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T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation,but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model,T-bet(-/-) nTreg,but not induced Treg,failed to prolong graft survival as effectively as wild-type Treg. T-bet(-/-) nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules,influencing nTreg distribution in tissues,so that T-bet(-/-) nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast,both wild-type and T-bet(-/-) CD4(+) conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet(-/-) nTreg displayed instability in the graft,failing to suppress Ag-specific CD4(+) T cells and prevent their infiltration into the graft and dLN. Thus,T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo. View Publication

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells,but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation,the process by which pathogen Ags are internalized,degraded,and presented by MHC class I,is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article,we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs,dendritic cells and macrophages,and CD4 T cells,three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities,reducing NADPH oxidase 2 activation,and lowering phagolysosomal reactive oxygen species production and pH,which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification,to our knowledge,of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV. View Publication

Natural killer (NK) cells play a key role in immunity,but how HLA class I (HLA-I) and killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1) polymorphism impacts disease outcome remains unclear. KIR3DL1 (*001/*005/*015) tetramers were screened for reactivity against a panel of HLA-I molecules. This revealed different and distinct hierarchies of specificity for each KIR3DL1 allotype,with KIR3DL1*005 recognizing the widest array of HLA-I ligands. These differences were further reflected in functional studies using NK clones expressing these specific KIR3DL1 allotypes. Unexpectedly,the Ile/Thr80 dimorphism in the Bw4-motif did not categorically define strong/weak KIR3DL1 recognition. Although the KIR3DL1*001,*005,and *015 polymorphisms are remote from the KIR3DL1-HLA-I interface,the structures of these three KIR3DL1-HLA-I complexes showed that the broader HLA-I specificity of KIR3DL1*005 correlated with an altered KIR3DL1*005 interdomain positioning and increased mobility within its ligand-binding site. Collectively,we provide a generic framework for understanding the impact of KIR3DL1 polymorphism on the recognition of HLA-I allomorphs. View Publication

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however,their influence on intracellular pathogens is less clear. We previously reported that CCR2(-/-) mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2(-/-) animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus,our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.Mucosal Immunology advance online publication,6 April 2016; doi:10.1038/mi.2016.26. View Publication

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury,although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation,respectively,attenuating the activity of the C3/C5 convertases and,consequently,avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study,we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly,FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture,resembling immature MoDCs,lower expression of the maturation marker CD83 and the costimulatory molecules CD40,CD80,and CD86,decreased production of key proinflammatory Th1-cytokines (IL-12,TNF-α,IFN-γ,IL-6,and IL-8),and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover,FH-treated MoDCs show low Ag uptake and,when challenged with LPS,display reduced CCR7 expression and chemotactic migration,impaired CD4(+) T cell alloproliferation,inhibition of IFN-γ secretion by the allostimulated T cells,and,conversely,induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus,this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity,transplantation,and autoimmunity. View Publication

Group 2 innate lymphoid cells (ILC2 cells) are important for type 2 immune responses and are activated by the epithelial cytokines interleukin 33 (IL-33),IL-25 and thymic stromal lymphopoietin (TSLP). Here we demonstrated that IL-1β was a critical activator of ILC2 cells,inducing proliferation and cytokine production and regulating the expression of epithelial cytokine receptors. IL-1β also governed ILC2 plasticity by inducing low expression of the transcription factor T-bet and the cytokine receptor chain IL-12Rβ2,which enabled the conversion of these cells into an ILC1 phenotype in response to IL-12. This transition was marked by an atypical chromatin landscape characterized by the simultaneous transcriptional accessibility of the locus encoding interferon-γ (IFN-γ) and the loci encoding IL-5 and IL-13. Finally,IL-1β potentiated ILC2 activation and plasticity in vivo,and IL-12 acted as the switch that determined an ILC2-versus-ILC1 response. Thus,we have identified a previously unknown role for IL-1β in facilitating ILC2 maturation and plasticity. View Publication

Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs),particularly staggered DSBs,in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID),subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However,consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung,we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA,but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases,such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA,primarily at dG/dC residues,the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA,as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG(-/-) mice and EndoG(-/-) B cells,we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro,as demonstrated by reduced cell surface IgG1,IgG2a,IgG3 and IgA,reduced secreted IgG1,reduced circle Iγ1-Cμ,Iγ3-Cμ,Iɛ-Cμ,Iα-Cμ transcripts,post-recombination Iμ-Cγ1,Iμ-Cγ3,Iμ-Cɛ and Iμ-Cα transcripts. In addition to reduced CSR,EndoG(-/-) mice showed a significantly altered spectrum of mutations in IgH J(H)-iEμ DNA. Impaired CSR in EndoG(-/-) B cells did not stem from altered B cell proliferation or apoptosis. Rather,it was associated with significantly reduced frequency of DSBs. Thus,our findings determine a role for EndoG in the generation of S region DSBs and CSR. View Publication

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision,RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study,we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing,revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision,thereby ensuring the utility of the postrevision repertoire. View Publication

In elderly subjects and in patients with chronic inflammatory diseases,there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype,whose origin and functional relevance has not been well characterized. In this study,we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells,and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence,intracellular cytokine expression,ability to interact with endothelial cells,and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p textless 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%,respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209,release of cytokines in response to low-intensity stimulus,and increased capacity to sustain lymphocyte proliferation. Finally,compared with CD14(++)CD16(-) cells,CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary,our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells,with increased inflammatory activity and ability to interact with endothelial cells. Therefore,accumulation of senescent monocytes may explain,in part,the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases. View Publication

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort,which,in concert with microRNAs,drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts,megakaryoblasts,monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes,which resulted in downregulation. Among the upregulated lineage-enriched microRNAs,hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate,grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate,modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation. View Publication

Lymphocyte activation is regulated by costimulatory and inhibitory receptors,of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably,it remains unclear how HVEM functions with each of its ligands during immune responses. In this study,we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT,Lymphotoxin-α,or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2,resulting in increased IFN-γ and TNF-α secretion,and tumor cell-expressed HVEM activated CD160 in a human NK cell line,causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast,HVEM activation of BTLA reduced cytolysis of target cells. Together,our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA. View Publication

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections,we investigated the function of the adhesion molecule,P-selectin glycoprotein ligand-1 (PSGL-1),that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably,this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically,PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1,leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs,PSGL-1 deficiency led to PD-1 downregulation,improved T cell responses,and tumor control. Thus,PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment. View Publication