Role for alpha1,2-fucosyltransferase and histo-blood group antigen H type 2 in resistance of rat colon carcinoma cells to 5-fluorouracil

5-Fluorouracil (5-FU) is a drug of standard use in chemotherapy of colon carcinoma. However, its efficacy is limited by inherent and acquired cell resistance. Major changes in histo-blood group antigenic expression, at times associated with poor prognosis, occur on colon cancer cells. To assess whether these antigens might play a role in the resistance to 5-FU, a rat model of colon carcinoma was used. We observed that in vivo treatment of tumors with the drug increased expression of antigen H type 2. The increase was also observed after in vitro short-term exposure to 5-FU, as well as on a cell-resistant variant selected by continuous exposure to the drug, and was accompanied by an increase in alpha1,2-fucosyltransferase activity, the key enzyme involved in synthesis of H antigens. Transfection of cells devoid of this enzymatic activity by an alpha1, 2-fucosyltransferase cDNA allowed expression of H type 2 antigen and increased resistance to 5-FU. Inversely, transfection of cells which possess enzymatic activity by a cDNA in anti-sense orientation reduced both H type 2 cell-surface antigen and resistance to the drug. These results demonstrate that, in this experimental model, alpha1,2-fucosyltransferase and H type 2 antigen are involved in cellular resistance to 5-FU.     

Human tumor cell line resistance to chemotherapeutic agents does not predict resistance to natural killer or lymphokine-activated killer cell-mediated cytolysis

Cancer cells selected for resistance to natural product chemotherapeutic agents typically display cross-resistance to a variety of structurally and mechanistically diverse agents, a phenomenon known as multidrug resistance. Preliminary studies involving cells selected for multidrug resistance in vitro have suggested that the development of resistance to these agents might simultaneously confer resistance to some forms of immunotherapy. Using human tumor cell line models, we have investigated the relationship between either intrinsic or selected multidrug resistance and sensitivity to natural killer (NK) or lymphokine-activated killer (LAK) cell-mediated cytolysis. We compared the NK and LAK cell susceptibility of three human tumor cell lines displaying distinct mechanisms of selected drug resistance with that of the parental drug-sensitive lines. We also evaluated the NK and LAK susceptibility of five established renal cell carcinoma lines, all of which were found to be intrinsically resistant to doxorubicin and vinblastine. The drug-resistant cell lines were variably sensitive to NK-mediated lysis. In contrast, all drug-resistant cell lines tested were LAK cell sensitive. The NK and LAK cell-mediated cytolytic sensitivities of the drug-resistant cell lines correlated well with those of the drug-sensitive parental lines, suggesting that susceptibility to lysis was related intrinsically to each tumor type, and not to the resistance phenotype. We attempted to correlated the NK sensitivity of these cells with the cell surface expression of Class I or II histocompatibility antigens, or the presence or absence of the membrane inhibitor of complement-mediated reactive lysis. None of these phenotypic markers were found to predict NK resistance. We therefore conclude that these cells, which are either spontaneously resistant to commonly utilized antitumor agents or are multidrug resistant as a result of drug exposure in vitro, remain sensitive to LAK cell-mediated cytolysis. Our studies suggest that interleukin 2-induced LAK cells may be useful in the therapy of some chemotherapy-resistant cancers.     

Synergistic suppressive effect of double transfection of tumor necrosis factor-alpha and interleukin 12 genes on tumorigenicity of Meth-A cells.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer / lymphocyte-activated killer (NK / LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK / LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK / LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-alpha and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.     

Synergistic Suppressive Effect of Double Transfection of Tumor Necrosis Factor-α and Interleukin 12 Genes on Tumorigenicity of Meth-A Cells

Tumor necrosis factor-α(TNF-α) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer/lymphocyteactivated killer (NK/LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK/LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK/LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-α and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.     

Loss of leukoregulin up-regulation of natural killer but not lymphokine-activated killer lymphocytotoxicity in human papillomavirus 16 DNA-immortalized cervical epithelial cells

The sensitivity of human cervical epithelial cells immortalized by transfection with human papillomavirus type 16 (HPV16) DNA, to lysis by natural killer (NK) and lymphokine-activated killer (LAK) lymphocytes was evaluated at progressive stages of transformation. Both early- (10-20 wk) and late- (greater than 30 wk) passage HPV16-immortalized cells were resistant to NK lymphocyte cytotoxicity but sensitive to LAK lymphocyte cytotoxicity at lymphocyte-to-cervical cell ratios ranging from 1:1 to 50:1 in a 4-hour 51Cr release assay. Treatment of early-passage HPV16 DNA-immortalized cells with 2.5 U/mL of the NK lymphocytotoxicity-sensitizing lymphokine, leukoregulin, for 1 hour induced modest sensitivity to NK cells (P less than .05) but markedly up-regulated LAK sensitivity twofold to threefold. At the later passages, leukoregulin up-regulation of sensitivity to NK was lost but remained to LAK lymphocytotoxicity. Similarly, an HPV16-positive human cervical carcinoma cell line, QGU, was also resistant to NK lymphocytotoxicity and sensitive to LAK lymphocytotoxicity; leukoregulin failed to confer sensitivity to the NK-resistant QGU tumor cells and increased their sensitivity to LAK lymphocytotoxicity 1.5-fold to twofold. Although the HPV-immortalized cervical cells containing integrated HPV16 DNA were not tumorigenic, they mimicked the response of established HPV16-positive cervical carcinoma cells. HPV16-immortalized cervical epithelial cells provide a useful model for the study of cytokine modulation of dysplastic and neoplastic cervical epithelial cell sensitivity to natural lymphocytotoxicity.     

Recombinant gamma interferon provokes resistance of human breast cancer cells to spontaneous and IL-2 activated non-MHC restricted cytotoxicity

Natural and lymphokine activated killer cells (NK and LAK) are believed to play an important role in the control of tumour progression and metastasis. Their specific receptors on tumours cells are still unknown. Several studies suggest that these cells recognise and eliminate abnormal cells with deleted or reduced expression of MHC class I molecules. Previous reports suggest that interferons (IFN), by increasing MHC class I expression on target cells, induce resistance to killing by NK cells. We investigated the role of MHC molecule expression by two human breast cancer cell lines T47D and ZR75-1 in their susceptibility to NK and LAK cells. These two cell lines spontaneously express low levels of HLA class I antigens but no HLA class II molecules. After IFN-gamma treatment they both overexpressed MHC class I and de novo expressed class II molecules as detected by flow cytometry, quantified by a radioimmunoassay and analysed by two-dimensional gel electrophoresis. Opposed to untreated cells these IFN-gamma treated cells were resistant to NK and LAK lysis. Furthermore, preincubation of IFN-gamma treated breast cancer cells with F(ab')2 fragments of monoclonal antibodies to HLA class I and HLA class II molecules was unable to restore lysis. In contrast, several complete monoclonal antibodies including anti-HLA class I and HLA class II induced the lysis of target cells whether or not they had been treated by IFN-gamma. The therapeutic use of monoclonal antibodies directed against antigens expressed on tumour cells (ADCC) in conjunction with interferon therapy should be discussed in lymphokine-based strategies for treatment of cancer patients.     

Increased tumorigenicity of rat colon carcinoma cells after α1,2‐fucosyltransferase FTA anti‐sense cDNA transfection

Accumulation of histo‐blood group antigens such as Lewis b, Lewis Y and H increases tumor cell motility and tumorigenesis. α1,2‐Fucosylation is a key step in the synthesis of these antigens. Two α1,2‐fucosyltransferases, expressed in colorectal carcinomas, have been characterized (FUT1 and FUT2 in humans, FTA and FTB in rats). To define the relative contribution of each of these enzymes in tumor cell behavior, we have used an anti‐sense transfection approach in rat colon carcinoma PROb cells, which synthesize mRNA encoding for both enzymes. We have previously reported that anti‐sense transfection of a cDNA fragment of the FTB enzyme decreased H antigenic cell‐surface levels and concomitantly decreased tumorigenicity. H antigens, detected by antibodies specific for H type 1, 3 or 4, were detected only on a splice variant of CD44 containing the product of exon v6. We now report the anti‐sense transfection of an FTA cDNA fragment into PROb cells, which resulted in decreased enzymatic activity on a type 2 precursor and decreased cell‐surface H type 2 antigen exclusively. Compared to controls, FTA anti‐sense‐transfected cells were significantly more tumorigenic in syngeneic animals but not in immunodeficient SCID mice. The UEA‐I lectin, specific for H type 2, revealed that these structures were present on the CD44v6 variant and on an uncharacterized 80‐kDa glycoprotein. Our results indicate that FTA and FTB fucosylate distinct glycan chains in the same cell, leading to opposite effects, under control of the immune system. Int. J. Cancer 80:606–611, 1999. © 1999 Wiley‐Liss, Inc.     

Increased susceptibility of IFN-gamma-treated neuroblastoma cells to lysis by lymphokine-activated killer cells: participation of ICAM-1 induction on target cells.

We have investigated the effect of interferon-gamma (IFN-gamma) treatment of neuroblastoma cells on the susceptibility to lysis by lymphokine-activated killer (LAK) cells and examined the participation of cell-adhesion molecules on the target cells in LAK cell lysis. Untreated neuroblastoma cells expressed lymphocyte-function-associated antigen 3 (LFA-3) and neural-cell-adhesion molecule (NCAM), but did not express MHC-class-I, MHC-class-II, or intercellular-adhesion molecule I (ICAM-I). IFN-gamma treatment of neuroblastoma cells induced the expression of MHC-class-I and ICAM-I antigens, but did not affect the expression of MHC-class-II, LFA-3, and NCAM. This was accompanied by an increased susceptibility to lysis by LAK cells. Anti-ICAM-I antibody inhibited partially the increased sensitivity of IFN-gamma-treated neuroblastoma cells to LAK cell lysis, and blocked completely the increase in binding of LAK cells observed after IFN-gamma treatment of the target cells. These results suggest that the increased LAK sensitivity of IFN-gamma-treated neuroblastoma cells is partially attributable to the induction of ICAM-I on neuroblastoma cells and indicate that post-binding events also play a role in the increased sensitivity to LAK cell lysis observed after IFN-gamma treatment.     

Lymphokine-activated killer (LAK) cells discriminate between Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells

We have generated in vitro lymphokine-activated killer (LAK) cells from healthy donors by stimulating their mononuclear leukocytes with recombinant interleukin-2 (rIL-2) (100 U/ml). After 6 days in culture, the lytic properties of the LAK cells were analyzed in the 51Cr-release assay by utilizing a target panel of 6 paired lines consisting of an Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line and an EBV-transformed lymphoblastoid cell line (LCL) from the same donor, the Raji BL line and the natural killer (NK) cell-sensitive K562 line. The patterns of lysis showed that the LAK cells discriminated between two categories of BL cell lines. Group I/II BL tumor cells which expressed the common acute lymphoblastic leukemia antigen (CALLA), the BL-associated glycolipid antigen (BLA) and phenotypically resembled biopsy cells were strongly lysed whereas group III BL cells which had assumed an LCL-like phenotype during culture and lacked the CALLA and BLA surface markers were only poorly lysed. The LCL targets were generally resistant to lysis but the K562 cell line was particularly sensitive. The outcome of cell depletion and monoclonal antibody (MAb) studies indicated that the LAK cell populations were phenotypically and functionally heterogeneous and consisted of at least 2 subpopulations of effector cells; a tumor-specific component and an NK-cell-mediated component.     

Analysis of factors associated with the tumorigenic potential of 12 tumor clones derived from a single rat colon adenocarcinoma.

We analyzed several factors which could influence the immunogenicity of colon tumor cells, using a series of clones derived from a single chemically induced rat adenocarcinoma cell line. These clones display variable tumorigenic potential in syngeneic immunocompetent animals, and it has been established that in this model the tumorigenicity of the cells depends on their ability to escape immune surveillance. The results show an absence of relationship between tumorigenicity and expression of MHC-class-I antigens, cell adhesion to rat fibroblasts or fibroblast extracellular matrix. The secretion of latent and active TGF beta I appeared to be quite variable from one clone to the other, but was unrelated to tumorigenicity. Unexpectedly, some regressive clones produced elevated levels of this cytokine, suggesting that in this model, spontaneous secretion of TGF beta I is not sufficient to impair the immune system of the host. In contrast, the more tumorigenic clones were more resistant than less tumorigenic ones to cytotoxicity mediated by NK or LAK cells. They also showed arrest of cell proliferation after reaching confluence, something not observed in the less tumorigenic clones. Finally, the strongest relationship with tumorigenicity was found for expression of blood-group carbohydrate antigens. Increased expression of blood-group-H antigen and, conversely, decreased expression of beta-galactoside precursors of this antigen correlated with increased tumorigenicity.     

The relationship between multi-drug resistance and resistance to natural-killer-cell and lymphokine-activated killer-cell lysis in human leukemic cell lines.

Results concerning a possible link between susceptibility to natural-cell-mediated immune cytolysis and the multi-drug resistance (MDR) phenotype are conflicting. We evaluated in human acute lymphocytic leukemia the relationship between acquired drug resistance and susceptibility to cytolysis mediated by endogenous, interferon-activated, and interleukin-2-activated natural cytotoxic cells. Eight human leukemia drug-resistant/sensitive cell line pairs were evaluated; drug-resistant sub-lines included those selected for primary resistance to adriamycin, etoposide, teniposide, vincristine, and vinblastine. A majority of P-glycoprotein-positive MDR sub-lines displayed slight but statistically significant resistance to endogenous and/or interferon-activated natural-killer(NK)-cell-mediated lysis, as compared with the drug-sensitive parental type. P-glycoprotein-negative sub-lines displayed variable NK susceptibility; within this group, the variants selected for primary etoposide resistance were more susceptible to NK cytolysis than parental cells. Results of cold-target-inhibition experiments suggest that altered NK susceptibility does not arise solely from modulation of NK target recognition and adherence structures. IL2-activated killer (LAK) cells lysed both drug-sensitive and drug-resistant lines. Two MDR lines selected for primary etoposide resistance displayed enhanced LAK susceptibility. In contrast, the 2 variants selected for resistance to adriamycin exhibited partial resistance to LAK-mediated killing, which could be overcome at high effector-to-target ratios. Our results support the development of interleukin-2/LAK immunotherapy for the treatment of leukemias with acquired drug resistance.     

Increased cytotoxicity against B-chronic lymphocytic leukemia by cellular manipulations: potentials for therapeutic use

B-cell chronic lymphocytic leukemia (CLL) is characterized by profound immune dysfunction and a marked resistance to apoptosis. Understanding the cellular biology of immune effector cells from CLL patients as well as leukemic target cells is essential to developing immune mediated therapeutic strategies for CLL. In this study, an immortal CLL cell line called WSU-CLL has been used to study the characteristics of B-cell CLL as a tumor target for natural killer (NK), activated natural killer, and lymphokine activated killer (LAK) cells. The WSU-CLL cells were significantly less (p<0.001) susceptible to NK cell mediated cytotoxicity compared to K562, a standard tumor target cell line. In vitro activation of effector cells with either short term, low dose IL-2 or long term, high dose IL-2 significantly increased the susceptibility of CLL cells for cell mediated killing. The addition of CD1a+/CD3-/CD4+/CD80+/CD83+ dendritic cells derived from human umbilical cord blood increased the cytotoxicity of LAK cells against WSU-CLL. There is an increased expression of Bcl-2 and decreased expression of Fas on WSU-CLL cells as determined by RT-PCR techniques indicating possible roles for these genes in exerting resistance to immune cell mediated lysis. When Bcl-2 expression was downregulated in WSU-CLL cells using gene specific antisense oligonucleotides, the susceptibility of WSU-CLL cells to the cytotoxicity of chemotherapeutic agent Fludarabine was increased. Thus, our results suggest that in vitro activation with cytokines, addition of accessory cell populations such as dendritic cells and/or manipulation of key gene expression i.e. down regulation of Bcl-2 might be potential strategies to increase the antitumor cytotoxicity against CLL cells.     

Interleukin-1 produced by tumorigenic fibroblasts influences tumor rejection.

Oncogene-transformed BALB/c-3T3 fibroblasts which spontaneously or upon immune-activation with cytokines and lipopolysaccharide (LPS) generate IL-1 alpha, were tested for their tumorigenicity as well as their interaction with natural immune defense by NK cells and macrophages. Oncogene-transformed fibroblasts were weakly tumorigenic, since not all mice developed tumors despite application of high doses of tumor cells. This was independent of the immune status of the host. However, in the immunocompetent host those transformed fibroblast lines which spontaneously produced IL-1 alpha grew only transiently and then regressed. After induction of IL-1 alpha production, a decrease in the rate of tumor take was noted and the rate of regression of developing tumors was increased. Regression of IL-1-producing transformed fibroblasts was strongly reduced but not completely abolished in sublethally irradiated mice. This indicated that IL-1 production may predominantly influence T-cell-mediated defense, but some influence on non-adaptive immunity could not be excluded a priori. IL-1 production did not influence susceptibility of transformed fibroblasts towards NK cells and macrophages. However, IL-1-producing transformed fibroblasts were most potent stimulators of NK cells and macrophages, the stimulatory effect being locally restricted. In conclusion, IL-1 producing, oncogene-transformed fibroblasts which generated the cytokine constitutively or upon immune-activation, were rejected from the tumor-bearing host following initial growth. Fibroblast-induced local activation of NK cells and macrophages was shown to play some role in tumor graft rejection. The influence of IL-1 production of transformed fibroblasts on T-cell-mediated defense is addressed in the accompanying report.     

Characteristics of human T-lymphotropic virus type-1 (HTLV-1)-infected cell line MT-2, which is not killed by a natural killer cell line NK-92 but is killed by lymphokine-activated killer cells

Natural killer (NK) cell-mediated cytolysis (NK-lysis) is triggered by costimulatory signals of adhesion molecules and is downregulated by negative signals of killer cell inhibitory receptors (KIRs). Recently, a NK cell line, NK-92, was established. This cell line can kill several tumor cells, which possess adhesion molecules CD54 and CD102. However, the NK-92 cannot kill a human T-lymphotropic virus type 1 (HTLV-1)-infected cell line, MT-2, although lymphokine-activated killer (LAK) cells can kill MT-2. In this report we investigated the reason for LAK sensitivity but NK-92 resistance of the MT-2. The MT-2 highly expressed CD54 and CD102, suggesting that the costimulatory signals may be intact. Then we tested the responsibility of the negative signals by determining HLA type of the MT-2 and KIRs of the effector cells. The MT-2 expressed HLA-A24, B40, B51, Cw3, and HLA-G. The NK-92 did not express KIR2DL1, KIR2DL2,3, nor KIR3DL1, but 24% of the cells weakly expressed CD94. The blocking tests against these HLA class I molecules and KIRs did not restore the NK-92 resistance, although blocking against HLA-G slightly increased its lysis. Finally, in order to eliminate the class I molecules from the cell surface, we treated the MT-2 using a buffered citric acid solution (pH 3.8). By using this treatment, the expression of class I molecules and HTLV-1 antigen decreased, and then the MT-2 was killed by the NK-92. These findings suggest that an aberrant class I molecule of the MT-2 transferred a negative signal to the NK-92 and induced the NK-92 resistance. It remains to be elucidated whether or not the HTLV-1 infection contributed to the alteration of the class I molecule.     

Intrasplenic vaccination against experimental melanoma

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.     

Inhibition of proliferation, activation and function of human natural-killer-cells in-vitro by secreted products of lovo, a colorectal-cancer cell-line

It has previously been suggested that some tumours may elaborate substances which can suppress the functional activity of lymphokine activated killer (LAK) cells. We have investigated the effects of secreted products from the colorectal cancer cell line LoVo on the pattern of expression of T lymphocyte subsets and natural killer cell phenotype and activation antigens during in vitro treatment of mixed populations of human peripheral blood mononuclear cells with recombinant human IL-2 (rhIL-2). Tumour cell line products inhibited the proliferation of cells expressing the NK cell antigen CD56 and of cells expressing the CD8 T cell subset antigen. Increased expression of the IL-2 receptor alpha chain component CD25, and the activation associated antigens CD71 and HLA-DR induced by rhIL-2 was inhibited by the presence of tumour cell products. Enhanced cytotoxic activity against standard target. cell lines Daudi and K562 induced by treatment with rhIL-2 was diminished by tumour cell supernatants. These mechanisms may contribute to the resistance of some tumours to immune response modulation treatment regimens which depend upon induction of lymphokine activation responses by rhIL-2.     

c-Ha-ras-I oncogene-induced differentiation and natural killer cell resistance in a human colorectal carcinoma cell line

Natural killer (NK) activity is primarily a peripheral blood function of a lymphocyte population capable of spontaneous lysis of many transformed and metastatic targets. However, NK-susceptible targets tend to be relatively poorly differentiated. We have previously shown that poorly differentiated human colorectal carcinoma are lysed by NK cells. Well-differentiated and chemically differentiated colorectal carcinomas are insensitive to NK lysis. The present study demonstrates that transfection of the c-Ha-ras-I oncogene into a poorly differentiated colorectal carcinoma cell line also renders it NK resistant. This resistance is accompanied by a more differentiated colorectal carcinoma phenotype. Two ras-transfected lines (Clone-A-5 and Clone-A-4) showed a 30-66% decrease in susceptibility to NK lysis as compared to the parental line in standard cytotoxicity assays. The resistance of these transfectants was strictly dependent on expression of the activated p21, the H-ras protein product. Studies to assess the integrity of the initial binding step in NK lysis showed a significant decrease in the ability of these transfectants to form conjugates with fresh NK cells. It is likely that transfection with c-Ha-ras-I has selectively modulated critical NK target recognition structures.     

NK细胞的生物学特性及临床应用研究进展

 目前NK细胞的临床治疗主要通过利用细胞因子体内扩增、激活NK细胞和体外产生LAK、CIK细胞杀伤自体肿瘤细胞,但并未取得实质性进展。随着自然杀伤细胞抑制性受体（KIR）及其他NK细胞受体（NKR）的发现,对NK细胞受体在识别和裂解肿瘤细胞的重要作用的不断了解,为肿瘤的免疫治疗提供了新的治疗策略。由KIR独特型不相容引发的NK细胞异源反应性在异基因造血干细胞移植中促进移植物植入,预防GVHD发生以及增强GVL作用,已经成为国内外学者研究的热点。就目前关于NK细胞的生物学特性及临床应用研究进展予以综述。     

Synergistic action of a plant rhamnogalacturonan enhancing antitumor cytotoxicity of human natural killer and lymphokine-activated killer cells: chemical specificity of target cell recognition.

The spontaneous natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity of highly purified CD56+CD3- NK cells (90 to 95%) against NK-sensitive and NK-insensitive target cells was drastically enhanced when a rhamnogalacturonan contained in a commercially available Viscum album extract was present during 4-h cytotoxicity assays. This enhancement correlated strictly with an increased formation of NK cell or LAK cell/tumor cell conjugate formation. Information on the chemical specificity of NK cell and LAK cell interaction with target cells and with the rhamnogalacturonan was obtained from inhibition studies. The most efficient inhibitors (100% inhibition at 5 mg/ml) were acetylated D-mannose and acetylated L-mannonic acid gamma-lactone. They specifically inhibited in a dose-dependent manner: (a) the cytotoxicity of NK cells against K562 cells and the formation of NK cell/K562 cell conjugates; (b) the cytotoxicity of LAK cells against K562 cells and Daudi cells as well as the formation of LAK cell/K562 cell and of LAK cell/Daudi cell conjugates; and (c) the synergistic effects of the rhamnogalacturonan in the cytotoxicity assays and the target cell-conjugate formation assays with NK cells and LAK cells. The inhibitory effects observed after pretreatment of NK cells or LAK cells with acetylated mannose were completely reversible, but that obtained with acetylated mannonic acid gamma-lactone was only partly reversible, and the degree of reversibility depended on the inhibitor concentration applied during pretreatment. Nonacetylated mannose or mannose derivatives up to concentrations of 20 mmol showed no inhibitory effects. A mechanistic model representing the interaction of NK cells and LAK cells with target cells and with rhamnogalacturonan is proposed.