Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H-31 and H-A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL-2) receptor (IL-2 R), anti-Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H-31 and H-A26 antibodies both reacted with only IL-2 R-positive cells, and they precipitated IL-2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T-cell leukemia virus type I (HTLV-I)-carrying MT-2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL-2 R with anti-Tac. Antibody-binding competition assays showed that H-31 and anti-Tac, and H-A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti-Tac also inhibited the binding of HIEI but not vice versa. H-31, like anti-Tac, strongly inhibited the IL-2-dependent proliferation of normal activated T-cells, absorption of IL-2 and direct binding of IL-2 to the cells, while H-A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV-I infection were different, as revealed by immunofluorescence studies. Human IL-2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL-2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL-2 R molecules. These observations indicate that H-31 and H-A26 recognize human IL-2 R molecules and that the antigenic sites on the IL-2 R molecule defined by H-31, H-A26, anti-Tac, and HIEI are different.     

The interaction of recombinant IL-2 with human resting lymphocytes: blocking effects of monoclonal antibodies to IL-2 receptors

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin-2 receptors (IL-2.R). Recombinant IL-2 (rIL-2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon-gamma (IFN-gamma) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool-passed non-T, non-B cells ("null cells" or T3- cells); in response to rIL-2, only Leu 11+T3- cells showed enhanced NK activity, and both Leu 11+T3- and Leu11-T3- cells showed predominantly AK activity, proliferation and production of IFN-gamma. These findings suggest that the T3- fraction (null cell fraction) contains predominantly cells expressing IL-2.R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL-2.R, including anti-Tac antibody at any dilution. These results indicate that IL-2.R on the resting T3- cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3- cells possess higher affinity IL-2.R than activated T or B cells. Other possibilities are also discussed.     

抗鸡白细胞介素4单克隆抗体的制备及鉴定

为了制备鸡白细胞介素4(chIL-4)单克隆抗体,将成熟的chIL-4基因亚克隆至原核表达载体pET-28a和pGEX-6P-1上,然后在大肠杆菌中分别诱导重组蛋白His-chIL-4和GST-chIL-4的表达,并纯化。将纯化后的His-chIL-4作为免疫原免疫BALB/c小鼠,经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将纯化后的GST-chIL-4作为筛选抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗chIL-4蛋白的杂交瘤细胞株,分别命名为1G11-3B、2E5-3D和1G11-5H。经ELISA检测,3株单克隆抗体的亚型均为IgG1,亲和力解离常数(Kd)分别为1.79×10~(–9)、1.61×10~(–9)和2.36×10~(–9)。经Western blotting及间接免疫荧光试验鉴定,3株单克隆抗体均能特异性识别原核和真核表达的chIL-4蛋白。Western blotting试验证明1G11-3B、2E5-3D和1G11-5H识别的抗原表位区域分别为chIL-4蛋白N端的第1–40、80–112和40–80位氨基酸。该单克隆抗体的制备为chIL-4的检测和生物学功能研究奠定了基础。     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone,prolactin, interleukin 2 and interleukin 6

Many membrane proteins are implicated in the regulation of cell functions by triggering specific signaling pathways. Porins are known potential modulators of cell proliferation and differentiation. We explored the possible involvement of this protein in signal transduction pathways in mouse gut macrophages. In the present work we have shown that porins can trigger signal transduction in mouse macrophages infected with S. typhimurium. Activation of macrophages by porins results in an increase in inositol trisphosphate and intracellular Ca2+ mobilization. There is a translocation of protein kinase C to the membrane which is accompanied by nitric oxide release within the macrophages. This effect is the outcome of the expression of nitric oxide synthase, which is dependent on Protein kinase C. Further, we observed that there is an increased binding of the porins on macrophages infected with S. typhimurium which results in activation of macrophages and triggering of specific signaling pathways. These results indicate that porins induce the production of nitric oxide via a protein kinase C dependent pathway. Nitric oxide plays a fundamental role in macrophage effector function where it has both communication and defensive function.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.     

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

In situ identification of cells in human leprosy granulomas with monoclonal antibodies to interleukin 2 and its receptor

Leprosy is a chronic granulomatous disease with an immunologic spectrum in which lepromatous leprosy patients have defective cell-mediated immune responses, in comparison to tuberculoid leprosy patients. Immunoregulatory aspects of this spectrum were investigated by using monoclonal antibodies to interleukin 2 (IL 2), IL 2 receptors (Tac), and T lymphocyte subpopulations with immunoperoxidase techniques on frozen sections of skin biopsy specimens from 10 tuberculoid and 10 lepromatous patients. A comparison of IL 2+ cells revealed markedly fewer IL 2+ cells in lepromatous specimens (lep. 0.028% +/- 0.02 vs tub. 0.46% +/- 0.28, p less than 0.001). These IL 2+ cells were large, exhibited cytoplasmic staining, and on double immunostaining were Leu-4+, Leu-3a+, Leu-2a-, Tac-, and OKT6-, consistent with the fact they are IL 2 producers. Equivalent numbers of Tac+ cells were observed in both lepromatous and tuberculoid granulomas (lep. 1.5% +/- 0.5 vs tub. 2.1% +/- 0.7, p, NS), suggesting that the responder cells are present in both conditions. The tuberculoid granuloma was highly organized, composed of a central core of mature macrophages, Leu-3a+ and Tac+ cells with a surrounding mantle of Leu-2a+, Leu-3a+, IL 2+, Tac+, and OKT6+ cells. In lepromatous granulomas, Leu-2a+, Leu-3a+, Tac+, and rare IL 2+ cells were randomly admixed with bacilli-laden macrophages. The defective cell-mediated immune responses in lepromatous leprosy appears to be associated with diminished IL 2 production and disorganization of the granuloma.     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies

Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild-type and glucocorticoid-resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of 'nuclear transfer deficient' (nt-) and 'increased nuclear transfer' (nti) phenotypes, respectively, while the third was of the 'receptorless' (r-) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild-type and nt- receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild-type and nt- receptors more efficiently after activation at 20 degrees C. By use of an immuno-competition assay we were able to detect cross-reacting material in considerable amounts in extracts of nti and r- cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild-type, nti and r- cells gave a major band of mol. wt. 94 000 upon SDS-gel electrophoresis while the steroid-binding polypeptides of wild-type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.     

Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies.

Two methods are described for the assay of low-density-lipoprotein (LDL) receptor protein based on the binding of receptor to microtitre plate wells coated with a specific monoclonal antibody or with LDL, followed by detection with radioactive antibody that recognizes a different part of the molecule. The two-antibody procedure detected approx. 2 ng of pure bovine receptor at twice background, and there was a linear relationship on a double-logarithm plot between radioactive antibody bound and the amount of receptor added, up to at least 500 ng of receptor protein per well. The procedure using immobilized LDL was less sensitive and the binding of receptor was inhibited by low concentrations of NaCl, which restricted its usefulness for routine assay of tissue extracts. LDL receptor protein could be readily assayed using the two-antibody procedure in normal human skin fibroblast extracts prepared by bulk-elution from small columns of DEAE-cellulose followed by rapid desalting. No radioactive antibody bound with extracts of cells from a receptor-negative familial hypercholesterolaemic subject. The LDL receptor content of normal fibroblasts preincubated with lipoprotein-deficient serum was estimated, using bovine receptor as standard, to be approx. 60 ng of receptor protein/mg of cell protein.     

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies

[This corrects the article on p. 1498 in vol. 3.].     

Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro

Several rat anti-mouse interleukin 3 (IL-3) monoclonal antibodies have been developed which inhibit the biologic activity of mouse IL-3. These antibodies were produced in rats immunized with preparations of purified, recombinant mouse IL-3, obtained from transiently transfected COS7 cell supernatant. Hybridomas secreting anti-IL-3 were selected initially either on the basis of their giving a positive signal in an indirect enzyme-linked immunosorbent assay, or for their ability to inhibit the proliferation of the IL-3-dependent mouse mast cell line, MC/9. Neutralizing rat monoclonal IgG1, IgG2a, and IgG2b antibodies have been identified; these also block IL-3-induced proliferation of the NFS-60 and IC2 cell lines. These antibodies also blocked the IL-3-induced proliferation of mouse bone marrow-derived colony-forming units-culture suggesting that the same epitopes on IL-3 influence receptor recognition for both the proliferation of factor-dependent cell lines as well as normal bone marrow cells. Fab fragments produced from certain of the IgG2a-neutralizing antibodies blocked as well as the parent IgG. Antibody cross-blocking studies identified one neutralizing antibody apparently recognizing an epitope that was spatially distinct from those recognized by the other blocking antibodies tested. The development of these neutralizing rat monoclonal antibodies to mouse IL-3 should facilitate further investigation on the role of this factor in hemopoietic regulation.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

Pathophysiology of estrogen receptors in mammary tissue by monoclonal antibodies

Identification of preneoplastic lesions of the breast has mainly rested on morphological grounds, supported by epidemiological data. These studies assign a definite precancerous potential to a group of atypical hyperplastic lesions and in situ carcinoma. In spite of much effort no criteria are yet available to understand which, among these lesions, is committed to infiltrative growth, in other words, to understand the risk to a single patient. Estrogens are know to play a critical role in the etiology of breast cancer. The hypothesis is investigated that this role is dependent on a modified expression of their receptor. To approach this question estrogen receptor expression was traced by specific monoclonal anti-receptor antibodies and immunocytochemistry, on a spectrum of breast tissue changes, from normal tissue to infiltrating cancer. Estrogen receptor expression is heterogeneous in normal tissue and in infiltrating cancer, and on the contrary is homogeneous in proliferative atypical lesions and in in situ carcinomas. Present results show that receptor expression is enhanced and becomes homogeneous, maybe constitutive, in atypical hyperplasia and in in situ carcinoma and that this phenomenon could subserve important changes of proliferative capacity which are necessary and possibly sufficient for autonomous growth.