Tolerogenic Dendritic Cells from Poorly Compensated Type 1 Diabetes Patients Have Decreased Ability To Induce Stable Antigen-Specific T Cell Hyporesponsiveness and Generation of Suppressive Regulatory T Cells.

Tolerogenic dendritic cells (tolDCs) may offer an interesting intervention strategy to re-establish Ag-specific tolerance in autoimmune diseases, including type 1 diabetes (T1D). T1D results from selective destruction of insulin-producing ß cells leading to hyperglycemia that, in turn, specifically affects a patient's immune system. In this study, we prepared monocyte-derived tolDCs modulated by dexamethasone and vitamin D2 from 31 T1D patients with optimal glycemic control and 60 T1D patients with suboptimal glycemic control and assessed their tolerogenic properties in correlation with metabolic state of patients. tolDCs differentiated from both groups of patients acquired a regulatory phenotype and an anti-inflammatory profile. Interestingly, tolDCs from well-controlled patients expressed higher levels of inhibitory molecules IL-T3 and PD-L1. Additionally, glutamic acid decarboxylase (GAD)65-loaded tolDCs from well-controlled patients decreased significantly primary Th1/Th17 responses, induced stable GAD65-specific T cell hyporesponsiveness, and suppressed markedly control DC-induced GAD65-specific T cell activation compared with poorly controlled patients. The ability of tolDCs from poorly controlled patients to induce durable GAD65-specific T cell hyporesponsiveness was reversed once the control of glycemia improved. In both groups of patients, tolDCs were able to induce regulatory T cells from autologous naive CD4+ T cells. However, regulatory T cells from well-controlled patients had better suppressive abilities. The functionality of tolDCs was confirmed in the adoptive transfer model of NOD-SCID mice where tolDCs delayed diabetes onset. These results suggest that metabolic control of T1D affects the functional characteristics of tolDCs and subsequent effector T cell responses. Metabolic control may be relevant for refining inclusion criteria of clinical trials in the settings of T1D.

Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family.

Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular ß-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding ß-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.

Poxvirus targeting of E3 ligase ß-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

14-3-3 protein masks the DNA binding interface of forkhead transcription factor FOXO4.

The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4.14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4.