New approaches to transplant immunosuppression.

Although considerable progress has been achieved using immunosuppressive drugs that inhibit lymphocyte activation and T-cell cytokine signal transduction pathways, the widespread tissue distribution of the molecular targets exploited to date, calcineurin, mammalian target of rapamycin, and inosine monophosphate dehydrogenase, engenders a constellation of collateral toxicities. One strategy to develop new immunosuppressants seeks to identify targets that are critical for and specific to the adaptive immune response. Three approaches have been used to guide this enterprise; molecular design based on steric resemblance of the antagonist to the natural ligand; construction of complementary DNA oligonucleotides that hybridize with the leader sequence of messenger RNA encoding the synthesis of the specific target, thereby preventing production of that protein; and functional comparisons based on similar inhibitory profiles of candidate compounds and a probe that blocks the target nonselectively. Use of these 3 technologies has led to identification of antagonists blocking selectins, intercellular adhesion molecule-1, or Janus kinase 3, respectively. These lead compounds have been tested for their effects on the alloimmune response and/or the ischemia-reperfusion injuries.

Molecular actions of sirolimus: sirolimus and mTor.

Recent therapeutic strategies to combat organ allograft rejection have focused on T-cell signaling pathways and the molecules that comprise them. The macrolide antibiotic produced by the bacterium Streptomyces hygroscopicus, known as sirolimus or rapamycin, has shown great therapeutic potential in the transplant setting. Sirolimus alone or in combination with other immunosuppressive agents can block acute rejection, chronic graft destruction, and promote permanent allograft acceptance. Sirolimus targets a unique serine-threonine kinase, mammalian target of rapamycin (mTor), which plays a key role in mitogenic and nutritional cells signals. Within T cells, mTor regulates a number of proteins likely dependent on T cell growth factors such as interleukin 2. This review is focused on the molecular mechanisms by which mTor may regulate T-cell signaling cascades and affect T-cell responsiveness, and how sirolimus likely uncouples this activity.

Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression.

Milk production remains suppressed in mammals during late pregnancy despite high levels of lactogenic polypeptide hormones. At parturition, associated with a precipitous fall in circulating progesterone, rising glucocorticoid levels synergize with prolactin to initiate copious milk production. This synergy is mediated at least in part through the coordinated activation of glucocorticoid receptors and transcription factor Stat5, particularly Stat5a. Here we show that two proline-juxtaposed serine residues within the transactivation domain of Stat5a are phosphorylated in the mammary gland during late gestation and lactation, and that these phosphorylation sites inhibit the transcriptional activity of Stat5a in the absence of glucocorticoid receptor costimulation. Specifically, transfection assays revealed that phosphorylation of residues S725 and S779 of Stat5a cooperatively suppressed prolactin-stimulated transcription from the beta-casein promoter in both COS-7 kidney and MCF-7 mammary cells. This suppression was associated with shortened duration and reduced amplitude of nuclear DNA binding activity of wild type Stat5a relative to that of the serine phosphorylation-defective Stat5 mutant. However, costimulation of glucocorticoid receptors completely reversed the suppressive effect of Stat5a serine phosphorylation on beta-casein gene transcription. We propose that serine phosphorylation within the transactivation domain may limit the activity of Stat5a in the absence of proper coactivation by glucocorticoid receptors.

Selective inhibition of IL-2 gene expression by IL-2 antisense oligonucleotides blocks heart allograft rejection.

PURPOSE: We tested the effects of selective inhibition of interleukin (IL)-2 gene expression by IL-2 antisense oligonucleotide (oligo) with phosphorothioate (PS)/phosphodiester (PD)/2'-methoxyethyl (ME) modifications (17359) on T-cell function and the survival of heart allografts in mice. METHODS: The PS- (17328) or PS/PD/ME- (17359) IL-2 oligo was electroporated to mouse T cell lymphoma cells (TIB 155) stimulated with concanavalin A (Con A). Expression of IL-2 was analyzed by an ELISA spot assay and a reverse transcript polymerase chain reaction method. C3H (H-2k) mice transplanted with BALB/c (H-2d) heart grafts were treated i.v. with a 7-day osmotic pump with 20 mg/kg 17359 alone or in combination with sirolimus (SRL). RESULTS: In comparison with untreated controls, 500 to 2000 nM 17328 inhibited IL-2 protein production by 21.8% to 47.2%, whereas 500 to 2000 nM 17359 did so by 35.5% to 83.5% (both P<0.001). In vivo, 20 mg/kg 17359 prolonged survivals to a mean survival time (MST) of 18.3+/-2.6 days (P<0.001) in comparison with only 8.2+/-0.8 days in untreated controls. Although 0.2 mg/kg SRL alone produced a MST of 18.8+/-6.0 days (P<0.01), addition of 20 mg/kg 17539 synergistically extended survivals to 54.3+/-12.1 days (P<0.001). As expected, IL-2 mRNA, but not IL-7, IL-9, or IL-15 mRNA, was reduced in allografts from recipients treated with 17359 compared with untreated controls. Lymph node cells from the same recipients displayed reduction in proliferative response to donor alloantigen and in generation of alloantigen-specific cytotoxic T cells. CONCLUSION: Selective inhibition of IL-2 mRNA in vivo inhibits T-cell function and extends allograft survival.

Concomitant inhibition of Janus kinase 3 and calcineurin-dependent signaling pathways synergistically prolongs the survival of rat heart allografts.

The cytoplasmic localized Janus tyrosine kinase 3 (Jak3) is activated by multiple cytokines, including IL-2, IL-4, and IL-7, through engagement of the IL-2R common gamma-chain. Genetic inactivation of Jak3 is manifested as SCID in humans and mice. These findings have suggested that Jak3 represents a pharmacological target to control certain lymphoid-derived diseases. Using the rat T cell line Nb2-11c, we document that tyrphostin AG-490 blocked in vitro IL-2-induced cell proliferation (IC(50) approximately 20 microM), Jak3 autophosphorylation, and activation of its key substrates, Stat5a and Stat5b, as measured by tyrosine/serine phosphorylation analysis and DNA-binding experiments. To test the notion that inhibition of Jak3 provides immunosuppressive potential, a 7-day course of i.v. therapy with 5-20 mg/kg AG-490 was used to inhibit rejection of heterotopically transplanted Lewis (RT1(l)) heart allografts in ACI (RT1(a)) recipients. In this study, we report that AG-490 significantly prolonged allograft survival, but also acted synergistically when used in combination with the signal 1 inhibitor cyclosporin A, but not the signal 3 inhibitor, rapamycin. Finally, AG-490 treatment reduced graft infiltration of mononuclear cells and Stat5a/b DNA binding of ex vivo IL-2-stimulated graft infiltrating of mononuclear cells, but failed to affect IL2R alpha expression, as judged by RNase protection assays. Thus, inhibition of Jak3 prolongs allograft survival and also potentiates the immunosuppressive effects of cyclosporin A, but not rapamycin.

Involvement of the ubiquitin-proteasome pathway in the degradation of nontyrosine kinase-type cytokine receptors of IL-9, IL-2, and erythropoietin.

The ubiquitin-dependent proteasome-mediated (Ub-Pr) degradation pathway has been shown to regulate a large variety of substrates, including nuclear, cytosolic, and membrane proteins. In mammalian systems, polyubiquitin modification has been identified in a number of cell surface receptors for more than a decade; however, its biological significance has remained unclear until recently. For growth factor receptors with intrinsic tyrosine kinase domains, polyubiquitination is believed to trigger the internalization and subsequent degradation via the lysosomal pathway. In this study we provide the first evidence that non-tyrosine kinase-type cytokine surface receptors, IL-9R alpha-chain, IL-2 receptor ss-chain, and erythropoietin receptor, can be polyubiquitinated and degraded by proteasomes. The Ub-Pr pathway regulates both the basal level turnover and the ligand-induced degradation of the receptors. A previously identified putative molecular chaperon, valosin-containing protein, undergoes tyrosine phosphorylation in a cytokine-dependent manner and associates with the receptor complexes following receptor engagement, suggesting that valosin-containing protein may target the ubiquitinated receptors to the proteasome for degradation.

Functional uncoupling of the Janus kinase 3-Stat5 pathway in malignant growth of human T cell leukemia virus type 1-transformed human T cells.

Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 microM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr(694)), and Stat5b (Tyr(699)); 2) serine phosphorylation of Stat5a (Ser(726)) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive beta-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-kappaB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.

Selective disruption of interleukin 4 autocrine-regulated loop by a tyrosine kinase inhibitor restricts activity of T-helper 2 cells.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4-mediated proliferation of both D10 and the IL-4-dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor alpha activation of NF-kappaB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4. (Blood. 2000;95:3816-3822)

Targeted disruption of stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4-driven T(H)2 cell response.

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates T(H)2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)-4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4-inducible cell proliferation of murine T(H)2 cells and primary human CD4(+) T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non-Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4-driven T(H)2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential. (Blood. 2000;95:1249-1257)

Tyrphostin AG-490 inhibits cytokine-mediated JAK3/STAT5a/b signal transduction and cellular proliferation of antigen-activated human T cells.

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)). Genetic inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid-derived diseases. Here we provide novel evidence that AG-490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use gamma(c). AG-490 also inhibited IL-2-mediated proliferative growth in human T cells with an IC50) = 25 microM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen-specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti-CD3 stimulation of human T cells. Taken together, these findings suggest that AG-490 inhibits the JAK3-mediated Type II signaling pathway but not the T cell receptor-derived Type I pathway and possesses therapeutic potential for T cell-derived pathologies such as graft-versus-host disease, allergy, and autoimmune disorders.

JAK3, STAT, and MAPK signaling pathways as novel molecular targets for the tyrphostin AG-490 regulation of IL-2-mediated T cell response.

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor, these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study, evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation, a non-JAK2-dependent signal, in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates, STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1, -3, -5a, and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways and their cellular consequences. Taken together, these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways.