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1.  Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects 
European journal of immunology  2013;43(5):1309-1321.
Macrophage migration inhibitory factor (MIF) and its receptor, CD74, are pivotal regulators of the immune system. Here we demonstrate for the first time that partial MHC class II constructs comprised of linked β1α1 domains with covalently attached antigenic peptides (also referred to as recombinant T-cell receptor ligands - RTLs) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also down-regulating CD74 cell-surface expression. This bi-functional inhibition of MIF/CD74 interactions blocked downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity and inhibition of random migration that all contribute to the reversal of clinical and histological signs of experimental autoimmune encephalomyelitis (EAE). Moreover, we demonstrate that enhanced CD74 cell surface expression on monocytes in mice with EAE and subjects with multiple sclerosis (MS) can be down-regulated by humanized RTLs, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity.
doi:10.1002/eji.201243162
PMCID: PMC3788583  PMID: 23576302
CD74; Macrophage migration inhibitory factor (MIF); multiple sclerosis; recombinant T-cell receptor ligand
2.  A novel HLA-DRα1-MOG-35-55 construct treats experimental stroke 
Metabolic brain disease  2014;29(1):37-45.
Chemoattraction of leukocytes into the brain after induction of middle cerebral artery occlusion (MCAO) increases the lesion size and worsens disease outcome. Our previous studies demonstrated that partial MHC class II constructs can reverse this process. However, the potential application of pMHC to human stroke is limited by the need to rapidly match recipient MHC class II with the β1 domain of the pMHC construct. We designed a novel recombinant protein comprised of the HLA-DRα1 domain linked to MOG-35-55 peptide but lacking the β1 domain found in pMHC and treated MCAO after 4 h reperfusion in humanized DR2 mice. Infarct volumes were quantified after 96 h reperfusion and immune cells from the periphery and CNS were evaluated for expression of CD74 and other cell surface, cytokine and pathway markers. This study demonstrates that four daily treatments with DRα1-MOG-35-55 reduced infarct size by 40 % in the cortex, striatum and hemisphere, inhibited the migration of activated CD11b+CD45high cells from the periphery to the brain and reversed splenic atrophy. Furthermore, DRα1-MOG-35-55 bound to CD74 on monocytes and blocked both binding and downstream signaling of macrophage migration inhibition factor (MIF) that may play a key role in infarct development. The novel DRα1-MOG-35-55 construct is highly therapeutic in experimental stroke and could be given to all patients at least 4 h after stroke onset without the need for tissue typing due to universal expression of DRα1 in humans.
doi:10.1007/s11011-013-9440-0
PMCID: PMC3975671  PMID: 24122483
Stroke; Inflammation; Immunotherapy; Recombinant T-cell receptor Ligand; MHC class II invariant chain
3.  A novel regulatory pathway for autoimmune disease: Binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance 
Journal of autoimmunity  2012;40:96-110.
Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands – RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b+ mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.
doi:10.1016/j.jaut.2012.08.004
PMCID: PMC3789252  PMID: 23026773
Partial MHC class II; CD74; EAE; MIF
4.  Intrastriatal B-cell administration limits infarct size after stroke in B-cell deficient mice 
Metabolic brain disease  2012;27(4):487-493.
Recent evidence emphasizes B-cells as a major regulatory cell type that plays an important role in limiting the pathogenic effects of ischemic stroke. The aim of the current study was to extend this initial observation to specifically examine the infiltration of regulatory B-cells and to determine if the effect of B-cells to limit the inflammatory response to cerebral ischemia is mediated by their action centrally or peripherally. Our data demonstrate the increased presence of a regulatory B-cell subset in the affected hemisphere of wild-type mice after middle cerebral artery occlusion (MCAO). We further explored the use of a novel method of stereotaxic cell delivery to bypass the blood brain barrier (BBB) and introduce CD19+ B cells directly into the striatum as compared to peripheral administration of B-cells. Infarct volumes after 60 minutes of MCAO and 48 hours of reperfusion were determined in B-cell deficient μMT−/− mice with and without replacement of either B-cells or medium. Infarct size was significantly decreased in cerebral cortex after intrastriatal transfer of 100,000 B-cells to μMT−/− mice vs. controls, with a comparable effect on infarct size as obtained by 50 million B-cells transferred intraperitoneally. These findings support the hypothesis that B-cells play a protective role against ischemic brain injury, and suggest that that B-cells may serve as a novel therapeutic agent for modulating the immune response in central nervous system inflammation after stroke.
doi:10.1007/s11011-012-9317-7
PMCID: PMC3427715  PMID: 22618587
regulatory B-cells; experimental stroke; intrastriatal transfer; uMT mice
5.  PD-L1 enhances CNS inflammation and infarct volume following experimental stroke in mice in opposition to PD-1 
Background
Stroke severity is worsened by recruitment of inflammatory immune cells into the brain. This process depends in part on T cell activation, in which the B7 family of co-stimulatory molecules plays a pivotal role. Previous studies demonstrated more severe infarcts in mice lacking programmed death-1 (PD-1), a member of the B7 family, thus implicating PD-1 as a key factor in limiting stroke severity. The purpose of this study was to determine if this protective effect of PD-1 involves either of its ligands, PD-L1 or PD-L2.
Methods
Central nervous system (CNS) inflammation and infarct volume were evaluated in male PD-L1 and PD-L2 knockout (-/-) mice undergoing 60 minutes of middle cerebral artery occlusion (MCAO) followed by 96 hours of reperfusion and compared to wild-type (WT) C57BL/6J mice.
Results
PD-L1-/- and PD-L2-/- mice had smaller total infarct volumes compared to WT mice. The PD-L1-/- and to a lesser extent PD-L2-/- mice had reduced levels of proinflammatory activated microglia and/or infiltrating monocytes and CD4+ T cells in the ischemic hemispheres. There was a reduction in ischemia-related splenic atrophy accompanied by lower activation status of splenic T cells and monocytes in the absence of PD-L1, suggesting a pathogenic rather than a regulatory role for both PD-1 ligands (PD-Ls). Suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice and there was decreased expression of CD80 on splenic antigen-presenting cells (APCs) as compared to the WT and PD-L2-/- mice.
Conclusions
Our novel observations are the first to implicate PD-L1 involvement in worsening outcome of experimental stroke. The presence of suppressor T cells in the right MCAO-inflicted hemisphere in mice lacking PD-L1 implicates these cells as possible key contributors for controlling adverse effects of ischemia. Increased expression of CD80 on APCs in WT and PD-L2-/- mice suggests an overriding interaction leading to T cell activation. Conversely, low CD80 expression by APCs, along with increased PD-1 and PD-L2 expression in PD-L1-/- mice suggests alternative T cell signaling pathways, leading to a suppressor phenotype. These results suggest that agents (for example antibodies) that can target and neutralize PD-L1/2 may have therapeutic potential for treatment of human stroke.
doi:10.1186/1742-2094-10-111
PMCID: PMC3846120  PMID: 24015822
Co-inhibitory pathway; Inflammatory states; MCAO; Programmed death-1 ligand 1 and 2
6.  PD-1 Interaction with PD-L1 but not PD-L2 on B-cells Mediates Protective Effects of Estrogen against EAE 
Increased remissions in multiple sclerosis (MS) during late pregnancy may result from high levels of sex steroids such as estrogen and estriol. Estrogen (E2=17β-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Treatment with relatively low doses of E2 can protect against clinical and histological signs of MOG-35-55 induced EAE through mechanisms involving the PD-1 coinhibitory pathway and B-cells. The current study evaluated the contribution of PD-1 ligands, PD-L1 and PD-L2, on B-cells in E2-mediated protection against EAE in WT, PD-L1−/− and PD-L2−/− mice. Unlike PD-L2−/− mice that were fully protected against EAE after E2 treatment, E2-implanted PD-L1−/− mice were fully susceptible to EAE, with increased numbers of proliferating Th1/Th17 cells in the periphery and severe cellular infiltration and demyelination in the CNS. Moreover, transfer of B-cells from MOG-immunized PD-L1−/− or PD-L2−/− donors into E2-preconditioned B-cell deficient μMT−/− recipient mice revealed significantly reduced E2-mediated protection against EAE in recipients of PD-L1−/− B-cells, but near-complete protection in recipients of PD-L2−/− B-cells. We conclude that PD-1 interaction with PD-L1 but not PD-L2 on B-cells is crucial for E2-mediated protection in EAE and that strategies that enhance PD-1/PD-L1 interactions might potentiate E2 treatment effects in MS.
doi:10.4172/2155-9899.1000143
PMCID: PMC3760596  PMID: 24009988
EAE; Multiple Sclerosis; Estrogen; PD-L; Regulatory B cells
7.  A Novel Hypothesis: Regulatory B Lymphocytes Shape Outcome from Experimental Stroke 
Translational stroke research  2012;3(3):324-330.
Although inflammatory immune cells clearly contribute to the development of middle cerebral artery occlusion (MCAO) in mice, the failure to block neutrophil-associated injury in clinical stroke trials has discouraged further development of immunotherapeutic approaches. However, there is renewed interest in a possible protective role for regulatory T- and B-cells that can suppress inflammation and limit central nervous system damage induced by infiltrating pro-inflammatory cells. Our failure to implicate CD4+FoxP3+ T-cells in limiting brain lesion volume after MCAO turned our focus towards regulatory B-cells known to mediate protection against other inflammatory CNS conditions. Our results clearly demonstrated that B-cell deficient mice developed larger infarct volumes, higher mortality and more severe functional deficits compared to wild-type mice, and had increased numbers of activated T-cells, macrophages, microglial cells, and neutrophils in the affected brain hemisphere. These MCAO-induced changes were completely prevented in B-cell-restored mice after transfer of highly purified WT B-cells but not IL-10-deficient B-cells. Our novel observations are the first to implicate IL-10-secreting B-cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B-cells might have application as a novel therapy for this devastating neurologic condition.
doi:10.1007/s12975-012-0187-4
PMCID: PMC3501272  PMID: 23175646
Experimental stroke; Bregs; IL-10; PD-1; immunotherapy
8.  Myelin specific cells infiltrate MCAO lesions and exacerbate stroke severity 
Metabolic Brain Disease  2011;27(1):7-15.
Although inflammatory responses increase stroke severity, the role of immune cells specific for central nervous system (CNS) antigens remains controversial. Disruption of the blood-brain barrier (BBB) during stroke allows CNS antigens to leak into the peripheral circulation and enhances access of circulating leukocytes to the brain, including those specific for CNS antigens such as myelin oligodendrocyte glycoprotein (MOG) that can induce experimental autoimmune encephalomyelitis (EAE). We here demonstrate for the first time that myelin reactive splenocytes specific for MOG transferred into severe combined immunodeficiency (SCID) mice can migrate into the infarct hemisphere of recipients subjected to 60 minutes middle cerebral artery occlusion (MCAO) and 96 hours reperfusion; moreover these cells exacerbate infarct volume and worsen neurological deficits compared to animals transferred with naïve splenocytes. These findings indicate that autoimmunity in the CNS can exert detrimental injury on brain cells and worsen the damage from ischemic stroke.
doi:10.1007/s11011-011-9267-5
PMCID: PMC3270145  PMID: 21989743
experimental stroke; myelin reactive splenocytes; inflammatory responses; neurologic deficit
9.  GPR30 FORMS AN INTEGRAL PART OF E2-PROTECTIVE PATHWAY IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS 
A major focus of our laboratory has been an in-depth evaluation as to how estrogens exert a pronounced protective effect on clinical and histological disease in the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). An important issue regarding their therapeutic application has been the undesirable estrogenic side effects thought to be mediated primarily through 17β-estradiol (E2) binding to intracellular estrogen receptor alpha (ERα). With the discovery and characterization of GPR30 as the putative membrane estrogen receptor, we sought to study whether signaling through GPR30 was sufficient to mediate protection against EAE without engagement of ERα. Treatment of EAE in WT mice with G-1, a selective GPR30 agonist, retained estradiol’s ability to protect against clinical and histological EAE without estrogenic side effects. G-1 treatment deviated cytokine profiles and enhanced suppressive activity of CD4+Foxp3+ Treg cells through a GPR30- and programmed death 1 (PD-1)-dependent mechanism. This novel finding was indicative of the protective effect of GPR30 activation in EAE and provides a strong foundation for the clinical application of GPR30 agonists such as G-1 in MS. However, future studies are needed to elucidate cross-signaling and evaluate possible additive effects of combined signaling through both GPR30 and ER-α. Deciphering the possible mechanism of involvement of GPR30 in estrogen-mediated protection against EAE may result in lowering treatment doses of E2 and GPR30 agonists that could minimize risks and maximize immunoregulation and therapeutic effects in MS. Alternatively, one might envision using E2 derivatives with reduced estrogenic activity alone or in combination with GPR30 agonists as therapies for both male and female MS patients.
PMCID: PMC3255092  PMID: 22247749
EAE; Estrogen; ER-α; ER-β; GPR30; G-1; G-15; 17β-estradiol; encephalitogenic T cells; Tregs; dendritic cells; inflammation; IL-10; IL-17; MS
10.  MHC Class II Derived Recombinant T Cell Receptor Ligands Protect DBA/1LacJ Mice from Collagen-Induced Arthritis1 
We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the α1 and β1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257–270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257–270 molecule could systemically reduce proinflammatory IL-17 and IFN-γ production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257–270 molecule could also selectively inhibit IL-1β, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.
PMCID: PMC3457790  PMID: 18178865
11.  Programmed death-1 pathway limits CNS inflammation and neurologic deficits in murine experimental stroke 
Background and Purpose
Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets that affect stroke outcome. Our goal was to evaluate the contribution of the well-described co-inhibitory pathway, Programmed Death (PD)-1, to the development of MCAO.
Methods
Infarct volumes, functional outcomes and effects on infiltrating immune cell populations were compared in wild type C57BL/6 versus PD-1 deficient mice after 60min MCAO and 96h reperfusion.
Results
The results clearly demonstrate a previously unrecognized activity of the PD-1 pathway to limit infarct volume, recruitment of inflammatory cells from the periphery, activation of macrophages and CNS microglia and functional neurological deficits. These regulatory functions were associated with increased percentages of circulating PD-Ligand (L)-1 and PD-L2 expressing CD19+ B-cells in blood, spleen and CNS with the capacity to inhibit activation of inflammatory T-cells and CNS macrophages and microglial cells through upregulated PD-1.
Conclusions
Our novel observations are the first to implicate PD-1 signaling as a major protective pathway for limiting CNS inflammation in MCAO. This inhibitory circuit would likely be pivotal in reducing stroke-associated TLR2- and TLR4-mediated release of neurotoxic factors by activated CNS microglia.
doi:10.1161/STROKEAHA.111.613182
PMCID: PMC3164218  PMID: 21737801
MCAO; inflammatory cells; Programmed Death-1; Co-inhibitory pathway
12.  Regulatory B-cells limit CNS inflammation and neurologic deficits in murine experimental stroke 
Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B-cells to the development of MCAO by comparing infarct volumes and functional outcomes in WT versus B-cell deficient μMT−/− mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits and increased numbers of activated T-cells, macrophages, microglial cells and neutrophils in the affected brain hemisphere of MCAO-treated μMT−/− vs. WT mice. These MCAO-induced changes were completely prevented in B-cell restored μMT−/− mice after transfer of highly purified WT GFP+ B-cells that were detected in the periphery, but not the CNS. In contrast, transfer of B-cells from IL-10−/− mice had no effect on infarct volume when transferred into μMT−/− mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B-cells to limit infarct volume, mortality rate, recruitment of inflammatory cells and functional neurological deficits 48h after MCAO. Our novel observations are the first to implicate IL-10-secreting B-cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B-cells might have application as a novel therapy for this devastating neurologic condition.
doi:10.1523/JNEUROSCI.1623-11.2011
PMCID: PMC3111929  PMID: 21653859
13.  Therapy with Recombinant T-cell Receptor Ligand reduces infarct size and infiltrating inflammatory cells in brain after middle cerebral artery occlusion in mice 
Metabolic brain disease  2011;26(2):123-133.
Stroke induces a biphasic effect on the peripheral immune response that involves early activation of peripheral leukocytes followed by severe immunosuppression and atrophy of the spleen. Peripheral immune cells, including T lymphocytes, migrate to the brain and exacerbate the developing infarct. Recombinant T-cell receptor (TCR) Ligand (RTL)551 is designed as a partial TCR agonist for myelin oligodendrocyte glycoprotein (MOG)-reactive T cells and has demonstrated the capacity to limit infarct volume and inflammation in brain when administered to mice undergoing middle cerebral artery occlusion (MCAO). The goal of this study was to determine if RTL551 could retain protection when given within the therapeutically relevant 4h time window currently in clinical practice for stroke patients. RTL551 was administered subcutaneously 4h after MCAO, with repeated doses every 24h until the time of euthanasia. Cell numbers were assessed in the brain, blood, spleen and lymph nodes and infarct size was measured after 24 and 96h reperfusion. RTL551 reduced infarct size in both cortex and striatum at 24h and in cortex at 96h after MCAO and inhibited the accumulation of inflammatory cells in brain at both time points. At 24h post-MCAO, RTL551 reduced the frequency of the activation marker, CD44, on T-cells in blood and in the ischemic hemisphere. Moreover, RTL551 reduced expression of the chemokine receptors, CCR5 in lymph nodes and spleen, and CCR7 in the blood and lymph nodes. These data demonstrate effective treatment of experimental stroke with RTL551 within a therapeutically relevant 4h time window through immune regulation of myelin-reactive inflammatory T-cells.
doi:10.1007/s11011-011-9241-2
PMCID: PMC3111858  PMID: 21472429
14.  GILT REQUIRED FOR RTL550-CYS-MOG TO TREAT EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS 
Metabolic Brain Disease  2012;27(2):143-149.
MHC class II-derived recombinant T cell receptor ligands (RTLs) modulate the behavior of pathogenic T cells and can reverse clinical and histological signs of autoimmune disease in experimental autoimmune encephalomyelitis (EAE), experimental autoimmune uveitis (EAU) and collagen-induced arthritis (CIA), and are currently in clinical trials for treatment of multiple sclerosis (MS). To expand the utility of these rationally-designed biologics and explore their mechanism(s) of activity in vivo, we have engineered RTL constructs bearing cysteine-tethered antigenic peptides and demonstrate that the appropriate cysteine-tethered RTLs effectively treat EAE. The data presented here suggests that the mechanism by which antigen-specific tolerance induction by RTLs bearing cysteine-tethered antigenic peptides in vivo involves delivery of RTL/antigen to endosomal compartments for processing and re-presentation by full-length MHC class II, with RTLs bearing cysteine-tethered antigenic peptides requiring gamma-interferon-inducible lysosomal thiol-reductase (GILT) for therapeutic activity.
doi:10.1007/s11011-012-9289-7
PMCID: PMC3348371  PMID: 22392628
EAE; GILT mice; RTL550-CYS-Mog; MHC Class II
15.  Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia 
Brain, behavior, and immunity  2011;25(4):685-695.
Stroke is a sexually dimorphic disease with male gender considered a disadvatage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B and CD11b+ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b+ blood cells solely in DHT-replaced animals at 24 h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96 h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.
doi:10.1016/j.bbi.2011.01.009
PMCID: PMC3081425  PMID: 21262338
Ischemia; Stroke; Hormone; dihydrotestosterone; testosterone; Immunosuppression; regulatory T lymphocyte; neuroprotection
16.  TCR-like antibodies distinguish conformational and functional differences in two vs. four-domain auto-reactive MHC II-peptide complexes 
European journal of immunology  2011;41(5):1465-1479.
SUMMARY
Antigen presenting cell-associated four-domain MHC class-II molecules play a central role in activating autoreactive CD4+ T-cells involved in Multiple Sclerosis (MS) and Type 1 Diabetes (T1D). In contrast, two-domain MHC-II structures with the same covalently-attached self peptide (Recombinant T-cell receptor Ligands=RTLs) can regulate pathogenic CD4+ T-cells and reverse clinical signs of experimental autoimmune diseases. RTL1000, comprised of the β1α1 domains of HLA-DR2 linked to the encephalitogenic human MOG-35-55 peptide, was recently shown to be safe and well-tolerated in a Phase I clinical trial in MS. To evaluate the opposing biological effects of four- vs. two-domain class-II structures, we screened phage Fab antibodies (Abs) for neutralizing activity of RTL1000. . Five different TCR-like Abs were identified that could distinguish between the two- vs. four-domain MHC peptide complexes, while the cognate TCR was unable to make such a distinction. Moreover, Fab detection of native two-domain HLA-DR structures in human plasma implies that there are naturally-occurring regulatory MHC-peptide complexes. These results demonstrate for the first time distinct conformational determinants characteristic of activating vs. tolerogenic MHC-peptide complexes involved in human autoimmunity.
doi:10.1002/eji.201041241
PMCID: PMC3085348  PMID: 21469129
Autoimmunity; Recombinant Antibodies; Immune tolerance; MHC class II
17.  Recombinant T-Cell Receptor Ligand (RTL) for Treatment of Multiple Sclerosis: A Double-Blind, Placebo-Controlled, Phase 1, Dose-Escalation Study 
Autoimmune Diseases  2012;2012:954739.
Background. Recombinant T-cell receptor ligand 1000 (RTL1000) is a single-chain protein construct containing the outer two domains of HLA-DR2 linked to myelin-oligodendrocyte-glycoprotein- (MOG-) 35–55 peptide. Analogues of RTL1000 induce T-cell tolerance, reverse clinical and histological disease, and promote repair in experimental autoimmune encephalomyelitis (EAE) in DR2 transgenic, C57BL/6, and SJL/J mice. Objective. Determining the maximum tolerated dose, safety, and tolerability of RTL1000 in multiple sclerosis (MS) subjects. Methods. This was a multicenter, Phase I dose-escalation study in HLA-DR2+ MS subjects. Consecutive cohorts received RTL1000 doses of 2, 6, 20, 60, 200, and 100 mg, respectively. Subjects within each cohort randomly received a single intravenous infusion of RTL1000 or placebo at a 4 : 2 ratio. Safety monitoring included clinical, laboratory, and brain magnetic resonance imaging (MRI) evaluations. Results. Thirty-four subjects completed the protocol. All subjects tolerated the 2–60 mg doses of RTL1000. Doses ≥100 mg caused hypotension and diarrhea in 3 of 4 subjects, leading to discontinuation of further enrollment. Conclusions. The maximum tolerated dose of RTL1000 in MS subjects is 60 mg, comparable to effective RTL doses in EAE. RTL1000 is a novel approach for MS treatment that may induce immunoregulation without immunosuppression and promote neural repair.
doi:10.1155/2012/954739
PMCID: PMC3328144  PMID: 22548151
18.  Estrogen-induced protection against experimental encephalomyelitis is abrogated in the absence of B cells 
European journal of immunology  2011;41(4):1165-1175.
SUMMARY
Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17β-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2-induced protective mechanisms in B cell deficient (µMT−/−) mice. EAE-protective effects of E2 were abrogated in µMT−/−) mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in the CNS compared to untreated controls. E2 treatment of WT mice selectively up-regulated expression of PD-L1 on B cells and increased the percentage of IL-10-producing CD1dhighCD5+ regulatory B cells. Upregulation of PD-L1 was critical for E2-mediated protection since E2 did not inhibit EAE in PD-L1−/− mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG35–55-specific T cells that required ERα. These results demonstrate for the first time a requirement for B cells in E2-mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD-1/PD-L1 negative co-stimulatory pathway. E2-primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B-cell depletion.
doi:10.1002/eji.201040992
PMCID: PMC3077096  PMID: 21413005
EAE; Multiple Sclerosis; Estrogen; Regulatory B cells
19.  CD4+FoxP3+ regulatory T-cells in cerebral ischemic stroke 
Metabolic brain disease  2010;26(1):87-90.
Experimental cerebral ischemic stroke is exacerbated by inflammatory T-cells and is accompanied by systemic increases in CD4+CD25+Foxp3+ regulatory T-cells (Treg). To determine their effect on ischemic brain injury, Treg were depleted in Foxp3DTR mice prior to stroke induction. In contrast to a recent Nature Medicine report, our results demonstrate unequivocally that Treg depletion did not affect stroke infarct volume, thus failing to implicate this regulatory pathway in limiting stroke damage.
doi:10.1007/s11011-010-9226-6
PMCID: PMC3070853  PMID: 21082336
Cerebral ischemia; T lymphocyte; T regulatory cells; middle cerebral artery occlusion; stroke
20.  RTL THERAPY FOR MULTIPLE SCLEROSIS: A Phase I Clinical Study 
Journal of neuroimmunology  2010;231(1-2):7-14.
A human Recombinant T-cell receptor Ligand (RTL1000) consisting of DR2 α1 and β1 domains linked covalently to MOG-35-55 peptide can reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE), and was evaluated for safety in a Phase 1 randomized, placebo-controlled, escalating dose study in 34 subjects with multiple sclerosis (MS). RTL1000 was safe and well tolerated at a dose of ≤60mg that is well within the effective dose range for EAE and did not cause worsening of MS disease at doses ≤200mg. RTL1000 represents a novel approach for the treatment of MS that promises potent immunoregulation and CNS repair without global immunosuppression.
doi:10.1016/j.jneuroim.2010.09.013
PMCID: PMC3026883  PMID: 20965577
Experimental Autoimmune Encephalomyelitis (EAE); Multiple Sclerosis (MS); Recombinant T cell receptor Ligand (RTL); neuroprotection; clinical trial
21.  Tissue-Dependent Expression of Estrogen Receptor β in 17β-Estradiol-Mediated Attenuation of Autoimmune CNS Inflammation 
The Open autoimmunity journal  2010;2:10.2174/1876894601002010197.
Treatment strategies using therapeutic estrogen are being developed and tested for multiple sclerosis (MS). MS is an autoimmune inflammatory disease that attacks the central nervous system, damages myelin and produces neurode-generative changes associated with periodic and chronic progression of functional neurological deficit. Experimental studies in chimeric bone marrow transplant mice treated with 17β-estradiol (E2) have revealed that the estrogen receptor-1 (Esr-1, or -alpha) expressed exclusively within the non-hematopoietic tissue compartment is sufficient for mediating a beneficial neuroprotective therapeutic response in mice lacking Esr-1 expression on T lymphocytes or other bone marrow-derived cells. Less is known regarding requirements for estrogen receptor-2 (Esr-2, or -beta) expression in E2-mediated therapy. Here, we tested and compared requirements for Esr-2 expression within distinct tissue compartments in bone marrow transplant mice. Our studies support a crucial role for Esr-1 in E2 treatment and demonstrate that Esr-2 expressed by non-bone marrow-derived cells plays a role in sustaining the neuroprotective response mediated through Esr-1.
doi:10.2174/1876894601002010197
PMCID: PMC3254147  PMID: 22242109
Autoimmune CNS inflammation; EAE; estrogen; gender
22.  Recombinant T Cell Receptor Ligands Improve Outcome After Experimental Cerebral Ischemia 
Translational stroke research  2011;2(3):404-410.
A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a therapeutically relevant timing after middle cerebral artery occlusion (MCAO) and verify functional benefit to complement histological outcome measures. We observed that the administration of mouse-specific RTL551 reduced infarct size and improved sensorimotor outcome when administered within a 3 h post-ischemic therapeutic window. RTL551 treatment reduced cortical, caudate putamen, and total infarct volume as compared to vehicle-treated mice. Using a standard behavioral testing repertoire, we observed that RTL551 reduced sensorimotor impairment 3 days after MCAO. Humanized RTL1000 (HLA-DR2 moiety linked to hMOG-35-55 peptide) also reduced infarct size in HLA-DR2 transgenic mice. These data indicate that this neuroantigen-specific immunomodulatory agent reduces damage when administered in a therapeutically relevant reperfusion timeframe.
doi:10.1007/s12975-011-0085-1
PMCID: PMC3181103  PMID: 21961027
Cerebral ischemia; Cerebral infarction; Inflammation; T lymphocyte; Immunotherapy; Mouse; Middle cerebral artery occlusion; Stroke
23.  Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis 
Journal of neuroimmunology  2010;225(1-2):52-61.
Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in antigen specific manner. We evaluated effects of RTL401 (I-As α1β1 + PLP-139-151) on splenocytes from mice with EAE to study RTL- T cell-tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-α1β1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RTL- incubated splenocytes to transfer EAE was likely mediated through macrophages/DC, since B cells were unnecessary for RTL treatment of EAE. These results demonstrate novel pathway of T cell regulation by RTL bound APCs.
doi:10.1016/j.jneuroim.2010.04.013
PMCID: PMC2924959  PMID: 20546940
EAE; RTLs; tolerance; APCs
24.  Estradiol and G1 Reduce Infarct Size and Improve Immunosuppression after Experimental Stroke 
Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-β–E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4+CD25+FoxP3+ T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.
doi:10.4049/jimmunol.0902339
PMCID: PMC3142781  PMID: 20304826
25.  RTL551 Treatment of EAE Reduces CD226 and T-bet+ CD4 T Cells in Periphery and Prevents Infiltration of T-bet+ IL-17, IFN-γ Producing T Cells into CNS 
PLoS ONE  2011;6(7):e21868.
Recombinant T cell receptor ligands (RTLs) that target encephalitogenic T-cells can reverse clinical and histological signs of EAE, and are currently in clinical trials for treatment of multiple sclerosis. To evaluate possible regulatory mechanisms, we tested effects of RTL therapy on expression of pathogenic and effector T-cell maturation markers, CD226, T-bet and CD44, by CD4+ Th1 cells early after treatment of MOG-35-55 peptide-induced EAE in C57BL/6 mice. We showed that 1–5 daily injections of RTL551 (two-domain I-Ab covalently linked to MOG-35-55 peptide), but not the control RTL550 (“empty” two-domain I-Ab without a bound peptide) or Vehicle, reduced clinical signs of EAE, prevented trafficking of cells outside the spleen, significantly reduced the frequency of CD226 and T-bet expressing CD4+ T-cells in blood and inhibited expansion of CD44 expressing CD4+ T-cells in blood and spleen. Concomitantly, RTL551 selectively reduced CNS inflammatory lesions, absolute numbers of CNS infiltrating T-bet expressing CD4+ T-cells and IL-17 and IFN-γ secretion by CNS derived MOG-35-55 reactive cells cultured ex vivo. These novel results demonstrate that a major effect of RTL therapy is to attenuate Th1 specific changes in CD4+ T-cells during EAE and prevent expansion of effector T-cells that mediate clinical signs and CNS inflammation in EAE.
doi:10.1371/journal.pone.0021868
PMCID: PMC3130056  PMID: 21750737

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