Two randomised phase II trials of subcutaneous interleukin-2 and histamine dihydrochloride in patients with metastatic renal cell carcinoma

Histamine inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). Two randomised phase II trials of IL-2 with or without histamine dihydrochloride (HDC) in patients with metastatic renal cell carcinoma (mRCC) were run in parallel. A total of 41 patients were included in Manchester, UK and 63 in Aarhus, Denmark. The self-administered, outpatient regimen included IL-2 as a fixed dose, 18 MIU s.c. once daily, 5 days per week for 3 weeks followed by 2 weeks rest. Histamine dihydrochloride was added twice daily, 1.0 mg s.c., concomitantly with IL-2. A maximum of four cycles were given. The Danish study showed a statistically significant 1-year survival benefit (76 vs 47%, P = 0.03), a trend towards benefit in both median survival (18.3 vs 11.4 months, P = 0.07), time to PD (4.5 vs 2.2 months, P = 0.13) and clinical benefit (CR + PR + SD) (58 vs 37%, P = 0.10) in favour of IL-2/HDC, whereas the UK study was negative for all end points. Only three patients had grade 4 toxicity; however, two were fatal. A randomised phase III trial is warranted to clarify the potential role of adding histamine to IL-2 in mRCC.     

Histamine: a novel approach to cancer immunotherapy

The functions of intratumoral lymphocytes in many human malignant tumors are inhibited by reactive oxygen species (ROS), generated by adjacent monocytes/macrophages (MO). In vitro data suggest that immunotherapeutic cytokines such as interleukin-2 (IL-2) or interferon-alpha (IFN-alpha) only weakly activate T cells or natural killer (NK) cells in a reconstituted environment of oxidative stress and that inhibitors of the formation of ROS or scavengers of ROS synergize with IL-2 and IFN-alpha to activate T cells and NK cells. In this review, we focus on the immunoenhancing properties of histamine, a biogenic amine. Histamine inhibits ROS formation in MO via H2-receptors; thereby, histamine protects NK cells from MO-mediated inhibition and synergizes with IL-2 and IFN-alpha to induce killing of NK cell-sensitive human tumor cells in vitro. Histamine also optimizes cytokine-induced activation of several subsets of T cells by affording protection against MO-inflicted oxidative inhibition. The putative clinical benefit of histamine as an adjunct to immunotherapy with IL-2 and/or IFN-alpha is currently evaluated in clinical trials in metastatic malignant melanoma and acute myelogenous leukemia.     

NK cell expression of natural cytotoxicity receptors may determine relapse risk in older AML patients undergoing immunotherapy for remission maintenance

In a phase IV trial, eighty-four patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin-2 (IL-2) to prevent relapse in the post-consolidation phase. Aspects of natural killer (NK) cell biology were analyzed before and during immunotherapy with focus on outcome in older patients. In younger (<60 years old, n = 37) and older patients (>60 years old, n = 47), treatment with HDC/IL-2 resulted in an expansion of CD56^bright and CD16⁺ NK cells in blood along with an increased NK cell expression of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. In older patients, a high expression of NKp30 or NKp46 on CD16⁺ NK cells before and during therapy predicted leukemia-free and overall survival. These results suggest that NK cell functions determine relapse risk and survival in older AML patients and point to biomarkers of efficacy in protocols for remission maintenance.     

Addition of histamine to interleukin 2 treatment augments type 1 T-cell responses in patients with melanoma in vivo: immunologic results from a randomized clinical trial of interleukin 2 with or without histamine (MP 104).

PURPOSE: Preclinical investigations suggest that histamine dihydrochloride (HDC) protects T cells and natural killer cells from inhibition by monocyte-derived reactive oxygen metabolites and synergizes with interleukin (IL) 2 in inducing T-cell activation. Here, we investigate whether this mechanism is operational in patients with melanoma treated with HDC as an adjunct to IL-2. EXPERIMENTAL DESIGN: Melanoma patients having liver metastases were treated with IL-2 with or without HDC within a randomized, multicenter, phase III trial. The effect of HDC on type 1 and type 2 T-cell cytokine production was investigated in peripheral blood samples from 19 patients with the use of intracellular cytokine flow cytometry. Melanoma-specific T-cell responses were analyzed in eight HLA-A2-positive patients. RESULTS: Frequencies of CD3+ T cells producing IFN-gamma (type 1 T cells) in response to phorbol myristate acetate/ionomycin increased (median, 1.8-fold) in patients receiving IL-2 plus HDC but not in those receiving IL-2 alone (P < 0.01 for comparison between arms). In contrast, the number of IL-13-producing type 2 T cells that increased in patients after treatment with IL-2 was not modulated by HDC. Melanoma- and tyrosinase-specific IFN-gamma and IL-13-producing T cells were detected in two of four HLA-A2-positive patients with melanoma following treatment with HDC + IL-2. CONCLUSIONS: Treatment of patients with stage IV melanoma with HDC in combination with IL-2 increases type 1 T-cell responses and may promote induction of melanoma-specific T cells. These effects are of relevance for tumor immunotherapy and provide a potential mechanism for the clinical efficacy of HDC added to IL-2.     

Cellular immune profile of patients with advanced cancer before and after taxane treatment

The purpose of this study is to determine immune recovery and function after treatment with docetaxel or paclitaxel. Peripheral blood mononuclear cells were harvested before chemotherapy and at weekly times afterwards for cycle 1. Leukocyte subsets ICD45hiCD14lo polymorphonuclear neutrophils, CD45hiCD14hi monocytes, CD45hiCD14- lymphocytes, CD3+CD4/CD8+ T cells, CD3-CD19+ B cells, CD3-CD16/CD56+ natural killer (NK) cells], and circulating cytokine levels [tumor necrosis factor-alpha, gamma-interferon (gamma-IFN), and interleukins (IL-2, IL-10, IL-12)] were followed. In addition, T-cell mitogenic function, NK function, and lymphokine activated killer (LAK) function was assessed. Ten patients were entered in the trial. T-cell frequency, B-cell frequency, and CD4/CD8 ratio did not change. IL-10 serum levels significantly decreased in paclitaxel-treated patients (4.4+/-1.3 pg/ml at week 4 versus 7.8+/-2.1 pg/ml at baseline; p < 0.05). IL-2, IL-12, and gamma-IFN levels were not detectable. NK cytotoxic activity decreased in docetaxel-treated patients. LAK cell activity was not altered. Four patients achieved a partial or complete response. They demonstrated higher than normal CD4:CD8 T-cell ratios and an improved phytohemagglutinin stimulation index (SI = 2.5). In conclusion, our findings suggest that immune function was affected more significantly after docetaxel treatment. Investigational approaches, which enhance cellular immunity, may be of greater relevance after treatment with docetaxel. Additional studies monitoring NK function after chemotherapy are recommended.     

Intratumoural and peripheral blood lymphocyte subsets in patients with metastatic renal cell carcinoma undergoing interleukin-2 based immunotherapy: association to objective response and survival

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-alpha and histamine. A total of 250 blood samples and 62 core needle biopsies from 23 and 19 of these patients, respectively, were analysed. After 2 weeks of treatment, a significant positive correlation between absolute number of peripheral blood lymphocytes (P=0.028), CD3 (P=0.017), CD57 (P=0.041) and objective response was demonstrated. There was no correlation between any peripheral blood leukocyte subsets and survival. Cytotoxicity of peripheral blood mononuclear cells was not correlated to objective response or survival. Within the tumour tissue at baseline, a significant positive correlation between CD4 (P=0.027), CD8 (P=0.028), CD57 (P=0.007) and objective response was demonstrated. After one month of immunotherapy, a significant positive correlation between intratumoral CD3 (P=0.026), CD8 (P=0.015), CD57 (P=0.009) and objective response was demonstrated. A significant positive correlation between intratumoral baseline CD4 (P=0.047), baseline CD57 (P=0.035), CD3 at one month (P=0.049) and survival was demonstrated. These data provide novel in vivo evidence of the possible contribution of lymphocyte subsets in the tumour reduction in responding patients during interleukin-2 based immunotherapy. Confirmation of the results requires further studies including a larger number of patients.     

IL-12 or IL-15, unlike IL-2, does not interact with histamine in augmenting cytotoxicity of splenocytes against melanoma cells and YAC-1 cells

It has been suggested that histamine by its ability to downregulate the production of macrophage-derived reactive oxygen species might effectively potentiate the cytotoxic activity of cytokine-stimulated NK cells. Histamine thus reverses negative regulation of NK cells treated with IL-2 and IFN-alpha in the presence of macrophages. We confirm that histamine potently enhances cytotoxic activity of IL-2-stimulated NK cell-enriched splenocytes admixed with macrophages against B16F10 melanoma cells and YAC-1 cells. This stimulation results in production of high amounts of INF-gamma and TNF-alpha. Interestingly, IL-15 by itself promotes production of reactive oxygen species. Although histamine decreased reactive oxygen species production from the cultures of IL-15-stimulated NK cell-enriched splenocytes admixed with macrophages, it did not potentiate the cytotoxicity of IL-15. Further, we demonstrate that histamine-mediated potentiation of cytotoxicity is not applicable to IL-12, another potent activator of NK cell activity.     

Lymphocyte subpopulations and interleukin levels in high-risk melanoma patients treated with high-dose interferon A-2B

Immunologic effects of high-dose interferon are still unclear. We have evaluated changes in blood lymphocyte subpopulations, immunoglobulins, and multiple interleukin in patients with high-risk cutaneous melanoma on adjuvant treatment with high-dose interferon and compared pretreatment values with normal controls. Samples were obtained before treatment, 1 month after induction treatment and at 3, 6, and 12 months of maintenance treatment from 24 patients with high-risk melanoma. Lymphocyte subpopulations were measured by flow cytometry and interleukin and immunoglobulin levels by radioimmunoassay. A statistically significant reduction in B-lymphocytes (p < 0.001), natural killer (NK) cells (p = 0.0004), and monocytes (p = 0.04), and an elevation in CD4/CD8 ratio (p < 0.0001) was observed after 1 month of intravenous interferon. No changes were seen in CD3, CD4, and CD8 lymphocytes. No changes in interleukin (IL)-2, -4, or -5 were observed during 1 year of treatment. IL-2 pretreatment levels were significantly lower than healthy blood donors (p = 0.001), and IL-5 pretreatment levels were significantly higher (p = 0.0056). IL-10 levels significantly dropped after 6 months of treatment (p = 0.01). Immunoglobulins (IgG, IgA, IgM) remained within normal ranges. Three patients had elevated pretreatment levels of IgE. There is a time- and dose-dependent impact of interferon on numbers of circulating B lymphocytes, NK cells, monocytes, and CD4/CD8 ratio. Defects in cellular and humoral immunity are suggested by the low IL-2 and high IL-5 levels, measured in patients with melanoma as compared with healthy controls.     

Antitumor effector mechanism of interleukin-1 beta at a distant site in the double grafted tumor system.

Recombinant human interleukin-1 beta (IL-1 beta) inhibited the growth of not only the right, but also the left non-treated tumor in a double grafted tumor system. Since the antitumor activity of IL-1 beta against the right and left tumors was not seen in nude mice, lymphocytes have a key role in the antitumor effect of intratumoral administration of IL-1 beta. TIL (tumor-infiltrating leukocytes) obtained from left and right side tumors treated with IL-1 beta were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from the right side clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. Spleen cells and right and left regional lymph node cells prepared from IL-1-treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes. The number of Lyt-1-positive lymphocytes increased in the spleen and in the right regional lymph nodes after intratumoral administration of IL-1. Isolated tumor cells obtained from the right tumor treated with IL-1 beta and the left side tumor on day 6 were cultured in RPMI 1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor and macrophage chemotactic factor activities were detected in the culture media from IL-1-treated tumor tissues cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated tumor tissues. These results suggest that intratumoral administration of IL-1 first induces neutrophils and macrophages in the right tumor, then Lyt-1-positive cells in the right regional lymph nodes and in the spleen, and subsequently induces macrophages in the left, non-treated tumor.     

Adoptive immunotherapy of solid tumors with activated macrophages: experimental and clinical results

Adoptive immunotherapy in cancer has been essentially restricted to the use of lymphoid effector cells (NK, TIL, LAK) stimulated with IL-2. Differentiated macrophages represent another key effector population even more important for the immune control of cancer. We have shown that activated murine macrophages reduced primary tumors and experimental metastases. Human macrophages differentiated from circulating monocytes and activated with IFN gamma (MAK) were cytotoxic in vitro for a variety of tumor cell and caused regression of human tumors implanted in nude mice. A large scale technology has been developed for the generation of antitumor macrophages. These MAK cells (10(8) to 10(9] were injected in cancer patients in pilot clinical trials and were well tolerated. MAK treatment is technically feasible, clinically safe and presents several advantages compared to other immunotherapies.     

Effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer activity

The enhancing effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer (LAK) activity was investigated in this study. Peritoneal macrophages significantly enhanced LAK activity generated from accessory cell-depleted splenic lymphocytes in both IL-2 and IL-4 cultures. This effect was dependent on the number of macrophages and was not replaced by a factor derived from macrophages or lymphocytes. Macrophages enhanced IL-2- and IL-4-induced LAK activity against both natural killer (NK)-sensitive (YAC-I, P388D1) and NK-resistant (P815) tumor cells. Negative selection of cells with antibodies and complement showed no differences in surface markers between IL-2 LAK effectors and IL-4 LAK effectors generated in the presence of macrophages. These results suggest that the same LAK effector subsets can be enhanced by macrophages in either IL-2 or IL-4 cultures.     

Enhancement of IL-2-induced cytotoxicity by interferon-alpha in renal cell carcinoma

Metastatic renal cell carcinoma (mRCC) treatment consists of molecular targeted agents and cytokines that have fundamentally different mechanisms of action. Clinical responses also differ; complete response is rare with molecular targeted agents but is sometimes achieved with cytokine therapies. Because of the relatively high efficacy of combination therapy with low-dose interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) against mRCC, it is important to reevaluate cytokine therapies in vitro. Here, we show that when IL-2 is administered in combination with IFN-alpha, a stronger cytotoxic effect of PBMCs on RCC cell lines is observed than when IL-2 is administered alone. The upregulation of TNF-related apoptosis-inducing ligand on NK cell by IL-2 and suppression of regulatory T cells (Tregs) by IFN-alpha were recognized at the same time when cytotoxicity of peripheral blood mononuclear cells (PBMCs) was enhanced. IL-2 is known to activate natural killer cell cytotoxicity; however, IL-2 also stimulates Treg expansion, which enhances immunosuppression. On the other hand, IFN-alpha negatively regulates Treg cells, thereby increasing the function of immune effector cells. Our in vitro results may explain, at least in part, the clinical efficacy of combination low-dose IL-2 and IFN-alpha therapy against mRCC.     

Human Alveolar Macrophages Augment Natural Killer Cell Stimulatory Factor (Interleukin-12)-inducible Killer Activity from Autologous Blood Lymphocytes

Interleukin-12 (IL-12), also known as natural killer cell stimulatory factor (NKSF), was found to induce cytotoxic activity from human blood T cells and NK cells. The present study was undertaken to examine the effect of human alveolar macrophages (AM) on induction by IL-12 cytotoxic cells from blood lymphocytes. AM were obtained by bronchoalveolar lavage from healthy donors. Highly purified lymphocytes (>99%) and monocytes (>90%) were also isolated by centrifugal elutriation from peripheral blood of the same donors. Cytotoxicity of lymphocytes was measured by 4-h ⁵¹Cr release assay. IL-12 stimulated blood lymphocytes to produce interferon γ (IFNγ) and tumor necrosis factor α (TNFα), and this effect was augmented by co-cultivation with monocytes or AM. AM-upregulated induction of cytotoxic lymphocytes was stimulated with IL-12, and this effect was significantly abrogated by addition of antibodies against IFNγ and TNFα. Induction by IL-12 of IFNγ production and cytotoxic activity of CD8⁺ cells was also augmented by co-cultivation with monocytes or AM. AM were more effective than monocytes in augmenting the cytotoxic activity of IL-12-stimulated lymphocytes and CD8⁺ cells. These observations suggest that in situ induction of IL-12-stimulated cytotoxic cells in the lung may be regulated by complex cytokine networks, depending on participation of monocytes and alveolar macrophages.     

Antitumor Effector Mechanism of Interleukin-lβ at a Distant Site in the Double Grafted Tumor System

Recombinant human interleukin-lβ (IL-lβ) Inhibited the growth of not only the right, but also the left non-treated tumor in a double grafted tumor system. Since the antitumor activity of IL-lβ against the right and left tumors was not seen in nude mice, lymphocytes have a key role in the antitumor effect of intratumoral administration of IL-lβ. TIL (tumor-infiltrating leukocytes) obtained from left and right side tumors treated with IL-1β were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from the right side clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. Spleen cells and right and left regional lymph node cells prepared from IL-1-treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes. The number of Lyt-1-positive lymphocytes increased in the spleen and in the right regional lymph nodes after intratumoral administration of IL-1. Isolated tumor cells obtained from the right tumor treated with IL-lβ and the left side tumor on day 6 were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor and macrophage chemotactic factor activities were detected in the culture media from IL-1-treated tumor tissues cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated tumor tissues. These results suggest that intratumoral administration of IL-1 first induces neutrophils and macrophages in the right tumor, then Lyt-1-positive cells in the right regional lymph nodes and in the spleen, and subsequently induces macrophages in the left, non-treated tumor.     

乌司他丁对食管癌手术患者CD14+肺泡巨噬细胞及免疫功能的影响

目的:探讨术前静脉输注乌司他丁对食管癌手术患者单肺通气时CD14+肺泡巨噬细胞、局部及全身炎性反应和免疫功能的影响。方法:前瞻性选取南京医科大学附属肿瘤医院2019年10月至2020年8月行胸腔镜下食管癌根治手术患者80例,数字随机表法分为两组:对照组和乌司他丁组各40例。乌司他丁组患者麻醉诱导前静脉输注20 KU乌司他丁,对照组给予生理盐水。检测两组患者术前和术后2 h外周静脉中血白介素-2（IL-2）和免疫细胞,并检测术中单肺通气1 h后非通气侧肺支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中CD14+巨噬细胞、IL-2、免疫细胞。结果:两组患者一般情况比较无差异。术后2 h乌司他丁组患者外周血中IL-2、CD14+单核细胞增高幅度小于对照组,差异有统计学意义（P＜0.05）,NK细胞、CD4+T细胞、CD8+T细胞及活化的T细胞亚群增高幅度高于对照组,差异有统计学意义（P＜0.05）。两组患者术前外周血IL-2、CD14+单核细胞及主要免疫细胞亚群差异无统计学意义（P>0.05）。与对照组相比,乌司他丁组患者单肺通气1 h后非通气侧肺BALF中IL-2和CD14+巨噬细胞增高幅度明显小于对照组,差异有统计学意义（P＜0.05）,而CD4+T细胞、CD8+T细胞高于对照组,差异有统计学意义（P＜0.05）,活化的T细胞也高于对照组,差异有统计学意义（P＜0.01）。结论:麻醉前给予乌司他丁能减少食管癌术中单肺通气时全身及肺部的炎性反应,保护患者的免疫功能,具有临床应用价值。     

Factors influencing haematological recovery following high-dose chemotherapy and peripheral stem-cell transplantation for haematological malignancies; 1-year analysis

Peripheral blood counts and factors influencing haematological recovery in 98 patients with a relapse-free survival of > or =1 year treated with high-dose chemotherapy (HDC) and peripheral stem-cell transplantation (PSCT) for haematological malignancies were analysed. One year after PSCT full haematological recovery was demonstrated for haemoglobin (Hb) in 47% of patients, for the white blood cell count (WBC) in 94%, and for platelets in 64%; 39% had a trilineage recovery. In the multivariate analysis, recovery was influenced by age (P=0.002), number of reinfused CD34+ cells (P=0.016), Hb at start of HDC (P=0.001), and platelets at start of HDC (P=0.008). One year following PSCT, 61% of patients still have subnormal values in one or more haematopoietic cell lineage, suggesting a limited bone-marrow reserve. Long-term recovery is highly dependent on age, blood counts at start of HDC and number of reinfused CD34+ cells without a threshold, all reflecting the residual function of bone marrow before HDC. Reinfusing more CD34+ cells can accelerate long-term haematological recovery.     

Outpatient treatment with subcutaneous histamine dihydrochloride in combination with interleukin-2 and interferon-alpha in patients with metastatic renal cell carcinoma: results of an open single-armed multicentre phase II study.

OBJECTIVES: Histamine inhibits formation and release of monocyte/macrophage-derived reactive oxygen metabolites and thereby protects natural killer (NK) and T cells against oxidative inhibition. Efficacy and safety of histamine, when given in combination with interleukin-2 (IL-2) and interferon-alpha (IFN-alpha), were evaluated in patients with metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Forty-eight mRCC patients were included. The self-administered, outpatient regimen included IFN-alpha, 3 MIU s.c., once daily for 1 week, followed by up to nine 4 week cycles of IFN-alpha, 3 MIU s.c., days 1-7, weeks 1-4; interleukin-2, 2.4 MIU/m2 s.c., b.i.d., days 1-5, weeks 1 and 2; and histamine dihydrochloride, 1 mg s.c., b.i.d. days 1-5, weeks 1-4. RESULTS: Forty-six patients were eligible. Forty-two patients were evaluable for response with four partial responses (9% of eligible patients, 10% of evaluable patients). Fifteen patients (36%) had stable disease. After subsequent surgery of residual tumours, three patients (7%) had no evidence of disease at 14+, 21+ and 21+ months. Median survival time for all patients was 16.3 months. One grade 4 toxicity (thrombocytopenia) was observed. Most frequent grade 3 toxicities were fatigue/malaise (26%), dyspnoe (11%), nausea (9%) and stomatitis (9%). Four patients discontinued due to treatment-related toxicity. There were no treatment-related deaths. CONCLUSIONS: The present combination of histamine with IL-2 and IFN-alpha. as self-administered outpatient therapy is a safe and well-tolerated regimen. However, histamine does not appear to add efficacy with respect to response in this low-dose schedule of IL-2 and IFN-alpha. Whether histamine might improve efficacy with higher doses of IL-2 and IFN-alpha requires further investigation.     

Adjuvant histamine in cancer immunotherapy

Interleukin-2 (IL-2) is an effective activator of lymphocytes with anti-neoplastic properties such as T-cells or natural killer cells, and this property of IL-2 has formed the basis for its widespread used as an immunotherapeutic agent in human neoplastic disease. In recent years, IL-2 therapy for solid neoplastic diseases and hematopoietic cancers has been supplemented with histamine dihydrochloride with the aim of counteracting immunosuppressive signals from monocytes/macrophages. Here we review the preclinical basis for the use of histamine as an adjunct to IL-2 in cancer immunotherapy.     

Heterogeneity of human lymphokine (IL-2)-activated killer (LAK) precursors and regulation of their LAK induction by blood monocytes

Highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated by CCE from peripheral blood of healthy donors. Blood lymphocytes were separated by this CCE into 9 subpopulations. The NK activities of these lymphocyte fractions against NK-sensitive K-562 cells and their LAK activities against NK cell-resistant target (Daudi) cells were assayed promptly or after incubation of the fractions for 4 days with or without an optimal concentration of IL-2. NK and LAK activities were measured by 4-hr 51Cr-release assay. On the basis of their NK and LAK activities, these lymphocyte fractions were classified into 3 subpopulations of LAK precursors: one lacking both NK and LAK activities (Fr.2), one with moderate NK activity but low LAK activity (Fr.5), and one possessing both NK and LAK activities (Fr.8). Addition of autologous fresh monocytes to the lymphocyte cultures resulted in a significant increase in induction of LAK activity in Fr.2 and Fr.5. This up-regulation of lymphocytes in Fr. 2 and Fr.5 by monocytes was confirmed in parallel experiments by measuring the blastogenic response of the lymphocytes to IL-2. Deletion of lymphocytes in Fr. 8 of CD16+ (Leu-11+) NK cells resulted in 74% reduction in LAK induction, whereas depletion of mixtures of monocytes and lymphocytes in Fr. 2 of cells reacting with CD3+ (OKT3+) antibody resulted in a 66% reduction in LAK induction. This up-regulation of LAK cell induction from LAK precursors by monocytes was confirmed using 4 lines of human lung cancer cells as targets for LAK activity. These results clearly indicate that human monocytes may cause up-regulation of the expression of IL-2-induced LAK activity in T cells and in a subpopulation of NK cells.