技术资料

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However,in some instances,the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative,but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein,we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important,hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells,such as dendritic cells (DC). Indeed,a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore,compared to immature or mature DC,Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether,these findings indicate that allogeneic hAC inhibit,rather than trigger,immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting. View Publication

CD4(+) T cells develop distinct and often contrasting helper,regulatory,or cytotoxic activities. Typically a property of CD8(+) T cells,granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However,the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling,we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs,which distinguishes them from other CD4(+) T helper (Th) cells,including Th1 cells,and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study,and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions. View Publication

During adaptive immune responses,CD8(+) T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study,we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity,whereas one day later,the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor,ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation,as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation,low-affinity effector CD8(+) T cells accumulated at efferent lymphatic vessels for egress,whereas high affinity-stimulated CD8(+) T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8(+) T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. View Publication

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However,the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic,whereas other responses,such as the ability to overcome tumor immunosuppression,are absent. Thus,the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles,biased T cell differentiation,and local delivery of non-native therapeutic payloads,such as antibodies,in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases. View Publication

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by self" MHC class I molecules in a process called "education." In humans� View Publication

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here,we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes,where they trap T cells by releasing the CCR7 ligand,CCL19,and to inflamed central nervous system,where they locally limit immunopathogenesis through interleukin-10 production,thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment,where proB cells develop,is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition,these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. View Publication

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo,we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood,spleen,and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes,which downregulate GFP expression on differentiation into macrophages in this model,CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages,allowing continued cell tracking during resolution of inflammation. In summary,this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study,we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak,whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin,and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction,exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin. View Publication

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication