In vivo boosting of lung natural killer and lymphokine-activated killer cell activity by interleukin-2: comparison of systemic, intrapleural and inhalation routes.

Natural killer (NK) cells are thought to play a role in host defence against malignancy and infection, in immunoregulation and as precursor cells in a generation of lymphokine-activated killer (LAK) cells which can lyse NK-resistant tumour cells. As the lung is a major site for malignancy and infection and as there are large numbers of lymphoid cells including NK cells in the interstitial compartment of the lung, we evaluated the capacity of interleukin-2 (IL-2), a lymphokine capable of augmenting NK activity in vitro, to augment lung NK cell activity in vivo, using different routes of IL-2 administration. We compared both systemic (i.v. and i.p.) and local (intrapleural and inhalation) routes of IL-2 administration (50,000 U/daily for 5 days) using CBA mice, assessing NK and LAK cell activity in the spleen (systemic) and in the lung. The target cells used for these studies were the YAC-1 (NK-sensitive) and P815, NO36 and HA56 (NK-resistant, LAK-sensitive) cell lines. Splenic NK activity was increased by 1.4-1.9-fold for i.v./i.p., respectively, compared with controls with both systemic routes of administration, and lung NK activity was increased 3.2-fold and 3.8-fold (i.v./i.p, respectively, P less than 0.05), to levels which were comparable to systemic (splenic) NK activity following the same therapy. Intrapleural IL-2 administration similarly enhanced lung NK activity (3.3-fold) and splenic NK activity (1.3-fold; P less than 0.05 versus controls for both). Surprisingly, inhaled IL-2 suppressed both splenic and lung NK cell activity (84 +/- 8% and 78 +/- 10% suppression, respectively, P less than 0.05). LAK cell activity was also enhanced in the lung by 1.8-8-fold in response to i.v., i.p. and intrapleural IL-2, whereas inhaled IL-2 was ineffective in generating LAK cell activity. These results suggest that the systemic and intrapleural administration of IL-2 effectively boost pulmonary NK and LAK activity whereas inhalation of IL-2 does not. Thus, in clinical situations where boosting of local lung NK or LAK cell activity is desired, these routes of IL-2 administration may be effective.     

Combined effect of cisplatin and lymphokine-activated killer cells on A549 cells of non-small cell lung cancer

We studied the ability of lymphokine-activated killer cells to lyse A549 human non-small cell lung cancer cells after preincubation with cisplatin. Lymphokine-activated killer cells obtained after incubation of human blood lymphocytes with interleukin-2 were characterized by high expression of natural killer cell antigens and activation molecules. Lymphokine-activated killer cells produced potent cytotoxic effect on intact A549 cells and lysed tumor cells survived after treatment with cisplatin in concentrations of IC₅₀ and IC₃₀. Cisplatin in noncytotoxic concentrations did not increase lytic activity of lymphokine-activated killer cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 198–201, August, 2007     

Appearance of neuropeptides in ascitic fluid after peritoneal therapy with interleukin-2 and lymphokine-activated killer cells for intraabdominal malignancy

Administration of intravenous interleukin-2 (IL-2), followed by intraperitoneal IL-2 and autologous lymphokine-activated killer (LAK) cells to six patients with colonic, ovarian, or endometrial carcinoma restricted to peritoneal spread increased significantly the ascitic fluid concentrations of the neuropeptides substance P (SP) and calcitonin-gene related peptide (CGRP). After intravenous IL-2 alone, the level of SP rose 10- to 140-fold, without a change in that of CGRP. Intraperitoneal IL-2 and LAK cells led to elevations in the concentrations of SP and CGRP to respective maximal means of 319 and 175 pM after 8 hr, which were maintained for 24–48 hr without alterations in the levels of vasoactive intestinal peptide or somatostatin. SP and CGRP from peritoneal fluid were chromatographically indistinguishable from synthetic neuropeptides. The increases in concentrations of SP and CGRP after IL-2 and LAK-cell therapy are the first demonstration of a neural response to a human cellular immunological reaction. The time course and magnitude of the neuropeptide response suggest a role in the vascular side effects of this form of treatment.     

Functional activity of natural killer cells and killer T cells in mice after ovarian transplantation

Tashkent Postgraduate Medical Institute, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Lopatkin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 9, pp. 273–275, September, 1991.     

Transcatheter arterial injection of autologous lymphokine-activated killer (LAK) cells into patients with liver cancers

We studied the effect of transcatheter arterial injection of lymphokine-activated killer (LAK) cells in conjunction with interleukin-2 on 13 patients with liver cancers, 9 primary hepatocellular carcinomas (HCC), and 4 metastatic liver cancers. Based on the fact that the LAK-cell phenomenon was observed to exist equally in these patients as in healthy controls, an injection of autologous LAK cells into these patients was performed. Of 13 patients receiving LAK cells plus interleukin-2, one had a partial response and three had minor responses. The response duration of this partial-response patient was around 6 months after the LAK-cell injection. Simultaneously, a reduction of serum AFP level was found in six of eight HCC patients. One patient having a minor response after receiving this therapy had severe side effects, such as dyspnea and hypotension. The others had only a transient fever which was easy to control by medication. Thus the present study indicates that a measurable tumor regression and a reduction of serum AFP level are observed in some liver cancers after undergoing this therapy.     

Whole-body hyperthermia decreases lung metastases in lung tumor-bearing mice,possibly via a mechanism involving natural killer cells

The effects of whole-body hyperthermia (WBH) on the course of the Lewis lung carcinoma (LLC) and B16 melanoma (B16) were examined. WBH was generated by microwave (2450 MHz) at an intraperitoneal temperature of 39.5–40.0°C and an intratumoral temperature of 40.0–40.5°C for 30 min once a week, ×3 (LLC) or ×6 (B16). The mice were sacrificed 21 days (LLC) or 42 days (B16) after tumor implantation and lung metastases were scored. Natural killer (NK)-cell activity was determined against the YAC-1 tumor target in WBH-treated tumorbearing mice as well as in tumor-bearing mice but untreated controls. The number of lung metastases was significantly reduced and NK-cell activity was higher in animals treated with WBH. Thus, this study suggests that WBH interferes with the spread of organ metastases, possibly through a mechanism involving NK cells.     

Optimal culture conditions for the generation of natural killer cell-induced dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines continue to be considered an attractive tool for cancer immunotherapy. DCs require an additional signal from the environment or other immune cells to polarize the development of immune responses toward T helper 1 (Th1) or Th2 responses. DCs play a role in natural killer (NK) cell activation, and NK cells are also able to activate and induce the maturation of DCs. We investigated the types of NK cells that can induce the maturation and enhanced function of DCs and the conditions under which these interactions occur. DCs that were activated by resting NK cells in the presence of inflammatory cytokines exhibited increased expression of several costimulatory molecules and an enhanced ability to produce IL-12p70. NK cell-stimulated DCs potently induced Th1 polarization and exhibited the ability to generate tumor antigen-specific cytotoxic T lymphocyte responses. Our data demonstrate that functional DCs can be generated by coculturing immature DCs with freshly isolated resting NK cells in the presence of Toll-like receptor agonists and proinflammatory cytokines and that the resulting DCs effectively present antigens to induce tumor-specific T-cell responses, which suggests that these cells may be useful for cancer immunotherapy.     

Asbestos fibres inhibit the in vitro activity of lymphokine-activated killer (LAK) cells from healthy individuals and patients with malignant mesothelioma.

Asbestos exposure is associated with an increased incidence of several malignancies, including malignant mesothelioma (MM). This study evaluates the relationship between asbestos exposure and the in vitro generation and function of LAK cells, an immune effector cell population with powerful lytic activity against MM cells. Both serpentine (chrysotile) and amphibole (amosite and crocidolite) forms of asbestos fibres suppress LAK cell generation, viability (by 5-11%, P less than 0.02) and cell recovery (by 13-15%, P less than 0.02). However, the LAK cells generated in the presence of the amphiboles were as effective as unexposed cells in lysing both standard tumour cell targets (K562, 56.4% lysis versus 61.5%, respectively, P greater than 0.5; NS; Daudi, 60.5% lysis versus 64.5% P greater than 0.5; NS), and MM tumour cell targets (mean of three MM cell lines 48.3% versus 46.3%, P greater than 0.5; NS), whereas the function of LAK cells generated in the presence of chrysotile was significantly reduced against three out of the five tumour cell targets tested (P less than 0.03). In the presence of asbestos fibres, LAK cell function was reduced against all five tumour cell targets (P less than 0.01), irrespective of whether the cell donors were healthy individuals or patients with MM. NK cell activity was also suppressed (P less than 0.01). The serpentine form of asbestos, chrysotile, was significantly more suppressive of both effector cell functions than either of the amphiboles (P less than 0.01). These findings suggest that asbestos exposure may suppress the function and in some instances the generation of immune effector cell mechanisms, thereby increasing the risk of disease and malignancy.     

Expression of natural killer cell activating receptors in patients with chronic lymphocytic leukaemia

Recent advances in chronic lymphocytic leukaemia (CLL) treatment, more particularly through upfront use of anti-CD20 monoclonal antibodies, have prolonged patient progression-free survival. Nonetheless, apart from allogeneic stem cell transplantation, no curative treatment is available. One possible explanation for the lack of cure in CLL could be a defective immune anti-tumour response. As the result of abnormal HLA class I molecule expression, CLL cells escape from specific T-lymphocyte immunity but should be the target for the innate natural killer (NK) cell-mediated immune response. Defective NK cytotoxicity as the result of decreased expression of the natural cytotoxicity receptors (NCRs) NKp30/NCR3, NKp44/NCR2 and NKp46/NCR1 has been described in haematological malignancies such as acute myeloid leukaemia. This prompted us to focus our attention on NCR expression on NK cells from patients with CLL. Although we failed to detect any difference between CLL patients and healthy age-matched controls, a precise analysis of clinical data showed a correlation between decreased NCR expression and poor prognosis factors such as low haemoglobin level, high (>30×10(9) per litre) lymphocyte count or elevated C-reactive protein. Together, these observations support the rationale for restoration of normal NK cell functions in patients with CLL, putatively through the use of immune therapy protocols that already have demonstrated some benefit in acute myeloid leukaemia such as interleukin-2 plus histamine dihydrochloride.     

Comparison of suppressor and cytotoxic activity of blood mononuclears during adaptive immunotherapy of cancer patients with lymphokine-activated killer cells with a low dose of recombinant interleukin-2

Laboratory of Cellular Immunity, Department of Blood Transfusion, All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 519–521, November, 1991.     

Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.     

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.     

Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells

We previously demonstrated a correlation between the frequency of CX3CR1-expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1(high) NK cells were more cytotoxic than their CX3CR1(neg/low) counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1(high), CX3CR1(neg/low) NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin-2 and promoted a strong up-regulation of the key co-stimulatory molecule CD40 on monocytes. Co-expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1(neg) CD56(bright), CX3CR1(neg) CD56(dim) and CX3CR1(high) CD56(dim) fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56(dim) discriminated between an intermediary CX3CR1(neg) CD56(dim) and fully mature CX3CR1(high) CD56(dim) NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.     

Peri-operative modulation of cellular immunity in patients with colorectal cancer.

The peri-operative cellular immune response is depressed in patients with gastrointestinal cancer, a factor which may facilitate malignant dissemination. We have investigated the effects of peri-operative rIL-2 and a combination of rIL-2 and interferon-alpha (IFN-alpha) on both peripheral blood lymphocyte function and number in patients undergoing surgical resection for colorectal cancer. Fifty-two patients were randomly allocated to either control, rIL-2 or rIL-2 with IFN-alpha treatment arms. In vitro studies were performed pre-operatively and on post-operative days 1, 4, 7 and 10. Natural killer (NK) and lymphokine-activated killer (LAK) cell function were profoundly depressed in control patients (P < 0.001; P < 0.01), an effect abrogated in both treatment groups; indeed NK function was augmented in the rIL-2 and IFN-alpha group on the first post-operative day in association with an increase in the percentage of cells expressing CD16 and CD56 (P < 0.01). Flow cytometric analysis of lymphocyte subsets in the control group was unremarkable, except for an early post-operative fall in numbers of lymphocytes. Treatment with either rIL-2 or rIL-2 and IFN-alpha produced an initial profound reduction in T lymphocyte numbers, followed by a 'rebound' lymphocytosis of activated CD3+ T cells, as demonstrated by a significant increase in co-expression of CD25, CD38 and CD45RO. No significant differences were observed between either of the treatment groups. Adjuvant immunotherapy affects peri-operative anti-tumour immune responses, and this may influence long term outcome in patients undergoing surgery for gastrointestinal cancer.     

Increased killer immunoglobulin-like receptor expression and functional defects in natural killer cells in lung cancer

Frequencies of natural killer (NK) cells from patients with non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) did not differ from healthy controls. A higher proportion of NK cells from NSCLC patients expressed the killer immunoglobulin-like receptor (KIR) CD158b than in controls (P = 0.0004), in the presence or absence of its ligand, HLA-C1. A similar result was obtained for CD158e in the presence of its ligand HLA-Bw4 in NSCLC patients (P = 0.003); this was entirely attributable to the Bw4I group of alleles in the presence of which a fivefold higher percentage of CD158e(+) NK cells was found in NSCLC patients than controls. Proportions of CD158b(+) NK cells declined with advancing disease in NSCLC patients. Expression of NKp46, CD25 and perforin A, and production of interferon-γ following stimulation with interleukin-12 and interleukin-18, were all significantly lower in NK cells from NSCLC patients than in controls. Both NK cell cytotoxicity and granzyme B expression were also reduced in lung cancer patients. Increased inhibitory KIR expression would decrease NK cell cytotoxic function against tumour cells retaining class I HLA expression. Furthermore, the reduced ability to produce interferon-γ would restrict the ability of NK cells to stimulate T-cell responses in patients with lung cancer.     

Activity of human natural killer cells against target cells differing in sensitivity to interferon

Department of Interferons, N. F. Gameleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110. No. 10, pp. 406–409, October, 1990.     

Diminished interleukin-2 activity production in cancer patients bearing solid tumors and its relationship with natural killer cells

Interleukin-2 (IL-2) activity production by stimulated peripheral blood lymphocytes (PBL) from solid tumor bearing cancer patients was lower than in normal subjects. Natural-killer (NK) cell activity in the PBL of cancer patients was very significantly correlated to IL-2 activity production (P less than 0.001). These results might suggest a central role for IL-2 in the immune dysfunctions in cancer patients, and the possible use of IL-2 as an immunological response modifier in these patients.     

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×10⁵ B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4⁺ cells and CD8⁺ cells apparently played some roles too. This revised version was published online in July 2006 with corrections to the Cover Date.     

树突状细胞诱导的CIK细胞对肺癌生长的抑制作用

目的探讨树突状细胞诱导的CIK细胞对肺癌移植瘤细胞生长的抑制作用。方法建立肺癌皮下移植瘤模型;分离患者外周血单个核细胞,培养成熟的DC及CIK细胞;用反复冻融肺癌组织的方法制备肿瘤细胞裂解物并负载DC,诱导DC-CIK细胞;进行肺癌移植瘤内注射,比较两组裸鼠移植瘤体积及重量,观察其抑瘤情况。结果 DC-CIK细胞对肺癌皮下移植瘤细胞的抑制率为70.3%,抑制作用明显强于DC或CIK细胞的抑制作用(P0.05)。结论树突状细胞诱导的CIK细胞能够有效抑制肺癌移植瘤生长。