Type I Interferon Signaling on Antigen-Presenting Cells Blunts Cell-Mediated Immunity toward Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen that has been used for decades to understand mechanisms of bacterial pathogenesis and both innate and adaptive immunity. L. monocytogenes is a potent activator of CD8+ T-cell-mediated immunity, yet how the innate immune response to infection modulates CD8+ T-cell responses is incompletely understood. Here, we address how two innate immune pathways triggered by L. monocytogenes, type I interferon (IFN) production and inflammasome activation, impact the CD8+ T-cell response. We utilized a combination of mutant mice and genetically engineered L. monocytogenes to address this question. Mice lacking the type I IFN receptor (IFNAR-/-) had the most robust T-cell response, while caspase-1-/- mice were not different from wild type (WT). Caspase-1-/-/IFNAR-/- mice had fewer T-cells than IFNAR-/- mice, suggesting a role for inflammasome activation in the absence of type I IFN. IFNAR-/- had more than twice as many memory precursors promoting enhanced protection from rechallenge. Importantly, short-lived effectors were equivalent in all strains of mice. L. monocytogenes strains genetically modified to induce lower type I interferon production yielded enhanced T-cell responses. IFNAR-/- dendritic cells induced more T-cells to proliferate than WT in ex vivo T-cell proliferation assays, suggesting deficits from type I interferon signaling may be dendritic cell intrinsic, rather than acting on T-cells. Thus, modulating type I IFN signaling during vaccination may lead to more potent T-cell-based vaccines. Importantly, this suggests innate immune signaling significantly impacts the CD8+ T-cell response and suggests CD8+ T-cell quantity and quality are important factors to consider during rational vaccine design.

Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment.

Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of "poised" cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work demonstrates that optimal PGE2 production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be to facilitate production of PGE2.

Expression of NrasQ61R and MYC transgene in germinal center B cells induces a highly malignant multiple myeloma in mice.

NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition-based therapies for treating advanced/relapsed MM.

Pumilio response and AU-rich elements drive rapid decay of Pnrc2-regulated cyclic gene transcripts.

Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of somites, or embryonic segments, which give rise to many mesodermal tissue types. This molecular oscillator generates cyclic gene expression with the same periodicity as somite formation in the presomitic mesoderm (PSM), an area of mesenchymal cells that give rise to mature somites. Molecular components of the clock include the Hes/her family of genes that encode transcriptional repressors, but additional genes cycle. Cyclic gene transcripts are cleared rapidly, and clearance depends upon the pnrc2 (proline-rich nuclear receptor co-activator 2) gene that encodes an mRNA decay adaptor. Previously, we showed that the her1 3'UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner, however, the molecular mechanism(s) by which cyclic gene transcripts are cleared remained largely unknown. To identify features of the her1 3'UTR that are critical for Pnrc2-mediated decay, we developed an array of transgenic inducible reporter lines carrying different regions of the 3'UTR. We find that the terminal 179 nucleotides (nts) of the her1 3'UTR are necessary and sufficient to confer rapid instability. Additionally, we show that the 3'UTR of another cyclic gene, deltaC (dlc), also confers Pnrc2-dependent instability. Motif analysis reveals that both her1 and dlc 3'UTRs contain terminally-located Pumilio response elements (PREs) and AU-rich elements (AREs), and we show that the PRE and ARE in the last 179 â€‹nts of the her1 3'UTR drive rapid turnover of reporter mRNA. Finally, we show that mutation of Pnrc2 residues and domains that are known to facilitate interaction of human PNRC2 with decay factors DCP1A and UPF1 reduce the ability of Pnrc2 to restore normal cyclic gene expression in pnrc2 mutant embryos. Our findings suggest that Pnrc2 interacts with decay machinery components and cooperates with Pumilio (Pum) proteins and ARE-binding proteins to promote rapid turnover of cyclic gene transcripts during somitogenesis.

Listeria monocytogenes cancer vaccines: bridging innate and adaptive immunity.

PURPOSE OF THE REVIEW: Immunotherapy has emerged as a promising cancer treatment, however success in only select clinical indications underscores the need for novel approaches. Recently Listeria monocytogenes-based vaccines have been developed to drive tumor specific T-cell responses. Here, we discuss recent preclinical studies using L. monocytogenes vaccines, innate immune pathways that influence T-cell priming, and new vaccine strategies in clinical trials. RECENT FINDINGS: Recent studies indicate that in addition to inducing antigen specific T-cell responses, L. monocytogenes vaccines remodel the TME. In addition, several innate immune pathways influence adaptive immune responses to L. monocytogenes and modulating these pathways holds promise to enhance anti-tumor T-cell responses. SUMMARY: The interplay between innate and adaptive immune responses to L. monocytogenes is poorly understood. Understanding these interactions will facilitate the design of better anti-cancer vaccines and improved use of combination therapies.

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis.

BACKGROUND: T-box genes encode a large transcription factor family implicated in many aspects of development. We are focusing on two related zebrafish T-box genes, tbx6l and tbx16, that are expressed in highly overlapping patterns in embryonic paraxial mesoderm. tbx16 mutants are deficient in trunk, but not tail, somites; we explored whether presence of tail somites in tbx16 mutants was due to compensatory function provided by the tbx6l gene. RESULTS: We generated two zebrafish tbx6l mutant alleles. Loss of tbx6l has no apparent effect on embryonic development, nor does tbx6l loss enhance the phenotype of two other T-box gene mutants, ta and tbx6, or of the mesp family gene mutant msgn1. In contrast, loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants. CONCLUSIONS: These data demonstrate that tbx6l and tbx16 genes function redundantly to direct tail somite development. tbx6l single mutants develop normally because tbx16 fully compensates for loss of tbx6l function. However, tbx6l only partially compensates for loss of tbx16 function. These results resolve the question of why loss of function of tbx16 gene, which is expressed throughout the ventral and paraxial mesoderm, profoundly affects somite development in the trunk but not the tail. Developmental Dynamics 246:759-769, 2017. © 2017 Wiley Periodicals, Inc.

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay.

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish.

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4-5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite-like cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.