HLA-DRα1 Constructs Block CD74 Expression and MIF Effects in Experimental Autoimmune Encephalomyelitis

Abstract

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.

The use of flow cytometry to assess a novel drug efficacy in multiple sclerosis

“…A similar hierarchy of efficacy was observed with the DRα1 and the DRα1–MOG constructs, in which the latter had a significantly more potent effect in down regulating CD74 on human CD11b + monocytes and was 50× more potent in treating EAE (Meza-Romero et al 2014). Subsequent comparisons established a significant structure-activity relationship (SAR) between CD74 levels on CNS-derived CD11b + cells and EAE severity measured by the cumulative disease index (CDI), which clearly established RTL342M as the most effective construct and predicted dose-dependent therapeutic activity of other constructs commensurate with their ability to modulate CD74 levels (Figure 3).…”

“…The use of flow cytometry based assays enabled us to identify the predominant cell population that bind these constructs and to follow the changes in the partial MHC class II native cell surface receptor, the class II invariant chain (CD74), during the course of EAE and correlate the treatment response with this potential disease biomarker (Benedek et al 2013). We further identified the DRα1 domain, but not the DR2β1 domain as the RTL component that binds and down-regulates CD74 (Meza-Romero et al 2014; Vandenbark et al 2013). …”

“…Because the DRα1 domain is invariable and would not be recognized as an immunologically foreign antigen, treatment with DRα1 constructs would not require HLA screening prior to injection. We demonstrated that these novel constructs could treat different central nervous system (CNS) diseases that involve migration of immune cells from the periphery to both the spinal cord (EAE) and brain (stroke) (Benedek et al 2014; Meza-Romero et al 2014). …”

“…These studies revealed a dose-dependent hierarchy of partial MHC class II constructs, the most active being those that are linked to the MOG-35–55 peptide (mouse or human). It is interesting to note that the DR2β1 domain, which does not bind CD74, did not down-regulate CD74 expression levels (Meza-Romero et al 2014; Vandenbark et al 2013). Furthermore, CD74 expression on human CD11b + monocytes from HC and MS subjects was down-regulated after treatment with RTL1000 (Figure 2C).…”

“…Blocking of MIF binding, by partial MHC class II construct, either to immunoprecipitated mouse CD74 or to human PBMC was detected by western blot or by flow cytometry, respectively, using Alexa-488 labeled recombinant MIF protein. This inhibition of MIF binding resulted in profound downstream effects on EAE, including inhibition of cell migration to the CNS, secretion of pro-inflammatory cytokines and enhanced survival of activated monocytes (Benedek et al 2013; Meza-Romero et al 2014). …”