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

Novel Humanized Recombinant T Cell Receptor Ligands Protect the Female Brain After Experimental Stroke

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

Characterization of human platelet binding of recombinant T cell receptor ligand

<p>Abstract</p> <p>Background</p> <p>Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.</p> <p>Methods</p> <p>We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function.</p> <p>Results</p> <p>Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.</p> <p>Conclusions</p> <p>Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.</p

Recombinant T-Cell Receptor Ligand (RTL) for Treatment of Multiple Sclerosis: A Double-Blind, Placebo-Controlled, Phase 1, Dose-Escalation Study

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

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

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

The splenic response to stroke: from rodents to stroke subjects

Abstract Background Stroke is the fifth leading cause of death and the leading cause of long-term disability in the USA, costing $40.2 billion in direct and indirect costs. Globally, stroke is the second leading cause of death and has a higher prevalence in lower- and middle-income countries compared to high-income countries. The role of the spleen in stroke has been studied in rodent models of stroke and is seen as a major contributor to increased secondary neural injury after stroke. Splenectomy 2 weeks prior to ischemic and hemorrhagic stroke in mice and rats shows decreased infarct volumes. Additionally, the spleen decreases in size following stroke in rodents. Pro-inflammatory mediators are also increased in the spleen and subsequently the brain after stroke. These data in preclinical models of stroke have led stroke neurologists to look at the splenic response in stroke subjects. The outcomes of these studies suggest the spleen is responding in a similar manner in stroke subjects as it is in animal models of stroke. Conclusion Animal models demonstrating the detrimental role of the spleen in stroke are providing strong evidence of how the spleen is responding during stroke in human subjects. This indicates treatments targeting the splenic immune response in animals could provide useful targets and treatments for stroke subjects

A novel neurotherapeutic for multiple sclerosis, ischemic injury, methamphetamine addiction, and traumatic brain injury

Abstract Neurovascular, autoimmune, and traumatic injuries of the central nervous system (CNS) all have in common an initial acute inflammatory response mediated by influx across the blood-brain barrier of activated mononuclear cells followed by chronic and often progressive disability. Although some anti-inflammatory therapies can reduce cellular infiltration into the initial lesions, there are essentially no effective treatments for the progressive phase. We here review the successful treatment of animal models for four separate neuroinflammatory and neurodegenerative CNS conditions using a single partial MHC class II construct called DRa1-hMOG-35-55 or its newest iteration, DRa1(L50Q)-hMOG-35-55 (DRhQ) that can be administered without a need for class II tissue type matching due to the conserved DRα1 moiety of the drug. These constructs antagonize the cognate TCR and bind with high affinity to their cell-bound CD74 receptor on macrophages and dendritic cells, thereby competitively inhibiting downstream signaling and pro-inflammatory effects of macrophage migration inhibitory factor (MIF) and its homolog, d-dopachrome tautomerase (D-DT=MIF-2) that bind to identical residues of CD74 leading to progressive disease. These effects suggest the existence of a common pathogenic mechanism involving a chemokine-driven influx of activated monocytes into the CNS tissue that can be reversed by parenteral injection of the DRa1-MOG-35-55 constructs that also induce anti-inflammatory macrophages and microglia within the CNS. Due to their ability to block this common pathway, these novel drugs appear to be prime candidates for therapy of a wide range of neuroinflammatory and neurodegenerative CNS conditions

GPR30, but not estrogen receptor-α, is crucial in the treatment of experimental autoimmune encephalomyelitis by oral ethinyl estradiol

Abstract Background Remission of multiple sclerosis during periods of high ovarian hormone secretion (such as pregnancy) has led to a great deal of interest in the potential for estrogens to treat autoimmune disease. Previous work has established that 17β-estradiol can inhibit onset of experimental autoimmune encephalomyelitis (EAE), while ethinyl estradiol (EE) can reduce the severity of established disease. In the current study, the influence of estrogen receptor-α (ERα) and the G-protein coupled estrogen receptor (GPR30 or GPER) on EE's ability to treat EAE was explored. Results EE reduced disease severity in wild-type and ERα knockout (ERKO) mice, but did not alter disease in the GPR30KO group. Production of anti-inflammatory IL-10 increased in EE-ERKO mice (which showed reduced disease) but not in EE-GPR30KO mice (who did not have improved disease). Conclusions Differential production of IL-10 following EE treatment in ERKO and GPR30KO animals may be responsible for the distinctly different effects on disease severity. Increased IL-10 in ERKO-EE compared to ERKO-Controls is likely to be an important factor in reducing established disease. The inability of EE to reduce disease in GPR30KO mice indicates an important but still undefined role for GPR30 in regulating immune reactivity.</p