Previous studies showed that single-chain fusion proteins comprised of GM-CSF and major encephalitogenic peptides of myelin, when injected subcutaneously in saline, were potent tolerogenic vaccines that suppressed experimental autoimmune encephalomyelitis (EAE) in rats and mice. These tolerogenic vaccines exhibited dominant suppressive activity in inflammatory environments even when emulsified in Complete Freund's Adjuvant (CFA). The current study provides evidence that the mechanism of tolerance was dependent upon vaccine-induced regulatory CD25+ T cells (Tregs), because treatment of mice with the Treg-depleting anti-CD25 mAb PC61 reversed tolerance. To assess tolerogenic mechanisms, we focused on 2D2-FIG mice, which have a transgenic T cell repertoire that recognizes myelin oligodendrocyte glycoprotein peptide MOG35-55 as a low-affinity ligand and the neurofilament medium peptide NFM13-37 as a high-affinity ligand. Notably, a single subcutaneous vaccination of GMCSF-MOG in saline elicited a major population of FOXP3+ Tregs that appeared within 3 days, was sustained over several weeks, expressed canonical Treg markers, and was present systemically at high frequencies in the blood, spleen, and lymph nodes. Subcutaneous and intravenous injections of GMCSF-MOG were equally effective for induction of FOXP3+ Tregs. Repeated booster vaccinations with GMCSF-MOG elicited FOXP3 expression in over 40% of all circulating T cells. Covalent linkage of GM-CSF with MOG35-55 was required for Treg induction whereas vaccination with GM-CSF and MOG35-55 as separate molecules lacked Treg-inductive activity. GMCSF-MOG elicited high levels of Tregs even when administered in immunogenic adjuvants such as CFA or Alum. Conversely, incorporation of GM-CSF and MOG35-55 as separate molecules in CFA did not support Treg induction. The ability of the vaccine to induce Tregs was dependent upon the efficiency of T cell antigen recognition, because vaccination of 2D2-FIG or OTII-FIG mice with the high-affinity ligands GMCSF-NFM or GMCSF-OVA (Ovalbumin323-339), respectively, did not elicit Tregs. Comparison of 2D2-FIG and 2D2-FIG-Rag1−/− strains revealed that GMCSF-MOG may predominantly drive Treg expansion because the kinetics of vaccine-induced Treg emergence was a function of pre-existing Treg levels. In conclusion, these findings indicate that the antigenic domain of the GMCSF-NAg tolerogenic vaccine is critical in setting the balance between regulatory and conventional T cell responses in both quiescent and inflammatory environments.
Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution x-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate and NAD(H) binding sites identify key residues in the active site for ligand interactions, including those in the 'aromatic box' that define the aldehyde binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.
Background: Tolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs. Methods: This study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35-55/MOG 35-55) and GMCSF-NFM (neurofilament medium peptide 13-37/NFM 13-37), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag −/− mice to reduce pre-existing Treg populations.
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