Soluble factors produced by macrophages/monocytes inhibit lymphokine-activated killer activity in rat splenocyte cultures

In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanolsensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Mycophenolate mofetil does not suppress the graft-versus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model

Background: Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. Aims: To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. Methods: MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h ³⁵S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c × C57BL/6] F₁ recipients) murine model. Results: A dosage regimen of 28–200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). Conclusions: These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.     

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of⁵¹Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.     

Heatstroke induces a reduction of natural killer cell activity in rats

Experiments were carried out to determine the changes of natural killer (NK) cell activity that occurred during heatstroke in rats pretreated with or without interleukin-1 (IL-1) receptor antagonist (IL-1ra). After the onset of heatstroke, all the splenic NK cell activity, the effector-target cell conjugation, and the NK cell numbers were decreased in rats. Additionally, an increase in the plasma IL-1 level was associated with arterial hypotension, cerebral ischemia and hyperthermia during rat heatstroke. Pretreatment with an IL-1ra reversed in part the heatstroke-induced inhibition of NK cell activity. Thus it appears that the inhibition of NK cell activity produced by activation of IL-1 receptor mechanism is associated with the increased susceptibility to infection that is well described in heatstroke.     

A regimen of surgical adjuvant immunotherapy for cancer with interleukin 2 and lymphokine-activated killer cells

We designed a unique regimen of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin 2 (rIL-2) for application with surgical adjuvant therapy of cancer. The regimen features the prolonged (6 consecutive days) s.c. administration of low-dose rIL-2 and the transfer ofex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. According to this regimen, 5 patients with primary lung cancer received immunotherapy about 2 weeks after surgery (pulmonary lobectomy). Clinical toxicities included fever(5/5), fatigue(5/5), slight(< 5%) weight gain(5/5), increase of pleural effusion at the lobectomy site(2/5), and edema formation(1/5). All toxicities reversed within 4 days after the completion of therapy. Rebound lymphocytosis after therapy ranged from 2.4 to 5.5-fold (mean, 4.3-fold) over the baseline. Peripheral blood lymphocytes obtained during this lymphocytosis exhibitedin vitro LAK activity in 4 of 5 patients. Thus, the regimen is considered to be well-tolerable and immunologically active in regard to the postoperative state of the patients.     

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells. Received: 26 February 1998 / Accepted: 4 August 1998     

The augmentation of lymphokine-activated killer activity following pulsing of human peripheral blood mononuclear cells with recombinant human interleukin-2

Summary The short-term exposure of peripheral blood mononuclear cells (PBMC) to recombinant human interleukin-2 (rhIL-2) at 37°C leads to the generation of lymphokine-activated killer (LAK) activity similar in magnitude to that obtained by the exposure of PBMC to rhIL-2 continuously for 3–5 days. In order to investigate whether the required signal for LAK induction occurred during the short exposure to rhIL-2 or at a later point in the induction phase, PBMC were exposed to rhIL-2 for 1 h at 4°C and then exposed to a low-pH wash to remove bound IL-2 from its receptor. PBMC treated in such a way showed increased LAK activity and proliferation compared to cells exposed to rhIL-2 alone. Expression of the p55 () subunit of the IL-2 receptor was also increased. In order to cause the augmentation, a lowering of the pH below 4.0 was necessary, and exposure of PBMC to low pH alone (in the absence of rhIL-2) failed to cause activation. Another relevant feature was a transient increase in the expression of the p75 subunit of the IL-2 receptor ( chain) immediately following the exposure to low pH and the release of interferon , tumour necrosis factor and IL-6; activation was blocked by the inclusion of neutralising antisera raised against rhIL-2 and interferon , thus demonstrating that the endogenous release of these cytokines is important for activation.     

Antitumor effects of interleukin-2 and mismatched double-stranded RNA,individually and in combination,against a human malignant melanoma xenograft

Summary The antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000–20 000 units) of rIL-2 and mismatched dsRNA (500 µg). The in vivo tumor growth was significantly inhibited by the combined treatments (P <0.05) and survival was significantly increased (P <0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer(NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P <0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P <0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.     

Enhancement of tumor cell susceptibility to lymphokine-activated killer cells by treatment with the streptococcal preparation OK432

We investigated whether tumor cell lysis by LAK cells was augmented by treatment with OK432in vitro. NK and LAK activity against K562 cells was not enhanced by their treatment with OK432. In contrast, the susceptibility of OK432-treated Daudi and KATO-III cells to lysis by LAK cells was enhanced. Succinate dehydrogenase activity and RNA synthesis were impaired in Daudi and KATO-III cells by treatment with OK432, and moreover the expression of HLA Class I antigen and ₂-microglobulin was inhibited in OK432-treated KATO-III cells. Thus, it is suggested that the enhancement of the susceptibility of OK432-treated tumor cells with regard to succinate dehydrogenase activity, RNA synthesis, and HLA Class I antigen expression.     

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.     

自剪切多肽P2A对自然杀伤细胞的高效诱导扩增作用的研究

目的改进现有过继性人自然杀伤（NK）细胞体外诱导、分化及扩增方法,实现高效定向分化及大量扩增NK细胞的目的。 方法利用自剪切多肽P2A连接IL-15和4-1BBL基因,通过慢病毒感染和药物筛选的方法制备稳定表达IL-15和4-1BBL的K562滋养层细胞株。该方法可刺激人PBMC细胞向NK细胞分化及高效增殖。实验结果以 ±s表示并用两样本t检验进行比较。 结果经该方法培养的CD3^- CD16⁺ 56⁺ NK细胞增殖倍数达到（4480.43±37.80）倍;CD3^- CD16⁺ 56⁺ NK细胞纯度达到94.79%;细胞内表达IFN-γ和TNF-α的NK细胞比例为（63.07±3.37）%和（54.85±2.04）%,分别高于对照组（16.28±2.86）%和（14.53±1.15）%（t = 62.25,22.66,P均< 0.01）;细胞分泌至培养上清液中的IFN-γ和TNF-α浓度为（111.39±6.95）?pg/ml和（32.76±3.23） pg/ml,分别高于对照组（44.99±4.74）pg/ml和（11.09±2.45）?pg/ml（t = 20.56,7.21,P均< 0.01）;在体外,该方法培养的NK细胞对K562细胞的杀伤效率为（78.52±7.36）%,高于对照组（48.53±6.66）%（t = 11.56,P < 0.01）;对H520细胞的杀伤效率为（65.03±3.27）%,高于对照组（35.85±3.99）%（t = 11.35,P < 0.01）。 结论利用自剪切多肽P2A构建稳定高表达的IL-15和4-1BBL的K562滋养层细胞株,从而提高了NK细胞增殖倍数、纯度和细胞毒性。     

A new human pancreatic carcinoma cell line developed for adoptive immunotherapy studies with lymphokine-activated killer cells in nude mice

Summary A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P=0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P<0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10⁶ cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4±4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.     

Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Experimental manipulations of afferent immune responses influence efferent immune responses to brain tumors

Tumors grow more readily in the brain than in the periphery, in part due to immune privilege. Differences in both afferent and efferent components of the immune response contribute to this lower level of responsiveness. On the afferent side, despite the lack of lymphatic vessels in the brain, antigens from brain arrive in lymph nodes and spleen by several routes, and the route taken may influence the type of response generated. Work with viruses and soluble antigens in mice has shown that the intracerebral location and the volume of the inoculation influence the strength of the cytotoxic T cell response. We examined whether these factors influence the T cell response against experimental brain tumors in mice. Placement of tumor cells in the cerebral ventricles instead of the parenchyma generated an immune response sufficient to increase survival time. A large volume of an intraparenchymal infusion of tumor cells caused spread of cells to the ventricles, and resulted in longer survival time relative to a small volume infusion. Infusion of the same dose of radiolabeled tumor cells in either a small volume or a large volume allowed tracking of potential tumor antigens to the periphery. Both modes of infusion resulted in similar levels of radioactivity in blood, spleen and kidney. Unexpectedly, cells infused intraparenchymally in a small volume, compared to a large volume, resulted in (1) more radioactivity in cervical lymph nodes (parotid and deep cervical lymph nodes), (2) a greater number of CD11b+/Gr1+ myeloid suppressor cells in the tumors, and (3) fewer CD8+ cells within the tumor mass. Consistent with these observations, providing a stronger afferent stimulus by giving a concurrent subcutaneous injection of the same tumor cells infused into the brain increased CD8+ T cell infiltration of the tumor in the brain. These results suggest that the immune response elicited by antigens that drain predominantly to the cervical lymph nodes may be less effective than responses elicited at other lymph nodes, perhaps due to immunosuppressive cells. Directing therapies to the optimal peripheral sites may improve immune responses against brain tumors.     

Graft-versus-host disease following interleukin-2/lymphokine-activated killer (LAK) cell immunotherapy in a patient with acute myelogenous leukaemia in second complete remission: autologous LAK cells following allogeneic bone marrow transplantation are donor-derived

A 48-year-old man was treated by allogeneic bone marrow transplantation (BMT) in first remission of M4 acute myelogenous leukaemia (AML). He experienced no graft-versus-host disease (GVHD) and 7 months later he relapsed. Following further chemotherapy, he entered a second complete remission; however, he refused a further allogeneic or autologous BMT but agreed to immunotherapy with interleukin-2 and autologous lymphokine-activated killer (LAK) cells. He tolerated this treatment well but went on to develop grade II skin GVHD. Polymerase chain reaction studies of DNA microsatellites of the autologous LAK cells showed that they were of donor origin. The patient remained well for 9 months until, immediately following the introduction of prednisolone for his persistent GVHD, he relapsed. He declined further active treatment and died 5 months later. The case shows that IL-2/LAK cells can be safely given to patients who have experienced no GVHD following allo-BMT and are likely to be effective through an ongoing graft-versus-leukaemia effect.     

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

Activin A regulates activities of peripheral blood natural killer cells of mouse in an autocrine and paracrine manner

Activin A, a multifunctional cytokine of transforming growth factor-β (TGF-β) superfamily, can be produced by the diverse immune cells. NK cells in peripheral blood are one of the major immune cells applied to cancer therapy in recent years. However, whether activin A can be produced by natural killer (NK) cells and be involved in regulation of peripheral blood NK cells activities of mouse are not well characterized. Here, we found that activin type IIA and IIB receptors and signaling molecules Smad2, 3 were expressed in peripheral blood NK cells of mouse by flow cytometry and RT-PCR. The cultured blood NK cells of mouse not only produced activin βA chain protein by intracellular cytokine staining, but also secreted mature activin A protein by enzyme-linked immunosorbent assay (ELISA), and the production was promoted by IL-2. In addition, IL-2 as a positive control obviously promoted IFNγ production of mouse blood NK cells in vitro. However, activin A suppressed IFNγ production, but enhanced IL-2 synthesis and did not alter IL-10 production. Moreover, we found that activin A significantly suppressed the ability of NK cells to lyse target cells. These data revealed that blood NK cells of mouse were not only the target cells in response to activin A, but also the source of activin A, suggesting that activin A may play an important role in regulation of NK cells activities of mouse in an autocrine / paracrine manner.     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.