技术资料

The youngest peripheral T cells (recent thymic emigrants [RTEs]) are functionally distinct from naive T cells that have completed postthymic maturation. We assessed the RTE memory response and found that RTEs produced less granzyme B than their mature counterparts during infection but proliferated more and,therefore,generated equivalent target killing in vivo. Postinfection,RTE numbers contracted less dramatically than those of mature T cells,but RTEs were delayed in their transition to central memory,displaying impaired expression of CD62L,IL-2,Eomesodermin,and CXCR4,which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge,indicating that the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus,the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity,driving the efficacy of the RTE response to that of mature T cells. View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (Gemtuzumab ozogamicin) have been shown to improve overall survival,validating CD33 as a target for antibody-based therapy of AML. Here we report the in vitro efficacy of BI 836858,a fully human,Fc-engineered,anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine,two hypomethylating agents that show efficacy in older patients,did not compromise BI 836858-induced NK cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a ten-day course of DAC (pre-DAC,days 4,11 and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared to pre-DAC treatment. Analysis of ligands (L) to activating receptors (NKG2D showed significantly increased NKG2DL expression in day 28 post-DAC samples compared to pre-DAC samples; when NKG2DL receptor was blocked using antibodies,BI 836858-mediated ADCC was significantly decreased,suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. View Publication

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade,including arrest,transmigration,and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2,which couples through G$\alpha$i2- and G$\alpha$i3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in G$\alpha$i2-deficient neutrophils. In this study,we show that G$\alpha$i3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested G$\alpha$i2- or G$\alpha$i3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. G$\alpha$i2-,but not G$\alpha$i3-,deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely,G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that G$\alpha$i2 controls arrest and G$\alpha$i3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils. View Publication

Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells,which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here,using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice,we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable,show increased expression of Treg signature genes such as Foxp3,CTLA4,IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. View Publication

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis,the animal model for MS,myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby,the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently,B cells were found to participate in the pathogenesis of CNS autoimmunity,with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore,myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy,leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this,short-boosted Ag-specific CD8 T cells continue to contract gradually over time,which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant,functional Ag-specific CD8 T cells that are poised for immediate protection; however,this is at the expense of forming stable long-term memory. View Publication

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies. View Publication

BACKGROUND: Conventional regulatory T cells (Tregs) can suppress human immunodeficiency virus type 1 (HIV-1)-specific immune responses but cannot control immune activation in primary HIV infection. Here,we characterized Treg subsets,using recently defined phenotypic delineation,and analyzed the relative contribution of cell subsets to the production of immunosuppressive cytokines in primary HIV infection. METHODS: In a longitudinal prospective study,ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline and month 6 of follow-up to characterize Treg subsets,immune activation,and cytokine production in isolated CD4(+) T cells. RESULTS: The frequency of CD4(+)CD25(+)CD127(low) Tregs and the distribution between the naive,memory,and activated/memory Treg subsets was similar in patients and healthy donors. However,Tregs from patients with primary HIV infection showed peculiar phenotypic profiles,such as elevated FoxP3,ICOS,and CTLA-4 expression,with CTLA-4 expression strikingly increased in all Treg subsets both at baseline and month 6 of follow-up. The great majority of interleukin 10 (IL-10)-producing CD4(+) T cells were FoxP3(neg) (ie,Tr1-like cells). In contrast to conventional Tregs,Tr1-like cells were inversely correlated with immune activation and not associated with lower effector T-cell responses. CONCLUSION: FoxP3(neg) Tr1-like cells-major contributors to IL-10 production-may have a beneficial role by controlling immune activation in early HIV infection. 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

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host. 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