Development of a nonhuman primate challenge model to evaluate CD8+ T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors.

Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4-1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

The Chromatin-Associated Phf12 Protein Maintains Nucleolar Integrity and Prevents Premature Cellular Senescence.

Pf1, also known as Phf12 (plant homeodomain [PHD] zinc finger protein 12), is a member of the PHD zinc finger family of proteins. Pf1 associates with a chromatin-interacting protein complex comprised of MRG15, Sin3B, and histone deacetylase 1 (HDAC1) that functions as a transcriptional modulator. The biological function of Pf1 remains largely elusive. We undertook the generation of Pf1 knockout mice to elucidate its physiological role. We demonstrate that Pf1 is required for mid- to late gestation viability. Pf1 inactivation impairs the proliferative potential of mouse embryonic fibroblasts (MEFs) and is associated with a significant decrease in bromodeoxyuridine incorporation; an increase in senescence-associated ß-galactosidase (SA-ß-Gal) activity, a marker of cellular senescence; and elevated levels of phosphorylated H2AX (γ-H2A.X), a marker associated with DNA double-strand breaks. Analysis of transcripts differentially expressed in wild-type and Pf1-deficient cells revealed the impact of Pf1 in multiple regulatory arms of the ribosome biogenesis pathways. Strikingly, assessment of the morphology of the nucleoli exposed an abnormal nucleolar structure in Pf1-deficient cells. Finally, proteomic analysis of the Pf1-interacting complexes highlighted proteins involved in ribosome biogenesis. Taken together, our data reveal an unsuspected function for the Pf1-associated chromatin complex in the ribosomal biogenesis and senescence pathways.

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex.

Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an ∼ 150-Å-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.

Monitoring F1651 P-like fimbria expression at the single-cell level reveals a highly heterogeneous phenotype.

F1651 and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors. They play a key role in establishing disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains in animals and humans. Both F1651 and Pap are under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Using the Gfp reporter system, we monitored in vitro the level of fluorescence intensity corresponding to the F1651 and Pap fimbrial synthesis. Monitoring individual Escherichia coli cells by flow cytometry and by real-time fluorescence microscopy, we identified cells associated with a low or high level of fluorescence intensity and a large amount of cells with partial levels of fluorescence, mostly present in the F1651 system. This mixed population identified through fluorescence intensity could be attributed to the high switching rate previously observed in F1651-positive bacteria. The fimbrial heterogeneous phenotype for these ExPEC could represent increased fitness in unpredictable environments. Our study illustrates that within the large repertoire of fimbrial variants such as the well-characterized Pap, F1651 is an exquisite example of regulatory expression that arms the bacterium with strategies for surviving in more than one particular environment.

Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell.

Senescence-associated SIN3B promotes inflammation and pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is strikingly resistant to conventional therapeutic approaches. We previously demonstrated that the histone deacetylase-associated protein SIN3B is essential for oncogene-induced senescence in cultured cells. Here, using a mouse model of pancreatic cancer, we have demonstrated that SIN3B is required for activated KRAS-induced senescence in vivo. Surprisingly, impaired senescence as the result of genetic inactivation of Sin3B was associated with delayed PDAC progression and correlated with an impaired inflammatory response. In murine and human pancreatic cells and tissues, levels of SIN3B correlated with KRAS-induced production of IL-1α. Furthermore, evaluation of human pancreatic tissue and cancer cells revealed that Sin3B was decreased in control and PDAC samples, compared with samples from patients with pancreatic inflammation. These results indicate that senescence-associated inflammation positively correlates with PDAC progression and suggest that SIN3B has potential as a therapeutic target for inhibiting inflammation-driven tumorigenesis.

Structural basis for molecular interactions involving MRG domains: implications in chromatin biology.

MRG15 is a member of the mortality family of transcription factors that targets a wide variety of multiprotein complexes involved in transcription regulation, DNA repair, and alternative splicing to chromatin. The structure of the apo-MRG15 MRG domain implicated in interactions with diverse proteins has been described, but not in complex with any of its targets. Here, we structurally and functionally characterize the interaction between MRG15 and Pf1, two constitutively associated subunits of the histone deacetylase-associated Rpd3S/Sin3S corepressor complex. The MRG domain adopts a structure reminiscent of the apo state, whereas the Pf1 MRG-binding domain engages two discrete hydrophobic surfaces on the MRG domain via a bipartite motif comprising an α-helix and a segment in an extended conformation, both of which are critical for high-affinity interactions. Multiple MRG15 interactors share an FxLP motif in the extended segment, but equivalent sequence/helical motifs are not readily evident, implying potential diversity in MRG-recognition mechanisms.

Lrp-DNA complex stability determines the level of ON cells in type P fimbriae phase variation.

F165(1) and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors that play a key role in the establishment of disease caused by extraintestinal Escherichia coli (ExPEC) strains. They are both under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Our present study demonstrates that the high level of ON cells found during F165(1) phase variation is due to altered stability of the DNA complex formed by the leucine-responsive regulatory protein (Lrp) at its repressor binding sites 1-3; after each cell cycle, complex formation is also modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp towards its activator binding sites 4-6. Furthermore, we identified two nucleotides (T490, G508) surrounding the Lrp binding site 1 that are critical to maintaining a high OFF to ON switch rate during F165(1) phase variation, as well as switching Pap fimbriae towards the OFF state.

Effects of DNA supercoiling and topoisomerases on the expression of genes coding for F165(1), a P-like fimbriae.

Pathogenic Escherichia coli 4787 (O115:KV165) causes septicemia in pigs and expresses the fimbriae F165(1) encoded by the foo operon that belongs to the P fimbrial family. fooI and fooB, encoding specific foo regulators, are divergently transcribed; their intergenic region is responsible for the regulation of foo expression. The role of global and local supercoiling (transcription-induced supercoiling within the intergenic region) on the regulation of foo expression was investigated. Expression of fooB was significantly altered when global negative supercoiling was reduced by a mutation that decreases DNA gyrase activity. Deletion of the topA gene, encoding for topoisomerase I that relaxes local negative supercoiling, further reduced fooB expression. This suggests that both global and local supercoiling can significantly affect fooB expression. Moreover, FooI, a positive regulator of fooB expression, has no effect on fooB expression in the topA null mutant. This study showed that divergent transcription from a strong promoter can significantly enhance fooB expression and compensate for the absence of FooI in a wild-type strain.

TLR2-dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness.

Streptococcus suis capsular type 2 is an important swine pathogen and an agent of zoonosis. Although meningitis is the most common form of disease, septicemia and septic shock are also frequently reported. Despite reports that CD14 is involved in the recognition of encapsulated S. suis by host cells, the mechanisms underlying exacerbated release of pro-inflammatory cytokines, which may have a negative impact on disease outcome, are unclear. Here, we demonstrated that stimulation of human monocytes by whole encapsulated S. suis or its purified cell wall components influences the relative expression of Toll-like receptor (TLR)-2 and CD14 mRNA. Moreover, this stimulation triggered the release of cytokines (tumor necrosis factor-alpha, IL-1beta and IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1), which was significantly reduced by antibody-mediated blocking of TLR2 but not TLR4. Mouse macrophages deficient in TLR2 also showed impaired cytokine responses to encapsulated bacteria. Given that this response was completely abrogated in myeloid differentiation factor 88 (MyD88)-deficient macrophages, other TLRs might also be involved. Furthermore, we demonstrated that the presence of capsular polysaccharide (CPS)-modulated S. suis interactions with TLRs. In the absence of CPS, uncovered cell wall components induced cytokine and chemokine production via TLR2-dependent as well as -independent pathways, whereas CPS contributes to MCP-1 production in a MyD88-independent manner. Overall, this study contributes to a better understanding of the inflammatory processes induced by an encapsulated pathogen and suggests that the relative expression of CPS, known to be modulated during bacterial invasion and dissemination in the host, might alter interactions with host cells and, consequently, the outcome of the inflammatory response.

A comparative study of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis extracellular proteomes.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are closely related species that share a similar genetic background but occupy different ecological niches. Virulence plasmids bearing genes coding for toxins, may explain, at least partly, this specialization. We have compared by 2-DE in the early stationary phase of growth the extracellular proteomes of three strains of these species that have lost their virulence plasmids. Proteins expected to be secreted or to belong to the cell wall or to the cytosol were found in the three proteomes. For the cell wall and cytosolic proteins located in the extracellular space, the three proteomes were similar. Cytosolic proteins included enolase, GroEL, PdhB, PdhD, SodA and others. Cell surface proteins were mainly autolysins, proteases, nucleotidases and OppAs. In contrast, the secreted proteins profiles of B. cereus and B. thuringiensis were quite different from that of B. anthracis. B. cereus and B. thuringiensis extracellular proteomes both contained large amounts of secreted degradative enzymes and toxins, including nine proteases, three phospholipases, two haemolysins and several enterotoxins. Most of the genes encoding these enzymes and toxins are controlled by the transcriptional activator PlcR. The extracellular proteome of the pXO1-, pXO2- B. anthracis 9131 strain contained only one secreted protein: the metalloprotease InhA1, also found in the proteomes of the two other strains and possibly involved in antibacterial peptide degradation.