Soluble CD25 imposes a low-zone IL-2 signaling environment that favors competitive outgrowth of antigen-experienced CD25high regulatory and memory T cells.

This study focused on soluble (s)CD25-mediated regulation of IL-2 signaling in murine and human CD4+ T cells. Recombinant sCD25 reversibly sequestered IL-2 to limit acute maximal proliferative responses while preserving IL-2 bioavailability to subsequently maintain low-zone IL-2 signaling during prolonged culture. By inhibiting IL-2 signaling during acute activation, sCD25 suppressed T-cell growth and inhibited IL-2-evoked transmembrane CD25 expression, thereby resulting in lower prevalence of CD25high T cells. By inhibiting IL-2 signaling during quiescent IL-2-mediated growth, sCD25 competed with transmembrane CD25, IL2Rßγ, and IL2Rαßγ receptors for limited pools of IL-2 such that sCD25 exhibited strong or weak inhibitory efficacy in IL-2-stimulated cultures of CD25low or CD25high T cells, respectively. Preferential blocking of IL-2 signaling in CD25low but not CD25high T cells caused competitive enrichment of CD25high memory/effector and regulatory FOXP3+ subsets. In conclusion, sCD25 modulates IL-2 bioavailability to limit CD25 expression during acute activation while enhancing CD25highT-cell dominance during low-zone homeostatic IL-2-mediated expansion, thereby 'flattening' the inflammatory curve over time.

Hypoxia-inducible factor-1 drives divergent immunomodulatory functions in the pathogenesis of autoimmune diseases.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric (HIF-1α/ HIF-1ß) transcription factor in which the oxygen-sensitive HIF-1α subunit regulates gene transcription to mediate adaptive tissue responses to hypoxia. HIF-1 is a key mediator in both regulatory and pathogenic immune responses, because ongoing inflammation in localized tissues causes increased oxygen consumption and consequent hypoxia within the inflammatory lesions. In autoimmune diseases, HIF-1 plays complex and divergent roles within localized inflammatory lesions by orchestrating a critical immune interplay sponsoring the pathogenesis of the disease. In this review, we have summarized the role of HIF-1 in lymphoid and myeloid immunomodulation in autoimmune diseases. HIF-1 drives inflammation by controlling the Th17/Treg /Tr1 balance through the tipping of the differentiation of CD4+ T cells in favour of pro-inflammatory Th17 cells while suppressing the development of anti-inflammatory Treg /Tr1 cells. On the other hand, HIF-1 plays a protective role by facilitating the expression of anti-inflammatory cytokine IL-10 in and expansion of CD1dhi CD5+ B cells, known as regulatory B cells or B10 cells. Apart from lymphoid cells, HIF-1 also controls the activation of macrophages, neutrophils and dendritic cells, thus eventually further influences the activation and development of effector/regulatory T cells by facilitating the creation of a pro/anti-inflammatory microenvironment within the autoinflammatory lesions. Based on the critical immunomodulatory roles that HIF-1 plays, this master transcription factor seems to be a potent druggable target for the treatment of autoimmune diseases.

Low-Zone IL-2 Signaling: Fusion Proteins Containing Linked CD25 and IL-2 Domains Sustain Tolerogenic Vaccination in vivo and Promote Dominance of FOXP3+ Tregs in vitro.

Low-zone IL-2 signaling is key to understanding how CD4+ CD25high FOXP3+ regulatory T cells (Tregs) exhibit dominance and overgrow conventional effector T cells (Tcons) that typically express lower levels of the IL-2 receptor alpha chain (i.e., CD25). Thus, modalities such as low-dose IL-2 or IL-2/anti-IL-2 antibody complexes have been advanced in the clinic to selectively expand Treg populations as a treatment for chronic inflammatory autoimmune diseases. However, more effective reagents that efficiently lock IL-2 signaling into a low signaling mode are needed to validate and exploit the low-zone IL-2 signaling niche of Tregs. This study focuses on CD25-IL2 and IL2-CD25 fusion proteins (FPs) that were approximately 32 and 320-fold less potent than IL-2. These FPs exhibited transient binding to transmembrane CD25 on human embryonic kidney (HEK) cells, had partially occluded IL-2 binding sites, and formed higher order multimeric conformers that limited the availability of bioactive IL-2. These FPs exhibited broad bell-shaped concentration ranges that favored dominant Treg outgrowth during continuous culture and were used to derive essentially pure long-term Treg monocultures (∼98% Treg purity). FP-induced Tregs had canonical Treg suppressive activity in that these Tregs suppressed antigen-specific proliferative responses of naïve CD4+ T cells. The in vivo administration of CD25-IL2/Alum elicited robust increases in circulating Tregs and selectively augmented CD25 expression on Tregs but not on Tcons. A single injection of a Myelin Oligodendrocyte Glycoprotein (MOG35-55)-specific tolerogenic vaccine elicited high levels of circulating MOG-specific Tregs in vivo that waned after 2-3 weeks, whereas boosting with CD25-IL2/Alum maintained MOG-specific CD25high Tregs throughout the 30-day observation period. However, these FPs did not antagonize free monomeric IL-2 and lacked therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). In conclusion, these data reveal that CD25-IL2 FPs can be used to select essentially pure long-term lines of FOXP3+ CD25high Tregs. This study also shows that CD25-IL2 FPs can be administered in vivo in synergy with tolerogenic vaccination to maintain high circulating levels of antigen-specific Tregs. Because tolerogenic vaccination and Treg-based adoptive immunotherapy are limited by gradual waning of Tregs, these FPs have potential utility in sustaining tolerogenic Treg responses in vivo.

Tolerogenic vaccines: Targeting the antigenic and cytokine niches of FOXP3+ regulatory T cells.

FOXP3+ regulatory T cells (Tregs) constitute a critical barrier that enforces tolerance to both the self-peptidome and the extended-self peptidome to ensure tissue-specific resistance to autoimmune, allergic, and other inflammatory disorders. Here, we review intuitive models regarding how T cell antigen receptor (TCR) specificity and antigen recognition efficiency shape the Treg and conventional T cell (Tcon) repertoires to adaptively regulate T cell maintenance, tissue-residency, phenotypic stability, and immune function in peripheral tissues. Three zones of TCR recognition efficiency are considered, including Tcon recognition of specific low-efficiency self MHC-ligands, Treg recognition of intermediate-efficiency agonistic self MHC-ligands, and Tcon recognition of cross-reactive high-efficiency agonistic foreign MHC-ligands. These respective zones of TCR recognition efficiency are key to understanding how tissue-resident immune networks integrate the antigenic complexity of local environments to provide adaptive decisions setting the balance of suppressive and immunogenic responses. Importantly, deficiencies in the Treg repertoire appear to be an important cause of chronic inflammatory disease. Deficiencies may include global deficiencies in Treg numbers or function, subtle 'holes in the Treg repertoire' in tissue-resident Treg populations, or simply Treg insufficiencies that are unable to counter an overwhelming molecular mimicry stimulus. Tolerogenic vaccination and Treg-based immunotherapy are two therapeutic modalities meant to restore dominance of Treg networks to reverse chronic inflammatory disease. Studies of these therapeutic modalities in a preclinical setting have provided insight into the Treg niche, including the concept that intermediate-efficiency TCR signaling, high IFN-ß concentrations, and low IL-2 concentrations favor Treg responses and active dominant mechanisms of immune tolerance. Overall, the purpose here is to assimilate new and established concepts regarding how cognate TCR specificity of the Treg repertoire and the contingent cytokine networks provide a foundation for understanding Treg suppressive strategy.

A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: Tolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs. METHODS: This study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35-55/MOG35-55) and GMCSF-NFM (neurofilament medium peptide 13-37/NFM13-37), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag-/- mice to reduce pre-existing Treg populations. RESULTS: This study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-ß, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG-Rag1-/- mice, which lacked pre-existing Tregs. CONCLUSIONS: This study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.

A GMCSF-Neuroantigen Tolerogenic Vaccine Elicits Systemic Lymphocytosis of CD4+ CD25high FOXP3+ Regulatory T Cells in Myelin-Specific TCR Transgenic Mice Contingent Upon Low-Efficiency T Cell Antigen Receptor Recognition.

Previous studies showed that single-chain fusion proteins comprised of GM-CSF and major encephalitogenic peptides of myelin, when injected subcutaneously in saline, were potent tolerogenic vaccines that suppressed experimental autoimmune encephalomyelitis (EAE) in rats and mice. These tolerogenic vaccines exhibited dominant suppressive activity in inflammatory environments even when emulsified in Complete Freund's Adjuvant (CFA). The current study provides evidence that the mechanism of tolerance was dependent upon vaccine-induced regulatory CD25+ T cells (Tregs), because treatment of mice with the Treg-depleting anti-CD25 mAb PC61 reversed tolerance. To assess tolerogenic mechanisms, we focused on 2D2-FIG mice, which have a transgenic T cell repertoire that recognizes myelin oligodendrocyte glycoprotein peptide MOG35-55 as a low-affinity ligand and the neurofilament medium peptide NFM13-37 as a high-affinity ligand. Notably, a single subcutaneous vaccination of GMCSF-MOG in saline elicited a major population of FOXP3+ Tregs that appeared within 3 days, was sustained over several weeks, expressed canonical Treg markers, and was present systemically at high frequencies in the blood, spleen, and lymph nodes. Subcutaneous and intravenous injections of GMCSF-MOG were equally effective for induction of FOXP3+ Tregs. Repeated booster vaccinations with GMCSF-MOG elicited FOXP3 expression in over 40% of all circulating T cells. Covalent linkage of GM-CSF with MOG35-55 was required for Treg induction whereas vaccination with GM-CSF and MOG35-55 as separate molecules lacked Treg-inductive activity. GMCSF-MOG elicited high levels of Tregs even when administered in immunogenic adjuvants such as CFA or Alum. Conversely, incorporation of GM-CSF and MOG35-55 as separate molecules in CFA did not support Treg induction. The ability of the vaccine to induce Tregs was dependent upon the efficiency of T cell antigen recognition, because vaccination of 2D2-FIG or OTII-FIG mice with the high-affinity ligands GMCSF-NFM or GMCSF-OVA (Ovalbumin323-339), respectively, did not elicit Tregs. Comparison of 2D2-FIG and 2D2-FIG-Rag1-/- strains revealed that GMCSF-MOG may predominantly drive Treg expansion because the kinetics of vaccine-induced Treg emergence was a function of pre-existing Treg levels. In conclusion, these findings indicate that the antigenic domain of the GMCSF-NAg tolerogenic vaccine is critical in setting the balance between regulatory and conventional T cell responses in both quiescent and inflammatory environments.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-ß elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-ß-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-ß-inducible "iTregs" because Tregs from 2D2-FIG Rag1-/- mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-ß fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy.

IFN-ß Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis.

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-ß and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-ß, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-ß + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35-55 in proteolipid protein- and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-ß/Alum-based vaccination exhibited hallmarks of infectious tolerance, because IFN-ß + OVA in Alum-specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-ß + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-ß and MOG35-55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti-TGF-ß and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-ß + MOG-induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-ß + NAg in Alum vaccines, vaccination with TGF-ß + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-ß + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-ß has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs.

Depletion of CD4+ CD25+ regulatory T cells confers susceptibility to experimental autoimmune encephalomyelitis (EAE) in GM-CSF-deficient Csf2-/- mice.

Previous studies established that GM-CSF-deficient (Csf2-deficient) mice exhibit profound resistance to experimental autoimmune encephalomyelitis. This study addressed whether the resistance of Csf2-deficient mice was a result of a requirement for GM-CSF in controlling the functional balance between effector and regulatory T cell subsets during experimental autoimmune encephalomyelitis. The main observation was that treatment with the anti-CD25 mAb PC61 rendered Csf2-deficient mice fully susceptible to severe, chronic experimental autoimmune encephalomyelitis, with disease incidences and severities equivalent to that of C57BL/6 mice. When both donors and recipients were treated with PC61 in a passive model of experimental autoimmune encephalomyelitis, adoptive transfer of myelin-specific Csf2-deficient T cells into Csf2-deficient recipients resulted in a nonresolving chronic course of severe paralytic experimental autoimmune encephalomyelitis. The peripheral Csf2-deficient T cell repertoire was marked by elevated CD3+ T cell frequencies that reflected substantial accumulations of naïve CD44null-low CD4+ and CD8+ T cells but essentially normal frequencies of CD4+ CD25+ forkhead box P3+ T cells among the CD3+ T cell pool. These findings suggested that Csf2-deficient mice had secondary deficiencies in peripheral T cell sensitization to environmental antigens. In accordance, myelin oligodendrocyte glycoprotein 35-55/CFA-sensitized Csf2-deficient mice exhibited deficient peripheral sensitization to myelin oligodendrocyte glycoprotein, whereas pretreatment of Csf2-deficient mice with PC61 enabled the robust induction of myelin oligodendrocyte glycoprotein-specific T cell responses in the draining lymphatics. In conclusion, the experimental autoimmune encephalomyelitis resistance of Csf2-deficient mice, at least in part, reflects a deficient induction of effector T cell function that cannot surmount normal regulatory T cell barriers. Experimental autoimmune encephalomyelitis effector responses, however, are unleashed upon depletion of regulatory CD25+ T cells.

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies.

Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated regions (3'UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3'UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund's adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund's adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6-7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.

GM-CSF-neuroantigen fusion proteins reverse experimental autoimmune encephalomyelitis and mediate tolerogenic activity in adjuvant-primed environments: association with inflammation-dependent, inhibitory antigen presentation.

Single-chain fusion proteins comprised of GM-CSF and neuroantigen (NAg) are potent, NAg-specific inhibitors of experimental autoimmune encephalomyelitis (EAE). An important question was whether GMCSF-NAg tolerogenic vaccines retained inhibitory activity within inflammatory environments or were contingent upon steady-state conditions. GM-CSF fused to the myelin oligodendrocyte glycoprotein MOG35-55 peptide (GMCSF-MOG) reversed established paralytic disease in both passive and active models of EAE in C57BL/6 mice. The fusion protein also reversed EAE in CD4-deficient and B cell-deficient mice. Notably, GMCSF-MOG inhibited EAE when coinjected adjacent to the MOG35-55/CFA emulsion. GMCSF-MOG also retained dominant inhibitory activity when directly emulsified with MOG35-55 in the CFA emulsion in both C57BL/6 or B cell-deficient models of EAE. Likewise, when combined with proteolipid protein 139-151 in CFA, GM-CSF fused to proteolipid protein 139-151 peptide inhibited EAE in SJL mice. When deliberately emulsified in CFA with the NAg, GMCSF-NAg inhibited EAE even though NAg was present at >30-fold molar excess. In vitro studies revealed that the GM-CSF domain of GMCSF-MOG stimulated growth and differentiation of inflammatory dendritic cells (DC) and simultaneously targeted the MOG35-55 domain for enhanced presentation by these DC. These inflammatory DC presented MOG35-55 to MOG-specific T cells by an inhibitory mechanism that was mediated in part by IFN-γ signaling and NO production. In conclusion, GMCSF-NAg was tolerogenic in CFA-primed proinflammatory environments by a mechanism associated with targeted Ag presentation by inflammatory DC and an inhibitory IFN-γ/NO pathway. The inhibitory activity of GMCSF-NAg in CFA-primed lymphatics distinguishes GMCSF-NAg fusion proteins as a unique class of inflammation-dependent tolerogens that are mechanistically distinct from naked peptide or protein-based tolerogens.

Tolerogenic vaccines for Multiple sclerosis.

Tolerogenic vaccines represent a new class of vaccine designed to re-establish immunological tolerance, restore immune homeostasis, and thereby reverse autoimmune disease. Tolerogenic vaccines induce long-term, antigen-specific, inhibitory memory that blocks pathogenic T cell responses via loss of effector T cells and gain of regulatory T cell function. Substantial advances have been realized in the generation of tolerogenic vaccines that inhibit experimental autoimmune encephalomyelitis in a preclinical setting, and these vaccines may be a prequel of the tolerogenic vaccines that may have therapeutic benefit in Multiple Sclerosis. The purpose here is to provide a snapshot of the current concepts and future prospects of tolerogenic vaccination for Multiple Sclerosis, along with the central challenges to clinical application.

Cytokine-neuroantigen fusion proteins as a new class of tolerogenic, therapeutic vaccines for treatment of inflammatory demyelinating disease in rodent models of multiple sclerosis.

Myelin-specific induction of tolerance represents a promising means to modify the course of autoimmune inflammatory demyelinating diseases such as multiple sclerosis (MS). Our laboratory has focused on a novel preclinical strategy for the induction of tolerance to the major encephalitogenic epitopes of myelin that cause experimental autoimmune encephalomyelitis (EAE) in rats and mice. This novel approach is based on the use of cytokine-NAg (neuroantigen) fusion proteins comprised of the native cytokine fused either with or without a linker to a NAg domain. Several single-chain cytokine-NAg fusion proteins were tested including GMCSF-NAg, IFNbeta-NAg, NAgIL16, and IL2-NAg. These cytokine-NAg vaccines were tolerogenic, therapeutic vaccines that had tolerogenic activity when given as pre-treatments before encephalitogenic immunization and also were effective as therapeutic interventions during the effector phase of EAE. The rank order of inhibitory activity was as follows: GMCSF-NAg, IFNbeta-NAg > NAgIL16 > IL2-NAg > MCSF-NAg, IL4-NAg, IL-13-NAg, IL1RA-NAg, and NAg. Several cytokine-NAg fusion proteins exhibited antigen-targeting activity. High affinity binding of the cytokine domain to specific cytokine receptors on particular subsets of APC resulted in the concentrated uptake of the NAg domain by those APC which in turn facilitated the enhanced processing and presentation of the NAg domain on cell surface MHC class II glycoproteins. For most cytokine-NAg vaccines, the covalent linkage of the cytokine domain and NAg domain was required for inhibition of EAE, thereby indicating that antigenic targeting of the NAg domain to APC was also required in vivo for tolerogenic activity. Overall, these studies introduced a new concept of cytokine-NAg fusion proteins as a means to induce tolerance and to inhibit the effector phase of autoimmune disease. The approach has broad application for suppressive vaccination as a therapy for autoimmune diseases such as MS.

Neuroantigen-specific, tolerogenic vaccines: GM-CSF is a fusion partner that facilitates tolerance rather than immunity to dominant self-epitopes of myelin in murine models of experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: Vaccination strategies that elicit antigen-specific tolerance are needed as therapies for autoimmune disease. This study focused on whether cytokine-neuroantigen (NAg) fusion proteins could inhibit disease in chronic murine models of experimental autoimmune encephalomyelitis (EAE) and thus serve as potential therapeutic modalities for multiple sclerosis. RESULTS: A fusion protein comprised of murine GM-CSF as the N-terminal domain and the encephalitogenic MOG35-55 peptide as the C-terminal domain was tested as a tolerogenic, therapeutic vaccine (TTV) in the C57BL/6 model of EAE. Administration of GMCSF-MOG before active induction of EAE, or alternatively, at the onset of EAE blocked the development and progression of EAE. Covalent linkage of the GM-CSF and MOG35-55 domains was required for tolerogenic activity. Likewise, a TTV comprised of GM-CSF and PLP139-151 was a tolerogen in the SJL model of EAE. CONCLUSION: These data indicated that fusion proteins containing GM-CSF coupled to myelin auto-antigens elicit tolerance rather than immunity.

Autoimmunity and asthma: The dirt on the hygiene hypothesis.

Self peptides shape T-cell development through selectional processes in the thymus and secondary lymphoid organs to promote a diverse and balanced repertoire of conventional and regulatory T cells. Foreign proteins and their derivative peptides permeate our mucosal tissues to constitute another diverse array of peptides that may specify and diversify the mucosal T-cell repertoire. Indeed, the distinction between self peptides and environmental foreign peptides may be academic if both are present constantly within the body. The premise here is that the plethora of foreign peptides, present ubiquitously in our environment and body, form homeostatic niches to foster highly diversified repertoires of conventional and regulatory T cells that recognize persistent environmental peptides as self. Highly diversified repertoires that recognize myriads of self and environmental foreign peptides as homeostatic ligands may be critical for adaptive distinctions of friend or foe in mucosal tissues. The change from our agrarian past to the highly sterile environments of today may adversely impact the diversity and concentrations of foreign peptides that shape the mucosal T-cell repertoire. Various hygiene hypotheses postulate that the lack of factors such as infectious pathogens, innate receptor engagement or Th1 bias is key to the marked increase in immunological disease in modern society. In this version of the hygiene hypothesis, highly diverse constellations of innocuous environmental peptides are postulated to be the critical factor for immune balance and homeostasis.

A GMCSF-neuroantigen fusion protein is a potent tolerogen in experimental autoimmune encephalomyelitis (EAE) that is associated with efficient targeting of neuroantigen to APC.

Cytokine-NAg fusion proteins represent an emerging platform for specific targeting of self-antigen to particular APC subsets as a means to achieve antigen-specific immunological tolerance. This study focused on cytokine-NAg fusion proteins that targeted NAg to myeloid APC. Fusion proteins contained GM-CSF or the soluble extracellular domain of M-CSF as the N-terminal domain and the encephalitogenic 69-87 peptide of MBP as the C-terminal domain. GMCSF-NAg and MCSF-NAg fusion proteins were approximately 1000-fold and 32-fold more potent than NAg in stimulating antigenic proliferation of MBP-specific T cells, respectively. The potentiated antigenic responses required cytokine-NAg covalent linkage and receptor-mediated uptake. That is, the respective cytokines did not potentiate antigenic responses when cytokine and NAg were added as separate molecules, and the potentiated responses were inhibited specifically by the respective free cytokine. Cytokine-dependent targeting of NAg was specific for particular subsets of APC. GMCSF-NAg and MCSF-NAg targeted NAg to DC and macrophages; conversely, IL4-NAg and IL2-NAg fusion proteins, respectively, induced an 1000-fold enhancement in NAg reactivity in the presence of B cell and T cell APC. GMCSF-NAg significantly attenuated severity of EAE when treatment was completed before encephalitogenic challenge or alternatively, when treatment was initiated after onset of EAE. MCSF-NAg also had significant tolerogenic activity, but GMCSF-NAg was substantially more efficacious as a tolerogen. Covalent GMCSF-NAg linkage was required for prevention and treatment of EAE. In conclusion, GMCSF-NAg was highly effective for targeting NAg to myeloid APC and was a potent, antigen-specific tolerogen in EAE.

Experimental autoimmune encephalomyelitis in Lewis rats: IFN-beta acts as a tolerogenic adjuvant for induction of neuroantigen-dependent tolerance.

Cytokine-Ag fusion proteins represent a novel approach for induction of Ag-specific tolerance and may constitute an efficient therapy for autoimmune disease. This study addressed whether a fusion protein containing rat IFN-beta and the encephalitogenic 73-87 determinant of myelin basic protein (i.e., the neuroantigen, or NAg) could prevent or treat experimental autoimmune encephalomyelitis (EAE) in Lewis rats. The optimal structure of the fusion protein was comprised of the rat IFN-beta cytokine as the N-terminal domain with an enterokinase (EK) linker to the NAg domain. Both cytokine and NAg domains had full biological activity. Subcutaneous administration of 1 nmol of IFNbeta-NAg fusion protein in saline on days -21, -14, and -7 before encephalitogenic challenge on day 0 resulted in a substantial attenuation of EAE. In contrast, administration of IFN-beta or NAg alone did not affect susceptibility to EAE. The covalent attachment of IFN-beta and NAg was not necessary, because separate injections of IFN-beta and NAg at adjacent sites were as effective as injection of IFNbeta-NAg for prevention of disease. When treatment was initiated after disease onset, the rank order of inhibitory activity was as follows: the IFNbeta-NAg fusion protein > or = a mixture of IFN-beta plus NAg > IFN-beta > NAg. The novel finding that IFN-beta acts as a tolerogenic adjuvant as well as a tolerogenic fusion partner may have significance for development of tolerogenic vaccines.

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II.

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-gamma, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC.

A fusion protein consisting of IL-16 and the encephalitogenic peptide of myelin basic protein constitutes an antigen-specific tolerogenic vaccine that inhibits experimental autoimmune encephalomyelitis.

To test a novel concept for the generation of tolerogenic vaccines, fusion proteins were constructed encompassing a tolerogenic or biasing cytokine and the major encephalitogenic peptide of guinea pig myelin basic protein (GPMBP; i.e., neuroantigen or NAg). The cytokine domain was predicted to condition APC while simultaneously targeting the covalently linked encephalitogenic peptide to the MHC class II Ag processing pathway of those conditioned APC. Rats were given three s.c. injections of cytokine-NAg in saline 1-2 wk apart and then at least 1 wk later were challenged with NAg in CFA. The rank order of tolerogenic activity in the Lewis rat model of EAE was NAgIL16 > IL2NAg > IL1RA-NAg, IL13NAg >or= IL10NAg, GPMBP, GP69-88, and saline. NAgIL16 was also an effective inhibitor of experimental autoimmune encephalomyelitis when administered after an encephalitogenic challenge during the onset of clinical signs. Covalent linkage of the NAg and IL-16 was required for inhibition of experimental autoimmune encephalomyelitis. These data identify IL-16 as an optimal cytokine partner for the generation of tolerogenic vaccines and indicate that such vaccines may serve as Ag-specific tolerogens for the treatment of autoimmune disease.