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1.  Preclinical Evaluation of Recombinant T-cell Receptor Ligand RTL1000 as a Therapeutic Agent in Ischemic Stroke 
Translational stroke research  2014;6(1):60-68.
Recombinant T-cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of single dose of RTL1000 administration and determined if RTL1000 affordslong-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60 min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6 or 8 h after MCAO, followed by 3 daily injections. In single dose study, one-time injection of RTL1000 was applied 4 h after MCAO. Cortical, striatal and hemispheric infarct sizes were measured 24 h or 96 h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference and novel object recognition was performed up to 28 days after stroke. Our data showed RTL1000 significantly reduced infarct size 96 h after MCAO when first injection was given 4 and 6, but not 8 h after the onset of stroke. A single dose of 400 µg or 100 µg RTL1000 also significantly reduced infarct size 24 h after MCAO. Behavioral testing showed RTL1000 treatment used 4 h after MCAO improved long-term cognitive outcome 28 days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.
PMCID: PMC4298461  PMID: 25270354
Ischemicstroke; Neurobehavioral evaluation; Immunotherapy; Recombinant T-cell receptor Ligand; HLA-DR2transgenic mice
2.  The role of the spleen in ischemic stroke 
This opinion piece highlights the scientific literature reporting that the peripheral immune response to ischemic stroke originates from the spleen. Removal of the spleen not only reduces stroke-induced neurodegeneration but also cellular degeneration in the body's other tissues when exposed to ischemic conditions.
PMCID: PMC4426754  PMID: 25465042
brain ischemia; macrophages; neuroinflammation
3.  Splenectomy reduces infarct volume and neuroinflammation in male but not female mice in experimental stroke 
Journal of neuroimmunology  2014;0:289-298.
The peripheral immune response contributes to neurodegeneration after stroke yet little is known about how this process differs between males and females. The current study demonstrates that splenectomy prior to experimental stroke eliminates sex differences in infarct volume and activated brain monocytes/microglia. In the periphery of both sexes, activated T cells correlate directly with stroke outcome while monocytes are reduced by splenectomy only in males. This study provides new information about the sex specific mechanisms of the peripheral immune response in neurodegeneration after stroke and demonstrates the need for representation of both sexes in basic and clinical stroke research.
PMCID: PMC4297719  PMID: 25434281
Experimental stroke; Sex difference; Immune response; Neuroinflammation; Infarct volume; Splenectomy
4.  Adoptive transfer of immune subsets prior to MCAO does not exacerbate stroke outcome in splenectomized mice 
The peripheral immune response contributes to neurologic impairment after stroke and the extent of initial damage is greater in males than females. We have previously shown that spleen cells directly contribute to ischemic damage in males, as splenectomy prior to experimental stroke eliminates the sex differences in infarct volume. This study aims to determine which specific subset of immune cells exert pathogenic effects when injected 24 hours before MCAO induction into splenectomized male and female WT mice. The results demonstrate that CD4/CD8/CD11b treated mice had no significant effect on infarct volumes vs. vehicle-treated control mice after MCAO. However, there were significant alterations to the resident peripheral immune composition. These results suggest that there are regulatory factors resulting from splenectomy or other possible influences that inhibit peripheral immune cell contribution to neuroinflammation and thus contributing to differential effects of the spleen on stroke outcome in males and female mice.
PMCID: PMC4664464  PMID: 26634148
inflammation; focal cerebral ischemia; middle cerebral artery occlusion; experimental stroke; CD4; CD8; CD11b; splenectomy; female mice; immune modulation
5.  OX40 Agonists can drive Treg expansion if the cytokine milieu is right 
We report that OX40 stimulation drives all lineages of CD4 T cell development including Treg and the plasticity of the response is dependant on local cytokines. In TGF-β1-treated cultures, OX40 agonist increased IFN-γ and IL-4 production and diverted T cells from the Treg lineage. However, cytokine blockade in the context of OX40 stimulation promoted also enhanced Treg accumulation. This observation was evident in naive mice, as OX40 engagement enhanced Treg proliferation and accumulation in vivo. Lastly, OX40 agonist administration influenced EAE disease severity in opposing directions depending on the timing of administration. Given during Ag priming, OX40 agonist drove Treg expansion and inhibited disease, whereas, given later it enhanced T cell effector cytokine production in the CNS and exacerbated disease. Hence, OX40 signaling can augment the accumulation of all CD4 T cell lineages; however its accentuation of immune responses may have vastly different biologic outcomes depending upon the local cytokine milieu.
PMCID: PMC4625917  PMID: 19786544
6.  Recombinant T-cell Receptor Ligand Treatment Improves Neurological Outcome in the Presence of Tissue Plasminogen Activator in Experimental Ischemic Stroke 
Translational stroke research  2014;5(5):612-617.
RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60 min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or 4 daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24h or 96 h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24 h but not at 96 h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96 h after MCAO and provides additional protection in t-PA treated mice at 24 h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA treated mice in experimental ischemic stroke.
PMCID: PMC4125526  PMID: 24953050
Ischemic stroke; Immunotherapy; Recombinant T-cell receptor Ligand; tissue plasminogen activator; HLA-DR2 transgenic mice
7.  Modelling immunity and inflammation in stroke: Don’t be afraid of mice? 
PMCID: PMC4146705  PMID: 25061079
8.  HLA-DRα1-mMOG-35-55 treatment of experimental autoimmune encephalomyelitis reduces CNS inflammation, enhances M2 macrophage frequency, and promotes neuroprotection 
DRα1-mouse(m)MOG-35-55, a novel construct developed in our laboratory as a simpler and potentially less immunogenic alternative to two-domain class II constructs, was shown previously to target the MIF/CD74 pathway and to reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in DR*1501-Tg mice in a manner similar to the parent DR2β1-containing construct.
In order to determine whether DRα1-mMOG-35-55 could treat EAE in major histocompatibility complex (MHC)-mismatched mice and to evaluate the treatment effect on central nervous system (CNS) inflammation, C57BL/6 mice were treated with DRα1-mMOG-35-55. In addition, gene expression profile was analyzed in spinal cords of EAE DR*1501-Tg mice that were treated with DRα1-mMOG-35-55.
We here demonstrate that DRα1-mMOG-35-55 could effectively treat EAE in MHC-mismatched C57BL/6 mice by reducing CNS inflammation, potentially mediated in part through an increased frequency of M2 monocytes in the spinal cord. Microarray analysis of spinal cord tissue from DRα1-mMOG-35-55-treated vs. vehicle control mice with EAE revealed decreased expression of a large number of pro-inflammatory genes including CD74, NLRP3, and IL-1β and increased expression of genes involved in myelin repair (MBP) and neuroregeneration (HUWE1).
These findings indicate that the DRα1-mMOG-35-55 construct retains therapeutic, anti-inflammatory, and neuroprotective activities during treatment of EAE across MHC disparate barriers.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-015-0342-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4481122  PMID: 26104759
Experimental autoimmune encephalomyelitis (EAE); Multiple sclerosis (MS); DRα1-mMOG-35-55 therapy; M2 macrophages; Neuroprotection
9.  HLA-DRα1 Constructs Block CD74 Expression and MIF Effects in Experimental Autoimmune Encephalomyelitis 
CD74, the cell surface form of the MHC class II invariant chain, is a key inflammatory factor that is involved in various immune mediated diseases as part of the Macrophage Migration Inhibitory Factor (MIF) binding complex. However, little is known about the natural regulators of CD74 in this context. In order to study the role of the HLA-DR molecule in regulating CD74, we utilized the HLA-DRα1 domain, which was shown to bind to and downregulate CD74 on CD11b+ monocytes. We found that DRα1 directly inhibited binding of MIF to CD74 and blocked its downstream inflammatory effects in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). Potency of the DRα1 domain could be destroyed by trypsin digestion but enhanced by addition of a peptide extension (MOG-35–55 peptide) that provided secondary structure not present in DRα1. These data suggest a conformationally-sensitive determinant on DRα1-MOG that is responsible for optimal binding to CD74 and antagonism of MIF effects, resulting in reduced axonal damage and reversal of ongoing clinical and histological signs of EAE. These results demonstrate natural antagonist activity of DRα1 for MIF that was strongly potentiated by the MOG peptide extension, resulting in a novel therapeutic, DRα1-MOG-35–55,that within the limitations of the EAE model may have the potential to treat autoimmune diseases such as multiple sclerosis.
PMCID: PMC4028955  PMID: 24683185
HLA-DRα1; CD74; Macrophage migration inhibitory factor (MIF); multiple sclerosis
10.  Treatment of experimental stroke with IL-10-producing B-cells reduces infarct size and peripheral and CNS inflammation in wild-type B-cell-sufficient mice 
Metabolic brain disease  2013;29(1):59-73.
Clinical stroke induces inflammatory processes leading to cerebral and splenic injury and profound peripheral immunosuppression. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B-cells led to larger infarct volumes and CNS damage after middle cerebral artery occlusion (MCAO) that could be prevented by transfer of IL-10+ B-cells. The purpose of this study was to determine if the beneficial immunoregulatory effects on MCAO of the IL-10+ B-cell subpopulation also extends to B-cell-sufficient mice that would better represent stroke subjects.
CNS inflammation and infarct volumes were evaluated in male C57BL/6J (WT) mice that received either RPMI or IL-10+ B-cells and underwent 60 min of middle cerebral artery occlusion (MCAO) followed by 96 hours of reperfusion.
Transfer of IL-10+ B-cells markedly reduced infarct volume in WT recipient mice when given 24 hours prior to or 4 hours after MCAO. B-cell protected MCAO mice had increased regulatory subpopulations in the periphery, reduced numbers of activated, inflammatory T-cells, decreased infiltration of T-cells and a less inflammatory milieu in the ischemic hemispheres of the IL-10+ B-cell-treated group. Moreover, transfer of IL-10+ B-cells 24 hours before MCAO led to a significant preservation of regulatory immune subsets in the IL-10+ B-cell protected group presumably indicating their role in immunomodulatory mechanisms, post-stroke.
Our studies are the first to demonstrate a major immunoregulatory role for IL-10+ regulatory B-cells in preventing and treating MCAO in WT mice and also implicating their potential role in attenuating complications due to post-stroke immunosuppression.
PMCID: PMC3944055  PMID: 24374817
MCAO; inflammatory cells; regulatory B-cells; IL-10
11.  Phenotypic changes in immune cell subsets reflect increased infarct volume in male vs. female mice 
Translational stroke research  2013;4(5):10.1007/s12975-013-0268-z.
Inflammatory responses in brain after cerebral ischemia have been studied extensively in male but not female mice, thus potentially giving a less-than-accurate view of gender-based pathological processes. In humans, cerebral infarcts are typically smaller in premenopausal females than age-matched males. In the current study, we confirmed smaller infarcts in female vs. male mice after middle cerebral artery occlusion and 96 hours of reperfusion. Moreover, we explored immunological alterations related to this difference and found that the percentage of CD4+ T lymphocytes was significantly higher in males than females in spleens with increased expression of the activation markers, CD69 and CD44. In contrast, the percentage of CD8+ T lymphocytes was significantly higher in females than males in spleens, leading to the identification of a small but distinct population of IL-10-secreting CD8+CD122+ T-suppressor cells that were also increased in females. Finally, we observed that males have a greater percentage of activated macrophages/microglia in brain than females, as well as increased expression of the VLA-4 adhesion molecule in both brain and spleen. This new information suggesting gender-dependent immunological mechanisms in stroke implies that effective treatments for human stroke may also be gender specific.
PMCID: PMC3811047  PMID: 24187596
Experimental stroke; gender bias; immune markers; activated T-cells; T-suppressor cells; ischemia
12.  IL-10-producing B-cells limit CNS inflammation and infarct volume in experimental stroke 
Metabolic brain disease  2013;28(3):375-386.
Clinical stroke induces inflammatory processes leading to cerebral injury. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B-cells led to larger infarct volumes and increased numbers of activated T-cells, monocytes and microglial cells in the brain, thus implicating a regulatory role of B-cell subpopulations in limiting CNS damage from stroke. The aim of this study was to determine whether the IL-10-producing regulatory B-cell subset can limit CNS inflammation and reduce infarct volume following ischemic stroke in B-cell deficient (µMT−/−) mice. Five million IL-10-producing B-cells were obtained from IL-10-GFP reporter mice and transferred i.v. to µMT−/− mice. After 24 h following this transfer, recipients were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 48 hours of reperfusion. Compared to vehicle-treated controls, the IL-10+ B-cell-replenished µMT−/− mice had reduced infarct volume and fewer infiltrating activated T-cells and monocytes in the affected brain hemisphere. These effects in CNS were accompanied by significant increases in regulatory T-cells and expression of the co-inhibitory receptor, PD-1, with a significant reduction in the proinflammatory milieu in the periphery. These novel observations provide the first proof of both immunoregulatory and protective functions of IL-10-secreting B-cells in MCAO that potentially could impart significant benefit for stroke patients in the clinic.
PMCID: PMC3737266  PMID: 23640015
MCAO; inflammatory cells; regulatory B-cells; IL-10
13.  Novel Humanized Recombinant T Cell Receptor Ligands Protect the Female Brain After Experimental Stroke 
Translational Stroke Research  2014;5(5):577-585.
Transmigration of peripheral leukocytes to the brain is a major contributor to cerebral ischemic cell death mechanisms. Humanized partial major histocompatibility complex class II constructs (pMHC), covalently linked to myelin peptides, are effective for treating experimental stroke in males, but new evidence suggests that some inflammatory cell death mechanisms after brain injury are sex-specific. We here demonstrate that treatment with pMHC constructs also improves outcomes in female mice with middle cerebral artery occlusion (MCAO). HLA-DR2 transgenic female mice with MCAO were treated with RTL1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide), HLA-DRa1-MOG-35-55, or vehicle (VEH) at 3, 24, 48, and 72 h after reperfusion and were recovered for 96 h or 2 weeks post-injury for measurement of histology (TTC staining) or behavioral testing. RTL1000- and DRa1-MOG-treated mice had profoundly reduced infarct volumes as compared to the VEH group, although higher doses of DRa1-MOG were needed for females vs. males evaluated previously. RTL1000-treated females also exhibited strongly improved functional recovery in a standard cylinder test. In novel studies of post-ischemic ultrasonic vocalization (USV), as measured by animal calls to their cage mates, we modeled in mice the post-stroke speech deficits common in human stroke survivors. The number of calls was reduced in injured animals relative to pre-MCAO baseline regardless of RTL1000 treatment status. However, call duration was significantly improved by RTL1000 treatment, suggesting benefit to the animal’s recovery of vocalization capability. We conclude that both the parent RTL1000 molecule and the novel non-polymorphic DRα1-MOG-35-55 construct were highly effective immunotherapies for treatment of transient cerebral ischemia in females.
PMCID: PMC4121525  PMID: 24838614
Cerebral ischemia; Gender; Sex; Immunotherapy; Partial MHC class II constructs; Stroke; Ultrasonic vocalization
14.  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.
PMCID: PMC3788583  PMID: 23576302
CD74; Macrophage migration inhibitory factor (MIF); multiple sclerosis; recombinant T-cell receptor ligand
15.  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.
PMCID: PMC3975671  PMID: 24122483
Stroke; Inflammation; Immunotherapy; Recombinant T-cell receptor Ligand; MHC class II invariant chain
16.  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.
PMCID: PMC3789252  PMID: 23026773
Partial MHC class II; CD74; EAE; MIF
17.  Targeting immune co-stimulatory effects of PD-L1 and PD-L2 might represent an effective therapeutic strategy in stroke 
Stroke outcome is worsened by the infiltration of inflammatory immune cells into ischemic brains. Our recent study demonstrated that PD-L1- and to a lesser extent PD-L2-deficient mice had smaller brain infarcts and fewer brain-infiltrating cells vs. wild-type (WT) mice, suggesting a pathogenic role for PD-ligands in experimental stroke. We sought to ascertain PD-L1 and PD-L2-expressing cell types that affect T-cell activation, post-stroke in the context of other known co-stimulatory molecules. Thus, cells from male WT and PD-L-deficient mice undergoing 60 min of middle cerebral artery occlusion (MCAO) followed by 96 h of reperfusion were treated with neutralizing antibodies to study co-stimulatory and co-inhibitory interactions between CD80, cytotoxic T-lymphocyte antigen-4 (CTLA-4), PD-1, and PD-Ls that regulate CD8+ and CD4+ T-cell activation. We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8+ and CD4+ T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation. Also, CD80/CD28 interactions played a prominent regulatory role for the CD8+ T-cells and the PD-1/PD-L2 interactions were dominant in controlling the CD4+ T-cell responses in WT mice after stroke. A suppressive phenotype in PD-L1-deficient mice was attributed to CD80/CTLA-4 and PD-1/PD-L2 interactions. PD-L2 was crucial in modulating CD4+ T-cell responses, whereas PD-L1 regulated both CD8+ and CD4+ T-cells. To establish the contribution of PD-L1 and PD-L2 on regulatory B-cells (Bregs), infarct volumes were evaluated in male PD-L1- and PD-L2-deficient mice receiving IL-10+ B-cells 4h post-MCAO. PD-L2- but not PD-L1-deficient recipients of IL-10+ B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs. These results imply that PD-L1 and PD-L2 differentially control induction of T- and Breg-cell responses after MCAO, thus suggesting that selective targeting of PD-L1 and PD-L2 might represent a valuable therapeutic strategy in stroke.
PMCID: PMC4127946  PMID: 25157219
MCAO; co-stimulatory pathway; programmed death ligand-1 and 2; T-cells; regulatory B cells
18.  Different immunological mechanisms govern protection from experimental stroke in young and older mice with recombinant TCR ligand therapy 
Stroke is a leading cause of death and disability in the United States. The lack of clinical success in stroke therapies can be attributed, in part, to inadequate basic research on aging rodents. The current study demonstrates that recombinant TCR ligand therapy uses different immunological mechanisms to protect young and older mice from experimental stroke. In young mice, RTL1000 therapy inhibited splenocyte efflux while reducing frequency of T cells and macrophages in the spleen. Older mice treated with RTL1000 exhibited a significant reduction in inflammatory cells in the brain and inhibition of splenic atrophy. Our data suggest age specific differences in immune response to stroke that allow unique targeting of stroke immunotherapies.
PMCID: PMC4174768  PMID: 25309326
experimental stroke; aging; RTL1000; therapy; immune response; neuroinflammation
19.  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.
PMCID: PMC3427715  PMID: 22618587
regulatory B-cells; experimental stroke; intrastriatal transfer; uMT mice
20.  PD-L1 enhances CNS inflammation and infarct volume following experimental stroke in mice in opposition to PD-1 
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.
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.
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.
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.
PMCID: PMC3846120  PMID: 24015822
Co-inhibitory pathway; Inflammatory states; MCAO; Programmed death-1 ligand 1 and 2
21.  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.
PMCID: PMC3760596  PMID: 24009988
EAE; Multiple Sclerosis; Estrogen; PD-L; Regulatory B cells
22.  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.
PMCID: PMC3501272  PMID: 23175646
Experimental stroke; Bregs; IL-10; PD-1; immunotherapy
23.  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.
PMCID: PMC3270145  PMID: 21989743
experimental stroke; myelin reactive splenocytes; inflammatory responses; neurologic deficit
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
25.  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

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