The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1+ T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28−/− recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-γ and IFN-γ-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.

The metabolic syndrome, a complex set of phenotypes typically associated with obesity and diabetes, is an increasing threat to global public health. Fundamentally, the metabolic syndrome is caused by a failure to properly sense and respond to cellular metabolic cues. We studied the role of the cellular metabolic sensor PAS kinase (PASK) in the pathogenesis of metabolic disease by using PASK ؊/؊ mice. We identified tissue-specific metabolic phenotypes caused by PASK deletion consistent with its role as a metabolic sensor. Specifically, PASK ؊/؊ mice exhibited impaired glucose-stimulated insulin secretion in pancreatic ␤-cells, altered triglyceride storage in liver, and increased metabolic rate in skeletal muscle. Further, PASK deletion caused nearly complete protection from the deleterious effects of a high-fat diet including obesity and insulin resistance. We also demonstrate that these cellular effects, increased rate of oxidative metabolism and ATP production, occur in cultured cells. We therefore hypothesize that PASK acts in a cell-autonomous manner to maintain cellular energy homeostasis and is a potential therapeutic target for metabolic disease.metabolism ͉ PAS domain ͉ nutrient sensing ͉ obesity B ecause of changes in diet and lifestyle, the incidence of obesity and type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes arises when pancreatic ␤-cells fail to secrete sufficient insulin to compensate for peripheral insulin resistance, a condition severely aggravated by obesity (1, 2). Type 2 diabetes is now widely viewed as a manifestation of a broader underlying metabolic disorder called the metabolic syndrome, which is characterized by hyperglycemia, hyperinsulinemia, dyslipidemia, hypertension, visceral obesity, and cardiovascular disease (3). The World Health Organization estimates that the current decade will witness a 46% increase in diabetes incidence worldwide (from 151 million to 221 million), with the vast majority of this increase being due to metabolic syndrome-related type 2 diabetes (4).Cellular energy and nutrient sensors determine how cells respond to excessive nutrients, and aberrant nutrient and energy sensing is a contributing factor to metabolic syndrome development (5, 6). AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are two well studied and evolutionarily conserved cellular energy and nutrient sensors. AMPK is activated in response to intracellular ATP depletion and acts to switch the cellular metabolic program from ATP consumption to ATP production (7). In contrast to AMPK, mTOR is activated by sufficient cellular energy or nutrients, particularly amino acids (8). Activation of mTOR stimulates cell growth by increasing protein synthesis through phosphorylation of ribosomal S6 kinase (S6K) and eIF4E-binding protein (9). Decreased AMPK activity and elevated mTOR activity have been linked with obesity, diabetes, and cancer (10-12).Like AMPK and mTOR, PAS kinase (PASK) is a nutrientresponsive protein kinase conserved from yeast to humans. Th...

Various adhesion molecules have been implicated in T lymphocyte binding to dermal vascular endothelium in psoriasis vulgaris, but the chemotactic signals that promote subsequent homing into the adjacent dermis and overlying epidermis are poorly defined. We studied chemokine receptor (CCR1-CCR5, CXCR1-CXCR3), chemokine (interferon-gamma inducible protein 10 [IP-10]), monokine induced by interferon-gamma (MIG), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and adhesion molecule (cutaneous lymphocyte antigen [CLA], E-selectin, lymphocyte function-associated antigen-1 [LFA-1], intercellular adhesion molecule-1 [ICAM-1], very late antigen 4 [VLA-4], vascular cell adhesion molecule-1 [VCAM-1], alphaEbeta7, and E-cadherin) expression in psoriasis by immunohistology, flow cytometry, and molecular techniques. CXCR3 and CCR4 were expressed by dermal CD3+ lymphocytes, and their chemokine ligands, IP-10, MIG, TARC, and MDC, were up-regulated in psoriatic lesions. Keratinocytes stimulated with tumor necrosis factor-alpha and interferon-gamma up-regulated expression of IP-10, MIG, and MDC mRNA, whereas dermal endothelial cells, similarly stimulated, up-regulated expression of IP-10, MDC, and TARC mRNA, suggesting that these cell types were sources of the chemokines detected in biopsies. There was enhanced expression of E-selectin, CLA, LFA-1, ICAM-1, VLA-4, VCAM-1, and alphaEbeta7 in psoriatic lesions versus nonlesional skin. Finally, intra-epidermal CLA+ and alphaEbeta7+ T lymphocytes selectively expressed the chemokine receptor CXCR3. Collectively, these data suggest that CXCR3 and CCR4 may be involved in T lymphocyte trafficking to the psoriatic dermis and that CXCR3 is selectively involved in subsequent T cell homing to the overlying epidermis.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

The ability of cells to recognize and respond to specific metabolic deficiencies is required for all forms of life. We have uncovered a system in the yeast S. cerevisiae that, in response to a perceived deficiency in cell wall glucan, alters partitioning of glucose toward glucan synthesis and away from glycogen synthesis. The paralogous yeast PAS kinases Psk1 and Psk2 phosphorylate UDP-glucose pyrophosphorylase (Ugp1), the primary producer of UDP-glucose, the glucose donor for glucan biosynthesis. Unexpectedly, phosphorylation of Ugp1 does not affect its catalytic activity but instead alters the terminal destination of the UDP-glucose it generates. Phosphorylated Ugp1 is required for intensive glucan production, and inability to phosphorylate Ugp1 is associated with a weak cell wall, decreased glucan content, and increased glycogen content. We provide data indicating that phosphorylation by Psk1 or Psk2 targets Ugp1 to the cell periphery, where the UDP-glucose it produces is in proximity to the site of glucan synthesis. We propose that regulation of glucose partitioning by altered enzyme and substrate localization is a rapid and potent response to metabolic deficiency.