技术资料

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes,including extracellular and intracellular pathogens. Therefore,understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this,we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless,these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1? and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common ?-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88,but not IL-1R-associated kinase 1/4. Thus,interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together,the de novo generation of pathogen-specific human Th17 requires complex,but complementary,actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens. View Publication

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics,the immune system,and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However,the capacity of IFNα/β to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here,we engineer human β-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4,resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet. View Publication

Immunotherapy treatments for cancer are becoming increasingly successful,however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy,precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1),MelanA,Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes,and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3),tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses,predict patient response to conventional therapy and direct personalised immunotherapy for patients. View Publication

In herpesvirus infections,the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection,we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist,we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly,persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice,indicating enhanced rather than diminished effector functions. Consistent with this,virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed,such as changes in Bcl-2 levels,interleukin-2 production,and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as classical" memory CD8(+) T cells." View Publication

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

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions,as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However,obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure� View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. View Publication

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement,these requirements are met by an increased glycolysis,due,at least in part,to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia,we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably,Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore,TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics,irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen,Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition,augmented proliferation,and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover,Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus,Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. View Publication

BACKGROUND AND OBJECTIVES Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples,potentially due to different temperatures during cell staining. MATERIALS AND METHODS Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients,and from 10 paediatric patients. After thawing,cells were prepared for analysis within 30 min between thawing and acquisition,at either 4°C or at room temperature. RESULTS For cell products cryopreserved in conventional freezing medium (10% final DMSO),viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed,CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT),followed by CD3,CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO),median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS The post-thaw environment and,potentially,the cryoprotectant impact the outcome of cell enumeration,and results from the analysis tube may not be representative of the cells infused into a patient. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Upon infection,antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor,the core component of mammalian target of rapamycin complex 2 (mTORC2),regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover,mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation,repression of T-bet,enhanced mitochondrial spare respiratory capacity,and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore,mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions. View Publication