技术资料

Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags,CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression,B cell survival and development,and enlarged peripheral lymphoid organs. However,it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article,we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically,CD40-induced AID expression was markedly impaired by B-TRAF2,but not B-TRAF3,deficiency. Moreover,B-TRAF2 deficiency causes defective activation of the NF-$\kappa$B1 complex in a CD40-autonomous manner,and restoring CD40-induced NF-$\kappa$B1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling. View Publication

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens,but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition,it was concentration dependent,and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling,thereby averting excessive immune responses. View Publication

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function,we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice,which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover,the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast,the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally,RAW cells overexpressing Ido2 did not alter nuclear factor $\kappa$B (NF-$\kappa$B) or stat1 expression,but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS,providing novel options for the treatment of immune disorders. View Publication

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints,such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor,is a powerful anticancer approach. However,many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker,but the complex mechanisms underlying its regulation are not completely understood. Here,we show that the eukaryotic translation initiation complex,eIF4F,which binds the 5' cap of mRNAs,regulates the surface expression of interferon-$\gamma$-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A,the RNA helicase component of eIF4F,elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus,eIF4A inhibitors,in development as anticancer drugs,may also act as cancer immunotherapies. View Publication

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans,including microcephaly in newborns and Guillain-Barr{\'{e}} syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here,we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing,NS1-specific mAbs can engage Fc$\gamma$R without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover,we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2-/- mice. Protection is Fc-dependent,as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen. View Publication

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as $\beta$-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex,consisting of purified Cas9 protein and guide RNA,into HSPCs. Because $\beta$-hemoglobinopathies are most prevalent in developing countries,it is desirable to have a reliable,efficient,easy-to-use and cost effective delivery method. With this goal in mind,we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF),a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale. View Publication

Despite showing success in treating melanoma and haematological malignancies,adoptive cell therapy (ACT) has generated only limited effects in solid tumors. This is,in part,due to a lack of specific antigen targets,poor trafficking/infiltration and immunosuppression in the tumor microenvironment. In this study,we combined ACT with oncolytic virus vaccines (OVV) to drive expansion and tumor infiltration of transferred antigen-specific T cells,and demonstrated that the combination is highly potent for the eradication of established solid tumors. Consistent with other successful immunotherapies,this approach elicited severe autoimmune consequence when the antigen targeted was a self-protein. However,modulation of IFN$\alpha$/$\beta$ signaling,either by functional blockade or rational choice of an OVV backbone,ameliorated autoimmune side effects without compromising antitumor efficacy. Our study uncovers a pathogenic role for IFN$\alpha$/$\beta$ in facilitating autoimmune toxicity during cancer immunotherapy and offers a safe and powerful combinatorial regimen with immediate translational applications. View Publication

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development,however,is restricted by significant off-tumor toxicities associated with Fc$\gamma$R interactions. Here,we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody,1D8N/CEGa1,consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly,treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc$\gamma$R interactions in the 4-1BB-agonist-associated immune abnormalities,and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. View Publication

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports,we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing,IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably,Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however,these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6,aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus,IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. View Publication

Pathogen immune responses are profoundly attenuated in fetuses and premature infants,yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic,metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-$\gamma$-regulated metabolic pathways,limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast,they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants,and suggest that the fetal immune system is metabolically programmed to avoid energetically costly,dispensable and potentially harmful immune responses during ontogeny. View Publication