技术资料

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

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

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

PURPOSE Tumor-associated macrophages (TAMs) and the hyperactivation of phosphoinositide 3-kinase(PI3K)/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma (HL) and affect disease outcome. Since the $\delta$ and $\gamma$ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME),we propose that the PI3K$\delta$/$\gamma$ inhibitor RP6530 might affect both HRS cells and TME,ultimately leading to an enhanced antitumor response. EXPERIMENTAL DESIGN HL cell lines (L-540,KM-H2 and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in HL cell line xenografts. RESULTS In vitro,RP6530 besides killing and inhibiting the proliferation of HL cells,downregulated lactic acid metabolism,switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing,we define tumor glycolysis as a specific PI3K$\delta$/$\gamma$-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator Pyruvate Kinase M2 (PKM2) as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore,we show in human tumor xenografts that RP6530 repolarizes TAMs into pro-inflammatory macrophages and inhibits tumor vasculature,leading to tumor regression. Interestingly,HL patients experiencing objective responses (CR and PR) in a phase 1 trial using RP6530 showed a significant inhibition of circulating MDSCs and an average mean reduction in serum TARC levels of 40{\%} (range,4-76{\%}). CONCLUSIONS Our results support PI3K$\delta$/$\gamma$ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat HL patients. View Publication

CRISPR pools are being widely employed to identify gene functions. However,current technology,which utilizes DNA as barcodes,permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities,we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate {\textgreater}100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies,we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR,we simultaneously analyzed multiple phenotypic markers,including phospho-signaling,on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes,the immunoproteasome component Psmb8 and a chaperone Rtp4,are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution. View Publication

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

Background: B cells play an important role in the development and maintenance of rheumatoid arthritis (RA). Although IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) able to suppress autoimmune and inflammatory responses,recent reports showed that B cell-mediated immune suppression may also occur independent of IL-10. For instance,B cells can modulate T cell immune responses through the expression of regulatory molecules such as PD-L1. So far,PD-L1-expressing B cells have not been analyzed in RA patients. Objective: To analyze the frequency of PD-L1-expressing B cells in the peripheral blood of RA patients compared to healthy controls (HC) matched for sex and age,their function on T cell response and their changes in response to therapy. Methods: Fresh peripheral blood B cells from RA patients and HC were characterized by flow cytometry and their functionality assessed in a co-culture system with autologous T cells. Results: The frequencies of CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells were significantly lower in untreated RA patients than in HC. In a follow-up study,the frequencies of PD-L1+ B cells (CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells) increased significantly after treatment in good responder patients,although the frequency of total CD24hiCD38hi B cells decreased. CD19+ B cells from untreated RA patients and HC upregulated PD-L1 expression similarly upon stimulation with CpG plus IL-2 and were able to suppress,in vitro,CD8+ T cell proliferation and cytokine production in a PD-L1-dependent manner. Conclusions: Our results show that PD-L1+ B cells exhibiting T cell suppressive capacity are significantly decreased in untreated RA patients but increase in response to successful treatment. PD-L1 expression on B cells from RA patients can be modulated in vitro and PD-L1+ B cells could thus provide new perspectives for future treatment strategies. View Publication

Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks,such as PD-1 expression,human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast,melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality,despite PD-1 expression,and suggest that they may be harnessed for effective immunotherapies. View Publication

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets,epidermal Langerhans cells (LCs) and dermal DCs,which can be distinguished by the expression of C-type lectin receptors,Langerin and DC-SIGN,respectively. Although peripheral blood monocytes differentiate into these distinct subsets,monocyte-derived LCs (moLCs) induced by coculture with GM-CSF,IL-4,and TGF-$\beta$1 coexpress both Langerin and DC-SIGN,suggesting that the environmental cues remain unclear. In this study,we show that LC differentiation is TGF-$\beta$1 dependent and that cofactors such as IL-4 and TNF-$\alpha$ promote TGF-$\beta$1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-$\alpha$-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs,dexamethasone-treated moLCs express CD1a,whereas monocyte-derived DCs (moDCs) express CD1b,CD1c,and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and $\alpha$-galactosylceramide,respectively. Strikingly,CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis,these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin. 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

The developmental pathways of broadly neutralizing antibodies (bNAbs) against HIV are of great importance for the design of immunogens that can elicit protective responses. Here we show the maturation features of the HIV-neutralizing anti-V1V2 VRC26 lineage by simultaneously sequencing the exon together with the downstream intron of VRC26 members. Using the mutational landscapes of both segments and the selection-free nature of the intron region,we identify multiple events of amino acid mutational convergence in the complementarity-determining region 3 (CDR3) of VRC26 members,and determine potential intermediates with diverse CDR3s to a late stage bNAb from 2 years prior to its isolation. Moreover,we functionally characterize the earliest neutralizing intermediates with critical CDR3 mutations,with some emerging only 14 weeks after initial lineage detection and containing only {\~{}}6{\%} V gene mutations. Our results thus underscore the utility of analyzing exons and introns simultaneously for studying antibody maturation and repertoire selection. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication