High K+ intake alleviates experimental autoimmune encephalomyelitis (EAE) and increases T regulatory cells.

Multiple sclerosis is believed to be triggered by the interplay between the environmental and genetic factors. In contrast to the Paleolithic diet, the modern Western diet is high in Na+ and low in K+. The present study was undertaken to determine whether high K+ intake alleviated experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Treatment of C57BL/6 or SJL mice for 7 days with a 5 % K+ diet prior to induction of EAE and maintaining mice on the diet until the end of experiments delayed the onset, reduced the peak, and accelerated the recovery of EAE in both strains compared with mice on a control diet (0.7 % K+), whereas feeding C57BL/6 mice with a 0.1 % K+ diet did the opposite. High K+ intake increased the splenic Treg cell frequency in the pretreatment and peak EAE. Thus, high K+ intake attenuates EAE, possibly by increasing the Treg cells.

NFAT5 contributes to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and decrease of T regulatory cells in female mice.

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.

CD8+ T cells: The past and future of immune regulation.

Regulation of the adaptive immune response is critical for health. Regulatory activity can be found in multiple components of the immune system, however, the focus on particular components of the immune regulatory network has left many aspects of this critical immune component understudied. Here we review the evidence for activities of CD8+ T cells in immune homeostasis and regulation of autoimmune reactivity. The heterogeneous nature of identified CD8+ cell types are examined, and common phenotypes associated with functional activities are defined. The varying types of antigen signal crucial for CD8+ T cell regulatory activity are identified and the implications of these activation pathways for control of adaptive responses is considered. Finally, the promising capacity for transgenic antigen receptor directed cytotoxicity as a mechanism for modulation of autoimmunity is detailed.

A novel CNS-homing peptide for targeting neuroinflammatory lesions in experimental autoimmune encephalomyelitis.

Using phage peptide library screening, we identified peptide-encoding phages that selectively home to the inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (MS). A phage peptide display library encoding cyclic 9-amino-acid random peptides was first screened ex-vivo for binding to the CNS tissue of EAE mice, followed by in vivo screening in the diseased mice. Phage insert sequences that were present at a higher frequency in the CNS of EAE mice than in the normal (control) mice were identified by DNA sequencing. One of the phages selected in this manner, denoted as MS-1, was shown to selectively recognize CNS tissue in EAE mice. Individually cloned phages with this insert preferentially homed to EAE CNS after an intravenous injection. Similarly, systemically-administered fluorescence-labeled synthetic MS-1 peptide showed selective accumulation in the spinal cord of EAE mice. We suggest that peptide MS-1 might be useful for targeted drug delivery to CNS in EAE/MS.

Targeting antigen presentation in autoimmunity.

Autoimmune diseases are heterogeneous group of disorders that together represent an enormous societal and medical problem. CD4+ T cells have critical roles in the initiation and pathogenesis of autoimmune disease. As such, modulation of T cell activity has proven to have significant therapeutic effects in multiple autoimmune settings. T cell activation is a complex process with multiple potential therapeutic targets, many of which have been successfully utilized to treat human disease. Current pharmacological treatment largely targets T cell intrinsic activities as a means of treating various autoimmune disorders. Here I review extant and potential therapeutic approaches that instead specifically target antigen presentation to CD4+ T cells as a critical checkpoint in autoimmune responses. In addition, the contribution of antigen modulation components in current therapeutic approaches is considered along with the impact of new antigen targeted treatment modalities. Finally, potential challenges are considered in the context of the potential for antigen specific targeting of the antigen presentation process.

Engineered MBP-specific human Tregs ameliorate MOG-induced EAE through IL-2-triggered inhibition of effector T cells.

Expanded polyclonal T regulatory cells (Tregs) offer great promise for the treatment of immune-mediated diseases. Inhibition by Tregs is under the control of the T-cell receptor (TCR). Therefore, we created Tregs with defined antigen specificity, using a recombinant T-cell receptor isolated from a myelin-basic protein specific T-cell clone of a multiple sclerosis (MS) patient (Ob2F3). We expressed this TCR using a retroviral expression vector in human Tregs from peripheral blood. We observed that transduced Tregs were activated in vitro in response to myelin basic protein (MBP) peptide on DR15 antigen-presenting cells (APC) and upregulated Treg markers, Foxp3, LAP and Helios. These engineered MBP-specific Tregs could suppress MBP-specific T effector cells, and were also able to suppress T cells with other specificities after Tregs had been activated through the TCR. Importantly, we showed that these engineered Tregs were able to function effectively in the presence of strong TLR-induced inflammatory signals, and that MBP-specific Tregs ameliorated EAE in myelin oligodendrocyte glycoprotein (MOG)-immunized DR15 transgenic mice. We further demonstrated in vitro that IL-2 produced by neighboring effector T cells activated MBP-specific Tregs, initiating contact-independent suppression to T effectors in local milieu. Mechanistic studies demonstrated that bystander suppression in vivo may involve transfer of soluble mediators, enhanced by cell contact between Tregs and effectors. Taken together, we show that engineered clonal MBP-specific Tregs are able to suppress autoimmune pathology in EAE. This approach may serve as a cellular therapy for MS patients with the common DR15 haplotype that is associated with disease susceptibility.

Progression of experimental autoimmune encephalomyelitis is associated with up-regulation of major sodium transporters in the mouse kidney cortex under a normal salt diet.

Recent demonstrations of exacerbation of experimental autoimmune encephalomyelitis (EAE) by high salt diets prompted us to study whether EAE stimulated Na absorption by the renal cortex, a primary regulatory site for Na balance, even under a normal NaCl diet. We found that as EAE progressed from mild to severe symptoms, there were parallel increases in the protein abundance of NHE3 and αENaC and the Na,K-ATPase activity with an affiliated elevation of its ß1-subunit protein. These effects are associated with increases in the protein levels of the well-known regulators SGK1 and scaffold NHERF2, and phosphorylation of ERK1/2. These effects of EAE could not be explained by reduction in water or food intake. We conclude that EAE progression is associated with up-regulation of major Na transporters, which is most likely driven by increased expression of SGK1 and NHERF2 and activation of ERK1/2. These data suggest that EAE progression increases Na absorption by the renal cortex.

RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis.

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.

Odorants specifically modulate chemotaxis and tissue retention of CD4+ T cells via cyclic adenosine monophosphate induction.

Retention of T cells within affected tissue is a critical component of adaptive immune inflammation. However, the mechanisms involved in T cell retention remain largely undefined. Previous studies revealed the capacity of cAMP signaling to regulate immune cell migration, as well as dynamic regulation of receptors that could induce cAMP production in immune cells. The potential for cAMP to act as a retention signal has been mostly unexplored, partially as a result of this second messenger's well-characterized inhibition of effector function in immune cells. Here, we report that cAMP regulates the tissue retention of mouse T cells at concentrations well below those that inhibited proliferation or decreased acquisition of an effector phenotype. Stimulation of CD4+ T cells with odorants known to be cognate ligands for T cell-expressed olfactory receptors induced cAMP and inhibited chemokine-driven chemotaxis without decreasing T cell proliferation or effector functions. Similar effects were observed following treatment with relatively low concentrations of the cAMP analog Sp-5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole-3',5'-monophosphorothioate. Furthermore, pretreatment with odorants or cAMP at concentrations that did not inhibit effector function induced T cell tissue retention in mice by inhibiting chemokine-dependent T cell egress from the footpad to the draining lymph node. Together, these results suggest that odorant receptor-mediated increases in intracellular cAMP can modulate T cell tissue trafficking and may offer new therapeutic targets for controlling T cell tissue accumulation.

Interferon gamma in autoimmunity: A complicated player on a complex stage.

Early views of autoimmune disease cast IFNγ as a prototypic pro-inflammatory factor. It is now clear that IFNγ is capable of both pro- and anti-inflammatory activities with the functional outcome dependent on the physiological and pathological setting examined. Here, the major immune modulatory activities of IFNγ are reviewed and current evidence for the impact of IFNγ on pathology and regulation of several autoimmune diseases and disease models is summarized.

Pre-existing central nervous system lesions negate cytokine requirements for regional experimental autoimmune encephalomyelitis development.

In region-specific forms of experimental autoimmune encephalomyelitis (EAE), lesion initiation is regulated by T-cell-produced interferon-γ (IFN-γ) resulting in spinal cord disease in the presence of IFN-γ and cerebellar disease in the absence of IFN-γ. Although this role for IFN-γ in regional disease initiation is well defined, little is known about the consequences of previous tissue inflammation on subsequent regional disease, information vital to the development of therapeutics in established disease states. This study addressed the hypothesis that previous establishment of regional EAE would determine subsequent tissue localization of new T-cell invasion and associated symptoms regardless of the presence or absence of IFN-γ production. Serial transfer of optimal or suboptimal doses of encephalitogenic IFN-γ-sufficient or -deficient T-cell lines was used to examine the development of new clinical responses associated with the spinal cord and cerebellum at various times after EAE initiation. Previous inflammation within either cerebellum or spinal cord allowed subsequent T-cell driven inflammation within that tissue regardless of IFN-γ presence. Further, T-cell IFN-γ production after initial lesion formation exacerbated disease within the cerebellum, suggesting that IFN-γ plays different roles at different stages of cerebellar disease. For the spinal cord, IFN-γ-deficient cells (that are ordinarily cerebellum disease initiators) were capable of driving new spinal-cord-associated clinical symptoms more than 60 days after the initial acute EAE resolution. These data suggest that previous inflammation modulates the molecular requirements for new neuroinflammation development.

Myeloid derived suppressor cells in transplantation.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of hematopoietic derived cell precursors that can suppress immune responses in a variety of inflammatory settings. Here we review recent studies detailing expansion of phenotypically and functionally disparate MDSC. Findings related to MDSC accumulation, activation, and mechanisms utilized in immune suppression are presented. Further, we discuss recent reports that suggest MDSC are expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Generation, persistence and plasticity of CD4 T-cell memories.

The development of immune memory mediated by T lymphocytes is central to durable, long-lasting protective immunity. A key issue in the field is how to direct the generation and persistence of memory T cells to elicit the appropriate secondary response to provide protection to a specific pathogen. Two prevailing views have emerged; that cellular and molecular regulators control the lineage fate and functional capacities of memory T cells early after priming, or alternatively, that populations of memory T cells are inherently plastic and subject to alterations in function and/or survival at many stages during their long-term maintenance. Here, we will review current findings in CD4 T-cell memory that suggest inherent plasticity in populations of memory CD4 T cells at all stages of their development--originating with their generation from multiple types of primed CD4 T cells, during their persistence and homeostatic turnover in response to T-cell receptor signals, and also following secondary challenge. These multiple aspects of memory CD4 T-cell flexibility contrast the more defined lineages and functions ascribed to memory CD8 T cells, suggesting a dynamic nature to memory CD4 T-cell populations and responses. The flexible attributes of CD4 T-cell memory suggest opportunities and mechanisms for therapeutic manipulation at all phases of immune memory development, maintenance and recall.

Encephalitogenic T-cells increase numbers of CNS T-cells regardless of antigen specificity by both increasing T-cell entry and preventing egress.

This study utilized an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induction in mice to characterize the mechanisms involved in CNS accumulation of transferred and host T-cells. Using a flow cytometric technique, we examined phenotypic characteristics of CNS T-cells following disease initiation and the role of T-cell activation in CNS invasion and retention. Host T-cell activation increased cell recruitment and EAE severity. CNS antigen specific T-cells were required to induce T-cell retention within the CNS. Once retention was initiated, CNS T-cells were retained regardless of specificity. This study characterizes mechanisms involved in CNS accumulation of T-cells during EAE pathogenesis.

IL-1R signaling within the central nervous system regulates CXCL12 expression at the blood-brain barrier and disease severity during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the CNS characterized by disruption of the blood-brain barrier (BBB). This breach in CNS immune privilege allows undeterred trafficking of myelin-specific lymphocytes into the CNS where they induce demyelination. Although the mechanism of BBB compromise is not known, the chemokine CXCL12 has been implicated as a molecular component of the BBB whose pattern of expression is specifically altered during MS and which correlates with disease severity. The inflammatory cytokine IL-1beta has recently been shown to contribute not only to BBB permeability but also to the development of IL-17-driven autoimmune responses. Using experimental autoimmune encephalomyelitis, the rodent model of MS, we demonstrate that IL-1beta mediates pathologic relocation of CXCL12 during the induction phase of the disease, before the development of BBB disruption. We also show that CD4, CD8, and, surprisingly gammadelta T cells are all sources of IL-1beta. In addition, gammadelta T cells are also targets of this cytokine, contributing to IL-1beta-mediated production of IL-17. Finally, we show that the level of CNS IL-1R determines the clinical severity of experimental autoimmune encephalomyelitis. These data suggest that T cell-derived IL-1beta contributes to loss of immune privilege during CNS autoimmunity via pathologic alteration in the expression of CXCL12 at the BBB.

Regional CNS responses to IFN-gamma determine lesion localization patterns during EAE pathogenesis.

The localization of inflammatory foci within the cerebellum is correlated to severe clinical outcomes in multiple sclerosis (MS). Previous studies of experimental autoimmune encephalomyelitis (EAE), a model of MS, revealed distinct clinical outcomes correlated with the capacity of the animal to produce IFN-gamma. Outcomes were linked to localization of inflammatory cells in either the spinal cord (wild type [WT]) or the cerebellum and brain stem (IFN-gamma deficient). We demonstrate, using an adoptive transfer system, that the ability of the central nervous system (CNS) to sense pathogenic T cell-produced IFN-gamma during EAE initiation determines the sites of CNS pathogenesis. Transfer of WT Th1 cells into IFN-gamma receptor-deficient mice results in pathogenic invasion of the brain stem and cerebellum with attendant clinical symptoms, which are identical to the disease observed after transfer of IFN-gamma-deficient T cells to WT hosts. Inflammation of the spinal cord associated with classical EAE is abrogated in both IFN-gamma-deficient systems. Cotransfer of CNS antigen-specific WT Th1 cells with IFN-gamma-deficient T cells is sufficient to restore spinal cord invasion and block cerebellar and brain stem invasion. These data demonstrate that interaction between IFN-gamma and host CNS cells during the initiation of EAE can selectively promote or suppress neuroinflammation and pathogenesis.

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines.

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.

A little stress is good: IFN-gamma, demyelination, and multiple sclerosis.

The exact role(s) of the cytokine IFN-gamma in the demyelinating disease multiple sclerosis remain controversial, with evidence suggesting both detrimental and protective effects of the cytokine in MS and MS models such as EAE. The study by Lin and coworkers in this issue of the JCI produces evidence that protective effects of IFN-gamma on mature oligodendrocytes during EAE induction are mediated via activation of the pancreatic ER kinase (PERK), resulting in induction of the endoplasmic reticular stress response pathway (see the related article beginning on page 448). Modulation of this stress pathway has what we believe to be novel therapeutic potential for MS.

T-cell trafficking competence is required for CNS invasion.

T-cell invasion of the CNS is critical for the induction of a variety of autoimmune mediated neuronal diseases. We utilized blood-brain barrier (BBB) mediated exclusion of anti-CD4 antibody to define populations of encephalitogenic T-cells recovered from mouse CNS preparations as either CNS invasive or non-invasive. This separation of cells allowed flow cytometric examination of the kinetics of encephalitogenic T-cell entry past the BBB. Further experiments examined the relative contribution of EAE inflammatory conditioning of the BBB to the kinetics of T-cell adherence and migration into the CNS. Inflammatory conditioning was found to have no effect on accumulation of T-cells at the vascular interface of the BBB, but was found to increase the entry of adoptively transferred T-cells into the CNS following their initial adherence to the BBB.