Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on itsin vitro andin vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.     

重组人GM—CSF／MCAF融合蛋白的变性,复性及纯化研究

人粒细胞巨噬细胞集落刺激因子(GM-CSF)和单核细胞趋化激活因子(MCAF)融合蛋白在大肠杆菌中高效表达后,表达产物以包涵体形式存在。包涵体经分离和洗涤后,探索了rhGM-CSF/MCAF变性和复性的合适条件。复性后的样品经Sephadex G-75凝胶过滤和CM-Sepharose FF离子交换两步层析,得到了具有生物学活性的SDS-PAGE纯的rhGM-CSF/MCAF。Western blot检测表明,纯化的rhGM-CSF/MCAF能分别与GM-CSF和MCAF抗体发生特异反应。     

Cooperative effect of 8-Cl-cAMP and rhGM-CSF on the differentiation of HL-60 human leukemia cells

In HL-60 leukemia cells the site-selective cAMP analog, 8-Cl-cAMP, at a dose of 5 microM produced growth inhibition with no signs of toxicity, whereas granulocyte-macrophage colony stimulating factor (GM-CSF) exerted an early transient increase of cell proliferation which was followed by differentiation toward monocytes. 8-Cl-cAMP in combination with GM-CSF blocked the growth stimulation due to GM-CSF and demonstrated a synergistic effect on the differentiation of HL-60 cells. The early proliferative effect of GM-CSF was correlated with an increased expression of type I regulatory subunit of cAMP-dependent protein kinase (RI alpha). Treatment with an RI alpha antisense oligodeoxynucleotide suppressed the GM-CSF-inducible cell proliferation and differentiation. Conversely, an RII beta antisense oligodeoxynucleotide, which suppresses the RII beta and causes a compensatory increase in RI alpha level, greatly enhanced the early proliferative input and the differentiation induced by GM-CSF. These results provide an insight into the mechanism of action of GM-CSF and the rationale for a combination differentiation therapy with 8-Cl-cAMP and GM-CSF.     

粒细胞-巨噬细胞集落刺激因子/白细胞介素-3融合蛋白的表达研究

利用PCR扩增得到粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)完整基因片段,将其分别克隆至pGEM-T,构建成GMCSF/IL-3融合蛋白基因,DNA序列与设计预期一致。将得到的融合蛋白基因克隆至T7RNA聚合酶表达载体pT7zz,得到表达质粒pFu,经转化至表达宿主E.coli BL21(DE3),在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS-PAGE电泳鉴定扫描分析,目的基因产物表达量占菌体总蛋白量的30%以上,目的基因表达产物以包涵体的形式表达。Westernblot鉴定表明,该表达产物可以与GM-CSF抗体及IL-3抗体特异性结合。目的基因表达产物经过包涵体变性、透析复性及柱层析纯化,用GM-CSF、IL-3依赖细胞株TF-1检测,具有明显的生物学活性。     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by glioblastoma cells. Despite the presence of inducing signals GM-CSF is not expressed in vivo.

One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production.     

Thrombin chemotactic stimulation of HL-60 cells: studies on thrombin responsiveness as a function of differentiation

Thrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well-known chemotactic agents such as fMet-Leu-Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL-60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP-alpha-thrombin, as well as the thrombin-derived chemotactic peptide CB67-129, elicits a dose-dependent chemotactic response in HL-60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL-60/mono), whereas no such response is evident in either undifferentiated HL-60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL-60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte-differentiated cells. In HL-60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15-20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL-60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL-60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL-60/mono (but HL-60/gran nor undifferentiated HL-60) are capable of significant specific 125-I-labeled alpha-thrombin-binding (ka approximately 20 nM), and possess an estimated 400,000 thrombin-binding sites per cell. Our findings further suggest that the thrombin response of HL-60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.     

Phospholipase D is required in the signaling pathway leading to p38 MAPK activation in neutrophil-like HL-60 cells, stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The differentiation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using phosphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies showed that increasing concentrations of ethanol or 1-butanol but not 2-butanol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p44/p42 activation. These data indicated that activation of phospholipase D (PLD) was required for activation of p38 but not p44/p42. We compared the effect of fMLP with those of tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that ethanol did not inhibit p38 phosphorylation upon stimulation with either GM-CSF or TNF alpha. These results suggested that in cells stimulated with fMLP, PLD was upstream of p38. To further test the involvement of PLD, we used antisense inhibition of human PLD1 expression. Treatment with antisense oligonucleotides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38 activation in neutrophil-like HL-60 cells. In addition, the results obtained with TNF alpha and GM-CSF demonstrated that p38 activation occurred independently of PLD activation.     

Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.     

Granulocyte-macrophage colony-stimulating factor as an autocrine survival-growth factor in human gliomas

We studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas.     

Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity

Anti-HER2/neu therapy of human HER2/neu-expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu IgG3 fusion protein containing GM-CSF. Anti-HER2/neu IgG3-(GM-CSF) expressed in myeloma cells was correctly assembled and secreted. It was able to target HER2/neu-expressing cells and to support growth of a GM-CSF-dependent murine myeloid cell line, FDC-P1. The Ab fusion protein activated J774.2 macrophage cells so that they exhibit an enhanced cytotoxic activity and was comparable to the parental Ab in its ability to effect Ab-dependent cellular cytotoxicity-mediated tumor cell lysis. Pharmacokinetic studies showed that anti-HER2/neu IgG3-(GM-CSF) is stable in the blood. Interestingly, the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistence observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu-expressing murine tumor CT26-HER2/neu and enhance the immune response against the targeted Ag HER2/neu. Anti-HER2/neu IgG3-(GM-CSF) is able to enhance both Th1- and Th2-mediated immune responses and treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors. Our results suggest that anti-HER2/neu IgG3-(GM-CSF) fusion protein is useful in the treatment of HER2/neu-expressing tumors.     

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.     

Big tumor regression induced by GM-CSF gene-modified 3LL tumor cells via facilitating DC maturation and deviation toward CD11c+CD8alpha+ subset

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a powerful immune-stimulating factor that helps to generate a systemic, strong, and long-lasting immune response. However, whether the transduction of GM-CSF to tumor cell results in tumor regression and optimizes local immune microenvironment remains to be investigated. In this study, using an experimental murine tumor model, we demonstrated that the in vivo growth of 3LL tumor cells modified with the GM-CSF gene (3LL-GM) was inhibited even when the tumor diameter was over 7 mm (big tumor), and mice inoculated with GM-CSF gene-modified 3LL cells survived over 90 days, whereas mice inoculated with control parental 3LL cells and 3LL cells transduced with control vector all succumbed to the tumor by day 17 after tumor inoculation. Further analysis showed that targeted expression of GM-CSF in 3LL tumor cells markedly enhanced the systemic antitumor effect, including specific lymphocytes proliferation, cytotoxicity against 3LL tumor, and increased production of IFN-gamma. GM-CSF gene-modified 3LL cells significantly protected the mice from the parental 3LL tumor challenge. More importantly, the percentage of dendritic cells (DCs) in tumor site was greatly increased and the DCs differentiated into CD11c(+)CD8alpha(+) cells, which were reported to be able to benefit the induction of CD8(+) cytotoxic T lymphocytes (CTLs) that contribute to tumor regression. Our research indicated that GM-CSF could optimize the immune microenvironment in the tumor site, which provides a potent approach for immunotherapy of tumors.     

重组人GM－CSF／MCAF融合蛋白抗血清的制备与鉴定

重组人粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人单核细胞趋化激活因子（ＭＣＡＦ）融合蛋白经ＳｅｐｈａｄｅｘＧ－７５和ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ两步柱层析,获得了电泳纯的ＧＭ－ＣＳＦ／ＭＣＡＦ融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ试验表明,该抗血清效价高、特异性好,可分别与ＧＭ－ＣＳＦ／ＭＣＡＦ、ＧＭ－ＣＳＦ和ＭＣＡＦ发生反应。     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.