.     

Characterization of Autoantibodies to Natural Killer Cells in HIV-Infected Patients

Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Autoantibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/μl) and antibody negative (141/μl) patients ( P  = 0.3). However, pre-incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells ( P  = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo . There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo