乳腺癌中神经激肽受体的表达及受体拮抗剂的作用研究*

目的:研究乳腺癌中全长型、截短型神经激肽1 受体（neurokinin 1 receptor,NK1R）和神经激肽2 受体（neurokinin 2 receptor,NK2R）的表达,及受体拮抗剂对乳腺癌细胞生长的影响。方法:采用免疫组织化学法检测天津医科大学肿瘤医院51例乳腺癌及其癌旁正常组织、30例乳腺良性病变组织总NK1R（包括NK1R-FL和NK1R-Tr）和NK2R 表达,采用实时定量PCR 和免疫印迹技术检测乳腺细胞系中NK1R-FL、NK1R-Tr和NK2R 表达,建立NK1R-FL和NK1R-Tr过表达的乳腺细胞系, 在NK1R 和NK2R 拮抗剂作用下测定细胞增殖和软琼脂集落形成能力。结果:乳腺癌及癌旁正常组织、乳腺良性病变组织中均总NK1R 过表达,乳腺癌组织中 NK1R-FL、NK2R 表达相比癌旁正常组织显著降低,并与乳腺癌分型、组织学分级、淋巴结转移及 Ki-67、HER-2、ER和PR表达相关。HBL-100 细胞中NK1R-FL和NK2R 过表达、NK1R-Tr低表达,MDA-MB-231、T-47D 和MCF-7 细胞中只表达NK1R-Tr。乳腺癌细胞中NK1R-Tr低表达、NK1R-FL表达增加其对NK1R 和NK2R 受体拮抗剂的敏感度。结论:乳腺组织中NK1R-FL、NK2R 共表达,乳腺癌细胞中NK1R-Tr过表达并负反馈调节NK1R-FL和NK2R 的表达,NK1R 和NK2R 受体可能成为乳腺癌治疗的新靶标。      

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.     

Diploid human lymphoblastoid and Burkitt lymphoma cell lines: susceptibility to murine NK cells and heterotransplantation to nude mice

Human lymphoid cell lines which had been classified on the basis of studies on clonality and morphological, on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell line (LCL) of presumed non-neoplastic origin and Burkitt lymphoma (BL) lines of proven malignant origin, were tested for susceptibility to natural killer (NK) cells obtained from the spleens of athymic nude mice. The 20 lines included normal diploid LCL and aneuploid BL lines. All cells carried the Epstein-Barr virus (EBV) genome. In addition, two EBV-negative BL lines were tested. The pronase-induced release of 14C-DNA from 14C-thymidine-labelled target cells was used to assess the sensitivity of the cell lines to NK activity. When attempts were made to correlate the growth of the EBV-positive LCL and the EBV-positive BL cell lines in the subcutaneous space of adult nude mice with their susceptibility to NK cells, no significant correlation was observed. The EBV-negative BL cell line, Ramos, however, could be transplanted subcutaneously in nude mice and was more resistant to NK activity than was the EBV-negative BL cell line, BJAB, which cannot be transplanted subcutaneously. Growth of heterotransplanted EBV-converted cell lines in the subcutaneous space of adult nude mice may be influenced by immune effectors other than NK cells.     

Human natural killing against ovarian carcinoma.

Natural killing (NK) by lymphocytes from normal individuals against primary and established ovarian carcinoma cell lines was tested in short-term chromium release assays. Two established cell lines and 5/6 primary cell lines tested showed significant susceptibility to NK. Primary cell lines are, in general, less sensitive to NK than long-term cultured target cells. A common NK recognition determinant on ovarian carcinoma cells and on the erythroleukaemic K562 cells was demonstrated by cold target inhibition assays. The recognition structure was also present on an ovarian cell line resistant to NK but not on the insensitive leukaemic cell line, SB. The activity against ovarian carcinoma cells was associated with the presence of large granular lymphocytes (LGL) previously shown to be the major effector cells against K562 targets. In fractions obtained by Percoll gradient centrifugation of lymphocytes, only fractions with a high content of LGL demonstrated good NK activity. LGL mediated NK against both non-adherent K562 and the adherent ovarian carcinoma target cells independent of monocytes.     

Suppression of NK activity of spleen cells by chicken fetal antigen present on Marek's disease lymphoblastoid cell line cells

Chicken fetal antigen (CFA) expressed on the cell surface of Marek's disease (MD) lymphoblastoid cell line (MDCC-MSBI) cells was purified by affinity chromatography using a monoclonal antibody (2H3). A 4-hr 51Cr release assay was performed using spleen cells as effector cells to investigate the role of the CFA in the immune response of chickens, especially in relation to natural killer (NK) activity. Spleen cells of specific-pathogen-free (SPF) chickens showed reduced NK activity in the presence of CFA in vitro, as did those cells treated with MSBI soluble antigen. NK activity of spleen cells from chickens treated with CFA was also suppressed when compared to the cells from untreated or ovalbumin-treated chickens. In addition, MSBI clo. 18 transplantable tumors grew progressively in some of the 14-day-old chickens treated with CFA, whereas the tumors regressed in age-matched chickens with or without treatment by ovalbumin.     

Sp1在 NK／T 细胞淋巴瘤细胞株中的表达及其对细胞侵袭的影响

目的：观察转录因子 Sp1在 NK／T 细胞淋巴瘤（NK／TCL）细胞株中的表达及其对细胞侵袭的影响。方法采用实时荧光定量聚合酶链反应（PCR）、免疫荧光和 Western blot 法测定 NK／TCL 细胞株 SNK-1、SNK-6和健康人 NK 细胞中 Sp1的表达;采用 Sp1抑制剂光神霉素 A（MIT）作用于 NK／TCL细胞株后,用实时荧光定量 PCR 和 Western blot 法检测 Sp1、胰岛素样生长因子1受体（IGF-1R）和基质金属蛋白酶2（MMP-2）的表达;采用 Transwell 实验观察 MIT 对细胞侵袭力的影响。结果 NK／TCL 细胞株SNK-1、SNK-6中 Sp1基因和蛋白高表达。其中基因的表达水平分别是健康人 NK 细胞的（9.4±0.3）倍和（10.6±0.3）倍（P＝0.0052,P＝0.0037）,蛋白的表达水平分别是健康人 NK 细胞的（5.4±0.3）倍和（8.6±0.5）倍（P＝0.0083,P＝0.0069）。100 nmol／L MIT 抑制 Sp1后,与二甲基亚砜处理的对照组相比,细胞株中 IGF-1R 的基因表达量分别下降了（83.9±3.7）%和（65.8±4.2）%（P＝0.0082,P＝0.0097）,蛋白表达量分别下降了（51.5±7.1）%和（49.6±9.1）%（P＝0.0178,P＝0.0155）。100 nmol／L MIT 处理细胞后 SNK-1、SNK-6细胞的侵袭率分别下降了（29.6±6.4）%和（37.2±7.6）%（P＝0.0418,P＝0.0372）,MMP-2蛋白表达量分别是对照组蛋白表达量的（52.7±4.7）%、（29.7±5.6）%（P＝0.0286,P＝0.0202）。结论 NK／TCL细胞株高表达 Sp1,其可能通过正调控 IGF-1R 增强 MMP-2的表达,进而提高 NK／TCL 细胞的侵袭力。     

Immunoprofiling of cell lines derived from natural killer-cell and natural killer-like T-cell leukemia-lymphoma

T-cells and natural killer (NK)-cells can be distinguished by their immunophenotype and molecular biological studies though there is overlap in T- and NK-cell antigen expression, function, and malignant diseases. The relatively new cell type of NKT-cells (also termed NK-like T-cells) represents a subpopulation of T-cells that share some characteristics with NK-cells. T- and NKT-cells have their T-cell receptor (TCR) genes rearranged while NK-cells are identified molecularly and immunologically by the absence of TCR gene rearrangements and TCR protein and lack of certain surface antigens. Various continuous malignant cell lines have been derived from patients with T-cell, NK- and NKT-cell neoplasms. These cell lines possess several traits typical of the respective diseases. Characterization of these cell lines which was the objective of this study will facilitate future studies of cell biology and therapeutics for which cell lines are indispensable models. In view of the imprecision of morphological criteria alone, we analyzed a series of seven NK-cell, five NKT-cell and five T-cell lines using functional and immunophenotypic tools. All T-cell lines were negative for the presence of azurophilic granules, NK activity and Epstein-Barr virus (EBV). In contrast, 7/7 NK-cell and 4/5 NKT-cell lines displayed the azurophilic granules but only three of these combined twelve NK/NKT-cell lines showed significant NK activity which may be explained by the functional immaturity of the cells. EBV was found in 5/7 NK-cell and in 1/5 NKT-cell lines. As expected, T-cell lines were commonly positive for T-cell surface antigens and negative for NK-cell markers, and NK-cell lines vice versa; nevertheless, a number of immunomarkers were shared between T- and NK-cell lines. NKT-cell lines express T-cell, NK-cell and markers shared between T- and NK-cells. Sets of markers distinctive for the three types of cell lines are presented. The composite data gained on the present panels of cell lines allow for the operational definition of typical NK- and NKT-cell line profiles. Such cell lines will prove invaluable as informative models for studies of normal and neoplastic NK- and NKT-cell biology.     

Functional Consequences of APO-1/Fas (CD95) Antigen Expression by Normal and Neoplastic Hematopoietic Cells

Murine monoclonal antibody (mAb) 7C11 binds to the same cell surface epitope as anti-APO-1 and anti-Fas and reacts specifically with cells transfected with a cDNA encoding the human Fas antigen. Furthermore, incubation with 7C11 causes death of hematopoietic cell lines that express APO-1/Fas but not APO-1/Fas-negative cell lines. 7C11 therefore recognizes the human APO- 1/Fas (CD95) antigen, a 40 to 50 kDa cell surface glycoprotein that can trigger apoptosis or programmed cell death. Expression of APO- 1/Fas antigen by normal and neoplastic hematopoietic cells was determined by flow cytometry using 7C11. APO-1/Fas is expressed by 30 to 40% of resting peripheral blood T cells, B cells, and monocytes and by -5% of resting NK cells and thymocytes. It was not detected on granulocytes, erythrocytes, or platelets. Approximately 80 to 90% of activated T cells, B cells, and thymocytes express APO-1/Fas, as do the majority of activated NK cells. Perturbation of APO-1/Fas by 7C11 does not affect the viability of resting lymphocytes or monocytes. In contrast, activated T cells and NK cells undergo apoptosis within 3 hours of exposure to 7C11. Other mAb that stimulate T cells or NK cells do not cause rapid induction of programmed cell death. APO-1/Fas antigen is expressed by many cell lines of lymphoid and myeloid lineage. However, this antigen was detected on neoplastic cells from only one of 69 patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or multiple myeloma. Only 3 out of 25 tumor samples from patients with non-Hodgkin's lymphoma were found to express APO-1/Fas. All three of these lymphomas harbored the bcl-2-Ig fusion gene associated with the chromosomal translocation t (14; 18). Conversely, only 27% of lymphomas that possessed the bcl-2-Ig gene were found to express the APO-1/Fas antigen. Like normal activated lymphocytes, leukemia and lymphoma cells that expressed APO-1/Fas antigen were found to undergo apoptosis in vitro after incubation with 7C11. The APO-1/Fas antigen appears to regulate the growth of normal hematopoietic cells, and the marked upregulation of this antigen on activated normal lymphocytes contrasts sharply with the absence of APO- 1/Fas on neoplastic cells of hematopoietic lineage. Defects in the apoptotic signal delivered through this antigen might contribute to the pathogenesis of hematopoietic neoplasms. Thus, the gene encoding APO- 1/Fas can be considered a novel type of tumor suppressor gene, just as bcl-2 can be considered a cellular proto-oncogene.     

Expression of a putative tumor-associated surface antigen on normal versus Marek's disease virus-transformed lymphocytes

Various avian tumor cell lines and normal spleen cells from 3 genetic strains of specific-pathogen-free (SPF) chickens were examined for expression of Marek's disease (MD) tumor-associated surface antigen (MATSA). Two anti-MATSA monoclonal antibodies (RPH 6 and EB 29) and a rabbit anti-MATSA antiserum were used in indirect fluorescent antibody tests, and cells were examined by fluorescence microscopy and with a fluorescence-activated cell sorter (FACS). Less than 5% MATSA-positive cells were observed in 2 non-MD tumor cell lines (LSCC-RP 9 and RECC-CU 60) with RPH 6, but 7-82% positive cells were observed with EB 29 or the rabbit antiserum. Five MD tumor cell lines (MDCC-CU 2, -CU 14, -CU 25, -CU 32, and -CU 41) had 12-72% positive cells detected with one or both monoclonals and 31-99% positive cells detected with the rabbit antiserum. Over 90% of cells in all MD lines were la and T3 positive, while values for the same parameters in LSCC-RP 9 were 100 and 3% and for RECC-CU 60, 48 and 51%, respectively. Evidence for cell-cycle-dependent expression of MATSA on MDCC-CU 2 was obtained from cell sorting experiments with the FACS and from examination of the MATSA-staining characteristics of 3 clones derived from the parent culture. Less than 5% MATSA-positive cells were observed in uncultured spleen cells from SPF chickens or in spleen cells stimulated for 48 hours with concanavalin A or phytohemagglutinin-M. However, with one exception, 10-53% of normal spleen cells were MATSA positive with RPH 6, after stimulation by mitogen for 24 or 48 hours followed by maintenance in conditioned medium (CM) for various times or after culture directly in CM for 3 days. More limited experiments with rabbit anti-MATSA antiserum yielded 55-85% MATSA-positive cells. From 60 to 97% of these MD virus-free, MATSA-positive cells were la-positive; and, in 2 cases, 89 and 90% were T3 positive.     

Induction of the EBV cycle in B-lymphocyte-derived lines is accompanied by increased natural killer (NK) sensitivity and the expression of EBV-related antigen(s) detected by the ADCC reaction

Human B-lymphocyte-derived lines were forced to enter the EBV-cycle by superinfection with the P3HR-l substrain of EBV or sodium butyrate treatment. The induced cells were used as targets for natural killing (NK) and EBV-specific, antibody-dependent cellular cytotoxicity (ADCC). Two Burkitt lymphoma lines, Raji and Daudi, and one normal adult derived lymphoblastoid cell line, NAD-7, were comparable in their ADCC-sensitivity after induction, but only the Burkitt lymphoma-derived lines showed a major increase in NK-sensitivity. The superinfection-induced membrane change, responsible for both NK and ADCC sensitivity, is an early function of the viral cycle, correlated with the appearance of early antigens (EA). Indirect evidence indicates that the NK and ADCC target sites are different but this problem requires further investigation. Sodium butyrate induced an increased NK sensitivity and EBV-related ADCC sensitivity in the Burkitt lymphoma-derived P3HR-l line. Lymphocyte effectors from different donors showed great differences in their NK and ADCC activity. Optimal ADCC could be demonstrated with effectors that were intermediate in their NK-activity.     

A murine model for B-cell lymphomagenesis in immunocompromised hosts: natural killer cells are an important component of host resistance to premalignant B-cell lines

The accompanying paper (D. W. Felsher et al., Cancer Res., 50:7042-7049, 1990) describes a new panel of cloned murine B-cell lines with a premalignant phenotype and in vivo-derived malignant variants. This paper assesses the contribution of immune mediated antitumor mechanisms which might account for host resistance to the tumorigenicity of these cell lines. Conventional T-cell-dependent responses did not appear to be critical to host resistance. In vivo elimination of T-helper cells with anti-L3T4 monoclonal antibody did not reduce host resistance to the tumorigenicity of these cell lines, nor did these cell lines elicit cytotoxic T-cell activity. However, a strong correlation was found between tumorigenicity and host natural killer (NK) activity. In vitro studies demonstrated that the cell lines were as NK sensitive as the prototypical NK target, YAC-1, whereas the malignant variants fully tumorigenic in normal hosts were greater than 20-fold less NK sensitive than were the parent cell lines. In vivo depletion of NK cells with anti-asialo-GM1 in BALB/c strongly diminished host resistance to cell line tumorigenicity, whereas polydeoxyinosinic-deoxycytidilic acid induction of NK cells enhanced host resistance. These findings indicate that NK function is a critical component to host resistance in this system and suggest that endogenous cellular mechanisms which overcome NK sensitivity could be a target for secondary transforming events in B-cell lymphomagenesis. They also raise the unexpected possibility that a non-antigen-dependent (versus immune cytotoxic T-lymphocytes) effector mechanism may be the key deficit promoting B-cell neoplasia in the setting of immunocompromised states.     

Natural killer (NK) cells as effectors of antibody-dependent cytotoxicity with chimeric antibodies reactive with human squamous-cell carcinomas of the head and neck

In patients with cancer, antibody-dependent cellular cytotoxicity (ADCC) may be used as a laboratory test or for enhancing immunotherapy with murine monoclonal or chimeric mouse/human anti-tumor antibodies (mMAbs or cMAbs, respectively). We have established an ADCC assay with IgG₁ cMAb SF-25, using human squamous-cell carcinoma of the head and neck (SCCHN) cell lines as targets. By flow cytometry, all SCCHN cell lines tested expressed the antigen recognized by cMAb SF-25. Trypsinization of the cell monolayers facilitated binding of cMAb SF-25 to the antigen on the cell surface of SCCHN targets. Using the PCI-50 SCCHN cell line as a target coated with this cMAb at the optimal concentration of 1.0 μg/ml, normal human peripheral blood mononuclear cells (PBMC, n = 28) were found to mediate ADCC at a mean level of 283 ± 42 (SEM) lytic units (LU₂₀/10₇ effector cells). Non-adherent monocyte-depleted PBMC and natural killer (NK) cells purified by negative selection mediated significantly higher levels of ADCC than unseparated PBMC against SCCHN targets. NK cells, defined as CD3-CD56' cells, could be effectively armed by cMAb SF-25, as confirmed by flow cytometry and ADCC assays. IL2-activated armed NK cells mediated higher levels of ADCC than non-armed NK cells. Binding of cMAb SF-25 to NK cells and their ADCC were enhanced by pre-incubation with polyethylene glycol. Arming of NK cells with chimeric antibodies should be considered in developing novel strategies for treatment of human SCCHN, especially in the adjuvant setting. © 1995 Wiley-Liss, Inc.     

Comparison between murine natural antibodies and natural killer cells: recognition of separate target structures as revealed by differential in vitro expression and dependence on glycosylation

The specificity of complement-fixing, cytotoxic antibodies against the YAC lymphoma in sera of normal young adult (A X C57BL)F1 mice was studied. In vivo-maintained, immunoselected sublines of the YAC lymphoma expressed low amounts of the natural antibody (NAb) target structure. These cell lines were also resistant to natural killer (NK) cell-mediated lysis. After 2-3 weeks of in vitro culture the immunoselected cell lines became NK sensitive, but they remained resistant to NAb. When several independently derived variants selected for low NK sensitivity were tested for their ability to absorb NAb, the degree of absorption varied considerably among the variants. NAb could be inhibited by purified C-type virus particles and also by bacterial sonicates and various glycoprotein preparations. Treatment of target cells with tunicamycin, an inhibitor of asparagine-linked glycosylation, decreased the sensitivity to NAb lysis but had no impact on NK sensitivity. Thus the results indicated that a) NAb and NK cells recognized separate target structures and b) the target structure(s) for NAb but not for NK cells were saccharides of the N-glycosidic type.     

Natural cytotoxicity to human leukemia mediated by mouse non-t cells

Natural killer (NK) cells cytotoxic for certain syngeneic or allogeneic tumor cells have been described in various species, including mouse, rat and man, as a new type of effector cell. In this paper, we show that mouse NK cells also function in a xenogeneic system. Established human hematopoietic cell lines were used as targets for normal mouse spleen effector cells in a short-term ⁵¹Cr release assay. Nine malignant lymphoma, two myeloma and four leukemia lines as well as six lympho-blastoid cell lines of presumed non-neoplastic origin were tested. Specific and reliable killing was obtained with the T leukemias MOLT-4, JM, CCRF-H-SB-2 and the myeloid leukemia K 562. Most other lines were sensitive to some degree but their susceptibility and the specificity of the reactions were of doubtful significance. The heterologous cytotoxic reactivity was shown to be caused by the same kind of murine effector cells that mediate NK activity against certain mouse lymphomas by the following criteria: (1) the differences in killing capacity found between spleen cell preparations from genetically “high” or “low” NK-reactive inbred mouse strains did apply in this heterologous system; (2) cytotoxicity was related in a typical manner to the age of the effector cell donor; (3) spleen cells from T-deficient nude mice were highly cytotoxic; (4) removal of T cells, B cells and phagocytic cells using standard fractionation procedures did not abrogate the lytic ability of the remaining spleen cell population. Specificity of lysis was assessed using unlabelled cells as competing targets together with labelled target cells. When competitor cells were identical to the target cells, good competition was achieved. Mouse or human cell lines insensitive to lysis by mouse NK cells had no competitive capacity for target cells of either species. Using NK sensitive lines, competition between mouse and human cells was found to be unidirectional with the most sensitive tumors being the best inhibitors. Thus, unlabelled mouse T lymphoma YAC-1 cells were efficient competitors for lysis of human T leukemia lines. Conversely, susceptible human cells used as competitors did not significantly inhibit killing of YAC-1 targets. The implications of these findings are discussed.     

Relationship between cell membrane potential and natural killer cell cytolysis in human hepatocellular carcinoma cells

One of the body's natural defense mechanisms against tumor cells is lysis of the invading cell by cytotoxic T-cells and natural killer (NK) cells. Five human hepatocellular carcinoma cell lines were found to have different sensitivities to killing by peripheral blood monocytes in a 51Cr release assay. This killing was demonstrated to be due to NK cell lysis. Electrical recording measurements of the membrane potentials of these five cell lines showed different values for each line, all below values reported for normal hepatocytes. Correlation between mean cell membrane potential, and sensitivity to NK lysis, revealed an inverse relationship. In this study we demonstrate that the lower the mean membrane potential of a human hepatocellular carcinoma cell line, the more sensitive it is to NK cell cytolysis. Cell surface positive potential did not correlate with NK cytolysis and only a weak correlation was found between cell membrane negative potential and cell surface positive potential between cell lines.     

Modulation of murine natural killer cells by a granulocytosis-inducing tumor

The nonmetastatic neutrophilia-inducing murine mammary carcinoma CE1460 has been shown previously to have profound effects on hemopoiesis and lymphopoiesis. In this report we examined the effects of progressive growth of CE1460 on natural killer (NK) cell activity both in the bone marrow, the site of primary NK cell production, and in a peripheral site, the spleen. (BALB/c x CE)F1 mice were injected subcutaneously with trypsinized cells from in vivo passaged CE1460 or from B66, a BALB/c mammary carcinoma that does not induce neutrophilia. 3 days posttumor implantation, NK activity in bone marrow cells or spleen cells was greatly enhanced compared to normal controls. In B66 tumor-bearing mice, NK activity returned to normal by Day 7 and remained there through Day 14. In contrast, however, NK activity in CE1460 tumor-bearing mice decreased to only 10-20% of normal by Day 14. Excision of the tumor on Day 14, when WBC counts were three times normal, was followed by a rapid return of the WBC count to the normal range. NK activity in bone marrow and in spleen cells recovered somewhat but was still significantly suppressed 7 days after tumor excision. Limiting dilution analysis revealed a 3-5-fold decrease in frequency of NK precursors in bone marrow cells of mice bearing CE1460 for 7 or 14 days. The dramatic changes in NK activity observed in these experiments may reflect perturbation in production as well as an initial activation and subsequent suppression of mature NK cells.     

Natural killer cells in children with malignant solid tumors. Effect of recombinant interferon-alpha and interleukin-2 on natural killer cell function against tumor cell lines

Natural killer (NK) cells and NK cell activity were determined in three groups (newly diagnosed [n = 21], on therapy [n = 21], and off therapy [n = 18]) of children with various types of malignant solid tumors and in a control group (n = 26) by means of Leu-7 and Leu-11b monoclonal antibodies and a 4-hour 51Cr-release assay, respectively. The erythroleukemia cell line K562 was used as a target cell. The newly diagnosed group included eight patients with localized disease (Stage I-II), ten with bulky but nonmetastatic disease (Stage III), and three with metastases (Stage IV). The mean percent of NK cell activity in the newly diagnosed group was significantly higher than that of the control group. Children with Stage III tumors at diagnosis had higher mean NK cell function than those with Stage I-II and Stage IV. On therapy patients had significantly fewer NK cells and lower NK cell cytotoxicity than those in the other groups studied. We also studied the following: (1) the in vitro effect of recombinant interferon-alpha (rIFN-alpha) and recombinant interleukin-2 (rIL-2) on NK cell function of peripheral blood lymphocytes (PBL) from children with solid malignancies; and (2) the susceptibility of neuroblastoma-derived (CHP-126 and SKNSH) and rhabdomyosarcoma-derived (A-204) cell lines to NK cell lysis. Both rIFN-alpha and rIL-2 enhanced NK cell activity of PBL from children with malignancies and healthy children against K562 and solid tumor cell lines. The enhancing effect or rIL-2 was greater than that of rIFN-alpha. CHP-126 and SKNSH cell lines were susceptible to NK cell lysis mediated by the PBL of children with neuroblastoma and the control group. The A-204 cell line was less sensitive than K562 to NK cell cytotoxicity. Our results suggest a potential therapeutic role for both cytokines in the treatment of malignant solid tumors of childhood.     

Natural killer cells in children with acute leukemia. The effect of interleukin-2

The purpose of this study was to determine (1) the number and activity of natural killer (NK) cells in children with acute leukemia at different stages of their disease; and (2) the effect of interleukin-2 (IL-2) in enhancing NK activity of these patients' cells. The mean percentage of Leu 11+ NK cells in patients at diagnosis (5% of peripheral blood (PB) mononuclear cells) was significantly lower than for patients on maintenance (23%), post-treatment (21%) and for normal children (20%). The mean PB NK cell cytotoxicity for patients at diagnosis (16% lysis versus K562) and during maintenance (20%) was significantly lower than for post-treatment (41%) and normal controls (40%). After NK cells were incubated for 5 days with IL-2, NK cells from 82% (36/44) of patients showed enhanced cytotoxicity toward K562 and several acute leukemia cell lines as well as toward autologous leukemic cells. Cytotoxicity toward autologous cells was very low (0% to 5%, 16 hour assay) before IL-2 stimulation, and significantly increased (23% to 69%) after stimulation, suggesting that IL-2 may be a useful agent for enhancing the antileukemic immune response.     

Natural killer cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory molecule-1-poliovirus receptor interaction

In the present study, we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor-ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts, similar to neuroblastoma cell lines, involves NKp46 and NKp30 (natural cytotoxicity receptors). However, freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover, a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably, susceptibility to lysis directly correlated with the surface expression, on neuroblasts, of poliovirus receptor [PVR (CD155)], a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed, PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover, monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus, assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing.     

Characterization of two novel cell lines, DERL-2 (CD56+/CD3+/Tcry5+) and DERL-7 (CD56+/CD3-/TCRgammadelta-), derived from a single patient with CD56+ non-Hodgkin's lymphoma.

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.