TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

Differential CD3/T cell antigen receptor-mediated IL-2 production in jurkat T cells. Dissociation of IL-2 response from total inositol phosphate and calcium responses

We used three anti-human anti-CD3 mAb each recognizing different surface CD3 epitopes to differentially perturb the CD3/TCR complex on the surface of Jurkat T cells. In the presence of phorbol ester, these anti-CD3 mAb triggered differential IL-2 production in Jurkat T cells, which could not be explained by differences in kinetics of IL-2 production, by differences in IL-2 adsorption caused by differential surface expression of p55 or p75 IL-2R, by effects on IL-2 secretion rather than actual synthesis, or by differential toxicities of the anti-CD3 mAb to Jurkat cells. In addition, this differential anti-CD3-induced IL-2 production could not be explained by differences in mAb isotype or in avidities of the anti-CD3 mAb for the Jurkat cells. Moreover, anti-CD3 mAb covalently immobilized onto beads also differentially induced IL-2 production in Jurkat cells, suggesting that the differential IL-2 response is not based on differential rates of anti-CD3-induced modulation of Jurkat cell surface CD3. Although differences among the anti-CD3 mAb in the initial rates of binding to Jurkat cell were observed, this was also believed unlikely to explain the differential IL-2 response. Regardless of the anti-CD3 mAb used, anti-CD3-induced total inositol phosphate (IP) production did not necessarily correlate with anti-CD3-induced IL-2 production. Nevertheless, despite the differences among the anti-CD3 mAb in inducing IL-2 production, the calcium responses were grossly similar. Taken together, these observations indicate that CD3/TCR-mediated IL-2 production in Jurkat cells can be dissociated from total IP generation, and the basis of differential CD3/TCR-mediated IL-2 production in these cells does not appear to be at the level of the initial activation-induced calcium response. These studies suggest that the nature of the CD3/TCR ligand (its physical form and/or the specific epitope it perturbs) can either directly influence intracellular events distal to the generation of IP and increase in intracellular free calcium leading to differential IL-2 production or can trigger IP-independent pathways that affect IL-2 production.     

L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.     

Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production in CD4+ T cells

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans, the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-, strain-, and species-specific, which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line, Jurkat, is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells, whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover, anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well, cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT, but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically, anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells, thereby blocking functions that are pivotal in the regulation of immune responses.     

Interaction of human MUC1 and beta-catenin is regulated by Lck and ZAP-70 in activated Jurkat T cells

The MUC1 transmembrane glycoprotein is aberrantly expressed by diverse hematologic malignancies, including those of the T cell lineage. The MUC1 cytoplasmic domain (CD) interacts with beta-catenin; however, the role of MUC1 in T cells is not known. In the present work, MUC1 was studied as a potential downstream effector of the Lck and ZAP-70 tyrosine kinases that are essential for T cell activation. The results demonstrate that anti-CD3-induced or PMA+ionomycin-induced activation of Jurkat T cells is associated with increased binding of MUC1 and Lck. Lck phosphorylates MUC1-CD on Y-46 and, in turn, stimulates the binding of MUC1 to beta-catenin. The results further demonstrate that MUC1 interacts with ZAP-70. In contrast to Lck, ZAP-70 phosphorylates MUC1-CD predominantly on Y-20. However, like Lck, ZAP-70-mediated phosphorylation of MUC1 Y-20 stimulates binding of MUC1 and beta-catenin. These findings indicate that MUC1 functions as a substrate for Lck and ZAP-70 in activated Jurkat T cells and that MUC1 integrates T cell receptor signaling with the beta-catenin pathway.     

Inhibition of the anti-CD3-induced T cell proliferation by crosslinking of stimulatory antibodies in the presence of PMA and interleukin-2.

Anti-CD3 antibodies are directed to the nonpolymorphic part of the T cell receptor complex and may activate human peripheral T cells. Under some circumstances crosslinked anti-CD3 has been described to augment the proliferative response. Here we demonstrate that crosslinking of stimulatory anti-CD3 antibodies by anti-IgG in cell suspension abolishes their effect on proliferation of human resting peripheral T cells in the presence of PMA and/or IL-2. This effect was observed within a wide range of anti-CD3 concentrations (1 ng/ml to 1 microgram/ml) independent of the presence of monocytes. The inhibition was not due to the induction of cell death, since cells remained propidium iodide-negative after treatment. Protein-tyrosine phosphorylation after anti-CD3 crosslinking was more pronounced than in the presence of noncrosslinked anti-CD3. This indicates that the signal was transmitted after anti-CD3 crosslinking, however, it was unable to induce T cell proliferation. Reduced IL-2 receptor expression after anti-CD3 crosslinking and the inability of exogenous IL-2 to restore the proliferative response might indicate a reduced susceptibility to IL-2 as a reason for the described phenomenon.     

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.     

Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B cell collaboration

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4(+) T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4(+) T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.     

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.     

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.     

Interleukin-1 stimulates diacylglycerol production in T lymphocytes by a novel mechanism

We have investigated the biochemical mechanism by which interleukin-1 (IL-1) serves as a comitogen with agents that directly activate the antigen receptor in T lymphocytes. We have studied the human T cell line Jurkat, which can be stimulated to produce Interleukin-2 by treatment with antibodies that bind to the CD3-antigen receptor complex and hence represents a model system for T cell activation. Using highly purified, recombinant human IL-1, we show that IL-1 stimulates rapid diacylglycerol and phosphorylcholine production from phosphatidylcholine (PC) in the absence of phosphatidylinositol turnover in Jurkat cells. This effect is also observed in peripheral blood T cells and a murine T cell line. The EC50 for IL-1 was 28 fM, and PC hydrolysis was detectable within 5 sec at 37 degrees C. The murine cell line had typical high-affinity IL-1 receptors (kd = 7 X 10(-11) M). However, we were unable to detect IL-1 binding to Jurkat cells. This reaction occurs via a novel mechanism and may explain the comitogenic activity of IL-1 in T lymphocyte activation as well as many of the pleiotropic biologic effects of this cytokine.     

TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.     

Expression of prohibitins on the surface of activated T cells

Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.     

Induction of human T cell proliferation by a monoclonal antibody to CD5.

A mouse mAb, TS 43, which recognized the human CD5 molecule, was found to induce the proliferation of human peripheral blood T cells. TS 43 mAb precipitated from 125I-radiolabeled T cells a 67-kDa band, which comigrated with the 67-kDa band precipitated by the anti-CD5 mAb OKT1. Preclearing of cell lysates with OKT1 mAb abolished the capacity of TS 43 mAb to precipitate radiolabeled material from T cell lysates. Furthermore, a mouse T cell hybridoma transfected with human CD5 was stained by TS 43 mAb. T cell proliferation mediated by TS 43 mAb was monocyte dependent, and was accompanied by IL-2R expression and by IL-2 synthesis. T cell activation by TS 43 mAb involved a rise in intracellular calcium level (CA2+)i and was dependent on the expression of the TCR/CD3 complex because no rise in (Ca2+)i was observed in a TCR-beta-deficient Jurkat T cell mutant. This study indicates that CD5 should be added to the list of surface molecules that can signal T cells to proliferate.     

Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity

TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.