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Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (&ggr;c). Because mutations in genes encoding &ggr;c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n=18) or its vehicle (controls, n=3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (± standard error of mean) in animals treated with CP-690,550 (53±7 days) was significantly longer than in control animals (7±1 days, P=0.0003) and was positively correlated with exposure to the drug (r=0.79, P<0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46±7 days from transplantation vs. 7±1 days in controls, P=0.0003). Persistent anemia, polyoma virus-like nephritis (n=2), and urinary calcium carbonate accretions (n=3) were seen in animals with high exposure. Natural killer cell and CD4+ and CD8+ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allografts.

Background. Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. Methods. Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-γ production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. Results. In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-γ production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 &mgr;M) and CD71 (IC50; 1.6 &mgr;M), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 &mgr;M). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8+ effector memory T-cell populations were unaffected. Conclusions. Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

Janus kinase 3 inhibition with CP-690,550 prevents allograft vasculopathy

Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common γ chain. The rationally designed inhibitor of JAK3, CP‐690,550, prevents acute allograft rejection in rodents and in nonhuman primates. Here we investigated the ability of CP‐690,550, to prevent allograft vasculopathy in a rodent model of aorta transplantation. Aortas from AxC Irish (RT1a) or Lewis (RT1l) rats were heterotopically transplanted into the infra‐renal aorta of Lewis recipients and harvested at 28 or 56u2003days. Treated recipients received CP‐690,550 by osmotic pumps (mean drug exposure of 110u2003±u200338u2003ng/ml). Significant intimal hyperplasia was demonstrated in untreated allografts when compared with isografts at 28u2003days (2.08u2003±u20030.85% vs. 0.43u2003±u20030.2% luminal obliteration, respectively, Pu2003=u20030.001) and 56u2003days (5.3u2003±u20032.4% vs. 0.38u2003±u20030.3%, Pu2003=u20030.002). Treatment caused a 51% reduction in intimal hyperplasia at day 56. CP‐690,550‐treated animals also had a significant reduction of donor‐specific IgG production and of the gene expression for suppressor of cytokine signaling‐3 and with unchanged levels of expression of RANTES, IP‐10 and transforming growth factor‐β1. These results are the first to show that JAK3 blockade by CP‐690,550 effectively prevents allograft vasculopathy in this rat model of aorta transplantation.

Immunomodulatory effects of docetaxel on human lymphocytes

Docetaxel is an antineoplastic taxoid that interferes with microtubule polymerization dynamics and is used clinically to treat advanced cancers. Because microtubules play significant roles in T lymphocyte activation and function we characterized the in vitro immunomodulatory properties of docetaxel. Effects of docetaxel on lectin-induced peripheral blood mononuclear cell (PBMC) proliferation were measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and proliferating cell nuclear antigen (PCNA) staining. In addition, apoptosis was measured by annexin V staining and cell activation by determination of CD25 and CD71 cell surface expression. Intracellular calcium kinetics in lectin-activated Jurkat T lymphocytes exposed to docetaxel were investigated. Th1 cytokine production was assessed in T lymphocytes by intracellular cytokine staining. Docetaxel significantly inhibited PBMC proliferation and promoted apoptosis of lectin-activated PBMCs. Docetaxel significantly decreased expression of CD71 but not that of CD25. Docetaxel altered intracellular calcium homeostasis but did not affect Th1 cytokine production in T lymphocytes. In conclusion we demonstrate that docetaxel, although exerting significant antiproliferative effects on lymphocytes and promoting activation-induced apoptosis does affect only partially lymphocyte activation and function and does not affect Th1 cytokine production. These results suggest maintenance of lymphocyte functions important for host tumor surveillance and suggest that this compound may have a role in the treatment of cancer arising organ transplant recipients.

Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123

Prenylated Ras GTPases transduce signals from the T cell receptor, CD28 costimulatory receptor and IL-2 receptor. Since signals from these receptors mediate T cell activation, proliferation and survival, we hypothesized that the prenylation inhibitor L-778,123 would impart immunomodulation.The effect of L-778,123 on T cell activation (CD71 or CD25 surface expression) was determined by flow cytometry. Peripheral blood mononuclear cell (PBMC) proliferation in the presence of L-778,123 and/or cyclosporine (CsA) was determined by [3H]thymidine incorporation. The ability of L-778,123 to inhibit IL-2 receptor signaling was investigated by measuring IL-2 induced proliferation in CTLL-2 cells and IL-2 prevention of apoptosis in activated human PBMC.L-778,123 inhibited lectin induced expression of CD71 and CD25 with IC50s of 6.48 ± 1.31 μM and 84.1 ± 50.0 μM, respectively. PBMC proliferation was inhibited by L-778,123 with an IC50 of 0.92 ± 0.23 μM, and addition of CsA did not increase the potency. L-778,123 did not inhibit IL-2 and IFN-γ production by T cells. L-778,123 abrogated IL-2 induced proliferation of CTLL-2 cells with an IC50 of 0.81 ± 0.44 μM. However, L-778,123 minimally reversed the prosurvival effect of IL-2 in activated lymphocytes.IL-2 ligand and receptor production during T cell activation are relatively unaffected by L-778,123. However, the activation and proliferative effects of IL-2 on T cells are potently blocked by L-778,123. These results reveal a selective blockade of the IL-2 cytokine axis distal to the IL-2 receptor by the L-778,123 and warrant evaluation of prenylation inhibitors in treating transplant rejection and autoimmune diseases.