ER Chaperone BiP/GRP78 Is Required for Myelinating Cell Survival and Provides Protection during Experimental Autoimmune Encephalomyelitis.

Myelinating cells synthesize large amounts of membrane protein through the secretory pathway, which makes these cells particularly sensitive to perturbations of the endoplasmic reticulum (ER). Ig binding protein (BiP), also known as glucose-regulated protein 78 (GRP78), is a critical ER chaperone that also plays a pivotal role in controlling the cellular response to ER stress. To examine the potential importance of BiP to myelinating cells, we used a conditional knock-out approach to BiP gene inactivation in oligodendrocytes during development, in adulthood, and in response to experimental autoimmune encephalomyelitis (EAE), an animal model of the inflammatory demyelinating disorder multiple sclerosis (MS). During development, mice lacking functional BiP gene expression in oligodendrocytes developed tremors and ataxia and died before reaching maturity. When BiP gene inactivation in oligodendrocytes was initiated in adulthood, the mice displayed severe neurological symptoms including tremors and hind-limb paralysis. The inactivation of BiP in oligodendrocytes during development or in adulthood resulted in oligodendrocyte loss and corresponding severe myelin abnormalities. Mice heterozygous for the oligodendrocyte-specific inactivation of BiP, which were phenotypically normal without evidence of neuropathology, displayed an exacerbated response to EAE that correlated with an increased loss of oligodendrocytes. Furthermore, mice in which the BiP gene was specifically inactivated in developing Schwann cells displayed tremor that progressed to hindlimb paralysis, which correlated with diminished numbers of myelinating Schwann cells and severe PNS hypomyelination. These studies demonstrate that BiP is critical for myelinating cell survival and contributes to the protective response of oligodendrocyte against inflammatory demyelination.

Genetic inactivation of PERK signaling in mouse oligodendrocytes: normal developmental myelination with increased susceptibility to inflammatory demyelination.

The immune-mediated central nervous system (CNS) demyelinating disorder multiple sclerosis (MS) is the most common neurological disease in young adults. One important goal of MS research is to identify strategies that will preserve oligodendrocytes (OLs) in MS lesions. During active myelination and remyelination, OLs synthesize large quantities of membrane proteins in the endoplasmic reticulum (ER), which may result in ER stress. During ER stress, pancreatic ER kinase (PERK) phosphorylates eukaryotic translation initiation factor 2α (elF2α), which activates the integrated stress response (ISR), resulting in a stress-resistant state. Previous studies have shown that PERK activity is increased in OLs within the demyelinating lesions of experimental autoimmune encephalomyelitis (EAE), a model of MS. Moreover, our laboratory has shown that PERK protects OLs from the adverse effects of interferon-γ, a key mediator of the CNS inflammatory response. Here, we have examined the role of PERK signaling in OLs during development and in response to EAE. We generated OL-specific PERK knockout (OL-PERK(ko/ko) ) mice that exhibited a lower level of phosphorylated elF2α in the CNS, indicating that the ISR is impaired in the OLs of these mice. Unexpectedly, OL-PERK(ko/ko) mice develop normally and show no myelination defects. Nevertheless, EAE is exacerbated in these mice, which is correlated with increased OL loss, demyelination, and axonal degeneration. These data indicate that although not needed for developmental myelination, PERK signaling provides protection to OLs against inflammatory demyelination and suggest that the ISR in OLs could be a valuable target for future MS therapeutics.

Overexpression of program death-1 in T cells has mild impact on allograft survival.

Program death-1 (PD-1), an inhibitory receptor upregulated on T cells upon TCR stimulation, has been shown to attenuate a number of immune responses in vivo, including acute allograft rejection. We tested whether constitutive expression of PD-1 would further inhibit allograft rejection. To this end, we generated transgenic mice expressing T-cell-restricted PD-1 under the control of the Lck proximal promoter and CD2 locus control. PD-1 transgenic (PD-1-Tg) mice did not develop gross abnormalities of thymic development and displayed normal numbers of thymocyte subsets and peripheral T cells. In vitro, PD-1-Tg T cells had reduced proliferative and cytokine secretion capacity upon TCR stimulation and cross-linking of PD-1 resulted in diminished phosphorylation of protein kinase C-theta and Akt, as well as increased activation of the phosphate and tensin homolog. However, only T-cell responses to minor but not major mismatches were reduced in vitro. Similarly, PD-1-Tg mice exhibited prolonged survival of cardiac allografts only in mice transplanted with heart allografts expressing multiple minor mismatches and treated with suboptimal doses of cyclosporine A. We conclude that genetic engineering of T cells to express PD-1 constitutively has only a mild impact on allograft survival.

Foxp3 controls autoreactive T cell activation through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection.

To elucidate the mechanisms of autoreactive T cell activation and expansion, we used endogenous viral superantigens (VSAg)-reactive T cells as a model of self-antigens in two strains of Foxp3-mutant mice. These two strains, together with wild-type mice, provided us with an advantage to simultaneously study the positively and negatively selected as well as rescued autoreactive T cells. We show here that while both VSAg-reactive and non-VSAg-reactive T cells are equally activated in Foxp3-mutant mice, only the VSAg-reactive T cells are preferentially expanded independently of their selected states in the thymus. The T cell activation appears to be controlled by Foxp3 through transcriptional regulation of early growth response (Egr) genes Egr-2 and Egr-3, and E3 ubiquitin (Ub) ligase genes Cblb, Itch and GRAIL, subsequently affecting degradation of two key signaling proteins, PLCgamma1 and PKC-theta. Physiologically, the positively, but not negatively selected VSAg-reactive T cells are spontaneously activated without significant expansion. The results suggest that autoreactive T cell activation is controlled by Foxp3 through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection.

Multiple sclerosis: expression of CD1a and production of IL-12p70 and IFN-gamma by blood mononuclear cells in patients on combination therapy with IFN-beta and glatiramer acetate compared to monotherapy with IFN-beta.

Current therapy of multiple sclerosis (MS) with interferon-beta (IFN-beta) or glatiramer acetate (GA) has modest effects on the course of MS. Both compounds affect several immune variables, like expression of cell surface molecules and cytokine levels. Here we compared untreated MS, therapy with IFN-beta alone and combined with GA, and healthy controls (HC), regarding expression on HLA-DR+ blood mononuclear cells (MNC) of CD1a that is a cell surface molecule with capacity to present glycolipids to T cells, and of CD80 and CD86 which are costimulatory molecules that activate Th1 and Th2 responses. Cytokine production by MNC was also measured. Flow cytometry and ELISA were used. Cross-sectional comparisons revealed that untreated MS patients had higher CD1a+ HLA-DR+ MNC and lower IL-10 production compared to patients treated with IFN-beta or IFN-beta+GA or HC. Untreated MS patients also had higher spontaneous IFN-gamma and IL-12p70 production compared to MS patients treated with IFN-beta+GA or HC, but not when compared to MS patients on monotherapy with IFN-beta. Low CD1a+ HLA-DR+ MNC and low spontaneous production of IL-12p70 and IFN-gamma were more pronounced in patients treated with IFN-beta+GA than with IFN-beta alone. In order to clarify whether these changes reflect disease activity or treatment effects, we performed a follow up study. Nineteen MS patients with disease progression, despite monotherapy with IFN-beta for more than one year; were re-examined after one to three and four to six months of treatment with IFN-beta+GA. This combination therapy was associated with normalization of CD1a+ HLA-DR+ MNC, IL-12p70 and IFN-gamma. It remains to be shown whether these immunological changes imply a clinical benefit. Follow up studies of immune variables versus clinical effects during combined therapy of MS with IFN-beta+GA are ongoing.