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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

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

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCRs),or pathogen-associated molecules through pattern-recognition receptors,such as Toll-like receptors (TLRs),leads to transcriptional and metabolic changes that ultimately transform B cells into antibody-producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton,tightly controlled by concerted action of multiple regulatory proteins,some of them exclusive to B cells. Here,we dissect the role of Missing-In-Metastasis (MIM),or Metastasis suppressor 1 (MTSS1),a cancer-associated membrane and actin cytoskeleton regulating protein,in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly,we found that MIM-/- B cells are defected in BCR signaling in response to surface-bound antigens but,on the other hand,show increased metabolic activity after stimulation with LPS or CpG. In vivo,MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells,demonstrates its regulatory role for B cell-mediated immunity,as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism,processes,which may also contribute to the poorly understood functions of MIM in cancer. View Publication