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Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen,we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study,we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore,we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition,deficiency in Kin17 affects the functionality of multiple DSB repair pathways,namely homologous recombination,non-homologous end-joining,and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR. View Publication

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study,we demonstrate that IFN regulatory factor 2 (IRF2),an ISG and a negative regulator of IFN signaling,influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2(-/-) mice after peripheral inoculation. Irf2(-/-) mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2(-/-) mouse brains despite the presence of peripheral neutralizing antibodies,suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection,yet WT B cells and serum are unable to rescue the Irf2(-/-) mice. Collectively,our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections. View Publication

The developmental pathway for human megakaryocytes remains unclear and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis,we have identified a cluster of cells within immature hematopoietic stem and progenitor cell populations that specifically express genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34(+)CD38(+)IL-3Rα(dim)CD45RA(-) common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte/macrophage potential,but exhibited robust differentiation into the megakaryocyte lineage at a high frequency,both in vivo and in vitro The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly,the CD41(+) CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis,independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia,the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus,the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis. View Publication

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however,the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic,epigenetic and immunological approaches,we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide,but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates,shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective,transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection. View Publication

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article,we show that EPCR-positive UM171-treated cells,as opposed to EPCR-negative cells,exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers,such as CD38,EPCR expression is maintained when cells are introduced in culture,irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity,its expression is required for the repopulating activity of human HSCs. Altogether,our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs. View Publication

The T cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the major histocompatibility complex (MHC). To convert this binding event into downstream signaling,the TCR complex contains immunoreceptor tyrosine-based activation motifs (ITAMs) that act as docking sites for the cytoplasmic tyrosine kinase ZAP-70. Unique among antigen receptors,the TCR complex uses 10 ITAMs to transduce peptide-MHC binding to the cell interior. Using synthetic,drug-inducible receptor-ligand pairs,it was found that greater ITAM multiplicity primarily enhanced the efficiency with which ligand binding was converted into an intracellular signal. This manifested as an increase in the fraction of cells that became activated in response to antigen,and a more synchronous initiation of TCR-proximal signaling,rather than direct amplification of the intracellular signals. Exploiting these findings,the potency and selectivity of chimeric antigen receptors targeted against cancer were substantially enhanced by modulating the number of encoded ITAMs. View Publication

Proteasome inhibitors bortezomib,carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM),but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites,$\beta$5i,$\beta$1i,and $\beta$2i,which are different from their constitutive $\beta$5c,$\beta$1c,and $\beta$2c counterparts. Bortezomib and carfilzomib block $\beta$5c and $\beta$5i sites. We report here that pharmacologically relevant concentrations of $\beta$5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition,increasing immunoproteasome expression by interferon-$\gamma$ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102,an inhibitor of $\beta$2 sites,dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus,immunoproteasome inhibitors,currently in clinical trials for the treatment of autoimmune diseases,should also be considered for the treatment of MM. View Publication

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. View Publication

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming,but systematic interrogation of transcription factors,and their interactions with each other,has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells,revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation,with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons,which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition. View Publication

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells,resulting in reversal of Treg suppression. Importantly,TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression,which may be used as a strategy to advance tumor immunotherapy. View Publication

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms,mouse and human cell infection models and human biopsy samples were employed. We show that the 'keystone' pathogen Porphyromonas gingivalis,disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway,involving pAKT1,pFOXO1,FOXP3,IDO1 and BIM,is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically,activation of this pathway,demonstrating FOXP3 as a direct FOXO1-target gene,was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly,FimA + P. gingivalis strain MFI invades OSCCs,inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4,plus pAKT1-pFOXO1. Conclusively,P. gingivalis,through Mfa1 and FimA fimbriae,promotes immunosuppression and oncogenic cell proliferation,respectively,through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi,activating a pAKT+hipFOXO1+hiBIM-lowFOXP3+hi and IDO+hi- driven pathway,likely to impact the prognosis of oral cancers in patients with periodontitis. View Publication