Histone/Protein Deacetylase 11 Targeting Promotes Foxp3+ Treg Function.

Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required. We report data from mice with constitutive or conditional deletion of HDAC11 within Foxp3+ Treg cells, and their use, along with small molecule HDAC11 inhibitors, in allograft models. Global HDAC11 deletion had no effect on health or development, and compared to WT controls, Foxp3+ Tregs lacking HDAC11 showed increased suppressive function, and increased expression of Foxp3 and TGF-β. Likewise, compared to WT recipients, conditional deletion of HDAC11 within Tregs led to long-term survival of fully MHC-mismatched cardiac allografts, and prevented development of transplant arteriosclerosis in an MHC class II-mismatched allograft model. The translational significance of HDAC11 targeting was shown by the ability of an HDAC11i to promote long-term allograft allografts in fully MHC-disparate strains. These data are powerful stimuli for the further development and testing of HDAC11-selective pharmacologic inhibitors, and may ultimately provide new therapies for transplantation and autoimmune diseases

CCAAT enhancer binding protein and nuclear factor of activated T cells regulate HIV-1 LTR via a novel conserved downstream site in cells of the monocyte-macrophage lineage.

Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage

Pichia pastoris-expressed dengue virus type 2 envelope domain III elicits virus-neutralizing antibodies

A tetravalent dengue vaccine that can protect against all four serotypes of dengue viruses is a global priority. The host-receptor binding, multiple neutralizing epitope-containing carboxy-terminal region of the dengue envelope protein, known as domain III (EDIII), has emerged as a promising subunit vaccine antigen. One strategy to develop a tetravalent dengue subunit vaccine envisages mixing recombinant EDIIIs, corresponding to the four dengue virus serotypes. Towards this objective, a recombinant clone of the methylotrophic yeast Pichia pastoris, harboring the EDIII gene of dengue virus type 2 (EDIII-2) for its intracellular expression, was created. Recombinant EDIII-2 protein, expressed by this clone was purified to near homogeneity by affinity chromatography, with final yields of >50 mg/l culture. Groups of Balb/c mice were immunized with this protein, separately formulated in two adjuvants, alum and montanide ISA 720. The EDIII-2 antigen, formulated in either adjuvant, elicited high levels of neutralizing antibodies to dengue virus type 2 in mice as analyzed by Plaque Reduction Neutralization Test (PRNT). This study demonstrates the feasibility of using P. pastoris to produce EDIII antigens capable of eliciting potent virus-neutralizing antibodies

An envelope domain III–based chimeric antigen produced in Pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes

There is currently no vaccine to prevent dengue (DEN) virus infection, which is caused by any one of four closely related serotypes, DEN-1, DEN-2, DEN-3 or DEN-4. A DEN vaccine must be tetravalent, because immunity to a single serotype does not offer cross-protection against the other serotypes. We have developed a novel tetravalent chimeric protein by fusing the receptor-binding envelope domain III (EDIII) of the four DEN virus serotypes. This protein was expressed in the yeast, Pichia pastoris and purified to near homogeneity in high yields. Antibodies induced in mice by the tetravalent protein, formulated in different adjuvants, neutralized the infectivity of all four serotypes. This, coupled with the high expression potential of the P. pastoris system and easy one-step purification, makes the EDIII-based recombinant protein a potentially promising candidate for the development of a safe, efficacious, and inexpensive, tetravalent DEN vaccine

Physical position and consensus sequence comparison of DS3 among HIV-1 subtypes A, B, C, and D and differential binding phenotype in CXCR4 and CCR5-utilizing viruses.

<p>(<b>A</b>) Genbank and LANL were searched for HIV-1 LTR sequences from subtypes A, B, C, and D resulting in 7,577 sequences. The MUSCLE alignment tool and TRANSFAC software were used to identify a predicted binding site for C/EBP factors from+158 to+172 with respect to the start site. This region was then used to construct a new consensus binding sequence for each of the four predominant subtypes in the dataset. (<b>B</b>) Comparison of nucleotide sequence deviations, from the consensus subtype B configuration, in important transcription factor binding sites in the LTR both upstream and downstream of the start site. (<b>C</b>) Sequence logos generated from 1,832 sequences which contained both LTR and envelope V3 sequence grouped by CXCR4 and CCR5 coreceptor utiliziation using WebPSSM. The differences observed were not statistically significant (Fischer’s exact test). (<b>D</b>) Survival function (1 - cdf) of the distribution of Jaspar binding scores for the NFAT and CEBP sites of the DS3 region for sequences in C grouped by predicted coreceptor utilization. The vertical black bars represent the binding threshold with a false positive rate of 0.01.</p

Experimental model.

<p>In basal conditions, DS3 stays in equilibrium between NFAT and C/EBP bound states. The affinity of NFATc2 is relatively higher than C/EBP isoforms for binding at DS3 (top panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088116#pone-0088116-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088116#pone-0088116-g004" target="_blank">4</a>). However, stimulation with cytokines including IL-6 results in activation of C/EBP isoforms and, which is evident from their ability to bind the DS3 region (middle panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088116#pone-0088116-g005" target="_blank">figure 5</a>). In conditions, where nuclear NFATc2 concentration was low, increased binding of C/EBPα to DS3 was possible (lower panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088116#pone-0088116-g004" target="_blank">figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088116#pone-0088116-g005" target="_blank">5</a>).</p

C/EBPα and C/EBPβ bind DS3 in the absence of NFAT.

<p>(<b>A</b>) Nuclear extracts of U-937 cells were incubated with labeled DS3 oligonucleotide (lane 4) and homologous cold competition was performed with unlabeled DS3 (lanes 5–7) or incubated with specified antibodies (lanes 8–11). (<b>B</b>) Western immunoblot showing overexpression of FLAG-tagged C/EBPβ in HEK 293T cells. (<b>C</b>) Nuclear extracts of HEK 293T transfected with either vector alone (lanes 4–8) or FLAG-C/EBPβ (lanes 9–13) were incubated with labeled DS3 and either homologous competition (lanes 6 and 10) or incubation was performed with indicated antibodies.</p

NFAT isoforms preferentially form complexes with DS3.

<p>(<b>A</b>) Nuclear extracts of IL-6-stimulated U-937 cells were incubated with labeled DS3 oligonucleotide and either homologous competition (lanes 5–7) or incubation with control IgG or specific antibody (lanes 8–14) was performed. (<b>B</b>) Nuclear extracts of IL-6-stimulated U-937 cells were incubated with labeled DS3 oligonucleotide and homologous competition was performed with unlabeled US2 (lanes 6–8), US1 (lanes 11–13), or consensus NFAT (lanes 16–18) oligonucleotides.</p