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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

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. View Publication

Chronic lymphocytic leukaemia (CLL) exhibits differences between Asians and Caucasians in terms of incidence rate,age at onset,immunophenotype,and genetic profile. We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG-binding domain sequencing (MBD-seq),as did five control subjects. Gene Ontology,pathway analysis,and network-based prioritization of differentially methylated genes were also performed. More regions were hypomethylated (2,062 windows) than were hypermethylated (777 windows). Promoters contained the highest proportion of differentially methylated regions (0.08{\%}),while distal intergenic and intron regions contained the largest number of differentially methylated regions. Protein-coding genes were the most abundant,followed by long noncoding and short noncoding genes. The most significantly over-represented signalling pathways in the differentially methylated gene list included immune/cancer-related pathways and B-cell receptor signalling. Among the top 10 hub genes identified via network-based prioritization,four (UBC,GRB2,CREBBP,and GAB2) had no known relevance to CLL,while the other six (STAT3,PTPN6,SYK,STAT5B,XPO1,and ABL1) have previously been linked to CLL in Caucasians. As such,our analysis identified four novel candidate genes of potential significance to Asian patients with CLL. View Publication

PURPOSE Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. PATIENTS AND METHODS Single-arm,phase II trial assessed the efficacy and safety of ensituximab in patients with advanced,refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10{\%} and 80{\%} power. Written informed consent was obtained from all patients. RESULTS Sixty-three patients with advanced,refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46{\%} of the patients were female. Among 57 evaluable patients,median OS was 6.8 months. No responses were observed,and stable disease was achieved in 21{\%} of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38{\%}),anemia (30{\%}),nausea (15{\%}),vomiting (11{\%}),increased bilirubin (9{\%}),constipation (8{\%}),decreased appetite (6{\%}),and diarrhea (6{\%}). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia,nausea,increased bilirubin,and hypoxia. No patients discontinued treatment due to drug-related AEs. CONCLUSIONS Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer. View Publication

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here,we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN,whereas without oil,antigen is distributed in the medullary region. Following oil immunization,CXCL10-producing inflammatory monocytes accumulate in the IFR,which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment,CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy. View Publication

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1),an actin bundling protein,resulted in an ocular commensal overgrowth,characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas,altered inflammatory responses,and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. Cre pos LPLfl/fl ) and S100A8 Cre (S100A8.Cre pos LPLfl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast,infected CD11c.Cre pos LPLfl/fl mice did not display elevated susceptibility to infection,implicating the myeloid L-plastin-sufficient cells (e.g.,macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically,the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa,augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively,our data highlight the importance of neutrophils in controlling ocular paucibacteriality,reveal molecular and cellular events involved in the process,and suggest a link between commensal exposure and resistance to infection. View Publication

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here,we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular,we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection,protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens,tumour-associated antigens,oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines. View Publication