Mutants of interleukin 13 with altered reactivity toward interleukin 13 receptors.

Interleukin 13 (IL13) belongs to a family of cytokines whose members exhibit structural homology, despite amino acid sequence dissimilarity. For example, while of limited sequence homology, IL13 and IL4 share a signaling receptor, IL13/4 receptor, on a variety of human normal cells. However, a subclass of IL4-independent IL13 receptors is overexpressed on certain transformed cells, including human malignant gliomas. We introduced mutations into human (h) IL13 to determine the site(s) involved in interaction with the shared receptor and/or the glioma-associated receptor. This analysis identified at least three protein regions that are needed for signaling through the shared receptor. These regions were localized to alpha-helices A, C, and D and were mainly separate from the region(s) needed to interact with the glioma-associated receptor. Glutamic acids at positions 13 and 16 in hIL13 alpha-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signaling since their specific mutation resulted in loss and/or gain of function phenomena. We demonstrate that the molecular requirements of hIL13 to interact with its respective receptors are generally distinct and can be controlled by mutagenesis of the cytokine.     

Production of an active anti‐CD20‐hIL‐2 immunocytokine in Nicotiana benthamiana

Anti‐CD20 murine or chimeric antibodies (Abs) have been used to treat non‐Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti‐CD20 Abs demonstrated to be effective in inducing regression of B‐cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti‐CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL‐2‐based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti‐CD20‐human interleukin‐2 (hIL‐2) immunocytokine (2B8‐Fc‐hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv‐Fc‐engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS‐PAGE and gel filtration. Purification yields using protein‐A affinity chromatography were in the range of 15–20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant‐type glycosylation. 2B8‐Fc‐hIL2 and the cognate 2B8‐Fc antibody, devoid of hIL‐2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody‐dependent cell‐mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8‐Fc‐hIL2, IL‐2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.     

Interleukin 27 induces differentiation of neural C6-precursor cells into astrocytes

Interleukin 6 (IL6)-type cytokines are major regulators of inflammation and thereby contribute to the neuropathology and pathophysiology associated with inflammation of the central nervous system (CNS). Furthermore, astrocyte development which is a key process in the development of the CNS is also controlled by cytokines of the IL6-family. Interleukin 27 (IL27) is a recently identified member of this family and has been implicated in the inhibition of TH₁₇ T-cell-responses. Here we show that IL27 and the HHV8 encoded viral IL6 (vIL6) induce C6 glioma cells to differentiate into an astrocyte-like state. Cytokine stimulation led to STAT-factor phosphorylation and consequently to protein expression of the astrocyte marker glial fibrillary acidic protein (GFAP). These data could be confirmed by GFAP-immunostaining of stimulated cells. Taken together, IL27 and vIL6 can be considered as new astrocyte-inducing cytokines of the brain.     

T细胞中IL-3基因表达调节的转录因子初探

主要利用凝胶迁移率试验和抗体超迁移试验,以ｈＩＬ－３基因５′侧翼含κＢ位点的序列为探针,探究ＮＦ－κＢ是否参与了ｈＩＬ－３基因的表达调节．结果表明,在Ｊｕｒｋａｔ细胞和人外周血Ｔ淋巴细胞中,都存在与此序列特异结合的可诱导蛋白;ＮＦ－κＢｐ６５亚基的单抗对ＤＮＡ－蛋白复合物的形成没有影响．这些结果揭示：正常细胞和肿瘤细胞中,确存在可特异识别ｈＩＬ－３基因５′侧翼的κＢ顺序的蛋白因子,而且该蛋白因子的表达是可诱导的;但ＮＦ－κＢ并未参与ＩＬ－３基因的表达调节．     

Soluble IL-6R alpha upregulated IL-6, MMP-1 and MMP-2 secretion in bone marrow stromal cells

IL-6 mediates its activity through a cell surface receptor composed of a signal transducing protein, CD130, and a ligand-binding protein which exists in membrane-bound form (CD126) or in soluble form (sIL-6R alpha). Interestingly, sIL-6R alpha combined with IL-6 is able to interact with CD130 leading to the intracellular cascade of activation. In the present study, using flow cytometry, we show that stromal cells from human bone marrow (BMSC) express CD130 but not CD126. We demonstrate that BMSC are responsive to IL-6 only in the presence of exogenous sIL-6R alpha. Indeed, exogenous sIL-6R alpha induces in BMSC the production of its own ligand, IL-6, and of both MMP-1 and MMP-2, two matrix metalloproteinases involved in bone resorption and in tumour spreading, respectively. Since myeloma cells release sIL-6R alpha in the close vicinity of BMSC, these data suggest a role for this factor in the pathophysiology of multiple myeloma, a B-cell malignancy dependent on IL-6 for its growth and characterized by bone destruction.     

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.     

Crosslinking of the CD5 antigen on human T cells induces functional IL2 receptors.

CD5 is a 67-kDa antigen that is expressed on the membrane of the majority of human T cells, and on a subset of B cells. Previous studies have demonstrated that anti-CD5 monoclonal antibodies (mAb) can provide a helper signal for T cell activation through the TCR/CD3 complex. We now demonstrate that when CD5 is crosslinked by immobilized anti-CD5 mAb in the absence of other activating stimuli, the T cells proliferate in response to recombinant interleukin 2 (rIL2) (but not to rIL4). Four different anti-CD5 mAb (anti-Leu1, 10.2, anti-T1, and OKT1) had a similar effect. IL2 responsiveness could be induced with immobilized anti-CD5 mAb in cultures of purified T cells, but was enhanced by the addition of monocytes, by monocyte culture supernatant, or by the combination of IL1 and IL6. Staining with an anti-IL2 receptor (p55) mAb demonstrated expression of IL2 receptors on about 10% of the anti-CD5-stimulated T cells. Both virgin (CD45RA+) and memory (CD45RO+) T cells were responsive. Our data provide further evidence for the involvement of CD5 in T cell activation.     

Regulation of interleukin 2 receptor expression on a human cytotoxic T lymphocyte clone, synergism between alloantigenic stimulation and interleukin 2

A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.     

Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL 1-alpha and IL 1-beta

The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1-alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.     

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.     

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.     

Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes

We previously described a cell surface antigen, termed Tp44, detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Analysis by SDS-polyacrylamide gel electrophoresis and isoelectric focusing demonstrated that this antigen consists of two identical 44 kilodalton glycopeptides that form a disulfide-linked homodimer. Competitive binding experiments showed that antibody 9.3 and an anti-CD3 antibody (64.1) recognize distinct antigenic determinants; furthermore, the binding of antibody 9.3 was unaffected by prior modulation of CD3. Thus, Tp44 has no detectable cell surface association with CD3. By itself, antibody 9.3 had no detectable effect on either IL 2 receptor expression or IL 2 release, and did not cause T cell proliferation even when monocytes were present and exogenous IL 2 was provided, indicating that binding of antibody 9.3 does not provide a primary signal for T cell activation. However, the proliferative responses of T lymphocytes activated by phytohemagglutinin, concanavalin A, or an anti-CD3 monoclonal antibody were strikingly enhanced in the presence of antibody 9.3, an effect associated with increased IL 2 receptor expression and increased IL 2 secretion. Antibody 9.3 enabled anti-CD3-Sepharose-activated T cells and anti-CD3 antibody-activated Jurkat cells to release IL 2 in the absence of monocytes. Fab fragments of antibody 9.3 had no effect on anti-CD3-induced IL 2 release by Jurkat cells, whereas F(ab')2 fragments had activity comparable to that of unmodified antibody, indicating that bivalent binding of Tp44 molecules is required for IL 2 secretion. Together, these results suggest that TP44 may function as a receptor for accessory signals in the activation of T cells.     

Development and characterization of a human B cell line that responds to B cell growth factor but not interleukin 2

The human lymphoblastoid cell line we present here proliferated in response to a 14,000 m.w. B cell growth factor (BCGF), and not to interleukin 2 (IL 2). This cell line, designated B-A3, was established by Epstein Barr virus (EBV) transformation of Staphylococcus aureus Cowan I (SAC)-activated spleen B cells, and has been maintained in RPMI 1640 medium complemented with 15% fetal calf serum (FCS) without the addition of other exogenous growth factors. A proliferative response, as measured by [3H]thymidine uptake of B-A3 cells was significantly induced by either commercial IL 2-free human BCGF preparations, or phytohemagglutinin-stimulated mixed lymphocyte culture supernatant at all FCS concentrations used in the assay. The most marked proliferation due to BCGF, however, was observed in the absence of FCS. This BCGF-induced proliferation was not influenced by IL 2 or interferon-gamma (IFN-gamma), because both recombinant IL 2 and IFN-gamma failed to induce proliferation. The response of B-A3 cells to a specific BCGF was additionally indicated by the responsiveness of this cell line to BCGF purified by a series of chromatographic steps. The BCGF to which B-A3 cells responded had a m.w. of 14,000 and was similar to low m.w. BCGF reported from other laboratories. Surface characterization of B-A3 cells, analyzed by flow cytometry with a panel of monoclonal antibodies, demonstrated that the majority of B-A3 cells were stained positively with Leu-12, HLA-DR, and surface IgG markers, whereas staining with surface IgM, IgD markers, pan T cell markers (Leu-4 and Leu-9), and IL 2 receptor (Tac) were consistently negative. Taken together, the human lymphoblastoid cell line we present here responded specifically to a low m.w. BCGF. This cell line may be of value in the purification of BCGF to homogeneity, in studies of the interactions of BCGF with human B cells, and in the identification of the BCGF receptor.     

A secreted form of the human interleukin 2 receptor encoded by an "anchor minus" cDNA

The DNA sequence encoding all of the putative intracytoplasmic domain and most of the trans-membrane domain of the human IL 2 receptor was deleted from a full length receptor cDNA. After expression in mouse L cells, the resultant "anchor minus" cDNA was found to direct the synthesis of a secreted rather than membrane-associated form of the IL 2 receptor. The secreted receptor protein (44,000 to 46,000 Mr) retained the capacity to bind both IL 2 and the monoclonal anti-Tac antibody, as evidenced by retention on IL 2 and anti-Tac affinity columns, inhibition of [3H]-anti-Tac binding to HUT 102B2 cells, and partial inhibition of IL 2-induced CTLL proliferation. Removal of these domains from the IL 2 receptor did not alter the posttranslational processing or rate of export of the truncated receptor protein. These data confirm the proposed membrane orientation of the IL 2 receptor (NH2 terminus out, COOH terminus in) and underscore the anchoring function of this carboxy terminal receptor segment. The availability of such anchor minus receptor cDNA constructs may facilitate purification of large quantities of receptor protein for further analysis of receptor structure, valency, and localization of the IL 2 binding site(s).