Study of the possibility to abolish the action of immunosuppressive factors of tumor cells]

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.     

Study of the action of tumor cell immunosuppressive factors on the production of cytokines by lymphocytes and macrophages and the mitogenic activity of interleukin-2

The effect of immunosuppressive factors (ISF) derived from tumor cell lines (mastocytoma P-815, leukosis EL-4 and melanoma B16) on the production of interleukin-1 (IL-1) by macrophages and of interleukin-2 (IL-2) by splenocytes of BALB/C mice was investigated. We could show that treatment of above cells by the tumor products resulted in strong decrease of IL-2 production but did not affect IL-1 secretion. ISF inhibited the proliferation of BALB/C lymphoblasts caused by recombinant IL-2. Thus, one of the significant mechanisms of ISF action seems to be disturbance of monolymphokine cascade of lymphocyte activation.     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment

Accumulating evidence indicates that a dynamic cross-talk between tumors and the immune system can regulate tumor growth and metastasis. Increased understanding of the biochemical nature of tumor antigens and the molecular mechanisms responsible for innate and adaptive immune cell activation has revolutionized the fields of tumor immunology and immunotherapy. Both the protective effects of the immune system against tumor cells (immunosurveillance) and the evasion of tumor cells from immune attack (tumor-immune escape) have led to the concept of cancer immunoediting, a proposal which infers that a bidirectional interaction between tumor and inflammatory/regulatory cells is ultimately responsible for orchestrating the immunosuppressive network at the tumor site. In this context, a major challenge is the potentiation or redirection of tumor antigen-specific immune responses. The success in reaching this goal is highly dependent on an improved understanding of the interactions and mechanisms operating during the different phases of the cancer immunoediting process. In this review, we discuss the multiple defense and counterattack strategies that tumors have devised in order to evade immune attack and to thwart the effectiveness of several immunotherapeutic approaches. Diego O. Croci, Mariano F. Zacarías Fluck contributed equally to this work. Gabriel A. Rabinovich, O. Graciela Scharovsky contributed equally to this work and should be considered as senior authors.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

Photoaffinity labeling of plasma membrane receptors for cytochalasins in Ehrlich tumor cells

Treatment of purified Ehrlich ascites cell plasma membranes either with [3H]cytochalasin B or [3H]19-O-acetylchaetoglobosin A under photolytic conditions produced several radioactive polypeptides which were characterized by SDS-PAGE analyses. The major proteins so photolabeled were in the 60,000-80,000 Da range, with less labeling found in polypeptides smaller than 43,000 and greater than 90,000 Da. Immunofluorescent staining failed to identify the major photolabeled component as actin. It is concluded, in keeping with prior investigations using other cell types, that the predominant proteins photolabeled by cytochalasins are affiliated with the glucose-transport system.     

Regulation of cytotoxic lymphocyte precursors. II. The effect of interleukin-2 and interferon-gamma on the apparent specificity of effector cells

The influence of lymphokines on the frequency and specificity of antigen-induced cytolytic T cells was investigated. Removal of an acid-labile factor(s) from supernatants produced by concanavalin A (Con A)-stimulated rat splenocytes was shown to increase the specificity of cytolytic T-cell clones as determined by their ability to distinguish between antigen modified and unmodified target cells. The overall frequency of cytolytic T cell precursors (antigen specific as well as nonspecific) was reduced in the presence of acid-pretreated rat Con A supernatant as compared to untreated rat Con A supernatant. Neither the addition of interleukin 2 nor interferon-gamma could substitute for this acid-labile factor. These data indicate that an additional lymphokine(s) contributes to the in vitro activation of nonspecific killer cells.     

Mechanisms of corticosteroid action on lymphocyte subpopulations: VI. Lack of correlation between glucocorticosteroid receptors and the differential effects of glucocorticosteroids on T-cell subpopulations

Glucocorticosteroid receptors were measured in subpopulations of human peripheral blood T cells identified by the presence of an Fc receptor for IgG (T_G) or an Fc receptor for IgM (T_M). T_M cells are selectively depleted from the circulation by in vivo administration of glucocorticosteroids as opposed to T_G cells which are relatively resistant to the lymphodepletive effects of these hormones. However, this selective lymphodepletive effect of glucocorticosteroids on T_M cells could not be explained on the basis of detectable differences in intracytoplasmic glucocorticosteroid receptors in these T-cell subpopulations since T_G and T_M cells had quantitatively similar glucocorticosteroid receptors as well as remarkably similar dexamethasone binding affinities and dissociation constants. Hence, the strikingly different effects of glucocorticosteroids on the circulatory kinetics of T_G and T_M cells must be explained by mechanisms other than those at the level of the glucocorticosteroid receptor.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Relation between interleukin-2 production and the time of irradiation and mitogenic lymphocyte activation

The effect of radiation on the production of interleukin 2 by activated lymphocytes was shown to be a function of time interval between irradiation and mitogen stimulation of cells. The most intensive production of interleukin 2 was noted in cells exposed 24 h following the effect of the mitogen. The enhancing effect of in vivo irradiation was less pronounced.     

Evidence for the production and action of interleukin-10 in pituitary cells

Summary 1. Interleukin-10 (IL-10) has a wide range of activities in the immune system such as modulation of interferon-gamma (IFN-) and antibody production. The neuropeptide hormone corticotropin (ACTH) has similar activities, suggesting that a bidirectional communication mechanism operates between the immune and the neuroendocrine system involving these two substances.2. Murine pituitary tumor cells (AtT-20) were found to produce up to 3 ng/ml of IL-10.3. Pituitary cell corticotropin production was enhanced by IL-10 treatment.4. IL-10 induced the production of ACTH in mouse splenocytes.5. Authenticity of pituitary-derived IL-10 was shown by the demonstration of identical neucleic acid sequences of reverse-transcribed, polymerase chain reaction amplified fragments of cDNA obtained from murine splenocytes, a murine pituitary tumor cell line, and freshly isolated murine pituitaries.     

Mitogenic receptors on human peripheral blood lymphocytes: the interaction of Phaseolus vulgaris erythroagglutinating phytohemagglutinin and anti-thymocyte globulin on the human peripheral blood lymphocyte membrane.

A horse anti-human thymocyte antibody (ATG) obtained from the Upjohn Company was shown to stimulate DNA synthesis in human lymphocytes with a time course and magnitude of radioactive thymidine uptake comparable to that seen with phytohemagglutinin (E-PHA) and concanavalin A (Con A). Low mitogenic or nonmitogenic concentrations of intact ATG or its Fab fragments inhibited E-PHA-induced mitogenesis, whereas the response to Con A was unaffected. Competitive binding studies with ATG and E-PHA revealed mutual inhibition of binding to lymphocytes suggesting that E-PHA and the ATG share a common receptor site on the cell surface. ATG binding was unaffected by Con A. From the analysis of the binding data and the inhibition of mitogenesis, it appears that at least part of the E-PHA response in human lymphocytes involves receptors that are not acted on by Con A.     

The stimulating action of lipopolysaccharide and muramyl dipeptide on the activation of mononuclear cells in human peripheral blood by recombinant interleukin-2 in vitro

Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were effective in augmentation of killer cells generation from human peripheral blood mononuclear cells (PBMC) in response to recombinant human interleukin-2 (IL-2). Pretreatment of PBMC with combination of LPS and MDP resulted in most significant their proliferation stimulated by IL-2. Thus our results show the enhancement of PBMC sensitivity to IL-2 by action of LPS, MDP and most of all by their combination in vitro.     

Lack of correlation between membrane lipid composition and thermotolerance in Chinese hamster ovary cells

Membrane composition and fluidity, and survival of Chinese hamster ovary fibroblasts have been examined following various thermal exposures. It has been found that enhanced thermal resistance following brief exposure to 43°C is not accompanied by detectable membrane lipid alterations. This is in contrast to membrane alterations that occur following adaptation to elevated temperatures compatible with growth (39°C and 41°C).     

Lack of generation of killer cells in the mixed lymphocyte reaction between mitogen-stimulated and unstimulated autologous lymphocytes.

The present study has demonstrated that the Con A-activated cell-mediated autologous mixed lymphocyte reaction (MLR) is not associated with the generation of cytotoxic effector cells that kill autologous targets. Thus, the suppression of antibody production of PWM stimulated lymphocytes by autologous Con A-activated suppressor cells cannot be explained by detectable cytotoxicity. We have further demonstrated that the stimulator cell in this system is a nonadherent non-T cell.     

IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

两种本地植物种子萌发对飞机草的化感耐受性及其相互竞争

为探讨本地物种假地豆和白饭树对入侵植物飞机草的替代控制潜力,利用培养皿法和同质园种植实验分别研究了两个本地物种种子萌发对飞机草的化感耐受性及其与飞机草的竞争关系。结果显示:除了假地豆的萌发率在高浓度(2.5%)的飞机草叶提取液下受到显著抑制外,两个本地种的萌发在不同浓度飞机草根、茎、叶提取液下均不受抑制。飞机草与假地豆混种时,飞机草的株高、地下生物量比及根冠比显著降低,假地豆的株高无显著变化,但生物量显著增加;飞机草的竞争参数相对产量(RY)显著小于1,竞争攻击力系数显著小于零,表明其竞争力弱于假地豆。飞机草与白饭树混种时,飞机草的根冠比也显著降低,但株高和生物量均显著增加,而白饭树的株高和生物量却显著降低;飞机草的相对产量(RY)显著大于1,竞争攻击力系数显著大于零,表明其竞争力强于白饭树。结合以上结果,本地植物假地豆可以一定程度上竞争抑制飞机草的生长,具有替代控制飞机草的潜力,而白饭树可以在清除飞机草后的入侵地辅助植被修复。实验结果为飞机草替代控制的目标物种的筛选提供了科学依据,对被飞机草入侵生境生物多样性的恢复和重建具有一定指导意义。