Jia- Wang

Expansion of Myeloid Suppressor Cells in SHIP-Deficient Mice Represses Allogeneic T Cell Responses

Previously we demonstrated that SHIP−/− mice accept allogeneic bone marrow transplants (BMT) without significant acute graft-vs-host disease (GvHD). In this study we show that SHIP−/− splenocytes and lymph node cells are poor stimulators of allogeneic T cell responses that cause GvHD. Intriguingly, SHIP−/− splenocytes prime naive T cell responses to peptide epitopes, but, conversely, are partially impaired for priming T cell responses to whole Ag. However, dendritic cells (DC) purified from SHIP−/− splenocytes prime T cell responses to allogeneic targets, peptide epitopes, and whole Ag as effectively as SHIP+/+ DC. These findings point to an extrinsic effect on SHIP−/− DC that impairs priming of allogeneic T cell responses. Consistent with this extrinsic effect, we found that a dramatic expansion of myeloid suppressor cells in SHIP−/− mice impairs priming of allogeneic T cells. These findings suggest that SHIP expression or its activity could be targeted to selectively compromise T cell responses that mediate GvHD and graft rejection.

Identification of a Novel Lipopolysaccharide-Inducible Gene with Key Features of Both a Kinase Anchor Proteins and chs1/beige Proteins

Mutations in chs1/beige result in a deficiency in intracellular transport of vesicles that leads to a generalized immunodeficiency in mice and humans. The function of NK cells, CTL, and granulocytes is impaired by these mutations, indicating that polarized trafficking of vesicles is controlled by CHS1/beige proteins. However, a molecular explanation for this defect has not been identified. Here we describe a novel gene with orthologues in mice, humans, and flies that contains key features of both chs1/beige and A kinase anchor genes. We designate this novel gene lba for LPS-responsive, beige-like anchor gene. Expression of lba is induced after LPS stimulation of B cells and macrophages. In addition, lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Strikingly, LBA-green-fluorescent protein (GFP) fusion proteins are localized to vesicles after LPS stimulation. Confocal microscopy indicates this protein is colocalized with the trans-Golgi complex and some lysosomes. Further analysis by immunoelectron microscopy demonstrates that LBA-GFP fusion protein can localize to endoplasmic reticulum, plasma membrane, and endocytosis vesicles in addition to the trans-Golgi complex and lysosomes. We hypothesize that LBA/CHS1/BG proteins function in polarized vesicle trafficking by guiding intracellular vesicles to activated receptor complexes and thus facilitate polarized secretion and/or membrane deposition of immune effector molecules.

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Regulating the Regulators: microRNA and Asthma

One obstacle to developing an effective therapeutic strategy to treat or prevent asthma is that the fundamental causes of asthma are not totally understood. Asthma is thought to be a chronic TH2 immune-mediated inflammatory disease. Epigenetic changes are recognized to play a role in the initiation and maintenance of a TH2 response. MicroRNAs (miRNAs) are key epigenetic regulators of gene expression, and their expression is highly regulated, therefore, deregulation of miRNAs may play an important role in the pathogenesis of asthma. Profiling circulating miRNA might provide the highest specificity and sensitivity to diagnose asthma; similarly, correcting potential defects in the miRNA regulation network may lead to new therapeutic modalities to treat this disease.

A simple method for profiling miRNA expression.

Here we describe a simple protocol that uses positively charged nylon membrane dot blot to profile miRNA expression. A library of 515 antisense oligodeoxynucleotides of human and mouse mature miRNAs was synthesized and spotted on GeneScreen Plus membrane using a dot-blot equipment. Total RNA or enriched small molecular weight RNAs (smwRNAs) were enzymatically radiolabeled by poly (A) polymerase and then hybridized to the nylon membrane oligo arrays. The spot signal intensity on the membrane was analyzed using phosphorimaging. This method offers a convenient and economic way to simultaneously detect the expression of hundreds of miRNAs.

LRBA is Essential for Allogeneic Responses in Bone Marrow Transplantation.

The PH-BEACH-WD40 (PBW) protein family members play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive-Beige-like Anchor (LRBA) is a PBW protein whose immune function remains elusive. Here we show that LRBA-null mice are viable, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone-marrow grafts. Further, we demonstrate that LRBA-null Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of IFN-γ by cytokines remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where GvHD is primed or initiated, and consistent with this LRBA-null mice also demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses and thus represents a unique and novel molecular target in transplant immunology.

Plasmid-encoded NP73-102 modulates atrial natriuretic peptide receptor signaling and plays a critical role in inducing tolerogenic dendritic cells

BackgroundAtrial natriuretic peptide (ANP) is an important endogenous hormone that controls inflammation and immunity by acting on dendritic cells (DCs); however, the mechanism remains unclear.ObjectiveWe analyzed the downstream signaling events resulting from the binding of ANP to its receptor, NPRA, and sought to determine what aspects of this signaling modulate DC function.MethodsWe utilized the inhibitory peptide, NP73-102, to block NPRA signaling in human monocyte-derived DCs (hmDCs) and examined the effect on DC maturation and induced immune responses. The potential downstream molecules and interactions among these molecules involved in NPRA signaling were identified by immunoprecipitation and immunoblotting. Changes in T cell phenotype and function were determined by flow cytometry and BrdU proliferation ELISA. To determine if adoptively transferred DCs could alter the in vivo immune response, bone marrow-derived DCs from wild-type C57BL/6 mice were incubated with ovalbumin (OVA) and injected i.v. into C57BL/6 NPRA-/- knockout mice sensitized and challenged with OVA. Lung sections were stained and examined for inflammation and cytokines were measured in bronchoalveolar lavage fluid collected from parallel groups of mice.ResultsInhibition of NPRA signaling in DCs primes them to induce regulatory T cells. Adoptive transfer of wild type DCs into NPRA-/- mice reverses the attenuation of lung inflammation seen in the NPRA-knockout model. NPRA is associated with TLR-2, SOCS3 and STAT3, and inhibiting NPRA alters expression of IL-6, IL-10 and TGF-β, but not IL-12.ConclusionsModulation of NPRA signaling in DCs leads to immune tolerance and TLR2 and SOCS3 are involved in this induction.

114 A Highly Sensitive and Specific Universal Mirna Profiling Method.

Background miRNAs can be used as robust biomarkers for diagnosis, staging, prognosis and the response to therapy in various diseases. Although a wide spectrum of miRNA detection techniques have been developed, none can accurately and sensitively perform genome-wide high-throughput miRNA profiling (Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, et al 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33:e179). This problem stems from that miRNAs are only ∼22 bases, and multiple species of nucleic acids that contain the mature miRNA sequences are present in the total RNA samples that are usually used for miRNA detection. Methods A novel RT-qPCR miRNA assay (UQmiR, universally quantitating miRNA) was developed to overcome the difficulty. This assay requires only one RT reaction and one universal set of multiple hydrolysis probes to detect all miRNAs, using one universal RT primer, a common reverse primer, and individual miRNA-specific forward primers. A computer program (MSPPD, miRNA-specific primer and probe designer) was developed for the assay. Results The UQmiR has the advantages, but not the disadvantages, of the 2 mostly used miRNA assays. It has the specificity of hydrolysis probe assay and the universal detection of SYBR Green assay. This assay is more sensitive and specific than the commercially available hydrolysis probe assay and SYBR Green assay. Using this method, we have successfully detected 91 out of 96 miRNAs in 0.8 mL of plasma for each miRNA. Conclusions This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.