技术资料

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

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

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs),thus preventing the development of durable antitumor immunity. Therefore,the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however,those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast,we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+),CD8(+),Tregs),B cells,and NK cells,which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells,B cells,and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4,TLR5L:flagellin,TLR4L:LPS,and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation,but not B cell proliferation. Besides CL097,TLR2L:PGN,CL075,and TLR9L:CpG-A,CpG-B,and CpG-C) were strong activators of NK cells. Importantly,we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation,2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg),and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients. View Publication

Treatment of HIV-infected patients with IFN-α results in significant,but clinically insufficient,reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2,which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs,the dominant Vpu sequences differed before and during treatment. In three patients,vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α,and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4,while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties. View Publication

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study,we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact,resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression,particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro,EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall,our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover,our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC. 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

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However,the activation requirements,the temporal aspects of the suppressive activity,and mode of action of human Tregs are subjects of controversy. In this study,we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously,whereas in the remaining donors,anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore,anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs,but could be fully obliterated by trypsin treatment,indicating that a cell surface protein is directly involved. By add-back of active,fixed Tregs at different time points after activation of responding T cells,the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly,we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation,indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells. 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

CD4+ T cells are required for immunity against many viral infections,including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1,whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors,although it is not known whether these factors are produced during primary antigen-specific responses. Here,we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of naïve CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22,CCL22),and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3),macrophage inflammatory protein-1 beta (CCL4),and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus. View Publication

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in resting lymphocytes was recently established,although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study,we used CD4(+) T cells isolated from PPARalpha(-/-) and wild-type mice,as well as cell lines that constitutively express PPARalpha,in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4(+) T cells lacking PPARalpha produce increased levels of IFN-gamma,but significantly lower levels of IL-2 when compared with activated wild-type CD4(+) T cells. Furthermore,we demonstrate that PPARalpha regulates the expression of these cytokines by CD4(+) T cells in part,through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4(+) T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation,and the presence of unliganded PPARalpha effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARalpha with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARalpha. View Publication

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore,we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2,IL-7,and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably,this outcome was not accompanied by increased cellular proliferation or activation. Moreover,IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus,IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals. View Publication