技术资料

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression,function,and/or tolerance. In this study,to explore whether specific types of BCR/TLR collaboration exist in CLL,we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM,imiquimod,and CpG oligodeoxynucleotide for BCR,TLR7,and TLR9,respectively,alone or in combination for different time points. After in vitro culture in the absence of stimulation,differences in p-ERK were identified at any time point,with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL,whereas BCR/TLR synergism was required in View Publication

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol,closely related natural products from the flavagline class of compounds,are able to preferentially kill functionally defined leukemia stem cells,while sparing normal stem and progenitor cells. In addition to efficacy as single agents,flavaglines sensitize leukemia cells to several anticancer compounds,including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis,leading to the reduction of short-lived antiapoptotic proteins. Notably though,treatment with flavaglines,alone or in combination with other drugs,yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines,which we propose contribute to their efficacy in targeting leukemia cells. Taken together,these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia. View Publication

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However,one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al.,Proc Natl Acad Sci USA,104,6283-6288,2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs,which contained instead nearby single amino acid changes in the HA head. Thus,either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover,this site is relevant for the human antibody response,as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance. View Publication

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1),suggesting a role in neutrophil migration. However,CD177pos neutrophils exhibit no clear migratory advantage in vivo,despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system,we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils,an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly,CD177 ligation enhanced its interaction with β2 integrins,as revealed by fluorescence lifetime imaging microscopy,leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity,impaired internalization of integrin attachments,and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. View Publication

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity,which links a given individual stimulus to a unique activated state. Here,we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells. View Publication

Mixed lineage kinase domain-like (MLKL)-dependent necroptosis is thought to be implicated in the death of mycobacteria-infected macrophages,reportedly allowing escape and dissemination of the microorganism. Given the consequent interest in developing inhibitors of necroptosis to treat Mycobacterium tuberculosis (Mtb) infection,we used human pharmacologic and murine genetic models to definitively establish the pathophysiological role of necroptosis in Mtb infection. We observed that Mtb infection of macrophages remodeled the intracellular signaling landscape by upregulating MLKL,TNFR1,and ZBP1,whilst downregulating cIAP1,thereby establishing a strong pro-necroptotic milieu. However,blocking necroptosis either by deleting Mlkl or inhibiting RIPK1 had no effect on the survival of infected human or murine macrophages. Consistent with this,MLKL-deficiency or treatment of humanized mice with the RIPK1 inhibitor Nec-1s did not impact on disease outcomes in vivo,with mice displaying lung histopathology and bacterial burdens indistinguishable from controls. Therefore,although the necroptotic pathway is primed by Mtb infection,macrophage necroptosis is ultimately restricted to mitigate disease pathogenesis. We identified cFLIP upregulation that may promote caspase 8-mediated degradation of CYLD,and other necrosome components,as a possible mechanism abrogating Mtb's capacity to coopt necroptotic signaling. Variability in the capacity of these mechanisms to interfere with necroptosis may influence disease severity and could explain the heterogeneity of Mtb infection and disease. View Publication

Priming of human NK cells with IL-2 is necessary to render them functionally competent upon NKG2D engagement. We examined the underlying mechanisms that control NKG2D responsiveness in NK cells and found that IL-2 upregulates expression of the amino acid transporters SLC1A5 and CD98. Using specific inhibitors to block SLC1A5 and CD98 function,we found that production of IFN-γ and degranulation by CD56bright and CD56dim NK cells following NKG2D stimulation were dependent on both transporters. IL-2 priming increased the activity of mTORC1,and inhibition of mTORC1 abrogated the ability of the IL-2-primed NK cells to produce IFN-γ in response to NKG2D-mediated stimulation. This study identifies a series of IL-2-induced cellular changes that regulates the NKG2D responsiveness in human NK cells. View Publication

Typhoid fever caused by Salmonella enterica serovar (S.) Typhi differs in its clinical presentation from gastroenteritis caused by S. Typhimurium and other non-typhoidal Salmonella serovars. The different clinical presentations are attributed in part to the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi,which prevents phagocytes from triggering a respiratory burst by preventing antibody-mediated complement activation. Paradoxically,the Vi antigen is absent from S. Paratyphi A,which causes a disease that is indistinguishable from typhoid fever. Here,we show that evasion of the phagocyte respiratory burst by S. Paratyphi A required very long O antigen chains containing the O2 antigen to inhibit antibody binding. We conclude that the ability to avoid the phagocyte respiratory burst is a property distinguishing typhoidal from non-typhoidal Salmonella serovars that was acquired by S. Typhi and S. Paratyphi A independently through convergent evolution. View Publication

The presence of persistent,latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models,which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses,which comprise the vast majority of all infected cells,and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here,we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA,but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus,ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus,our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection. View Publication