Effects of Heat Shock on Cytolysis Mediated by NK Cells, LAK Cells, Activated Monocytes and TNFs α and β

We have recently shown that a heat treatment of a murine target cell line, WEHI 164, induces resistance to lysis mediated by tumour necrosis factors alpha (TNF-α) and beta (TNF-β). In the present study the effect of the heat shock of target cells on cytotoxicity mediated by natural killer cells (NK cells), lymphocyte-activated killer cells (LAK cells), activated monocytes, TNF-α, and TNF-β was investigated, First, WEHI 164 cell line and six human cell lines (ME 180, K 562, U 937, HeLa, MCF 7, and SK-OV 3) were screened for their sensitivity to different forms of lysis, and then sensitive cell lines were heat-treated. Pretreatment of target cells at 42° C for 45-60 min also rendered human target cell lines more resistant to lysis by rTNFs, and the acquired resistance was accompanied by an increased resistance to activated monocytes, but not to NK cells or LAK cells. Thus, the heat-induced resistance mechanisms capable of protecting target cells from lysis by rTNFs and by activated monocytes do not elicit resistance to lysis by NK cells and LAK cells, supporting the hypothesis that mediators other than TNFs are involved in NK cell-and LAK cellmediated killing.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.     

Correlation between killing activity towards the murine L929 cell line and expression of membrane-associated lymphotoxin-related molecule of human lymphokine-activated killer cells.

Human lymphokine-activated killer (LAK) cells expressed a membrane-associated lymphotoxin-related molecule (mLT) which was detected by flow cytometric analysis with anti-lymphotoxin antibody. Upon removal of exogenous interleukin-2 from LAK cell culture medium and another 24 h cultivation, the expression of mLT was decreased. Corresponding to the decrease of mLT expression, the killing activity of LAK cells towards L929 cells was remarkably reduced and killing of MIA PaCa-2 and U937 cells was moderately reduced, whereas killing of Daudi and K562 cells was fully restored. The supernatant of mLT-expressing LAK cells had no cytotoxic activity towards L929 cells in the absence of actinomycin D. Moreover, not only the killing of L929 cells but also that of human tumor cell lines (MIA PaCa-2, U937) by mLT-expressing LAK cells was partially inhibited in the presence of anti-lymphotoxin antibody. These results suggest an involvement of mLT in the killing of some tumor target cells by LAK cells.     

Suppression of Target Cell Proliferation by Natural Killer Cells

The cytolytic effects of natural killer (NK) cells have been extensively studied in recent years. In the present study we have investigated the cytostatic effects of NK cells. Human peripheral blood lymphocytes from healthy volunteers were used as a source of effector cells, and the cell lines K562, U937, U1285, and Molt-4 were used as target cells. Effector cells were enriched for NK cells using Percoll gradients and depleted of NK cells on Percoll gradients or by using Leu-19 antibodies and magnetic beads. By monitoring cell numbers during co-culture of effector cells and K562, it was found that after an initial phase of cell killing for 3 h target cell numbers remained stable during the following 24-48 h. In a microcytotoxicity assay measuring inhibition of uptake of [3H]thymidine, the four target cell types were shown to have different NK sensitivity; inhibition of greater than or equal to 80% was obtained for K562 and U937 at an effector to target cell (E/T) ratio of 30:1, 50% for U1285, and 30% for Molt-4. This inhibition was shown to be partly a direct effect on DNA synthesis for all cell lines, as incorporation of [3H]thymidine was decreased in cocultured target cells compared with an equal number of target cells alone. Inhibition of DNA synthesis was thus not directly related to cell death and was also observed for the Molt-4 cell line that was not killed. A cell division assay, with target cells in agarose and effector cells in a liquid upper layer, showed a decline in the rate of target cell divisions. Effects on the cell cycle were studied on latent-phase cells. It was shown that effector cells delayed the onset of DNA synthesis. This anti-proliferative effect was observed for several days, but cell growth then gradually resumed. The effector cells were identified as CD56-positive large granular lymphocytes (LGL). Double-layer cultures and experiments using effector cell supernatants demonstrated that the growth-inhibitory effect could be mediated by soluble factors, and the production of such factors was stimulated by exposure to a small proportion of target cells (50:1). Studies with specific antibodies indicated that growth inhibition was not mediated by alpha interferon (IFN-alpha) but it was partly mediated by tumour necrosis factor alpha (TNF-alpha). It is concluded that NK cells have a growth-inhibitory effect that is distinct from the cytolytic effect and this activity is probably mediated by several soluble factors including TNF-alpha.     

Comparative susceptibility of peripheral blood leucocytes and related cell lines to killing by T-cell perforin.

The comparative susceptibility of lymphocyte subsets, monocytes and polymorphonuclear leucocytes (PMN) to killing by murine perforin was measured using physical separation techniques, cell-surface phenotyping and scatter characteristics to isolate cell types, together with propidium iodide (PI) uptake as a measure of cell death. In the majority of individuals, PMN were more resistant to perforin than other peripheral blood cells including natural killer (NK) cells and CD8+ lymphocytes. Among the lymphocytes, CD4+ cells were the most susceptible subset, followed by CD19+, CD8+ and CD56+ lymphocytes respectively. The human promyelocytic leukaemia cell line, HL-60, and the human histiocytic lymphoma cell line, U937, were readily killed by perforin. When HL-60 were differentiated to either macrophage- or neutrophil-like end cells, and U937 differentiated to macrophage-like end cells, there was no difference between differentiated and undifferentiated cells in their relative susceptibility to perforin. The relative resistance of PMN to perforin may be important in protecting them from damage in in vivo situations where both NK cells and neutrophils are localized in the same inflammatory areas.     

Clinical Progression of HIV Infection: Role of NK Cells

The authors determined the natural killer (NK) and lymphokine-activated killer (LAK) activities of peripheral blood mononuclear cells (PBMCs) from a severely immunocompromised (CD4 cell counts <100/mm³) group of AIDS patients, using K562 and U937 target cells. An increase in cytotoxicity was observed in the PBMCs of all 17 patients following a 48 h incubation with the combination of 400 U/ml of recombinant gamma interferon plus 20 U/ml of natural interleukin-2. Although NK and LAK activities were significantly higher in healthy controls than in patients, patients' LAK activity was higher than the NK activity of controls. The authors also demonstrated that the use of medium containing fetal bovine serum, when compared with medium containing autologous serum, increases NK activity without affecting LAK activity. Lymphokine augmentation of cytotoxicity is achievable in severely immunocompromised AIDS patients and might be of benefit in delaying opportunistic infections and malignancies.     

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.     

Modulation of natural killing by tumor promoters: the regulatory influence of adherent cells varies with the type of target cell

We have analyzed the regulatory effects of two classes of tumor promoters, phorbol diesters and indole alkaloids, on human natural killer (NK) cell activity in vitro. In accordance with previous reports, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited natural killing against K 562 targets by unseparated mononuclear cells. Here, suppression of NK required the presence of adherent cells (macrophages). Contrary to the results obtained with K 562, tumor promoter-induced suppression of NK activity tested against U 937, another cell line of known NK susceptibility, was independent of the presence of adherent cells. Thus, NK cytotoxicity of effector cells rigorously depleted of adherent and Ia-positive cells was still inhibited when assayed against U 937, while it was generally enhanced when tested against K 562. Identical results were obtained with teleocidin and dihydroteleocidin B, two members of the recently discovered indole alkaloid class of tumor promoters. Therefore, we demonstrate that the regulatory effect of tumor promoters on human NK activity (suppression or stimulation) is determined not only by macrophages at the effector cell level but also by the type of target cell under study.     

Evidence that Protein Kinase C Activation is Essential for Killing by IL-2-Activated Lymphocytes

Previous pharmacological evidence has suggested that activation of protein kinase C (PKC) is necessary for T and natural killer (NK) killing of different target cells. In the present study we find, using interleukin 2 (IL-2)-activated lymphocytes (LAK cells), that phosphorylation of a well-characterized 80-kDa PKC substrate increases during conjugation to target cells. Furthermore, down-regulation of PKC by pretreatment with the active phorbol esters PDB (24 h) or PMA (2 h), but not with the inactive phorbolester PDD, simultaneously inhibits killing by LAK cells. H-7, an inhibitor of PKC, also inhibited LAK-cell killing without affecting the target-effector cell conjugate formation. We also demonstrate that pretreatment of target cells with phorbol ester (PMA) decreases killing, suggesting that PKC activation in the target cell population may also influence killing although the effect may vary depending on the particular target cell used. We conclude that PKC activation is essential for triggering of lysis in LAK cells.     

Engagement of MHC Class I Proteins on Natural Killer Cells Inhibits their Killing Capacity

We have studied whether engagement of MHC class I (MHC—I) molecules on natural killer (NK) cells can influence the NK killing activity. Human NK effector cells, enriched by nylon wool passage, were incubated with monoclonal antibodies (MoAb) to MHC—I followed by cross-linking with secondary rabbit anti mouse Ig or streptavidin. Cross linking of MHC—I molecules on NK cells resulted in a clear inhibition of the NK activity against the target cells K562, Molt-4 and U937. The inhibitory effect was selective for MHC—I and was not seen with MoAb to MHC—II or CD56 molecules. The inhibition was not mediated via Fc receptors since F(ab)₂ fragments of the MHC—I MoAb W6/32 were as effective as the intact antibody. The best inhibition of NK activity was obtained using biotin-labelled F(ab)₂ fragments of W6/32 and streptavidin as a cross-linker, where up to 70 % reduction in NK cell activity was observed. Antibody dependent cellular cytotoxicity (ADCC) was also inhibited by cross-linking MHC—I molecules on the effector cells.
The results show that antibody mediated cross-linking of MHC—I proteins on NK cells can inhibit their killing capacity. This indicates that MHC—I molecules on NK cells can be involved in the regulation of NK cytotoxicity, perhaps by transmitting inhibitory signals into the NK cell.     

Resistance of different tumor cells to lysis by lymphokine activated killer cells can be mediated by distinct mechanisms.

Lymphokine activated killer (LAK) cells have been shown to exert a potent cytotoxic effect on many histologically different tumors and virally infected targets. Most normal cells but very few tumors have proven resistant to LAK lysis. The availability of two LAK resistant tumors, P815r, a murine mastocytoma, and SNUC-1, a human colon carcinoma, allowed us to study the phenomenon of LAK lysis. We examined the role of surface molecules on targets, which mediate binding to LAK cells, by cold target competition experiments and lectin dependent cellular cytotoxicity assays. The results showed that in the murine system, P815r cells do not compete for lysis of the LAK sensitive target B16 whereas other LAK sensitive murine targets compete. Alternatively, in the human system, SNUC-1 cells compete for lysis of the LAK sensitive target SNUC-4 as do other LAK sensitive human tumor cells. Furthermore, inducing binding of target and effector cells with lectin reverted the resistance of P815r but not SNUC-1 targets to lysis by LAK cells. These results imply that distinct stages of the lytic pathway might be involved in the resistance of different tumors to killing by LAK cells. The murine cell line is resistant to lysis because it cannot bind LAK cells. The human target, which does bind LAK, was insensitive to the effects of tumor necrosis factor alpha (TNF-alpha), a lymphokine released by LAK effectors and possibly involved in their lysis. Resistance to TNF-alpha was not mediated by the presence of endogenous short-lived proteins in the SNUC-1 targets. The elucidation of mechanisms of resistance may provide a tool to improve current protocols of adoptive immunotherapy as well as insights as to how tumor cells are or are not killed by LAK effectors.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

N-α-Benzyloxycarbonyl-l-Lysine Thiobenzyl Ester (BLT) Serine Esterase in Human Cytolytic Effector Cells and Cell Line Targets

The granules of in vitro primed cytotoxic mouse T cells and cytotoxic cell lines have been shown to contain high levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase. The enzyme activity has been suggested to be associated with the cytotoxic capacity of killer cells. We investigated human leucocytes and found that neutrophils, monocytes, cytotoxic T lymphocytes (CTL), natural killer (NK) cells [large granular lymphocytes (LGL)], and interleukin 2 activated killer (LAK) cells, which all display efficient cytotoxic capacity, show only marginal BLT esterase activity. The low BLT esterase activity in human lymphocytes increases about twofold when cells are stimulated in vitro with interleukin 2 (IL-2), phytohaemagglutinin (PHA), or cultured in mixed lymphocyte culture (MLC). Mouse T lymphocytes have about 20 times more BLT esterase activity than human T lymphocytes. The BLT activity in mouse T cells also increases about twofold in MLC. The human leukaemia cell lines (K562, U937, MOLT-4, Jurkat) and the mouse mastocytoma line (P815), which are frequently used as target cells, contain more BLT esterase activity than human resting or activated lymphocytes. We did not find a direct correlation between the cytotoxic capacity and the BLT esterase activity of killer cells.     

Indirect inhibition of generation of murine lymphokine-activated killer cell activity in splenocyte cultures by interferon-gamma.

The effect of mouse recombinant interferon-gamma (rIFN-gamma) on murine lymphokine-activated killer (LAK) cell activity was investigated using natural killer (NK)-resistant, spontaneously developed, weakly immunogenic and highly tumourigenic, syngeneic murine mammary adenocarcinoma, JC, mimicking that of human disease, as the target. Murine YAC-1 also was used as a target cell line. rIFN-gamma, when used in combination with recombinant interleukin-2 (rIL-2), was shown to exhibit a suppressed effect on LAK cell activity generated from BALB/c mouse splenocytes, compared to that with rIL-2 alone. The decrease in LAK cell activity was rIFN-gamma dose-dependent. Kinetic study revealed that this inhibitory effect was demonstrated only when rIFN-gamma was added to the medium at the early phase of rIL-2 culture. The inhibitory effect on LAK cell generation by rIFN-gamma was completely abrogated when the nylon-wool-treated non-adherent 'macrophage-free' splenocytes were incubated with rIL-2 and rIFN-gamma. These results indicated that the LAK cell activity generated from murine splenocytes cultured with rIL-2 could be depressed by rIFN-gamma, and that the macrophages may be involved as mediators.     

Different functional domains on the transferrin receptor molecule defined by monoclonal antibodies.

Three monoclonal antibodies (mAb) FG 1/5, FG 1/6 and FG 2/12, specific for the human transferrin receptor molecule (TR), have been used to define epitopes on the TR molecule and to block natural killer lysis. FG 2/12 mAb but not FG 1/5 or FG 1/6 blocked [125I-] transferrin binding to the cellular receptor. Furthermore, FG 1/5 and FG 1/6 mAbs competed out the binding of each other to the cells but not significantly that of FG 2/12. As expected, the binding of F2/12 but not of FG 1/5 or FG 1/6 was inhibited by transferrin. In addition, FG 2/12 inhibited in a dose-dependent manner the NK activity of purified T3- large granular lymphocyte effector cells against HeLa or Molt-4 cells but not against K-562 or U937 cells. FG 1/5 preferentially inhibited NK activity against HeLa cells and FG 1/6 mAb was completely uneffective. These inhibitions were stronger at low effector to target cell (E:T) ratios than at high E:T ratios, suggesting that NK cells and anti-TR mAbs compete for the same site in the target cell. It was shown that FG 1/5 and FG 2/12 mAbs blocked cells' conjugate formation by acting at the target cell level. Our results confirm the role of TR as a one of the target structures in NK lysis and suggest that the epitope recognized by NK cells is close to but different from the transferrin binding site.     

Human invariant valpha24+ natural killer T cells activated by alpha-galactosylceramide (KRN7000) have cytotoxic anti-tumour activity through mechanisms distinct from T cells and natural killer cells

Human Valpha24 + NKT cells, a subpopulation of natural killer cell receptor (NKR-P1A) expressing T cells with an invariant T-cell receptor (TCR; Valpha24JalphaQ) are stimulated by the glycolipid, alpha-galactosylceramide (KRN7000), in a CD1d-dependent, TCR-mediated fashion. Little is known about Valpha24 + NKT-cell function. The murine counterpart, Valpha14 + NKT cells, appear to have an important role in controlling malignancy. There are no human data examining the role of Valpha24 + NKT cells in controlling human malignancy. We report that Valpha24 + NKT cells have perforin-mediated cytotoxicity against haemopoietic malignancies. Valpha24 TCR, CD1d and alpha-galactosylceramide may all play a role in cytotoxicity but are not absolute requirements. The greatest cytotoxicity was observed against the U937 tumour cell line (95 +/- 5% lysis). THP-1, Molt4, C1R cells and allogeneic mismatched dendritic cells were also sensitive to Valpha24 + NKT cytotoxicity but neither the NK target, K562, nor lymphokine-activated killer-sensitive Daudi cells, were sensitive. These results indicate a killing pattern distinct from conventional major histocompatibility complex-restricted T cells, NK cells and other cytotoxic lymphoid cells previously described. We conclude that human Valpha24 + NKT cells have cytotoxic anti-tumour activity against haemopoietic malignancies through effector mechanisms distinct from conventional T cells and NK cells and that their specific stimulator KRN7000 may have therapeutic potential.     

Interferon-Induced NK Augmentation in Humans

By combining a single-cell cytotoxicity assay in agarose with estimations of the maximal natural killer (NK) cell potential (Vmax) by 51Cr release, the mechanism behind interferon augmentation of human NK cells were analysed. The number of target-binding cells (TBCs) the fraction of active TBCs and NK cell recycling were studied after short-term interferon treatment. The results demonstrate a dual effect of interferon on human NK cells: effector cell recruitment and increased effector cell recycling. Both of these variables were increased when NK cells were tested against the standard target K-562 and against Daudi and BJAB cells, derived from B-type lymphomas. However, when T cell lines derived from acute lymphocytic leukaemia (Molt-4 and 1310) were used as targets, a larger fraction of active NK cells were found among untreated TBCs, whereas interferon treatment only resulted in increased effector cell recycling and not in effector cell recruitment. No increase in TBCs after interferon treatment could be detected with any cell line tested. The difference seen between T and non-T cell lines with regard to interferon-induced effector cell recruitment is discussed in relation to known characteristics of the human NK system.     

Capacity of CD8+ T Cells to Reject Immunogenic Variants of a Spontaneous Murine Carcinoma: Lack of Non-Specific (NK1.1+) Effector Mechanisms

Class I MHC-expressing (Ia-) immunogenic (imm +) variants, which elicit a strong syngeneic host immune rejection response, can be isolated following 5-azacytidine treatment from the MHC-negative non-immunogenic (imm-) murine carcinoma cell line SP1 (10.1 subclone). In the present study, we have shown that CD4-depleted CD8+ T cells are both necessary and sufficient for the rejection process. Treatment of semi-syngeneic B6 X CBA F1 mice with anti-NK1.1 antibodies had no effect on the rejection of immunogenic variants, although the splenic NK (natural killer) activity of recipients was fully abrogated. Thus NK1.1+ effectors, which include most NK and LAK (lymphokine activated killer) cells, are most likely not involved in the rejection process. This finding was supported by a complete lack of NK susceptibility of SP1 cells in vitro, although variable killing by LAK and poly-I: C-induced killer cells was observed. To assess the role of NK1.1-LAK and other non-T killers (e.g. cytolytic macrophages) in vivo, we determined the specificity of the rejection process. We examined the ability of immune animals to reject a mixture of non-immunogenic parent tumour cells (or cells of an unrelated syngeneic tumour) and of the variant tumour cells used for the initial immunization. Growth of the parent tumour cells was unaffected while the same animals rejected the immunogenic tumour cells. Our findings support a primary role of tumour-specific CD8+ T cells in the rejection of imm+ variants with no detectable involvement of non-specific effector cells in the tumour destruction process.     

健康人外周血单个核细胞诱导的NK细胞对脑胶质瘤杀伤作用的探讨

目的 对体外培养的NK细胞杀伤脑胶质瘤细胞进行探讨,为脑胶质瘤的免疫细胞疗法提供理论基础.方法 从4个健康人外周血提取单个核细胞,在KRATM培养环境下,诱导产生NK细胞和LAK细胞.分别用细胞计数法和流式细胞仪检测NK细胞增殖和细胞表面特异性标志CD3/CDl6/CD56,并用MTr法检测NK细胞及LAK细胞对脑胶质瘤细胞的杀伤.结果 经过体外培养,平均可得到7.93×109个以上细胞.和LAK细胞相比,NK细胞对脑胶质瘤细胞LNI8、LN229、T98G和U87MG的敏感性更高.结论 NK细胞能够对脑胶质瘤细胞产生有效免疫应答,为过继性免疫细胞治疗脑胶质瘤提供可能.