The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

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 identifies 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.

Tolerogenic vaccines: Targeting the antigenic and cytokine niches of FOXP3+ regulatory T cells.

FOXP3+ regulatory T cells (Tregs) constitute a critical barrier that enforces tolerance to both the self-peptidome and the extended-self peptidome to ensure tissue-specific resistance to autoimmune, allergic, and other inflammatory disorders. Here, we review intuitive models regarding how T cell antigen receptor (TCR) specificity and antigen recognition efficiency shape the Treg and conventional T cell (Tcon) repertoires to adaptively regulate T cell maintenance, tissue-residency, phenotypic stability, and immune function in peripheral tissues. Three zones of TCR recognition efficiency are considered, including Tcon recognition of specific low-efficiency self MHC-ligands, Treg recognition of intermediate-efficiency agonistic self MHC-ligands, and Tcon recognition of cross-reactive high-efficiency agonistic foreign MHC-ligands. These respective zones of TCR recognition efficiency are key to understanding how tissue-resident immune networks integrate the antigenic complexity of local environments to provide adaptive decisions setting the balance of suppressive and immunogenic responses. Importantly, deficiencies in the Treg repertoire appear to be an important cause of chronic inflammatory disease. Deficiencies may include global deficiencies in Treg numbers or function, subtle 'holes in the Treg repertoire' in tissue-resident Treg populations, or simply Treg insufficiencies that are unable to counter an overwhelming molecular mimicry stimulus. Tolerogenic vaccination and Treg-based immunotherapy are two therapeutic modalities meant to restore dominance of Treg networks to reverse chronic inflammatory disease. Studies of these therapeutic modalities in a preclinical setting have provided insight into the Treg niche, including the concept that intermediate-efficiency TCR signaling, high IFN-ß concentrations, and low IL-2 concentrations favor Treg responses and active dominant mechanisms of immune tolerance. Overall, the purpose here is to assimilate new and established concepts regarding how cognate TCR specificity of the Treg repertoire and the contingent cytokine networks provide a foundation for understanding Treg suppressive strategy.

A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE).

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/MOG35-55) and GMCSF-NFM (neurofilament medium peptide 13-37/NFM13-37), 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. RESULTS: This study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-ß, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG-Rag1-/- mice, which lacked pre-existing Tregs. CONCLUSIONS: This study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.

A GMCSF-Neuroantigen Tolerogenic Vaccine Elicits Systemic Lymphocytosis of CD4+ CD25high FOXP3+ Regulatory T Cells in Myelin-Specific TCR Transgenic Mice Contingent Upon Low-Efficiency T Cell Antigen Receptor Recognition.

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.