Selective formation of ErbB-2/ErbB-3 heterodimers depends on the ErbB-3 affinity of epidermal growth factor-like ligands

EGF-like growth factors activate their ErbB receptors by promoting receptor-mediated homodimerization or, alternatively, by the formation of heterodimers with the orphan ErbB-2 through an as yet unknown mechanism. To investigate the selectivity in dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/transforming growth factor alpha chimera T1E as the template molecule because it binds to ErbB-3 homodimers with low affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many phage variants were selected with enhanced binding affinity for ErbB-3 homodimers, indicating that C-terminal residues contribute to the interaction with ErbB-3. These variants were also potent ligands for ErbB-2/ErbB-3 heterodimers despite negative selection for such heterodimers. In contrast, phage variants positively selected for binding to ErbB-2/ErbB-3 heterodimers but negatively selected for binding to ErbB-3 homodimers can be considered as "second best" ErbB-3 binders, which require ErbB-2 heterodimerization for stable complex formation. Our findings imply that epidermal growth factor-like ligands bind ErbB-3 through a multi-domain interaction involving at least both linear endings of the ligand. Apparently the ErbB-3 affinity of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes or also ErbB-3 homodimers. Because no separate binding domain for ErbB-2 could be identified, our data support a model in which ErbB heterodimerization occurs through a receptor-mediated mechanism and not through bivalent ligands.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

Interleukin 2 receptor blockade by anti-Tac antibody inhibits IFN-gamma induction

Anti-Tac antibody, which binds to the interleukin 2 (IL-2) receptor and thus blocks IL-2 binding to and activation of T lymphocytes, was used to investigate the role of IL-2 in interferon-gamma (IFN-gamma) production. Three T-cell mitogens (phytohemagglutinin, concanavalin A, and the pan-T monoclonal antibody OKT3) were used as IFN-gamma inducers. In each case, anti-Tac antibody clearly inhibited IFN-gamma production. This occurred even under conditions where cellular proliferation (as measured by incorporation of [3H]thymidine) was only slightly inhibited. The inhibitory effects of anti-Tac were reversed by the addition of purified IL-2. Therefore, endogenous production of IL-2 and its binding to the IL-2 receptor are needed for maximum IFN-gamma production.     

A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses

CD4＋ helper T （TH） cells play crucial roles in immune responses. Recently a novel subset of TH cells, termed THIL-17, TH 17 or inflammatory TH （THi）, has been identified as critical mediators of tissue inflammation. These cells produce IL-17 （also called IL-17A） and IL-17F, two most homologous cytokines sharing similar regulations. Here we report that when overexpressed in 293T cells, IL-17 and IL-17F form not only homodimers but also heterodimers, which we name as IL-17A/F. Fully differentiated mouse THi cells also naturally secrete IL-17A/F as well as IL-17 and IL-17F homodimeric cytokines. Recombinant IL-17A/F protein exhibits intermediate levels of potency in inducing IL-6 and KC （CXCL 1） as compared to homodimeric cytokines. IL-17A/F regulation of IL-6 and KC expression is dependent on IL-17RA and TRAF6. Thus, IL-17A/F cytokine represents another mechanism whereby T cells regulate inflammatory responses and may serve as a novel target for treating various immune-mediated diseases.     

Tac antigen forms disulfide-linked homodimers

Interleukin 2 (IL-2) is responsible for stimulating T-cell proliferation via interaction with specific, high-affinity membrane receptors. Although an IL-2-binding protein has been identified by virtue of its reactivity with a monoclonal antibody that competes with IL-2 for binding (anti-Tac), the complete and precise structure of functional IL-2 receptors is still unknown. To define further the composition of IL-2 receptors, both IL-2 itself and anti-Tac were used as ligands to adsorb membrane proteins for analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). A variety of experimental approaches yield results indicating that the Tac antigen, which migrates as a single protein on NaDod-SO4/PAGE under reducing conditions, is also expressed as disulfide-linked homodimers and oligomers. Examined under nonreducing conditions, both activated normal human T cells and cell lines from patients with adult T cell leukemia (HUT-102; MT-1) express Tac antigen homodimers (Mr 105,000) in addition to monomers (Mr 54,000). Formation of the disulfide bond is not a consequence of the experimental procedures used to isolate the proteins for analysis, inasmuch as identical results are obtained when the receptor proteins are iodinated and extracted in the presence of N-ethylmaleimide or prepared for electrophoresis in the absence of heat denaturation. Accordingly, these findings point to Tac antigen associating with itself preferentially. The physiologic significance of homodimer and oligomer formation, especially as it relates to the formation of high-affinity IL-2 receptors, is presently unknown.     

Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2

The mechanism whereby noncognate contact with activated IL-2-producing Type 1 helper T cells (TH1) induces B cell activation was examined. Small resting B cells from C57B1/6 mice were cultured, in the absence of any ligand for surface Ig, with irradiated cells of the hapten-specific, CBA-derived, F23.1+ TH1 clone E9.D4 in F23.1 (anti-T cell receptor V-beta 8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-gamma mAb partially inhibited Ig secretion, without affecting entry into cycle. LPS responses or T cell activation. Other antibodies (anti-IL-3, IL-4, IL-5, Thy-1.2, CD5) were not inhibitory. After 2 days of culture with F23.1-activated T cells, B cells appeared to have become responsive to IL-2, in that they could be driven to immunoglobulin production by the addition of IL-2. Flow cytometry showed no expression by these B cells of 55-kDa (Tac) IL-2 receptors. Also, rigorous removal of T cells from 2-day cocultures prevented the response to IL-2, and readdition of T cells restored it. Because the reconstituted responses were inhibited both by anti-IL-2 and by anti-Tac, IL-2 must have acted indirectly, via the T cells that were present in these cultures. Continued contact with T cells was therefore necessary for the progression of B cells to antibody secretion.     

Developmental and functional analyses of CD8(+) NK1.1(+) T cells in class-I-restricted TCR transgenic mice.

Using a class-I-restricted T cell receptor (TCR) transgenic mice (Tgm), 2C (Valpha3.1/Vbeta 8.2, specific for L(d) + LSPFPFDL), the development and cytokine production of tg-TCR(+) NKT cells were analyzed. We found that CD8(+) or double negative (DN) NKT cells constituted a major population of NKT cells in the H-2(b/b) 2C Tgm (positive selecting background) or the H-2(b/d) 2C Tgm (negative selecting background), respectively. Virtually no NKT cells were generated in the H-2(k/k) 2C Tgm (neutral selecting background). CD8(+) NKT cells in the H-2(b/b) 2C Tgm expressed CD8alphabeta heterodimers, whereas those in the H-2(b/d) 2C Tgm expressed CD8alphaalpha homodimers. These findings suggest that development of a subpopulation of NKT cells is influenced by the H-2 molecules. Upon stimulation with anti-CD3 mAb, tg-TCR(+) NKT cells generated in the H-2(b/b) and H-2(b/d) backgrounds produced IFN-gamma, but not IL-4.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Stepwise activation of T cells. Role of the calcium ionophore A23187

The calcium ionophore A23187, at a concentration of 1 microgram/ml, is able to stimulate proliferation of freshly isolated peripheral blood lymphocytes, CD4+-enriched cells, or CD8+-enriched cells as measured by [3H]thymidine incorporation. This proliferation is accompanied by an increase in interleukin 2 (IL-2) receptor expression but not by a detectable up-regulation in (IL-2) production or the development of cytotoxicity. Proliferation can be blocked by anti-CD3, CD4, or CD8 monoclonal antibodies, but not by anti-Tac. If CD8+-enriched cells are activated for 3 days with A23187 and the blasts present on day 3 are sorted and returned to culture, they rapidly develop cytolytic activity in the presence of recombinant IL-2 but not recombinant interferon-gamma. CD4+ enriched cells, after activation with A23187, do not become cytotoxic in the presence of either recombinant IL-2 or recombinant interferon-gamma. These findings permit study of the stepwise maturation of T cells in this alternative pathway by using "minimal signals" that do not, by themselves and as used in these studies, stimulate precursor Tc to mature to full effector cytotoxic function. These findings are consistent with the model that A23187 drives T cells only part way along a pathway of maturation and that an additional second signal must be given to effect maturation of cytotoxic status.     

Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity

Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

在CHO细胞中表达具有高效成骨活性的BMP-2/7异源二聚体

PCR扩增BMP-2与BMP-7的编码基因, 利用重叠PCR以柔性肽(Gly4Ser)5编码序列将二者串连并克隆到质粒pIRESneo3上, 转染CHO-K1细胞得到混合稳定克隆。ELISA检测培养液中BMP-2/7异源二聚体蛋白的表达水平为230.75±13.34 ng/mL, 以此为条件培养基处理成骨细胞株MC3T3, 对照组为分别含有CHO-K1细胞及大肠杆菌表达的BMP-2同源二聚体以及PBS的条件培养基。结果发现碱性磷酸酶染色与茜素红染色差异明显, 定量RT-PCR显示分子指标OC、ALP、Runx2与Osx的转录水平明显增高(P<0.05), Luciferase报告基因检测BMP/Smad通路活性较对照组升高明显(P<0.05)。首次设计构建了BMP-2/7异源二聚体蛋白, 其成骨活性显著高于BMP-2同源二聚体。     

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.