Beth A. Tamburini

Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair.

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. We find that Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

Gene Expression Profiles of Sporadic Canine Hemangiosarcoma Are Uniquely Associated with Breed

The role an individuals genetic background plays on phenotype and biological behavior of sporadic tumors remains incompletely understood. We showed previously that lymphomas from Golden Retrievers harbor defined, recurrent chromosomal aberrations that occur less frequently in lymphomas from other dog breeds, suggesting spontaneous canine tumors provide suitable models to define how heritable traits influence cancer genotypes. Here, we report a complementary approach using gene expression profiling in a naturally occurring endothelial sarcoma of dogs (hemangiosarcoma). Naturally occurring hemangiosarcomas of Golden Retrievers clustered separately from those of non-Golden Retrievers, with contributions from transcription factors, survival factors, and from pro-inflammatory and angiogenic genes, and which were exclusively present in hemangiosarcoma and not in other tumors or normal cells (i.e., they were not due simply to variation in these genes among breeds). Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) was among genes preferentially enriched within known pathways derived from gene set enrichment analysis when characterizing tumors from Golden Retrievers versus other breeds. Heightened VEGFR1 expression in these tumors also was apparent at the protein level and targeted inhibition of VEGFR1 increased proliferation of hemangiosarcoma cells derived from tumors of Golden Retrievers, but not from other breeds. Our results suggest heritable factors mold gene expression phenotypes, and consequently biological behavior in sporadic, naturally occurring tumors.

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

BackgroundThe etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.MethodsWe first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.ResultsEach of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).ConclusionsThe data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.

Antigen capture and archiving by lymphatic endothelial cells following vaccination or viral infection

Antigen derived from viral infections with influenza and Vesicular Stomatitis Virus (VSV) can persist after resolution of infection. Here we show that antigen can similarly persist for weeks following viral challenge and vaccination. Antigen is captured by Lymphatic Endothelial Cells (LECs), under conditions that induce LEC proliferation. Consistent with published data showing that viral antigen persistence impacts on the function of circulating memory T cells, we find that vaccine elicited antigen persistence, found on LECs, positively influences the degree of protective immunity elicited by circulating memory CD8+ T cells. The coupling of LEC proliferation and antigen capture identifies a mechanism by which the LECs store, or “archive”, antigens for extended periods of time after antigen challenge, thereby increasing IFNγ/IL-2 production and enhancing protection against infection. These findings therefore have the potential to impact future vaccination strategies and our understanding of the role for persisting antigen in both vaccine and infectious settings.

T cell responses: naïve to memory and everything in between

THE MAMMALIAN IMMUNE SYSTEM can be broadly divided into two main arms: innate and adaptive immunity. As its name implies, the cells and receptors of the innate immune system are critical for the rapid recognition of the infectious agent and initiating a proinflammatory response. While the inflammation generated by innate immune cells [neutrophils, macrophages, monocytes, natural killer (NK) cells, dendritic cells (DCs), etc.] is important in the initial containment of the infection, it also informs and directs the expansion and differentiation of adaptive immune cells. Responding to the inflammatory environment created by the innate response, cells of the adaptive arm of the immune response (B cells, T cells, and T cells) are stimulated to expand in number (proliferate) and to differentiate into cells with a range of functions appropriate for the immunological challenge. Upon elimination of the invading pathogen, the majority of adaptive cells die and leave behind an (evergrowing) array of memory cell subsets. These memory cells offer a diversity of migratory properties and functions, collectively mediating a rapid and protective immune response upon reinfection. Thus, the major advantages of an adaptive response to the host are twofold. First, it allows the host to form an immune response that is specifically tailored to the invading pathogen. Second, it forms a pool of memory cells from these specific effectors that can last for many years, capable of protecting the host against reinfection by their rapid response. This combination of specificity and memory are the mechanistic underpinnings for the clinical success of vaccination. Critical to almost all functions of the adaptive immune response is the activation and programming of T cells from their naive/resting state. Although there is much more to be learned, we now have a good basic understanding of the signals and cell types involved in the various stages of the T cell response initiated within the secondary lymphoid organs (SLOs). To provide a comprehensive overview, this review will summarize the T cell response broken down into three major stages: activation, differentiation, and memory formation. We will then assemble these components into a description of the anatomy of an immune response and its relationship to productive immune protection.

Functional Conservation and Specialization among Eukaryotic Anti-Silencing Function 1 Histone Chaperones

ABSTRACT Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1s role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.

Dominant Mutants of the Saccharomyces cerevisiae ASF1 Histone Chaperone Bypass the Need for CAF-1 in Transcriptional Silencing by Altering Histone and Sir Protein Recruitment

Transcriptional silencing involves the formation of specialized repressive chromatin structures. Previous studies have shown that the histone H3–H4 chaperone known as chromatin assembly factor 1 (CAF-1) contributes to transcriptional silencing in yeast, although the molecular basis for this was unknown. In this work we have identified mutations in the nonconserved C terminus of antisilencing function 1 (Asf1) that result in enhanced silencing of HMR and telomere-proximal reporters, overcoming the requirement for CAF-1 in transcriptional silencing. We show that CAF-1 mutants have a drastic reduction in DNA-bound histone H3 levels, resulting in reduced recruitment of Sir2 and Sir4 to the silent loci. C-terminal mutants of another histone H3–H4 chaperone Asf1 restore the H3 levels and Sir protein recruitment to the silent loci in CAF-1 mutants, probably as a consequence of the weakened interaction between these Asf1 mutants and histone H3. As such, these studies have identified the nature of the molecular defect in the silent chromatin structure that results from inactivation of the histone chaperone CAF-1.

Antigen archiving by lymph node stroma: A novel function for the lymphatic endothelium.

Secondary lymphoid stroma performs far more functions than simple structural support for lymphoid tissues, providing a host of soluble and membrane‐bound cues to trafficking leukocytes during inflammation and homeostasis. More recently it has become clear that stromal cells can manipulate T‐cell responses, either through direct antigen‐mediated stimulation of T cells or more indirectly through the retention and management of antigen after viral infection or vaccination. In light of recent data, this review provides an overview of stromal cell subsets and functions during the progression of an adaptive immune response with particular emphasis on antigen capture and retention by follicular dendritic cells as well as the recently described “antigen archiving” function of lymphatic endothelial cells (LECs). Given its impact on the maintenance of protective immune memory, we conclude by discussing the most pressing questions pertaining to LEC antigen capture, archiving and exchange with hematopoetically derived antigen‐presenting cells.

T cells compete by cleaving cell surface CD27 and blocking access to CD70-bearing APCs.

T cells compete against each other for access to molecules on APCs in addition to peptide/MHC complexes. However, the identity of cell surface molecules that influence T‐cell competition, other than peptide/MHC, have yet to be defined. Here, we identify CD70, a TNF ligand expressed on activated APCs, as an important mediator of T‐cell competition for APCs. Upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the surface of the interacting CD8+ T cell and captured by CD70 expressing dendritic cells. The capture of CD27 effectively masks CD70 on APCs, disallowing the interaction with CD27 on other competing T cells. Collectively, our data indicate that T cells compete against each other for access to the TNF‐ligand CD70, an interaction that affects the duration and potency of T cell/DC interactions, thus influencing the repertoire of responding CD8+ T cells to self or foreign antigens.

IL-6 inducing whole yeast-based immunotherapy directly controls IL-12 dependent CD8 T cell responses

In current clinical trails, whole yeast-based immunotherapy expressing hepatitis C viral antigens demonstrated statistically significant improvement in end of treatment responses when combined with type I interferon based standard of care, even in standard of care resistant patients. Although preclinical data suggest yeast vaccination, such as type I interferon, facilitates CD8 T-cell immunity, the capacity of yeast to generate immunity in patients resistant to type I interferon calls into question the mechanism(s) underpinning the efficacy of this approach. We show yeast and a Toll-like receptor exclusive agonist, Pam3Cys, differ in CD8 T-cell generation when combined with an agonistic CD40 antibody. Although both yeast and PamCys were largely Toll-like receptor dependent, the primary CD8 response generated by yeast was significantly less than Pam3Cys in wild-type hosts even in a CD4 T-cell-deficient setting. In addition, immunization of IL6−/− mice with yeast produced a 3-fold to 6-fold increased CD8 response while the Pam3Cys response was unaffected. The yeast but not Pam3Cys-driven CD8 response was inhibited in both wild-type and IL-6−/− hosts by blocking interleukin (IL)-12. In addition, IL6−/− mice had increased CD86 expression on their dendritic cells after yeast immunization also inhibited by IL-12 blockade. Collectively, our results indicate the CD8 T-cell response to yeast but not Pam3Cys is influenced by IL-6-mediated control of IL-12 critical for dendritic cell activation. To our knowledge this is the first demonstration that yeast directly influence IL-12-associated CD8 T-cell immunity providing an additional route whereby recombinant yeast may provide efficacy independent of type I interferon.