Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms.

Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

Immunologic and Virologic Parameters Associated With Human Immunodeficiency Virus (HIV) DNA Reservoir Size in People With HIV Receiving Antiretroviral Therapy.

BACKGROUND: A better understanding of the dynamics of human immunodeficiency virus (HIV) reservoirs in CD4+ T cells of people with HIV (PWH) receiving antiretroviral therapy (ART) is crucial for developing therapies to eradicate the virus. METHODS: We conducted a study involving 28 aviremic PWH receiving ART with high and low levels of HIV DNA. We analyzed immunologic and virologic parameters and their association with the HIV reservoir size. RESULTS: The frequency of CD4+ T cells carrying HIV DNA was associated with higher pre-ART plasma viremia, lower pre-ART CD4+ T-cell counts, and lower pre-ART CD4/CD8 ratios. During ART, the High group maintained elevated levels of intact HIV proviral DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA. HIV sequence analysis showed no evidence for preferential accumulation of defective proviruses nor higher frequencies of clonal expansion in the High versus Low group. Phenotypic and functional T-cell analyses did not show enhanced immune-mediated virologic control in the Low versus High group. Of considerable interest, pre-ART innate immunity was significantly higher in the Low versus High group. CONCLUSIONS: Our data suggest that innate immunity at the time of ART initiation may play an important role in modulating the dynamics and persistence of viral reservoirs in PWH.

A Population Genomic Assessment of Three Decades of Evolution in a Natural Drosophila Population.

Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality-encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality-most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. We find a genome-wide excess of outliers for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. We also find evidence for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales.

Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.

Correction: Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

[This corrects the article DOI: 10.1371/journal.ppat.1009395.].

Clinical exome sequencing of 1000 families with complex immune phenotypes: Toward comprehensive genomic evaluations.

BACKGROUND: Prospective genetic evaluation of patients at this referral research hospital presents clinical research challenges. OBJECTIVES: This study sought not only a single-gene explanation for participants' immune-related presentations, but viewed each participant holistically, with the potential to have multiple genetic contributions to their immune phenotype and other heritable comorbidities relevant to their presentation and health. METHODS: This study developed a program integrating exome sequencing, chromosomal microarray, phenotyping, results return with genetic counseling, and reanalysis in 1505 individuals from 1000 families with suspected or known inborn errors of immunity. RESULTS: Probands were 50.8% female, 71.5% were ≥18 years, and had diverse immune presentations. Overall, 327 of 1000 probands (32.7%) received 361 molecular diagnoses. These included 17 probands with diagnostic copy number variants, 32 probands with secondary findings, and 31 probands with multiple molecular diagnoses. Reanalysis added 22 molecular diagnoses, predominantly due to new disease-gene associations (9 of 22, 40.9%). One-quarter of the molecular diagnoses (92 of 361) did not involve immune-associated genes. Molecular diagnosis was correlated with younger age, male sex, and a higher number of organ systems involved. This program also facilitated the discovery of new gene-disease associations such as SASH3-related immunodeficiency. A review of treatment options and ClinGen actionability curations suggest that at least 251 of 361 of these molecular diagnoses (69.5%) could translate into ≥1 management option. CONCLUSIONS: This program contributes to our understanding of the diagnostic and clinical utility whole exome analysis on a large scale.

Markers of endothelial glycocalyx dysfunction in Clarkson disease.

BACKGROUND: Clarkson disease (monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome, ISCLS) is a rare idiopathic condition marked by transient, relapsing-remitting episodes of systemic microvascular hyper-permeability, which liberates plasma fluid and macromolecules into the peripheral tissues. This pathology manifests clinically as the abrupt onset of hypotensive shock, hemoconcentration, and hypoalbuminemia. METHODS: We analysed endothelial glycocalyx (eGCX)-related markers in plasma from patients with ISCLS during acute disease flares and convalescence by ELISA and comprehensive proteomic profiling. We evaluated eGCX-related components and gene expression in cultured endothelial cells using RNA-sequencing, real-time PCR, and fluorescence staining. RESULTS: Serum levels of eGCX-related core components including hyaluronic acid (HA) and the core proteoglycan soluble syndecan-1 (sCD138) were elevated at baseline and during acute ISCLS flares. Serial measurements demonstrated that sCD138 levels peaked during the recovery (post-leak) phase of the illness. Proteomic analysis of matched acute and convalescent ISCLS plasma revealed increased abundance of eGCX-related proteins, including glypicans, thrombospondin-1 (TSP-1), and eGCX-degrading enzymes in acute compared to remission plasma. Abundance of endothelial cell damage markers did not differ in acute and baseline plasma. Expression of several eGCX-related genes and surface carbohydrate content in endothelial cells from patients with ISCLS did not differ significantly from that observed in healthy control cells. CONCLUSIONS: eGCX dysfunction, but not endothelial injury, may contribute to clinical symptoms of acute ISCLS. Serum levels of of eGCX components including sCD138 may be measured during acute episodes of ISCLS to monitor clinical status and therapeutic responses.

Heterogeneity in refractory acute myeloid leukemia.

Successful clinical remission to therapy for acute myeloid leukemia (AML) is required for long-term survival to be achieved. Despite trends in improved survival due to better supportive care, up to 40% of patients will have refractory disease, which has a poorly understood biology and carries a dismal prognosis. The development of effective treatment strategies has been hindered by a general lack of knowledge about mechanisms of chemotherapy resistance. Here, through transcriptomic analysis of 154 cases of treatment-naive AML, three chemorefractory patient groups with distinct expression profiles are identified. A classifier, four key refractory gene signatures (RG4), trained based on the expression profile of the highest risk refractory patients, validated in an independent cohort (n = 131), was prognostic for overall survival (OS) and refined an established 17-gene stemness score. Refractory subpopulations have differential expression in pathways involved in cell cycle, transcription, translation, metabolism, and/or stem cell properties. Ex vivo drug sensitivity to 122 small-molecule inhibitors revealed effective group-specific targeting of pathways among these three refractory groups. Gene expression profiling by RNA sequencing had a suboptimal ability to correctly predict those individuals resistant to conventional cytotoxic induction therapy, but could risk-stratify for OS and identify subjects most likely to have superior responses to a specific alternative therapy. Such personalized therapy may be studied prospectively in clinical trials.

Correlation Between TIGIT Expression on CD8+ T Cells and Higher Cytotoxic Capacity.

Persistent exposure to antigen leads to T-cell exhaustion and immunologic dysfunction. We examined the immune exhaustion markers T cell immunoglobulin and ITIM domain (TIGIT) and programmed cell death protein 1 (PD-1) in human immunodeficiency virus (HIV)-infected and healthy individuals and the relationship with cytotoxic CD8+ T-lymphocyte activity. Frequencies of TIGIT but not PD-1 were positively correlated with CD8+ T-lymphocyte activity in HIV-aviremic and healthy individuals; however, there was no correlation in HIV-viremic individuals. Transcriptome analyses revealed up-regulation of genes associated with antiviral immunity in TIGIT+CD8+ versus TIGIT-CD8+ T cells. Our data suggest that TIGIT+CD8+ T cells do not necessarily represent a state of immune exhaustion and maintain an intrinsic cytotoxicity in HIV-infected individuals.

Low-level variant calling for non-matched samples using a position-based and nucleotide-specific approach.

BACKGROUND: The widespread use of next-generation sequencing has identified an important role for somatic mosaicism in many diseases. However, detecting low-level mosaic variants from next-generation sequencing data remains challenging. RESULTS: Here, we present a method for Position-Based Variant Identification (PBVI) that uses empirically-derived distributions of alternate nucleotides from a control dataset. We modeled this approach on 11 segmental overgrowth genes. We show that this method improves detection of single nucleotide mosaic variants of 0.01-0.05 variant allele fraction compared to other low-level variant callers. At depths of 600 × and 1200 ×, we observed > 85% and > 95% sensitivity, respectively. In a cohort of 26 individuals with somatic overgrowth disorders PBVI showed improved signal to noise, identifying pathogenic variants in 17 individuals. CONCLUSION: PBVI can facilitate identification of low-level mosaic variants thus increasing the utility of next-generation sequencing data for research and diagnostic purposes.

Experimental Babesia rossi infection induces hemolytic, metabolic, and viral response pathways in the canine host.

BACKGROUND: Babesia rossi is a leading cause of morbidity and mortality among the canine population of sub-Saharan Africa, but pathogenesis remains poorly understood. Previous studies of B. rossi infection were derived from clinical cases, in which neither the onset of infection nor the infectious inoculum was known. Here, we performed controlled B. rossi inoculations in canines and evaluated disease progression through clinical tests and whole blood transcriptomic profiling. RESULTS: Two subjects were administered a low inoculum (104 parasites) while three received a high (108 parasites). Subjects were monitored for 8 consecutive days; anti-parasite treatment with diminazene aceturate was administered on day 4. Blood was drawn prior to inoculation as well as every experimental day for assessment of clinical parameters and transcriptomic profiles. The model recapitulated natural disease manifestations including anemia, acidosis, inflammation and behavioral changes. Rate of disease onset and clinical severity were proportional to the inoculum. To analyze the temporal dynamics of the transcriptomic host response, we sequenced mRNA extracted from whole blood drawn on days 0, 1, 3, 4, 6, and 8. Differential gene expression, hierarchical clustering, and pathway enrichment analyses identified genes and pathways involved in response to hemolysis, metabolic changes, and several arms of the immune response including innate immunity, adaptive immunity, and response to viral infection. CONCLUSIONS: This work comprehensively characterizes the clinical and transcriptomic progression of B. rossi infection in canines, thus establishing a large mammalian model of severe hemoprotozoal disease to facilitate the study of host-parasite biology and in which to test novel anti-disease therapeutics. The knowledge gained from the study of B. rossi in canines will not only improve our understanding of this emerging infectious disease threat in domestic dogs, but also provide insight into the pathobiology of human diseases caused by Babesia and Plasmodium species.

Genome sequence, transcriptome, and annotation of rodent malaria parasite Plasmodium yoelii nigeriensis N67.

BACKGROUND: Rodent malaria parasites are important models for studying host-malaria parasite interactions such as host immune response, mechanisms of parasite evasion of host killing, and vaccine development. One of the rodent malaria parasites is Plasmodium yoelii, and multiple P. yoelii strains or subspecies that cause different disease phenotypes have been widely employed in various studies. The genomes and transcriptomes of several P. yoelii strains have been analyzed and annotated, including the lethal strains of P. y. yoelii YM (or 17XL) and non-lethal strains of P. y. yoelii 17XNL/17X. Genomic DNA sequences and cDNA reads from another subspecies P. y. nigeriensis N67 have been reported for studies of genetic polymorphisms and parasite response to drugs, but its genome has not been assembled and annotated. RESULTS: We performed genome sequencing of the N67 parasite using the PacBio long-read sequencing technology, de novo assembled its genome and transcriptome, and predicted 5383 genes with high overall annotation quality. Comparison of the annotated genome of the N67 parasite with those of YM and 17X parasites revealed a set of genes with N67-specific orthology, expansion of gene families, particularly the homologs of the Plasmodium chabaudi erythrocyte membrane antigen, large numbers of SNPs and indels, and proteins predicted to interact with host immune responses based on their functional domains. CONCLUSIONS: The genomes of N67 and 17X parasites are highly diverse, having approximately one polymorphic site per 50 base pairs of DNA. The annotated N67 genome and transcriptome provide searchable databases for fast retrieval of genes and proteins, which will greatly facilitate our efforts in studying the parasite biology and gene function and in developing effective control measures against malaria.

Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Antigenic Restimulation of Virus-Specific Memory CD8+ T Cells Requires Days of Lytic Protein Accumulation for Maximal Cytotoxic Capacity.

In various infections or vaccinations of mice or humans, reports of the persistence and the requirements for restimulation of the cytotoxic mediators granzyme B (GrB) and perforin (PRF) in CD8+ T cells have yielded disparate results. In this study, we examined the kinetics of PRF and GrB mRNA and protein expression after stimulation and associated changes in cytotoxic capacity in virus-specific memory cells in detail. In patients with controlled HIV or cleared respiratory syncytial virus (RSV) or influenza virus infections, all virus-specific CD8+ T cells expressed low PRF levels without restimulation. Following stimulation, they displayed similarly delayed kinetics for lytic protein expression, with significant increases occurring by days 1 to 3 before peaking on days 4 to 6. These increases were strongly correlated with, but were not dependent upon, proliferation. Incremental changes in PRF and GrB percent expression and mean fluorescence intensity (MFI) were highly correlated with increases in HIV-specific cytotoxicity. mRNA levels in HIV-specific CD8+ T-cells exhibited delayed kinetics after stimulation as with protein expression, peaking on day 5. In contrast to GrB, PRF mRNA transcripts were little changed over 5 days of stimulation (94-fold versus 2.8-fold, respectively), consistent with posttranscriptional regulation. Changes in expression of some microRNAs, including miR-17, miR-150, and miR-155, suggested that microRNAs might play a significant role in regulation of PRF expression. Therefore, under conditions of extremely low or absent antigen levels, memory virus-specific CD8+ T cells require prolonged stimulation over days to achieve maximal lytic protein expression and cytotoxic capacity.IMPORTANCE Antigen-specific CD8+ T cells play a major role in controlling most virus infections, primarily by perforin (PRF)- and granzyme B (GrB)-mediated apoptosis. There is considerable controversy regarding whether PRF is constitutively expressed, rapidly increased similarly to a cytokine, or delayed in its expression with more prolonged stimulation in virus-specific memory CD8+ T cells. In this study, the degree of cytotoxic capacity of virus-specific memory CD8+ T cells was directly proportional to the content of lytic molecules, which required antigenic stimulation over several days for maximal levels. This appeared to be modulated by increases in GrB transcription and microRNA-mediated posttranscriptional regulation of PRF expression. Clarifying the requirements for maximal cytotoxic capacity is critical to understanding how viral clearance might be mediated by memory cells and what functions should be induced by vaccines and immunotherapies.

The mutational landscape of histiocytic sarcoma associated with lymphoid malignancy.

Histiocytic sarcoma and tumors with dendritic cell differentiation (HDT) are uncommon neoplasms often with an aggressive clinical course that may occur in association with another hematologic malignancy or mediastinal germ cell tumor (secondary HDT, sHDT). Previous studies have shown mutations in the RAS/MAPK pathway in HDT and have demonstrated a clonal relationship between HDT and associated lymphoid malignancies through common translocations or identical immunoglobulin or T-cell receptor gene rearrangements. We performed whole exome sequencing on 16 cases of sHDT to further evaluate the spectrum of mutations that occur in sHDT in the context of an associated lymphoid malignancy, including cases associated with follicular lymphoma (FL), chronic lymphocytic leukemia/small lymphocytic lymphoma, B- and T-cell acute lymphoblastic leukemia/lymphoma and peripheral T-cell lymphoma, NOS. In addition, we assessed the clonal relationship between the HDT and the associated lymphoid malignancy in three cases for which matched samples were available. We found mutations in RAS/MAPK pathway genes in 14/16 cases of sHDT associated with diverse mature and precursor B-cell and T-cell neoplasms, involving KRAS (8/16), BRAF (2/16), NRAS (2/16), MAP2K1 (1/16), and NF1 (1/16). In addition, we note that FL-associated sHDT frequently shares a similar mutational profile to the associated malignancy, identifying mutations in CREBBP or KMT2D in all cases and "aberrant" somatic hypermutation in 5/6 cases. Our study confirms the role of the RAS/MAPK pathway in the pathogenesis of sHDT, provides further evidence of a common neoplastic precursor and, in the case of FL, gives additional insight into the stage in lymphomagenesis at which transdifferentiation may occur.

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers.IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.

Genomic profiling of primary histiocytic sarcoma reveals two molecular subgroups.

Histiocytic sarcoma is a rare malignant neoplasm that may occur de novo or in the context of a previous hematologic malignancy or mediastinal germ cell tumor. Here, we performed whole exome sequencing and RNA-sequencing (RNA-Seq) on 21 archival cases of primary histiocytic sarcoma. We identified a high number of genetic alterations within the RAS/RAF/MAPK pathway in 21 of 21 cases, with alterations in NF1 (6 of 21), MAP2K1 (5 of 21), PTPN11 (4 of 21), BRAF (4 of 21), KRAS (4 of 21), NRAS (1 of 21), and LZTR1 (1 of 21), including single cases with homozygous deletion of NF1, high-level amplification of PTPN11, and a novel TTYH3-BRAF fusion. Concurrent NF1 and PTPN11 mutations were present in 3 of 21 cases, and 5 of 7 cases with alterations in NF1 and/or PTPN11 had disease involving the gastrointestinal tract. Following unsupervised clustering of gene expression data, cases with NF1 and/or PTPN11 abnormalities formed a distinct tumor subgroup. A subset of NF1/PTPN11 wild-type cases had frequent mutations in B-cell lymphoma associated genes and/or clonal IG gene rearrangements. Our findings expand the current understanding of the molecular pathogenesis of this rare tumor and suggest the existence of a distinct subtype of primary histiocytic sarcoma characterized by NF1/PTPN11 alterations with predilection for the gastrointestinal tract.