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Radiotherapy (RT) allows for tumor control through the cytotoxic action of ionizing radiation (IR). Although modern technologies permit precise IR delivery to the tumor mass while minimizing exposure of surrounding healthy tissues, the efficacy of RT remains limited by the intrinsic or acquired radioresistance of many tumors. There is thus an ongoing search for agents that augment the sensitivity of tumor cells to IR cytotoxicity, with recent interest in targeting components of signaling pathways involved in tumor growth and radioresistance. Here, we review the evidence suggesting that disabling one of these components, the mechanistic target of rapamycin (mTOR) kinase, may enhance RT efficacy. This molecule constitutes the catalytic subunit of the mTORC1 and mTORC2 protein complexes, which regulate cell growth and other processes implicated in tumorigenesis. Much work has focused on mTORC1 because it is selectively blocked by the microbial product rapamycin and its analogs (collectively designated rapamycins) that are approved for cancer treatment, and is frequently hyperactivated in malignant cells. In several, but not all human cancer cell lines, rapamycins increased IR cytotoxicity in vitro, apparently through multiple mechanisms, including the promotion of autophagic cell death. Rapamycins also potentiated fractionated RT in tumor xenograft models, in part by suppressing tumor angiogenesis. Synthetic kinase inhibitors that simultaneously target PI3K and both mTOR complexes also enhanced RT in vitro and in vivo, but with greater efficiency than rapamycins. These encouraging data have led to early clinical trials of rapamycins and catalytic mTOR inhibitors combined with RT in various cancers.

Introduction: Autophagy is an intracellular process of self-digestion involving the lysosomal degradation of cytoplasmic organelles and macromolecules. It occurs at low basal levels to perform housekeeping functions and is dramatically augmented upon nutrient depletion or exposure to other stresses, thus maintaining cellular homeostasis and energy balance and providing cytoprotective responses to adverse conditions. Mounting evidence that autophagy malfunction contributes to the pathogenesis of diverse human diseases has stimulated efforts to identify pharmacological agents that modulate autophagy in potentially beneficial ways. Here, we review the progresses accomplished toward this goal in recent years, as reflected by the patent literature., Areas Covered: Patent applications published from 2008 to mid-2012 that pertain to the pharmacological modulation of autophagy are reviewed and their potential therapeutic utilities are discussed., Expert Opinion: Of 40 patents related to autophagy, 21 claim novel enhancers or inhibitors of autophagy. One of the most promising applications of these compounds concerns cancer therapy, a few of them being already considered for clinical evaluation. Further work is, however, needed to identify compounds that target unique molecular effectors/regulators of autophagy to selectively modulate its various stages in different tissues and to design therapeutic interventions applicable to a broad variety of dysfunctional autophagy-associated disorders.

Protein Design Labs is developing fontolizumab, a humanized anti-interferon gamma monoclonal antibody, for the potential treatment of autoimmune diseases. By February 2003, the company was actively seeking to outlicense the development and commercialization rights to fontolizumab outside of the US and Canada.

Mitsubishi Pharma Corp and Novartis AG are developing fingolimod, an orally active immunosuppressant affecting lymphocyte re-circulation, for the potential prevention of transplant rejection and the treatment of autoimmune diseases, including multiple sclerosis. Fingolimodis a synthetic sphingosine analog that becomes phosphorylated in vivo and acts as a sphingosine-1-phosphate receptor agonist.

Bristol-Myers Squibb is developing the fusion protein abatacept for the potential treatment of various immunological disorders, including rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. Abatacept is undergoing phase III clinical trials.

ISAtx-247 is a ciclosporin A analog under development by Isotechnika and Roche as an immunosuppressant for the potential prevention of organ rejection after transplantation, and for the potential treatment of autoimmune diseases such as rheumatoid arthritis, psoriasis and type 1 diabetes. A phase II renal transplantation study had been completed by June 2003.

CXCR6, the receptor for the membrane-anchored chemokine, CXCL16, is expressed on a subset of CCR5-bearing memory T cells, and may play a role in recruiting these cells to sites of inflammation. Here, we set out to determine the effect of T cell activation on CXCR6 expression. Highly purified human peripheral blood T cells were cultured for 7-8 days in presence of IL-2 (400 U/ml) to enhance CXCR6 expression. Overnight stimulation with anti-CD3 mAb+anti-CD28 mAb, which resulted in CD69 induction and cytokine (IL-2 and IFN-gamma) production, reduced cell surface expression of CXCR6 by 85% and that of CCR5 by 76%. The Ca(2+) ionophore, ionomycin (125-500 ng/ml), also markedly diminished CXCR6 expression (85%), but without inducing CD69 expression or cytokine production, and reduced CCR5 expression by only 40%. In contrast, the phorbol esters, PdBu or PMA had little effect on CXCR6 expression (23% reduction) but induced CD69 expression and caused a profound down-regulation (92%) of CCR5 expression. Moreover, CCR7, whose expression was low on CXCR6(+) T cells, was little affected by any of these modes of activation. The down-regulation of CXCR6 expression induced by CD3/CD28 activation was blocked by the broad kinase inhibitor, staurosporine, and by the src kinase inhibitor, PP2, but not by the MEK1 inhibitor, U0106. Most interestingly, the calcineurin inhibitor, FK506, consistently inhibited CD3/CD28-induced CXCR6 down-regulation. FK506 also blocked the decrease of CXCR6 expression caused by ionomycin, whereas staurosporine or PP2 had no effect on this decrease. Altogether, these data indicate that CXCR6 expression is down-regulated, independent of CCR5 or CD69 expression and of cytokine induction, by T cell activation signals that involve predominantly the Ca(2+)-dependent calcineurin pathway.

IDEC, in collaboration with Eisai, is developing IDEC-131 (E6040), a humanized monoclonal antibody (mAb) against CD154, the ligand for CD40 also called CD40L or gp39, for the potential treatment of several autoimmune diseases. IDEC-131 is based on technology that IDEC licensed from Dartmouth Medical School where researchers demonstrated the biological effects of the anti-CD154 antibody in animal models of autoimmunity. In January 2001, phase II trials in psoriasis and idiopathic thrombocytopenic purpura (ITP) were initiated. By january 2002, a phase II trial in Crohn's disease was also ongoing. A pilot, multicenter, multiple-dose phase I trial in moderate-to-severe psoriasis was initiated in January 2001. This trial was ongoing in January 2002. IDEC, in collaboration with Dartmouth Medical School had also initiated a phase I trial in multiple sclerosis by March 1999. IDEC-131 was also previously being developed for systemic lupus ezythematosus (SLE), although no further development for this indication has been reported since the disclosure of disappointing phase II results in April 2000. Analysts at Morgan Stanley predicted in February 2002, that the product would be launched in 2005, with sales of US $25 million, rising to US $75 million in 2006.

CAMPATH (CAMPATH-1H, alemtuzumab, MabCAMPATH), is a lymphocyte-depleting humanized monoclonal antibody that was recently approved in the USA and Europe for the treatment of chronic lymphocytic leukemia (CLL). It targets CD52--a small glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed on normal T- and B-lymphocytes and on a large proportion of lymphoid cell malignancies--but not on hematopoietic progenitor cells. CAMPATH was shown to be effective against CLL refractory to chemotherapy with an acceptable toxicity profile. CAMPATH is also active against T-cell prolymphocytic leukemia and has been extensively used to prevent graft-versus-host disease associated with bone marrow transplantation. CAMPATH is owned by ILEX Pharmaceuticals LP and distributed by Schering AG and its US affiliate Berlex Laboratories.

Everolimus (RAD-001, SDZ RAD, Certican), an analog of sirolimus, is an oral immunosuppressant that inhibits growth factor-induced cell proliferation, under development by Novartis as a potential treatment for transplant rejection. Phase III trials were initiated by the end of 1998 [319337] and were ongoing in February 2001 [400448]. At the end of 2000, Novartis was hoping to file for approval of the compound in 2001 [392881], with a possible launch in mild-2002 [392881], [401979]. Completion of phase III trials in heart transplant patients is expected this year and lung and liver transplants by 2003. In 1999, American Home Products (AHP) initiated an action for infringement of the patent EP-00401747, which covers the use of sirolimus in transplantation in the UK, the Netherlands and Germany, seeking to restrain the clinical trial program for everolimus. Novartis subsequently filed a counterclaim for invalidity. In December 1999, the UK High Court of Justice ruled that everolimus infringes the British counterpart of EP-00401747 [349637]. In contrast, in April 2000, the District Court of The Hague ruled that everolimus does not infringe patent rights licensed to AHP [362823] and in July 2000, The Court of Appeal in the UK came to the same conclusion [376559]. In February 2001, the Opposition Board at the European Patent Office upheld Novartis' European patent for everolimus, which the Board held to be 'inventive' [400448]. In July 2000, Vontobel estimated sales of SFr 80 million in 2002, rising to SFr 800 million in 2004 [378871]. In February 2001, Merrill Lynch predicted sales of SFr 125 million rising to SFr 661 million in 2005 [411704].

Due to the improvement in the understanding of the anti-allogenic immune response, the success of transplantation medicine has increased rapidly over the last two decades. The knowledge that the T-lymphocyte played an integral role in transplant rejection, brought cyclosporine A and FK-506 to the fore as therapeutic immunosuppressants. However, the current mainstays in transplant rejection are not without their problems and many drug companies are exploring the possibilities of improving the available therapies by developing drugs with reduced toxicity, improved long-term survival and efficacy against chronic rejection and improved immunosuppressive selectivity. The advances in the understanding of T-cell activation and lymphocyte trafficking has highlighted ways to improve the existing therapies and more selective immunosuppressant targets.

Alemtuzumab, a lymphocyte-depleting humanized monoclonal antibody, is being developed by Millennium Pharmaceuticals Inc and ILEX Oncology for the potential treatment of chronic lymphocytic leukemia (CLL) [274580]. The utility of the compound for treating bone marrow (BM) stem cell transplantation-associated graft-versus-host disease (GVHD) [372946] and for ex vivo purging of BM to remove malignant T-cells [244056] is also being investigated. Additional potential therapeutic areas for which clinical trials are planned or ongoing include vasculitis, multiple sclerosis [288762] and organ transplantation [338304]. A Biologics License Application (BLA) was filed with the FDA in December 1999 by ILEX and Millennium [351523], [351524], [373873]. The FDA accepted the application for filing in February 2000 [355775] and returned a complete response letter in June 2000 [372172]. Millennium and ILEX submitted a response to the FDA in August 2000 [379766]. Alemtuzumab has received Fast Track designation [304771] and orphan drug status from the FDA [288762], and the drug was reviewed by the FDA's Oncologic Drugs Advisory Committee on 14 December, 2000 [387228]. The committee voted 14 to 1 to recommend accelerated approval of alemtuzumab for patients with CLL who have been treated with alkylating agents and who have failed fludarabine therapy [393778], [393894]. In March 2000, Millennium and ILEX also submitted a Marketing Authorization Application (MAA) for alemtuzumab to the European Agency for the Evaluation of Medicinal Products (EMEA) [363595]. In October 2000, EMEA accepted the MAA for alemtuzumab under the agency's centralized approval procedure [387228]. Alemtuzumab was originally synthesized by Herman Waldmann and colleagues at Cambridge University and licensed to Burroughs Wellcome (BW) via the British Technology Group (BTG) [162622]. BW conducted phase I and II trials for a broad range of indications, but then discontinued development because of disappointing results in phase II rheumatoid arthritis trials [326848]. In April 1997, LeukoSite licensed rights to the antibody from BTG for the treatment of CLL and prolymphocytic leukemia, plus an option to develop it for other indications. BW agreed to supply LeukoSite with intellectual property [244056], [326848]. In May 1997, LeukoSite entered into a joint venture with ILEX Oncology for the further development of alemtuzumab [245986]. By the end of 1999, Millennium acquired LeukoSite with commitment to pursue development of the compound through the joint venture Millennium & ILEX Partners LP [351523], [370237]. In August 1999, Schering AG and its US affiliate Berlex Laboratories obtained exclusive worldwide marketing rights for alemtuzumab, excluding Japan and East Asia. In the US, Berlex, Millennium and ILEX will divide profits from alemtuzumab sales equally [337702], [338837].

The macrolactam natural product, FK506 (Tacrolimus), acts as a powerful and clinically useful immnosuppressant through disruption of signaling events mediated by the calcium-dependent serine/threonine protein phosphatase, calcineurin (CaN), in T lymphocytes. Its mechanism of action involves the formation of a molecular complex with the intracellular FK506-binding protein-12 (FKBP12), thereby acquiring the ability to interact with CaN and to interfere with its access to and dephosphorylation of various substrates. Among the CaN substrates whose activity is altered by FK506, the nuclear factors of activated T cells (NFAT), a family of transcription factors regulating lymphokine gene expression, have been shown to play a prominent role in FK506-induced immunosuppression. Over the past few years, additional members of the FKBP and NFAT families of proteins have been identified, providing further insights into the complexity of FK506 biological effects. Furthermore, it has become clear that, predominantly as a result of CaN inhibition, FK506 alters multiple biochemical processes in a variety of cells besides lymphocytes. This may account for the adverse side effects of the drug, including neurotoxicity and nephrotoxicity. Extensive medicinal chemistry efforts have been devoted to the generation of analogs of FK506 with the hope of identifying compounds with an improved therapeutic index, that could have broader therapeutic utility than the parent drug. These efforts yielded several compounds with unique biochemical attributes, showing evidence for a dissociation between immunosuppressive and toxic properties, which may pave the way towards designing safer FK506-related immunosuppressants.

Bristol-Myers Squibb is developing CTLA4-Ig, an immunosuppressant immunoglobulin, for the potential treatment of various immunological disorders, including graft versus host disease, lupus erythematosus and psoriasis. A phase II trial has commenced for psoriasis. The compound is also in development for inflammation, rheumatoid arthritis and allergy. A collaboration with Genzyme Transgenics covers the following indications: psoriasis; organ transplant rejection; and several autoimmune disorders.

Hypothemycin, a resorcylic acid lactone antibiotic, was identified as active in a screen for inhibitors of T cell activation. It was found to inhibit the proliferation of mouse and human T cells stimulated with anti-CD3 mAb + PMA and of human PBMC stimulated with anti-CD3 mAb alone. This inhibition was partially reversed by exogenous IL-2 indicating that it is not due to non-specific toxicity. Hypothemycin potently suppressed the production of IL-2 (IC50: 9 nM) but affected IL-2-induced proliferation to a lesser extent (IC50: 194 nM). Hypothemycin also inhibited IL-6, IL-10, IFN-gamma and TNF-alpha production. By contrast, it markedly enhanced the production of IL-4, IL-5 and IL-13. These effects were seen both at the mRNA and protein secretion levels. Analysis of the effect of hypothemycin on CD69 induction suggested that it disrupts calcineurin-independent rather than calcineurin-dependent signaling. Furthermore, hypothemycin was able to inhibit the phosphorylation of ERK1/2 induced by PMA treatment of T cells. Therefore, hypothemycin represents an inhibitor of T cell activation with a novel mode of action and unique modulatory activity on cytokine production.

The synthesis of C32-O-arylethyl ether derivatives of ascomycin that possess equivalent immunosuppressant activity but reduced toxicity, compared to FK-506, is described.

A tether length study of C32-O-phenalkyl ether derivatives of ascomycin was conducted wherein it was determined that a 2-carbon tether provides optimum in vitro immunosuppressive activity. Oxygen-bearing substituents along the 2-carbon tether can further increase the potency of this design.

Cytokine production upon T cell activation results from the integration of multiple signaling pathways from TCR/CD3 and from costimulatory molecules such as CD28. Among these pathways, the possible role of p38 mitogen activated protein kinase (MAPK) is the least understood. Here, we used a highly specific p38 MAPK inhibitor, the SB203580 compound, to examine the role of this enzyme in the induction of various cytokines in human T cells stimulated with anti-CD3 and anti-CD28 mAb together or in combination with PMA. Cytokine induction was monitored by ELISA and at the mRNA level. While SB203580 had little effect on IL-2 production and proliferation, it significantly reduced the production of several other cytokines. The secretion of IL-4, IL-5, IL-13, and TNF-alpha was inhibited by 20-50% with modes of T cell activation involving the CD28 pathway, whereas their mRNA expression was little affected. In contrast, IFN-gamma induction via CD28/PMA or CD3/CD28, but not CD3/PMA, was markedly diminished both at the protein and at the mRNA levels. Most interestingly, SB203580 also suppressed IL-10 secretion and mRNA induction via CD28-dependent activation by 75-85% (IC50 approximately 0.2 microM). Subset analysis suggested that this inhibition did not reflect a differential effect on T cell subsets. Therefore, p38 MAPK activity appears to contribute to cytokine production, mostly via CD28-dependent signaling. Moreover, IL-10 seems to rely more on this activity than other cytokines for its induction in T cells., (Copyright 1999 Academic Press.)

Calcineurin (CaN) controls the production of multiple cytokines, including IL-2 and TNF-alpha, during T cell activation. However, its role in chemokine production is unclear. Here, we used the CaN inhibitor FK506 to probe for the contribution of CaN in MIP-1alpha, MIP-1beta, and RANTES production at the protein and mRNA levels in human T cells stimulated via CD3/PMA or CD3/CD28. With both modes of activation, FK506 inhibited RANTES production only partially and late during a 3-day culture, whereas it suppressed both MIP-1alpha and MIP-1beta production throughout the culture. However, FK506 inhibition was more pronounced on MIP-1beta than MIP-1alpha, especially in CD3/CD28-activated T cells. Surprisingly, FK506 also significantly reduced MIP-1beta induction by PMA alone. Furthermore, comparison with IL-2 and TNF-alpha revealed that both were more potently inhibited by the drug upon CD3/PMA or CD3/CD28 induction than either MIP-1alpha or MIP-1beta. These differences in FK506 sensitivity were also observed in CD4(+) and CD8(+) T cell subsets. Therefore, all three chemokines are affected by FK506 distinctly from one another and from IL-2 and TNF-alpha, suggesting that CaN participates to different extents in the induction of these cytokines during T cell activation. Further evidence that this induction relies on distinctive mechanisms, depending on the cytokine, came from analyses of the kinetics and cycloheximide sensitivity of cytokine mRNA expression., (Copyright 1998 Academic Press.)

A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.

Productive T cell activation leading to cytokine secretion requires the cooperation of multiple signaling pathways coupled to the TCR and to costimulatory molecules such as CD28. Here, we utilized two pharmacophores, PD98059 and FK506, that inhibit, respectively, mitogen-activated protein (MAP) kinase kinase 1 (MEK 1) and calcineurin, to determine the relative role of the signaling pathways controlled by these enzymes in T cell activation. Although the two compounds had distinctive effects on CD69 induction, they both suppressed T cell proliferation induced by anti-CD3 mAb, in a manner reversible by exogenous IL-2, suggesting that PD98059, like FK506, affects the production of, rather than the responsiveness to growth-promoting cytokines. Accordingly, IL-2 production by T cells stimulated with anti-CD3 mAb in conjunction with PMA or with anti-CD28 mAb was inhibited by both compounds. However, these compounds differentially affected the production of other cytokines, depending on the mode of activation. PD98059 inhibited TNF-alpha, IL-3, granulocyte-macrophage (GM)-CSF, IFN-gamma, and to a lesser extent IL-6 and IL-10 production but enhanced IL-4, IL-5, and IL-13 production induced by CD3/PMA or CD3/CD28. FK506 suppressed CD3/PMA-induced production of all cytokines examined here but to a lesser extent IL-13. FK506 also reduced CD3/CD28-induced production of IL-3, IL-4, IL-10, TNF-alpha, and IL-6 but augmented that of GM-CSF, IL-5, IFN-gamma, and IL-13. Therefore, the biochemical targets of PD98059 and FK506 contribute differently to the production of various cytokines by T cells, which may have implications for the therapeutic manipulation of this production.

Background: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility., Methods: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity., Results: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats., Conclusions: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Two genetically engineered mutant strains of Streptomyces sp. MA6548 produced two FK506 analogs, 9-deoxo-31-O-demethylFK506 and 31-O-demethylFK506. The structures were determined by a combination of NMR and mass spectrometry. These compounds exhibited immunosuppressive and antifungal activities, albeit reduced, compared to FK506. Both compounds contain a free hydroxyl group at C-31 for the synthesis of novel FK506 derivatives.

The immunosuppressant cyclosporin A (CsA) exerts its pharmacologic actions by inhibiting calcineurin function. Here, we investigated the effect of CsA on the DNA-binding activity of the transcription factor, AP-1, in YAC-1 cells. We found that elevation of intracellular Ca2+ by ionomycin increased AP-1 DNA-binding activity in these cells. CsA treatment upregulated the ionomycin-induced, but not the basal AP-1 DNA-binding activity. In contrast, a CsA analog, MeVal4CsA, that does not inhibit calcineurin, failed to enhance ionomycin-induced AP-1 DNA-binding activity. This activity was shown to involve c-Fos, c-Jun and JunB. CsA consistently augmented ionomycin-induced c-fos mRNA expression and more variably that of JunB. Therefore, calcineurin negatively regulates Ca(2+)-stimulated AP-1 activity principally at the c-fos induction level. By inhibiting calcineurin, CsA shifts the balance between positive and negative AP-1 regulation. Since AP-1 controls the transcription of many genes, this finding may have implications for both the immunosuppressive and toxic effects of CsA.

FK506 blocks T cell activation by preventing the transcription of lymphokine genes through binding to the intracellular protein FKBP12 and formation of complex that inhibits the phosphatase activity of calcineurin. Beside exerting potent suppressive activity on cellular and humoral immune responses, in vivo treatment with FK506 in rodent models induces thymic alterations characterized by a selective reduction of mature CD4+8- cells. The potential relationship between such thymic alterations and the immunosuppressive and calcineurin inhibitory activities of FK506 has not been defined. Here, we took advantage of the availability of FK506 analogs with different immunosuppressive potencies to address this question. Intravenous daily administration of FK506 in Sprague-Dawley rats for 4 days was found to be sufficient to cause a depletion of CD4+8- thymocytes with an ED50=0.06 mg/kg/day. Under the same conditions, L-683,590 which is 2-3-fold less potent than FK506 in inhibiting T cell activation and calcineurin function gave an ED50=0.17 mg/kg/day. In contrast, the nonimmunosuppressive, calcineurin noninhibitory antagonist L-685,818, failed to deplete the CD4+8- thymocyte subset but could reverse the reducing effect of FK506 on this subset. Another analog, L-688,617, which does not completely inhibit T cell activation in vitro, also behaved as a partial agonist of CD4+8- cell depletion. Therefore, the ability of FK506 analogs to deplete the CD4+8- thymocytes subset correlates with their immunosuppressive and calcineurin inhibitory potencies. This suggests that calcineurin is involved in the intra-thymic maturation processes of CD4+8- T cells. Moreover, the short-term treatment protocol described here provides a rapid and quantitative assay to determine the immunosuppressive potency of FK506-like compounds in vivo

FK-506 blocks T cell activation by preventing lymphokine gene transcription through formation of a complex with FKBP12 that inhibits calcineurin phosphatase activity. Immunosuppressive FK-506 analogs (agonists) have been generated whose potency correlates with calcineurin inhibition. Nonimmunosuppressive antagonist analogs have also been identified, including L-685,818, which binds to FKBP12 but does not inhibit calcineurin. We describe a novel property of FK-506 analog, characterized as a mixed agonist/antagonist immunosuppressive activity. It is displayed by L-688,617, the 32 O-methoxyethoxymethyl derivative of the agonist L-683,590 (C21-ethyl). Although it binds to FKBP12 similarly to L-683,590, L-688,617 incompletely suppressed T cell proliferation induced by optimal activation and enhanced that induced by supraoptimal activation. In the latter situation, L-688,617 suppressed IL-2 production only partially but blocked activation-driven cell death. Moreover, a 1000-fold molar excess of L-688,617 antagonized the immunosuppressive activity of L-683,590. L-688,617 inhibited calcineurin phosphatase activity in cells only partially. The unique agonist/antagonist activity of L-688,617 may therefore reflect its high affinity for FKBP12, combined with a reduced ability of the drug-FKBP12 complex to inhibit calcineurin function. However, in a cell-free system, L-688,617 completely blocked this function when a large excess of FKBP12 over calcineurin was present, suggesting that the intracellular concentration of FKBP12 may be a limiting factor that prevents full agonist activity of L-688,617 in cells.

Rapamycin has potent immunosuppressive properties reflecting its ability to disrupt cytokine signaling that promotes lymphocyte growth and differentiation. In IL-2-stimulated T cells, rapamycin impedes progression through the G1/S transition of the proliferation cycle, resulting in a mid-to-late G1 arrest. Two major biochemical alterations underlie this mode of action. The first one affects the phosphorylation/activation of the p70 S6 kinase (p70s6k), an early event of cytokine-induced mitogenic response. By inhibiting this enzyme, whose major substrate is the 40S ribosomal subunit S6 protein, rapamycin reduces the translation of certain mRNA encoding for ribosomal proteins and elongation factors, thereby decreasing protein synthesis. A second, later effect of rapamycin in IL-2-stimulated T cells is an inhibition of the enzymatic activity of the cyclin-dependent kinase cdk2-cyclin E complex, which functions as a crucial regulator of G1/S transition. This inhibition results from a prevention of the decline of the p27 cdk inhibitor, that normally follows IL-2 stimulation. To mediate these biochemical alterations, rapamycin needs to bind to intracellular proteins, termed FKBP, thereby forming a unique effector molecular complex. However, neither(p70s6k) inhibition, nor p27-induced cdk2-cyclin E inhibition are directly caused by the FKBP-rapamycin complex. Instead, this complex physically interacts with a novel protein, designated "mammalian target of rapamycin" (mTOR), which has sequence homology with the catalytic domain of phosphatidylinositol kinases and may therefore be itself a kinase. mTOR may act upstream of (p70s6K) and cdk2-cyclin E in a linear or bifurcated pathway of growth regulation. Molecular dissection of this pathway should further unravel cytokine-mediated signaling processes and help devise new immunosuppressants.

The immunosuppressive macrolide, rapamycin, impedes the G1 to S cell cycle progression in cytokine-stimulated normal lymphocytes and in certain autonomously proliferating cell lines. Here, we found that the rapamycin-induced growth arrest augments homotypic aggregation in the YAC-1 T cell lymphoma. The growth arrest and increased aggregation were both blocked by the rapamycin antagonist, L-685,818, which interacts with the intracellular binding proteins mediating rapamycin's biochemical action. Moreover, rapamycin-induced aggregation was not seen in YAC-1 cells mutants selected for resistance to the drug's antiproliferative effect. Although the inhibition of G1/S progression induced by serum deprivation also resulted in increased cellular aggregation, cell cycle blockade in late G1 by mimosine, early S phase by hydroxyurea, or G2/M by nocodazole all failed to do so. Furthermore, the aggregation induced by rapamycin was blocked by antibodies to the alpha (CD11a) or beta (CD18) subunits of the integrin, LFA-1, or to its ligands, ICAM-1 and ICAM-2, and did not occur in LFA-1-deficient YAC mutants. However, the surface expression of LFA-1, ICAM-1, or ICAM-2 was not augmented in cells aggregated by rapamycin. Finally, the serine/threonine protein phosphatase inhibitor, okadaic acid, was found to abrogate rapamycin-induced aggregation. Therefore, rapamycin's impairment of YAC-1 cell growth in G1 is accompanied by enhanced LFA-1-mediated homotypic cell adhesion that may reflect an increase of the integrin's avidity for its ligands and may involve protein phosphorylation/dephosphorylation events. This suggests the existence of a link between cell cycle progression and "inside-out" LFA-1 signaling, possibly regulated by rapamycin's biochemical targets.

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.

The regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells of Bacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived from Bacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) exert distinct biological effects: inhibition of interleukin-2 production and inhibition of interleukin-2-induced proliferation, respectively, through binding to intracellular receptors, termed FKBPs. Although the interaction of these drugs with purified FKBPs in vitro has been well characterized, little is known about their interaction with FKBPs in living cells. Here, we used [3H]-dihydro-FK-506 as a probe to examine the binding of these macrolides in both normal mouse splenic T-cells and the human Jurkat T-cell lymphoma. These cells were found to accumulate the radioligand, predominantly in the cytosol, to a saturable level corresponding to an estimated concentration of 6 to 7 microM. Half-maximal suppression of T-cell activation was shown to require radioligand occupancy of only 3 to 5% of the pool of available intracellular binding sites (FKBPs). Moreover, the binding and immunosuppressive effect of the radioligand could not be removed by extensive washing and remained stable for at least 6 hr upon incubation of the cells at 37 degrees C. However, a molar excess of either FK-506 or RAP was found to rapidly displace [3H]-dihydro-FK-506 from its cellular binding sites. Consistently, FK-506 and RAP were able to antagonize mutually their immunosuppressive activities even when added several hr after each other to T-cell cultures. We took advantage of the reciprocal antagonism of FK-506 and RAP to define their apparent affinities for the functionally relevant cellular receptors by Schild analysis. This indicated that the drugs compete for a single cellular receptor with similar KdS and, therefore, may mediate their immunosuppressive action upon interaction with similar or highly related FKBPs.

31-O-Desmethylimmunomycin O: methyltransferase (DIMT), an enzyme involved in the biosynthesis of immunomycin (ascomycin/FR-900520), was used to synthesize three analogs of this immunosuppressant compound. These compounds were assigned the following structures: 13-O-desmethyl-, 15-O-desmethyl- and 13,15-O-bisdesmethyl-immunomycins. Two of these compounds, namely, 15-O-desmethyl- and 13,15-O-bismethyl-immunomycins have novel structures and were examined for possible immunosuppressive activity by in vitro T-cell proliferation assay. The results showed that methylation of the C-15 hydroxyl is critical for full biological activity of the immunomycin.

Rapamycin (RAP) exerts potent immunosuppressive effects that are thought to reflect primarily an inhibition of T cell proliferation driven by growth-promoting cytokines. In the present study, we examined whether RAP would be able to alter other T cell responses mediated by cytokines, such as lymphokine production. As a model system, we used the murine T cell lymphoma, YAC-1, that can be induced to produce IFN-gamma when activated with either ionomycin + PMA or IL-1 alpha + PMA. As previously found in other systems, induction of this lymphokine by ionomycin + PMA was not inhibited by RAP, although it was highly sensitive to inhibition by FK-506, an immunosuppressive macrolide structurally related to RAP. In contrast, the induction of the same lymphokine by IL-1 alpha + PMA was highly sensitive to RAP but resistant to FK-506. This inhibition of IFN-gamma production by RAP was detectable at the mRNA level. Such an inhibition was specific as it did not occur in a mutant clone selected for resistance to the antiproliferative effects of RAP. Moreover, kinetics analysis revealed that RAP still inhibited IFN-gamma mRNA accumulation in YAC-1 cells, when added as late as 12-18 h after initial stimulation of the cells with IL-1 alpha + PMA. While the inhibitory action of RAP could not be removed by extensive washing of the cells, it was readily reversed by a hundred-fold excess of FK-506 added to the cultures simultaneously with RAP or up to 16-18 h later. Altogether, these data indicate that the action of RAP in T cells is not limited to the inhibition of proliferation but also affects the regulation of lymphokine gene expression mediated by IL-1 alpha. The inhibition of lymphokine gene expression by RAP takes place at a late stage of the inductive response, and through a mechanism that involves interaction with the same cellular binding proteins as FK-506. Such an inhibitory effect of RAP may contribute significantly to its ability to suppress immune responses.

FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.

CD45 is a transmembrane tyrosine phosphatase implicated in T cell antigen receptor (TCR)-mediated activation. In T cell variants expressing progressively lower levels of CD45 (from normal to undetectable), CD45 expression was inversely related to spontaneous tyrosine phosphorylation of multiple proteins, including the TCR zeta chain, and was directly correlated with TCR-driven phosphoinositide hydrolysis. The Ca2+ response in these cells was altered in an unexpected fashion. Unlike wild-type cells, stimulated CD45- cell populations did not manifest an early increase in intracellular Ca2+, but did exhibit a delayed and gradual increase in mean intracellular Ca2+. Computer-aided fluorescence imaging of individual cells revealed that CD45- cells experienced late Ca2+ oscillations that were not blocked by removal of extracellular Ca2+. CD45 revertants had the signaling properties of wild-type cells. Thus, CD45 has a profound influence on both TCR-mediated signaling and phosphotyrosine homeostasis, and its loss reveals a novel role for this tyrosine phosphatase in Ca2+ regulation.

CsA, FK-506, and rapamycin are microbial products with potent immunosuppressive properties that result primarily from a selective inhibition of T lymphocyte activation. Although chemically unrelated, CsA and FK-506 affect a similar subset of calcium-associated signaling events involved in the regulation of lymphokine gene expression, activation-driven T-cell death and exocytosis. Rapamycin has structural similarity with FK-506 but suppresses T-cell activation at a different level, mainly through inhibition of proliferation induced by growth-promoting lymphokines. CsA interacts with an abundant 17 kDa protein, termed cyclophilin, that possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity. Additional, minor cyclophilin-like molecules have been identified. Both FK-506 and rapamycin interact with FKBP, a 12 kDa protein, which, although unrelated to cyclophilin, is also abundant and ubiquitous, has a similar enzymatic activity, and is a member of a larger family of FKBPs. All three immunosuppressants inhibit the PPIase activity of their respective binding proteins. However, nonimmunosuppressive analogs of CsA and FK-506 are also inhibitory, indicating that inhibition of PPIase activity is not directly implicated in immunosuppression. Moreover, only a small fraction of the cellular pool of the major forms of cyclophilin or FKBP needs to be occupied by the drugs in order to achieve maximal immunosuppression. These observations suggest that complexes formed between the drugs and their major binding proteins may affect the function of other, unidentified, molecules or, alternatively, that minor binding proteins may play a role in the drugs' action. Further characterization of the biochemical processes altered by CsA, FK-506, and rapamycin should yield important insights into the signal transduction pathways involved in T-cell activation and should help in the development of novel immunosuppressive agents.

The cell surface Ly-6E antigen, known to play a role in T cell activation, is up-regulated by IFNs. In the present study, we investigated the possible interactions between IFNs and other cytokines in this regulation. As a model system, we used the YAC T cell lymphoma, in which Ly-6E is normally absent but can be highly induced both at the mRNA and surface protein levels by IFN-gamma or IFN-alpha/beta. The combination of the two IFNs was found to result in markedly synergistic Ly-6E induction in this cell line. Moreover, mutants of YAC cells were isolated that did not respond to the Ly-6E-inducing action of IFN-gamma or IFN-alpha/beta alone but did respond to their combination. Such a synergistic interaction is consistent with the notion that the two IFN types utilize different intracellular mechanisms to induce Ly-6E expression. Ly-6E induction mediated by IFN-gamma or IFN-alpha/beta was also enhanced by cotreatment with TNF-alpha or IL-1 alpha, which by themselves had no detectable Ly-6E-inducing effect. These two cytokines similarly synergized with IFNs to trigger a response in several Ly-6E-induction-deficient mutants. However, their action could be dissociated in one mutant (B54) where the response to IFN-alpha/beta was enhanced by TNF-alpha, but not by IL-1 alpha. Altogether, these data indicate that Ly-6E antigen expression is regulated by the interaction of several inflammatory cytokines, which may provide a mechanism for the local modulation of T cell activation. The YAC cell mutants described here should facilitate further analysis of the molecular bases of Ly-6E regulation.

This study was initiated to examine the role of cyclic nucleotides in the regulation of the expression of the Ly-6E cell surface Ag by IFN. As a model system, we used the YAC T cell lymphoma where this Ag is constitutively absent but is highly inducible by both IFN-gamma and IFN-alpha/beta. Treatment with cAMP or cGMP analogs did not cause Ly-6E expression in the absence of IFN. However, treatment with cAMP analogs, but not with cGMP analogs, markedly altered Ly-6E expression triggered by IFN, both at the mRNA and at the cell surface protein levels. Interestingly, these effects depended on whether Ly-6E induction was mediated by IFN-gamma or IFN-alpha/beta. Thus, the response to IFN-gamma was enhanced up to tenfold, whereas the response to IFN-alpha/beta was suppressed by 90-95%. Similar differential modulations of Ly-6E induction were also exerted by forskolin and cholera toxin, which are known to elevate intracellular cAMP concentration through distinct mechanisms. A YAC cell variant (C10) was isolated, where cAMP analogs or cAMP inducers could not modify Ly-6E induction. Although resistant to the biological effect of cAMP, the C10 mutant exhibited normal IFN-mediated Ly-6E responses. Moreover, in this mutant, Ly-6E induction was still affected by the PKC activator PMA, as in wild-type YAC cells. Altogether, our data demonstrate that cAMP (and cGMP) is not directly involved as second messenger in Ly-6E induction mediated by IFNs. However, a rise of cAMP modulates in an opposite fashion the Ly-6E-inducing actions of IFN-gamma and IFN-alpha/beta, which suggests that the two types of IFN utilize separate biochemical pathways to regulate Ly-6E expression.

Cyclosporin A (CsA) and FK-506 inhibit lymphokine gene activation in T-cells. In the present study, we investigated the effects of these immunosuppressants on the regulation of a non-lymphokine molecule, the Ly-6E surface antigen, in the YAC-1 T-cell lymphoma. These cells do not normally express Ly-6E mRNA or Ly-6E surface molecules but are induced to do so upon treatment with IFN-gamma. At submicromolar concentrations, CsA or FK-506 did not alter this induction. However, at higher concentrations (1-12 microM), they both increased the induction of Ly-6E mRNA expression. Cyclosporin A or FK-506 also markedly affected Ly-6E induction when the cultures were co-treated with the calcium ionophore, ionomycin. In the absence of CsA or FK-506, ionomycin suppressed Ly-6E induction by IFN-gamma. Both immunosuppressants reversed this inhibitory effect and increased Ly-6E mRNA and Ly-6E surface expression to levels that were 2- to 3-fold higher than in cells induced with IFN-gamma alone. In this system, the two immunosuppressants were active at pharmacologically relevant concentrations, similar to those inhibiting normal T-cell activation, with FK-506 being 30- to 50-fold more potent than CsA. The ability of CsA analogs to enhance Ly-6E induction in the presence of ionomycin also correlated with their immunosuppressive activity. Therefore, through mechanisms apparently related to those involved in their immunosuppressive action, both CsA and FK-506 convert the negative effect of ionomycin on IFN-gamma-mediated Ly-6E induction into an overall positive effect. The YAC-1 cell model, described here, provides a unique example of upregulation of gene expression by these two immunosuppressants.

Activation of certain T-cell lines induces, besides lymphokine production, a suicide process (apoptosis) mediated by fragmentation of the cell's genome. This also occurs intrathymically during negative selection of the T-cell receptor (TcR) repertoire. Cyclosporin A (CsA) has been shown to block activation-driven T-cell apoptosis, an effect which may account for the perturbations of TcR repertoire selection caused by this agent in vivo. Recently, the macrolide FK-506 was demonstrated to suppress T-cell activation by inhibiting lymphokine production in a manner apparently similar to CsA. Thus, it seemed important to determine whether FK-506 would also prevent T-cell apoptosis. For the purpose of comparison, we also investigated rapamycin (RAP), a macrolide structurally related to FK-506, but that does not block lymphokine production and antagonizes the immunosuppressive action of FK-506. The DO-11.10 T-cell hybridoma stimulated with ionomycin plus PMA was used as a model system. FK-506 (1.2 nM) totally prevented DNA fragmentation detectable by agarose gel electrophoresis at 16 h of culture. FK-506 still inhibited this phenomenon when added 2 h after the initiation of the cultures but not later. In contrast, concentrations of RAP as high as 1 microM failed to block apoptosis. However, RAP (110 nM) reversed the apoptosis-inhibitory effect of FK-506, even if added 1-2 h after the latter to the cultures. Consistent with this antagonism, RAP also reversed the binding of a radiolabeled derivative of FK-506 in DO-11.10 cells. Therefore, FK-506 interferes with an early event of T-cell activation that leads to apoptosis whereas RAP does not.(ABSTRACT TRUNCATED AT 250 WORDS)