T cell activation. II. Activation of human T lymphoma cells by cross-linking of their MHC class I antigens

The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cai2+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Cai2+ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when Cai2+ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CD3-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules.     

Role of uncultured human melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes.

The role of uncultured melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes (CTLs) was investigated. Uncultured autologous tumor cells by themselves induced modest, but significant, proliferation in 10 of 13 (77%) CTL clones and in only two of nine non-CTL clones. Uncultured allogenic melanoma cells mostly failed to induce CTL proliferation. Autologous tumor-induced CTL proliferation declined with increasing age of the culture. It did not correlate with IL-2 receptor-alpha expression or was not inhibited by addition of anti-IL-2 antibody to the culture. It was inhibited by pretreatment of tumor cells with anti-MHC class II, but not -MHC class I mAb. IL-2 alone was sufficient for the potent proliferation of five of nine CTL clones. In all these five CTL clones, autologous tumor cells suppressed IL-2-induced proliferation. The remaining four CTL clones, however, required both uncultured autologous melanoma cells and IL-2 for the proliferation. IL-4 or IL-6, in particular IL-6, facilitated IL-2-induced CTL proliferation, but not their cytotoxicity. In summary, uncultured melanoma cells by themselves induced modest levels of CTL proliferation in the context of MHC class II antigens, whereas they suppressed IL-2-induced CTL proliferation in more than half of the clones.     

Class I-specific antibodies inhibit proliferation in primary but not secondary mouse T cell responses.

Murine T and B splenocytes were incubated with antibodies that recognize CD3 or surface IgM. These antibodies induced proliferation of their respective target cells. Once stimulated via their receptors, the proliferation of both CD4+ and CD8+ T but not B lymphocytes was inhibited by class I-specific antibodies or their monovalent Fab' fragments. The inhibition of proliferation was dependent on the site on class I molecules recognized by the antibodies used, with the alpha 1/alpha 2 domains of H-2K molecules representing the major site for inhibition. Only soluble antibody-mediated proliferation could be inhibited by class I-directed antibodies; proliferation induced by CD3-specific antibody immobilized on plastic was not inhibited. Primary allogeneic MLR was also inhibited by class I-specific antibodies. In contrast, neither secondary allogeneic MLR, secondary Ag-specific responses, nor proliferation of CTL clones or tumor cell lines were inhibited by class I-specific antibodies. These results suggest a role for class I molecules in regulation of TCR/CD3- but not surface IgM-mediated cell signaling, which depends on the form of stimulation and the stage of differentiation of T cells.     

Murine T helper cell clones secrete granulocyte-macrophage colony-stimulating factor (GmCSF) by both interleukin-2-dependent and interleukin-2-independent pathways

Granulocyte-macrophage colony-stimulating factor (GmCSF) is a lymphokine secreted by class II major histocompatibility complex (MHC)-restricted T cells after lectin or antigen stimulation. To investigate the relationship between interleukin-2 (IL-2) and GmCSF production, we utilized long-term cultures of porcine myelin basic protein (PMBP)-specific T helper cell clones that were maintained with IL-2 in the absence of antigen or irradiated antigen-presenting cells (APC). We have found that supernatants of these T cell clones contained GmCSF activity after IL-2 stimulation. Inhibition of cell proliferation by irradiation failed to stop GmCSF production. When these clones were stimulated with PMBP and irradiated APC in the presence of anti-IL-2 receptor antibody, the T cell supernatants still contained GmCSF activity. These results indicate that (1) GmCSF production by T helper clones after IL-2 stimulation is independent of cell proliferation and (2) antigen/MHC-stimulated GmCSF production by T cell clones can occur by an IL-2-independent pathway.     

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.     

Involvement of ERK, p38 MAP kinase, and PKC in MHC class II-mediated signal transduction in a resting B cell line

Substantial evidence suggests that MHC class II molecules play a critical role in transducing signals during B cell activation and differentiation. In addition, we previously found that cross-linking of MHC class II molecules using anti-MHC class II antibodies inhibited NF-kappaB activation in resting B cells isolated from mouse spleen. In this study, we investigated the mechanism of anti-MHC class II antibody-mediated inhibition of LPS-induced NF-kappaB activation using a resting B cell line, 38B9. We found that treatment with a corresponding anti-MHC class II antibody reduced the activation of NF-kappaB in LPS-stimulated 38B9 cells, treatment of the antibody mediated down-regulation of PKC and ERK/p38 MAP kinase pathways, and treatment with PKC inhibitors caused down-regulation of ERK and p38 MAP kinase activities in LPS-stimulated 38B9 cells. Our results suggest that the PKC and ERK/p38 MAP kinase pathways are regulated by anti-MHC class II antibodies, and that MHC class II molecules are actively involved in the signal transduction pathway in the resting B cell line, 38B9. Consequently, disruption of these pathways might contribute to the inhibition of LPS-induced NF-kappaB activation in 38B9 cells.     

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

HLA-DR antigens render resting T cells sensitive to interleukin-2 and induce production of the growth factor in the autologous mixed lymphocyte reaction

Monoclonal anti-HLA-DR (anti-Ia) antibodies inhibited autologous mixed lymphocyte reactions (AMLR) when added from the initiation of the cultures, but not 72 hr later. The suppressive principle was removed by the stimulator non-T cells, but not by the responding T cells. Antibody-treated non-T cells lost their ability to activate T cells, whereas antibody-treated T cells could still respond to untreated non-T cells. The anti-DR antibodies prevented T cells from acquiring responsiveness to Interleukin-2 (IL-2). However, T cells previously activated by AMLR responded to IL-2 even in the presence of the anti-DR antibodies. OKT4⁺ lymphocytes synthesized IL-2 in the AMLR while OKT8⁺ cells did not. Anti-DR antibodies caused OKT4⁺ cells to become unresponsive to Interleukin-1 stimulation and inhibited the production of IL-2. Interleukin-1 (IL-1) promoted the synthesis of IL-2 in non-anti-DR-treated AMLR cultures. Since resting T cells are unresponsive to IL-2 and resting OKT4⁺ lymphocytes are unable to produce IL-2 even in the presence of IL-1, it is concluded that HLA-DR antigens render resting T cells sensitive to IL-2 and enable OKT4⁺ lymphocytes to respond to IL-1 and subsequently, to produce Interleukin-2.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

Differential effects of interleukin-2 and interleukin-4 on protein tyrosine phosphorylation in factor-dependent murine T cells

Interleukin-2 (IL-2) is a requisite factor for growth and proliferation of IL-2-dependent T cells. At present, the mechanism by which the high-affinity IL-2-IL-2 receptor interaction transmits a mitogenic signal to the cellular interior remains unclear. In this report we have used three murine T cell clones to demonstrate that IL-2 stimulates rapid tyrosine phosphorylation of several proteins. Two of these clones, CTLL-2 and CT6, exhibit a cytotoxic T cell phenotype, while the third, HT-2, was derived from a helper T cell line. All three T cell clones proliferated in response to IL-2 stimulation, but HT-2 cells also proliferated in response to interleukin-4 (IL-4). We comparatively examined the effects of IL-2 and IL-4 on protein tyrosine phosphorylation in these cells by immunoaffinity purification of phosphotyrosyl substrates with an anti-phosphotyrosine monoclonal antibody. Stimulation with concentrations of IL-2 resulting in maximal (10-30 U/ml) or sub-maximal (1-5 U/ml) proliferation caused the rapid tyrosine phosphorylation of 97 and 57 kDa proteins in all three cell lines. The 97 kDa protein was localized in the cytosol, while the 57 kDa protein was detected in both cytosolic and crude membrane fractions. IL-2-dependent tyrosine phosphorylation of an 86 kDa cytosolic protein was observed only in CT6 cells. Tyrosine phosphorylation of 22, 23 and 200 kDa proteins was also observed, but only in the cytotoxic T cell clones. Phosphoamino acid analyses revealed that the 97, 86 and 57 kDa proteins contained phosphotyrosine and phosphoserine residues. Concentrations of IL-2 below the threshold concentration for induction of a proliferative response correspondingly failed to stimulate protein tyrosine phosphorylation. In contrast, growth stimulation of HT-2 cells by IL-4 was not preceded by early changes in protein tyrosine phosphorylation, suggesting that protein tyrosine phosphorylation may not be essential for the induction of IL-4-dependent cell-cycle progression. These results demonstrate that high-affinity IL-2 receptors are coupled to tyrosine kinase activity(s) in T cells. However, the failure of IL-4 to stimulate protein tyrosine phosphorylation in the same cells indicates that enhanced protein tyrosine phosphorylation may not be requisite for growth factor-dependent T cell proliferation.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

Specificity of interleukin-2 receptor gamma chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway.

Interleukins 9 (IL-9) and 4 are cytokines within the IL-2 receptor gamma chain (IL-2R gamma) superfamily that possess similar and unique biological functions. The signaling mechanisms, which may determine cytokine specificity and redundancy, are not well understood. IRS proteins are tyrosine-phosphorylated following IL-9 and IL-4 stimulation, a process in part mediated by JAK tyrosine kinases (Yin, T. G., Keller, S. R., Quelle, F. W., Witthuhn, B. A., Tsang, M. L., Lienhard, G. E., Ihle, J. N., and Yang, Y. C. (1995) J. Biol. Chem. 270, 20497--20502). In the present study, we used 32D cells stably transfected with insulin receptor (32D(IR)), which do not express any IRS proteins, as a model system to study the requirement of different structural domains of IRS proteins in IL-9- and IL-4-mediated functions. Overexpression of IRS-1 and IRS-2, but not IRS-4, induced proliferation of 32D(IR) cells in response to IL-9. The pleckstrin homology (PH) domain of IRS proteins is required for IRS-mediated proliferation stimulated by IL-9. The phosphotyrosine binding and Shc and IRS-1 NPXY binding domains are interchangeable for IRS to transduce the proliferative effect of IL-4. Therefore, the PH domain plays different roles in coupling IRS proteins to activated IL-9 and IL-4 receptors. The role of IRS proteins in determining cytokine specificity was corroborated by their ability to interact with different downstream signaling molecules. Although phosphatidylinositol 3' -kinase (PI3K) and Grb-2 interact with tyrosine-phosphorylated IRS proteins, Shp-2 only binds to IRS proteins following IL-4, but not IL-9, stimulation. Although PI3K activity is necessary for the IRS-1/2-mediated proliferative effect of IL-9 and IL-4, Akt activation is only required for cell proliferation induced by IL-4, but not IL-9. These data suggest that IRS-dependent signaling pathways work by recruiting different signaling molecules to determine specificity of IL-2R gamma superfamily cytokines.