Gil Benedek

Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects.

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 downregulating CD74 cell‐surface expression. This bifunctional 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 EAE. Moreover, we demonstrate that enhanced CD74 cell‐surface expression on monocytes in mice with EAE and subjects with multiple sclerosis can be downregulated 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.

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

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.

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 used 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 (myelin oligodendrocyte glycoprotein [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.

Estrogen induces multiple regulatory B cell subtypes and promotes M2 microglia and neuroprotection during experimental autoimmune encephalomyelitis.

Sex hormones promote immunoregulatory effects on multiple sclerosis. The current study evaluated estrogen effects on regulatory B cells and resident CNS microglia during experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of multiple regulatory B cell markers indicative of IL-10 dependent as well as IFN-γ dependent pathways. Moreover, although estrogen pretreatment of EAE mice inhibited the infiltration of pro-inflammatory cells into the CNS, it enhanced the frequency of regulatory B cells and M2 microglia. Our study suggests that estrogen has a broad effect on the development of regulatory B cells during EAE, which in turn could promote neuroprotection.

IL-10 producing B cells partially restore E2-mediated protection against EAE in PD-L1 deficient mice

Women with multiple sclerosis (MS) often experience clinical improvement during pregnancy, indicating that sex hormones might have therapeutic effects in MS. Our previous studies have demonstrated that B cells and PD-L1 are crucial for E2 (17β-estradiol)-mediated protection against experimental autoimmune encephalomyelitis (EAE). We here demonstrate that the transfer of IL-10(+) B cells into E2-treated PD-L1(-/-) mice after EAE induction could partially restore E2-mediated protection and decrease the frequency of pro-inflammatory cells in the CNS compared to E2/saline treated PD-L1(-/-) mice. Hence, co-administration of IL-10(+) B cells and E2 might have a powerful therapeutic potential for treatment of EAE.

HLA-DRα1-mMOG-35-55 treatment of experimental autoimmune encephalomyelitis reduces CNS inflammation, enhances M2 macrophage frequency, and promotes neuroprotection

BackgroundDRα1-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.MethodsIn 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.ResultsWe 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).ConclusionThese findings indicate that the DRα1-mMOG-35-55 construct retains therapeutic, anti-inflammatory, and neuroprotective activities during treatment of EAE across MHC disparate barriers.

Novel feedback loop between M2 macrophages/microglia and regulatory B cells in estrogen-protected EAE mice

Immunoregulatory sex hormones, including estrogen and estriol, may prevent relapses in multiple sclerosis during pregnancy. Our previous studies have demonstrated that regulatory B cells are crucial for estrogen-mediated protection against experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of alternatively activated (M2) macrophages/microglia that results in an increased frequency of regulatory B cells in the spinal cord of estrogen treated mice with EAE. We further demonstrate that cultured M2-polarized microglia promote the induction of regulatory B cells. Our study suggests that estrogen neuroprotection induces a regulatory feedback loop between M2 macrophages/microglia and regulatory B cells.

Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

MIF and D-DT are potential disease severity modifiers in male MS subjects

Significance The biological processes that are involved in the progression of multiple sclerosis (MS) are far from complete. Macrophage migration inhibitory factor (MIF) and its homolog, D-dopachrome tautomerase (D-DT), are immunoregulatory cytokines known to be involved in the worsening of various autoimmune disorders. We demonstrate that genetically controlled high MIF expression (and D-DT) promotes MS progression in males, suggesting that these two factors are sex-specific disease modifiers. In addition, we show that MIF or D-DT deficiency ameliorates the disease severity of the murine model of MS. Our data suggest that targeting CD74, the common receptor for MIF and D-DT, with therapies such as partial MHC class II constructs could be therapeutically beneficial for inhibiting MS clinical progression in selected patients. Little is known about mechanisms that drive the development of progressive multiple sclerosis (MS), although inflammatory factors, such as macrophage migration inhibitory factor (MIF), its homolog D-dopachrome tautomerase (D-DT), and their common receptor CD74 may contribute to disease worsening. Our findings demonstrate elevated MIF and D-DT levels in males with progressive disease compared with relapsing-remitting males (RRMS) and female MS subjects, with increased levels of CD74 in females vs. males with high MS disease severity. Furthermore, increased MIF and D-DT levels in males with progressive disease were significantly correlated with the presence of two high-expression promoter polymorphisms located in the MIF gene, a −794CATT5–8 microsatellite repeat and a −173 G/C SNP. Conversely, mice lacking MIF or D-DT developed less-severe signs of experimental autoimmune encephalomyelitis, a murine model of MS, thus implicating both homologs as copathogenic contributors. These findings indicate that genetically controlled high MIF expression (and D-DT) promotes MS progression in males, suggesting that these two factors are sex-specific disease modifiers and raising the possibility that aggressive anti-MIF treatment of clinically isolated syndrome or RRMS males with a high-expresser genotype might slow or prevent the onset of progressive MS. Additionally, selective targeting of MIF:CD74 signaling might provide an effective, trackable therapeutic approach for MS subjects of both sexes.

Partial MHC class II constructs as novel immunomodulatory therapy for stroke

The worldwide prevalence of stroke continues to rise despite recent successes in treating acute ischemic stroke. With limited patient eligibility and associated risk of tPA and mechanical thrombectomy, new preventive and therapeutic modalities are needed to stave the rising wave of stroke. Inflammation plays a key role in brain damage after cerebral ischemia, and novel therapies that target pro-inflammatory cells have demonstrated promise for treatment for stroke. Partial MHC class II constructs have been shown to prevent and/or reverse clinical signs of various inflammatory diseases such as experimental autoimmune encephalomyelitis, collagen-induced arthritis and experimental autoimmune uveitis, by reducing the number and frequency of activated cells in the damaged CNS. Herein, we review the use of partial MHC class II constructs as a novel treatment for ischemic stroke. These constructs have been shown to reduce infarct volume and neurological deficit in various cerebral ischemia models in young adult and aging male and female mice. In addition, partial MHC class II constructs were shown to reverse stroke-associated splenic atrophy and promote a protective M2 macrophage/microglia phenotype in the CNS which contributes to tissue repair and recovery after stroke. By addressing remaining STAIR criteria, such as efficacy in large animal models of stroke, these constructs will be prime candidates for clinical trials of acute ischemic stroke.