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Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However,the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study,we investigated the possibility to circumvent these limitations by engineering Stat3 -deficient CD8(+) T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3in CD8(+) T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation,resulting in increased tumor antigen-specific T-cell activity and tumor growth inhibition. For potential clinical translation,we combined adoptive T-cell therapy with a Food and Drug Administration-approved tyrosine kinase inhibitor,sunitinib,in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells and reduced conversion of transferred FoxP3(-) T cells to tumor-associated regulatory T cells while increasing transferred CD8(+) T-cell infiltration and activation at the tumor site,leading to inhibition of primary tumor growth. These data show that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3-silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors. View Publication

The T-cell functions of a proliferation-inducing ligand (APRIL,also known as TNFSF13) remain largely undefined. We previously showed that APRIL suppressed Th2 cytokine production in cultured CD4(+) T cells and Th2 antibody responses. Here we show that APRIL suppresses allergic lung inflammation,which is associated with diminished expression of the transcription factor c-maf. Mice deficient in the April gene (April(-/-) mice) had significantly aggravated lung inflammation compared with WT mice in the ovalbumin-induced allergic lung inflammation model. Likewise,blockade of APRIL in WT mice by the APRIL-receptor fusion protein,transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)-Ig,enhanced lung inflammation. Transfer of APRIL-sufficient,ovalbumin-specific,TCR-transgenic CD4(+) T (OT-II) cells to April(-/-) mice restored the suppressive effect of APRIL on lung inflammation. Mechanistically,the expression of the Th2 cytokine transcription factor c-maf,but not GATA-3,was markedly enhanced in April(-/-) CD4(+) T cells at the RNA and protein level and under non-polarizing (Th neutral,ThN) and Th2-polarizing conditions. Since c-maf transactivates the IL-4 gene,the increased c-maf expression in April(-/-) mice readily explains increased Th2 cytokine production. Independent of its effect on IL-4,APRIL suppressed IL-13 expression. APRIL thus may regulate lung inflammation in a dual way,by acting on c-maf expression and by directly controlling IL-13 production. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication

We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using free,open source software to accurately quantitate neutrophil NETosis,a recently discovered process involved in multiple human diseases. CNNs achieved {\textgreater}94{\%} in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology,CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways,making them a precise tool for NETosis detection. Furthermore,by using CNNs and tools to determine object dispersion,we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus,we demonstrate the design,performance,and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science. View Publication

Despite the clinical success of cancer immunotherapies,the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here,we conducted a series of unbiased,genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-$\gamma$ (IFN-$\gamma$) signaling protect tumor cells from NK cell killing. Indeed,Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-$\gamma$-driven transcriptional events that regulate MHC I expression. Importantly,tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together,we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells,unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape. View Publication

Recurrent oncogenic mutations of MyD88 have been identified in a variety of lymphoid malignancies. Gain-of-function mutations of MyD88 constitutively activate downstream NF-$\kappa$B signaling pathways,resulting in increased cellular proliferation and survival. However,whether MyD88 activity can be aberrantly regulated in MyD88-wild-type lymphoid malignancies remains poorly understood. SPOP is an adaptor protein of CUL3-based E3 ubiquitin ligase complex and frequently mutated genes in prostate and endometrial cancers. In this study,we reveal that SPOP binds to and induces the nondegradative ubiquitination of MyD88 by recognizing an atypical SPOP-binding motif in MyD88. This modification blocks Myddosome assembly and downstream NF-$\kappa$B activation. SPOP is mutated in a subset of lymphoid malignancies,including diffuse large B-cell lymphoma (DLBCL). Lymphoid malignancies-associated SPOP mutants exhibited impaired binding to MyD88 and suppression of NF-$\kappa$B activation. The DLBCL-associated,SPOP-binding defective mutants of MyD88 escaped from SPOP-mediated ubiquitination,and their effect on NF-$\kappa$B activation is stronger than that of wild-type MyD88. Moreover,SPOP suppresses DLBCL cell growth in vitro and tumor xenograft in vivo by inhibiting the MyD88/NF-$\kappa$B signaling. Therefore,SPOP acts as a tumor suppressor in DLBCL. Mutations in the SPOP-MyD88 binding interface may disrupt the SPOP-MyD88 regulatory axis and promote aberrant MyD88/NF-$\kappa$B activation and cell growth in DLCBL. View Publication

Our previous studies have demonstrated that a previously unrecognized role of CFTR in hematopoiesis and acute leukemia. Here,we show that CFTR inhibitor CFTR-inh172 possesses ability to inhibit human T-cell acute lymphoblastic leukemia cells. In detail,CFTR-inh172 inhibited cell proliferation,promoted apoptosis and arrested the cell cycle in human T-cell acute lymphoblastic leukemia cell CCRF-CEM,JURKAT and MOLT-4. Furthermore,transcriptome analysis reveals that CFTR-inh172 induces significant alteration of gene expression related to apoptosis and proliferation. These findings demonstrate the potential of CFTR inhibitor CFTR-inh172 in human T-cell acute lymphoblastic leukemia treatment. View Publication

Despite the key role that antibodies play in protection,the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria,we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3,both of which contribute to the impaired GC polarization observed in the absence of T-bet,resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics,thereby promoting the differentiation of B cells with increased affinity for antigen. View Publication

Latent proviruses persist in central (TCM),transitional (TTM),and effector (TEM) memory cells. We measured the levels of cellular factors involved in HIV gene expression in these subsets. The highest levels of acetylated H4,active nuclear factor $\kappa$B (NF-$\kappa$B),and active positive transcription elongation factor b (P-TEFb) were measured in TEM,TCM,and TTM cells,respectively. Vorinostat and romidepsin display opposite abilities to induce H4 acetylation across subsets. Protein kinase C (PKC) agonists are more efficient at inducing NF-$\kappa$B phosphorylation in TCM cells but more potent at activating PTEF-b in the TEM subset. We selected the most efficient latency-reversing agents (LRAs) and measured their ability to reverse latency in each subset. While ingenol alone has modest activities in the three subsets,its combination with a histone deacetylase inhibitor (HDACi) dramatically increases latency reversal in TCM cells. Altogether,these results indicate that cellular HIV reservoirs are differentially responsive to common LRAs and suggest that combination of compounds will be required to achieve latency reversal in all subsets. View Publication

CRISPR/Cas is a transformative gene editing tool,that offers a simple and effective way to target a catalytic Cas9,the most widely used is derived from Streptococcus pyogenes (SpCas9),with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology,and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore,the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines,which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally,we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall,the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology,and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity. View Publication

CD16a (Fc$\gamma$RIIIa) mediates the antibody dependent cellular cytotoxicity (ADCC) and is important for anti-tumor activities of many therapeutic antibodies. Bispecific antibody targeting natural killer (NK) cells has been studied for cancer therapy. In this work,anti-CD16a single-domain antibodies were identified from hCD16a immunized camel. Bispecific antibodies are then constructed by fusing these single domain antibodies with an anti-CEA single domain antibody. These bispecific antibodies can recruite NK cells to kill CEA-positive tumor cells,and inhibit tumor growth in vivo,suggesting that these anti-CD16a single domain antibodies are powerful tools to engaging NK cells for cancer therapy. View Publication

PURPOSE The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. EXPERIMENTAL DESIGN A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR,EpCAM,and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. RESULTS The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells,each expressing a single antigen,and displayed synergistic activity when tumor cells expressed more than one target antigen. CONCLUSIONS DARPins can serve as high-affinity recognition motifs for CAR design,and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape. View Publication