Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.     

Ultraviolet light-induced increase in tumor cell susceptibility to TNF-dependent and TNF-independent natural cell-mediated cytotoxicity

Previously we demonstrated that two consecutive in vitro irradiations of MCA 102 cells with high doses of UVC light (610 and 457 J/m2) resulted in a selection of a permanent line MCA 102UV that manifested high sensitivity to natural cell-mediated cytotoxicity (NCMC). In the present study analysis of the effector cells involved in lysis of these tumor cells was performed by comparing the cytotoxicity of normal spleen cells which mediated both NK and NC cell activity with (a) normal spleen cells in which NC activity was neutralized by anti-TNF Abs (NK+,NC-), (b) NK-depleted or NK-deficient spleen cells (NK-,NC+), and (c) NK-deficient or -depleted spleen cells with NC activity neutralized by anti-TNF Abs (NK-,NC-). Results of these studies indicate that lysis of the original MCA 102 tumor cells was relatively low and was mediated by NC cells. UV irradiation significantly increased MCA 102 tumor cell sensitivity to lysis by both NK and NC cells. Analysis of the mechanisms involved in UV-induced NK sensitivity revealed that UV irradiation increased tumor cell susceptibility to lytic NK-derived granules. NC sensitivity of MCA 102UV tumor cells was associated with their increase in sensitivity to TNF and selection of MCA 102UV cells for resistance to rTNF resulted in a decrease in their susceptibility to NC cells. To determine how fast UV-induced sensitivity to NCMC and rTNF can be established, 51Cr-labeled MCA 102 cells were irradiated in vitro with 38-304 J/m2 of UVC light and their sensitivity to lysis by spleen cells and rTNF was tested immediately in an 18-hr cytotoxicity assay. UV treatment with the same doses was repeated 12 days later. The data obtained showed that tumor cell sensitivity to NCMC and TNF appeared shortly after UV irradiation, was stable, and was further substantially augmented by the second round of UV treatment. Thus, in vitro UV irradiation of tumor cells could be an effective modulator of tumor cell sensitivity to TNF-dependent and TNF-independent cell-mediated cytotoxicity.     

Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

The effects of hexose 6-O-sulfate esters on human natural killer cell lytic function

Natural cell-mediated cytotoxicity (NCMC) is inhibited by some neutral hexoses and hexose phosphates at 25 to 100 mM concentrations. In this study we describe the effects of hexose 6-O-sulfate esters on NCMC against K-562 target cells. Mannose 6-sulfate, galactose 6-sulfate, N-acetylglucosamine 6-sulfate, and N-acetylgalactosamine 6-sulfate inhibit NCMC in a dose-dependent manner at concentrations of 10 mM and below. Inhibitory effects of mannose 6-sulfate and galactose 6-sulfate were evident at concentrations as low as 1.25 mM. The neutral forms of these sugars, glucose and glucose 6-sulfate, did not inhibit NCMC over this range of concentrations. Comparison of the inhibitory effects of sulfated and phosphorylated forms of mannose and galactose indicated that the sulfated forms are much more potent inhibitors. Formation of effector cell:target cell conjugates was unaffected by the presence of sugar sulfates. Calcium pulse experiments demonstrated that inhibitory effects of sugar sulfates were exerted after the Ca++-dependent triggering step in the NK lytic process. Kinetic studies showed that addition of sugars as long as 60 min after initiation of cultures yielded potent inhibitory effects. Sugar sulfates were not toxic for effector cell populations and effectors were not refractory for lytic function after removal of sugars. Sugar sulfates were inhibitory against multiple tumor types in both human and murine NK lytic assays. These results suggest that the sugar sulfates inhibit NK cells at a postconjugation, posttriggering step involving lectin-like receptors or lectin-like molecules.     

Natural killer (NK)-resistant human lung cancer cells are lysed by recombinant interleukin-2-activated NK cells

Natural killer (NK) cells are active in host defence against tumors. In order to determine if NK cells have the capacity to lyse human lung cancer cells, we evaluated blood NK cell activity against human lung carcinoma lines representing each of the commonest histological types of lung cancer, NCI-H157 (large cell), LICM107 and NCI-H146 (small cell), NCI-H226 (squamous cell), and LICM26 (adeno), and compared the results to their activity against a standard NK-sensitive target, K562, using a 16-hr 51Cr-release assay. At effector to target (E:T) ratios up to 50:1, NK activity was very low against each of the lung cancer cell lines compared to the K562 cells (NCI-H157 10 +/- 2%, LICM107 12 +/- 2%, NCI-H146 14 +/- 5%, NCI-H226 8 +/- 5%, and LICM26 7 +/- 3%, compared to K562 60 +/- 3%, P less than 0.001, for each compared to K562 cells). Recombinant interleukin 2 (IL-2) produced a dose-dependent augmentation of NK activity against each of the lung cancer cell lines, with doses as low as 0.25 U/ml being effective. The highest level of boosting was seen against NCI-H157 cells where NK activity (E:T, 50:1, IL-2, 250 U/ml) increased from 9 +/- 2 to 56 +/- 7%, P less than 0.001). Only brief exposure to IL-2 was necessary for augmentation to occur, with as little as 5 min being required for activation, although increased exposure times produced increased levels of augmentation. NK cells appeared to be the IL-2-responsive lytic cell population in these experiments as Leu 11b-depleted lymphocytes expressed little IL-2-mediated augmentation of activity against these target cells, and most of this IL-2-mediated augmentation of activity was located in the large granular lymphocyte-enriched fraction of the lymphocyte population. We conclude that normal blood NK cell activity against human lung cancer cell lines is low but that this activity can be markedly augmented by brief exposure of NK cells to low doses of recombinant IL-2, suggesting a potential role for IL-2 in the immunotherapy of human lung cancer.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Analysis of cytotoxic effector cell function in patients with leukemia or aplastic anemia before and after marrow transplantation

Cytotoxic effector cells mediating natural killing (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC) were studied in patients with leukemia or aplastic anemia before and/or after marrow transplantation. Before transplantation, about one-third to one-half of the patients were deficient in cytotoxic activity. In patients with leukemia, this was most likely due to large numbers of circulating blast cells diluting or replacing the effector cells. In patients with aplastic anemia there was an apparent absence of the effector cells in a proportion of the patients. After marrow transplantation, cytotoxic activity in all three systems returned to normal rapidly, by 30 days, and remained so through 100 days. However, about 20% of patients studied beyond 1 year were deficient in these functions. There were no significant associations between cytotoxic activity and important clinical parameters including infections, graft-vs-host disease, and recurrence of leukemia. Our findings do not support an immunosurveillance role for NK against leukemia after marrow transplantation. Furthermore, they point out the need for new in vitro approaches for meaningful monitoring of marrow transplant patients. Finally, our results showed a significant discordance between NK, ADCC, and LDCC activities in these immunologically perturbed individuals, indicating that either different cell populations or different cellular mechanisms are involved in these cytotoxic functions.     

Indomethacin does not influence natural cell-mediated cytotoxic response to endurance exercise.

Natural cell-mediated cytotoxicity (NCMC) has been shown to be attenuated during recovery from high-intensity or prolonged exercise. Two theories have been proposed to explain the transient suppression of NCMC: prostaglandin-induced inhibition of natural killer (NK) cell activity or a numerical redistribution of NK cells. This study was designed to examine the effects of oral indomethacin (a prostaglandin inhibitor) on NCMC before and after 1 h of high-intensity running (85% maximal oxygen uptake). A secondary purpose was to compare whole blood and isolated peripheral blood mononuclear cell assay procedures for assessing NCMC. Ten male distance runners completed two trials that were preceded by either 48 h of indomethacin (Indo; 150 mg/day) or no treatment (control). NK (CD3(-)/CD16(+)/CD56(+)) cell concentrations were significantly elevated postexercise but were not affected by Indo. NCMC was significantly suppressed at 1.5 h of recovery relative to preexercise only with the whole blood assay procedure. Indo was not found to influence NCMC, leukocyte, or lymphocyte subset concentrations. Mean cytotoxic response was significantly greater with the whole blood method.     

Delayed hypersensitivity and nonspecific cellular immunity. The cytotoxic activity of macrophages, natural and antibody-dependent killer cells

The experiment on (BALB/cXC57BL)F1 mice, showing a high level of delayed hypersensitivity (DH) when sensitized with BCG vaccine and Staphylococcus aureus strain B-243, has demonstrated the influence of such sensitization and DH reaction induced by the injection of a specific antigen (old tuberculin or staphylococcal phagolysate) into the sensitized animals on the cytotoxicity of macrophages, natural killers (NK) and antibody-dependent killers (ADK). Sensitization with BCG vaccine alone results in an insignificant rise in the activity of these effector cells, and sensitization with S. aureus produces no changes at all. The pronounced activation of the cytotoxicity of macrophages, NK and, to a lesser extent, ADK has been observed in DH reaction induced by the injection of a specific antigen into the sensitized mice. In the course of DH reaction a rise in the activity of NK and ADK not only against tumor target cells, but also against microbial ones (Candida albicans and S. aureus) has been found to occur.     

Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle

Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target Cell Cycle.     

Augmentation of mouse natural killer cell activity by interferon and interferon inducers.

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.     

Suppression of human natural and antibody-dependent cytotoxicity by soluble factors from unstimulated normal lymphocytes

Serum-free culture supernatants of unstimulated normal human peripheral blood mononuclear cells contain soluble suppressor factor(s) (SSF) that significantly inhibit natural (NK) and antibody-dependent cellular cytotoxic (ADCC) activities of allogenic lymphocytes against a variety of target cells. Lymphocytes precultured with increasing concentrations of SSF showed a dose-dependent suppressive effect on these cytotoxic functions that was optimal at a concentration of 20% volume/volume. Adherent cells were not required for the production of SSF. Suppression was evident even at higher effector: target cell ratios and the inhibition was not reversed by washing lymphocytes. SSF was not itself cytotoxic, was stable at 56 degrees C, and its suppressive effect was maximal after 72 hr of incubation with effector lymphocytes. Initial estimate of the molecular weight of SSF by ultra-filtration was less than 20,000 daltons. Gel filtration of SSF on Sephacryl S-200 resulted in the elution of two peaks of activity; one in the region between markers of 13,700 and 25,000 daltons, and the other less than 13,700 daltons. Both fractions demonstrated significant suppressive activity on NK and ADCC functions of allogenic lymphocytes. SSF inhibition of NK activity could be partially reversed by incubating lymphocytes for 1 hr with human leukocyte interferon (IF) and almost completely reversed after 24 hr of IF treatment. A few selected monosaccharides (alpha-methyl-D-mannoside, L-fucose and L-rhamnose) showed a dose-dependent blocking effect on SSF activity, which suggests that SSF may act via receptor sites recognized by these sugars. As demonstrated for other lymphocyte functions, NK and ADCC activities may also be modulated by SSF elaborated by normal PBL.     

Decreased natural killer activity in thalassemia major: a possible consequence of iron overload

We have investigated the natural killer (NK) activity of both fractionated (Percoll density gradient) and unfractionated mononuclear cells from patients with beta-thalassemia major who are iron overloaded as a consequence of chronic transfusion therapy. These patients were found to have significantly decreased NK activity against K562 targets at all effector:target ratios tested (p less than 0.001). Both splenectomized and nonsplenectomized patients had normal proportions of Leu-11b-staining (NK) cells. Since they had normal to elevated absolute white cell and lymphocyte counts, a change in the absolute number of NK cells could not account for the decreased killing. We also found that the decrease in NK activity was transfusion related (r = -0.603, p less than 0.005). To determine whether or not this decrease in NK activity could be related to iron overload, we preincubated patient effector cells with desferrioxamine (DFO) or 2,3-dihydroxybenzoic acid (DHB) for 6 hr before addition of K562 targets. Both of these iron-chelating agents consistently increased the NK activity of cells from thalassemia patients. DHB had the greater effect, being able to increase patient NK activity to virtually normal levels. On the other hand, preincubation of cells from normal controls with DHB caused only a slight increase in NK activity, while similar treatment with DFO had little or no effect. When target cells were preincubated with the chelating agents before addition of either normal or patient effector cells, no change in cytotoxicity was seen, demonstrating that the chelating agents act at the effector cell level. Furthermore, if the chelating agents were saturated with iron prior to preincubation with the effectors, no increase in the cytotoxicity of thalassemic NK cells was observed. These results indicate that thalassemia patients have a reversible, transfusion-related decrease in NK function which may arise as a consequence of iron overload.     

BCG-induced murine effector cells. II. Characterization of natural killer cells in peritoneal exudates.

The cytotoxic activity in peritoneal exudates harvested from C57BL/6 mice 4 to 6 days after they had received viable Bacillus Calmette-Guérin (BCG) organisms i.p. was associatee with a nonadherent, nonphagocytic cell. The cytotoxic cell lacked demonstrable surface immunoglobulin and Thy 1 antigen and bore no readily detectable Fc receptors. Lytic activity was labile at 37 degrees C and was diminished after trypsinization of the effector cells. Preincubating effector cells with immune complexes was without effect on lytic expression. These features make it likely that the cytolytic activity was associated with "natural killer" (NK) cells, previously described in unimmunized mouse spleens and mesenteric lymph nodes. Whether BCG induced "activation" of resident NK cells, the de novo production of NK cells, or "homing" into the peritoneum of cells normally resident in other lymphoid tissue is not known.     

Long-term influence of adjuvant therapy on natural killer cell activity in breast cancer

Summary Unstimulated IFN- and IL2-stimulated (NK) cell activities were investigated in patients with breast cancer who had received either local radiotherapy alone or adjuvant chemotherapeutic treatment with CMF combined with radiotherapy 12 to 18 months previously. When tested against the primarily NK-sensitive K562 cell line, patients who had received adjuvant chemotherapeutic treatment with CMF were shown to have a significantly decreased unstimulated and IFN-stimulated NK cell activity, as compared to both patients after radiotherapy only (P<0.002) and P<0.005, respectively) and healthy control persons (P<0.05). The former group of patients also had a significantly decreased IFN-stimulated NK cell activity, when tested against the primarily NK-insensitive Chang hepatoma cell line, as compared to patients after radiotherapy only (P<0.005) and healthy controls (P<0.05). Moreover, patients after radiotherapy only proved to have a significantly increased unstimulated (P<0.01) and IFN-stimulated NK cell activity (K562: P<0.05; Chang hepatoma cell line: P<0.05), as compared to healthy control individuals. In contrast, no difference in IL2-stimulated NK cell activity was detected. The investigation for the expression of CD3 and/or Leu 19 antigens as phenotypic markers of cells with non-MHC restricted cytotoxicity showed a significantly lower percentage of cells with the CD3+ phenotype in patients with breast cancer, irrespective of the chosen post-operative treatment, as compared to healthy controls (P<0.01). Finally, patients with breast cancer who had received radiotherapy only had a significant trend towards an increased percentage of CD³+/Leu 19+ PMNC, as compared to both patients after CMF treatment (P<0.05) and healthy controls (P<0.025). We conclude that patients with breast cancer vary on a long-term basis in their NK activity and in the phenotype of their PMNC depending on their post-operative adjuvant management.Abbreviations NK natural killer cells - IFN interferon - IL 2 interleukin 2 - CMF cyclophosphamide, methotrexate, fluorouracil - ER oestrogen receptor - PMNC peripheral blood mononuclear cells - MHC major histocompatibility complex - CTL cytotoxic T lymphocytes     

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4⁺ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.     

Lymphocytes from children and infants mediate natural cell mediated cytotoxicity, (cytotoxicity)

Summary Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRF-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lymphocytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in development is consistent with early mobilization of the policing mechanism.This work was supported by Grant CA 25765 from the National Cancer InstitutePartially supported by a Grant from the Concern Foundation.