Cyclophilin A (CypA/Ppia) is a peptidyl-prolyl isomerase (PPIase) that binds the immunosuppressive drug cyclosporine. The resulting complex blocks T cell function by inhibiting the calcium-dependent phosphatase calcineurin. To identify the native function of CypA, long suspected of regulating signal transduction, we generated mice lacking the Ppia gene. These animals develop allergic disease, with elevated IgE and tissue infiltration by mast cells and eosinophils, that is driven by CD4+ T helper type II (Th2) cytokines. Ppia(-/-) Th2 cells were hypersensitive to TCR stimulation, a phenotype consistent with increased activity of Itk, a Tec family tyrosine kinase crucial for Th2 responses. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in the SH2 domain of Itk disrupted interaction with CypA and specifically increased Th2 cytokine production from wild-type CD4+ T cells. Thus, CypA inhibits CD4+ T cell signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.

T o regulate the immune response and dampen the potential for autoimmunity, the immune system has evolved several mechanisms to down-regulate and control the outgrowth and differentiation of activated CD4 ϩ T cells. One level of control, mediated during the initial interaction of the CD4 ϩ T cell with MHC͞peptide complexes on the surface of antigen-presenting cells, determines whether T cell activation, anergy, or apoptosis will ensue (1-3). A second level of control, mediated by cytokines, regulates the growth and differentiation of activated CD4 ϩ T cells. Different cytokines secreted by CD4 ϩ or CD8 ϩ T cells either stimulate or inhibit CD4 ϩ T cell proliferation and determine whether a naïve T helper (TH) precursor cell differentiates as an IFN-␥-producing TH1 cell or as an IL-4-and IL-10-producing TH2 cell (4-6). A third level of control resides in the regulatory T cells including both CD4 ϩ (7) and CD8 ϩ (8) T cell populations. For example, ample data demonstrate the ability of CD8 ϩ T cells to regulate CD4 ϩ T cell responses (9-13). These effects of CD8 ϩ T cells have been mostly attributed, in recent years, to the CD8 ϩ T cells' secretion of cytokines (14).In addition to identifying cytokines as potential effectors of immune regulation by CD8 ϩ T cells, other studies have identified specific cognate interactions between regulatory CD8 ϩ T cells and activated CD4 ϩ T cells. For example, during antigen-or superantigen-driven CD4 ϩ T cell responses in vivo, CD8 ϩ T cells emerge that specifically regulate CD4 ϩ T cells in a T cell antigen receptor (TCR) V␤-specific manner (15, 16). These CD8 ϩ T cells preferentially recognize antigen-activated CD4 ϩ T cell clones expressing certain TCR V␤ molecules and are restricted by the class I-b MHC molecule Qa-1. Unlike conventional MHC class I-a molecules, Qa-1 molecules are expressed only at low levels on resting T cells but are increased after antigen activation (17). These data are consistent with a model of specific immunoregulation in which after antigen activation CD4 ϩ T cells express membrane Qa-1͞TCRV␤ motifs that are recognized by the ␣␤ TCR expressed by precursor regulatory CD8 ϩ T cells. These CD8 ϩ T cells are induced to differentiate and down-regulate CD4 ϩ T cells expressing the particular Qa-1͞TCRV␤ motifs.A prediction of this model is that Qa-1-restricted, V␤-specific regulatory CD8 ϩ T cells will be induced by ''vaccination'' of animals with antigen-activated CD4 ϩ T cells, using T cell vaccination (TCV) protocols known to prevent autoimmune disease in animal models (18,19). In this regard, we have shown that Qa-1-restricted, V␤-specific CD8 ϩ cytotoxic T cell lines are induced by TCV (16). Moreover, we isolated a CD8 ϩ T hybridoma clone from a T cell-vaccinated mouse that preferentially recognizes CD4 ϩ V␤8 ϩ but not CD4 ϩ V␤6 ϩ myelin basic protein (MBP)-reactive clones in a Qa-1-restricted fashion (16). In this current study, we further confirmed this prediction by investigating an experimental autoimmune encephalomyelitis (EAE) model system in wh...

No abstract

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia−/− CD4+ T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia−/− mice to allogeneic challenge. Rag2−/− mice reconstituted with Ppia−/− splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia−/− immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine.

An American option grants the holder the right to select the time at which to exercise the option, so pricing an American option entails solving an optimal stopping problem. Difficulties in applying standard numerical methods to complex pricing problems have motivated the development of techniques that combine Monte Carlo simulation with dynamic programming. One class of methods approximates the option value at each time using a linear combination of basis functions, and combines Monte Carlo with backward induction to estimate optimal coefficients in each approximation. We analyze the convergence of such a method as both the number of basis functions and the number of simulated paths increase. We get explicit results when the basis functions are polynomials and the underlying process is either Brownian motion or geometric Brownian motion. We show that the number of paths required for worst-case convergence grows exponentially in the degree of the approximating polynomials in the case of Brownian motion and faster in the case of geometric Brownian motion.

Effector CD4+ T cells rapidly activate high-level cytokine expression following TCR stimulation. Consistent with accelerated protein production in these cells, global mRNA profiles revealed that, after cytokines, the most impressive cluster of activated genes encode rRNA-maturation factors. Activation of these genes was ERK-MAPK dependent, accompanied by increased rRNA transcription and faster maturation kinetics, and much greater in effector CD4+ T cells than in naive cells. Ribosomal protein subunit (RPS) synthesis was also ERK-MAPK dependent and increased to match rRNA production, but without evident increase in RPS mRNA. Instead, stimulation promoted polysome loading of RPS mRNA via cis-acting, 5'-terminal oligopyrimidines. These results demonstrate how, in response to extracellular signals, effector CD4+ T cells coordinately increase multiple ribosomal components to accommodate burgeoning cytokine production.

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the αTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37°C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in μl ⋅ h−1 ⋅ cm−2) 3.3 ± 0.3 for αTN4 cells ( n = 27) and 4.7 ± 1.0 for bovine layers ( n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 ± 0.62 μl ⋅ h−1 ⋅ lens−1( n = 5) and along the same pressure head at 12.5 ± 1.1 μl ⋅ h−1 ⋅ lens−1( n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.

The development of novel photosensitizer with high phototoxicity, low dark toxicity, and good water solubility is a challenging task for photodynamic therapy (PDT). A series of chlorin p6-based water-soluble amino acid conjugates were synthesized and investigated for antitumor activity. Among them, aspartylchlorin p6 dimethylester (7b) showed highest phototoxicity against melanoma cells with weakest dark toxicity, which was more phototoxic than verteporfin while with less dark toxicity. It also exhibited better in vivo PDT antitumor efficacy on mice bearing B16-F10 tumor than verteporfin. The biological assays revealed that 7b was localized in multiple subcellular organelles and could cause both cell necrosis and apoptosis after PDT in a dose-dependent manner, resulting in more effective cell destruction. As a result, 7b represents a promising photosensitizer for PDT applications because of its strong absorption in the phototherapeutic window, relatively high singlet oxygen quantum yield, highest dark toxicity/phototoxicity ratio, good water solubility, and excellent in vivo PDT antitumor efficacy.