技术资料

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless,many details of the immunopathogenesis of TRALI,particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast,34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia,severe pulmonary edema,and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8(+) T lymphocytes or CD4(+) T cells but not CD19(+) B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia,lung damage,and mortality,suggesting that T lymphocytes were responsible for the protective effect. Taken together,these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions. View Publication

RATIONALE: Chitin is a ubiquitous polysaccharide in fungi,insects,allergens,and parasites that is released at sites of infection. Its role in the generation of tissue inflammation,however,is not fully understood. OBJECTIVES: We hypothesized that chitin is an important adjuvant for adaptive immunity. METHODS: Mice were injected with a solution of ovalbumin and chitin. MEASUREMENTS AND MAIN RESULTS: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo,ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response,Th2 cytokine elaboration,IgE induction,and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2,MyD88,and IL-17A played critical roles in the chitin-induced responses,and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro,CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments,CD4(+) T-cell proliferation,IL-5,IL-13,IFN-γ,and IL-17A production were appreciated. Toll-like receptor-2,MyD88,and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation,whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. CONCLUSIONS: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2,Th1,and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2,MyD88,and IL-17A. View Publication

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects,including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments,with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However,current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover,FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific,whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations,we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs,and maintained the capacity to suppress conventional T cell responses directed against tyrosinase,as well as bystander T cell responses. Using this methodology in a model tumor system,murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy. 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

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance,but they can also play a detrimental role by preventing antitumor responses. Here,we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution,focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently,Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall,our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival,higher migratory capacity,and selective T-effector suppressive ability. View Publication

Exposure to Mycobacterium tuberculosis (Mtb) results in heterogeneous clinical outcomes including primary progressive tuberculosis and latent Mtb infection (LTBI). Mtb infection is identified using the tuberculin skin test and interferon-gamma (IFN-gamma) release assay IGRA,and a positive result may prompt chemoprophylaxis to prevent progression to tuberculosis. In the present study,we report on a cohort of Ugandan individuals who were household contacts of patients with TB. These individuals were highly exposed to Mtb but tested negative disease by IFN-gamma release assay and tuberculin skin test,'resisting' development of classic LTBI. We show that 'resisters' possess IgM,class-switched IgG antibody responses and non-IFN-gamma T cell responses to the Mtb-specific proteins ESAT6 and CFP10,immunologic evidence of exposure to Mtb. Compared to subjects with classic LTBI,'resisters' display enhanced antibody avidity and distinct Mtb-specific IgG Fc profiles. These data reveal a distinctive adaptive immune profile among Mtb-exposed subjects,supporting an expanded definition of the host response to Mtb exposure,with implications for public health and the design of clinical trials. View Publication

Missing in Metastasis (MIM),or Metastasis Suppressor 1 (MTSS1),is a highly conserved protein,which links the plasma membrane to the actin cytoskeleton. MIM has been implicated in various cancers,however,its modes of action remain largely enigmatic. Here,we performed an extensive in silico characterisation of MIM to gain better understanding of its function. We detected previously unappreciated functional motifs including adaptor protein (AP) complex interaction site and a C-helix,pointing to a role in endocytosis and regulation of actin dynamics,respectively. We also identified new functional regions,characterised with phosphorylation sites or distinct hydrophilic properties. Strong negative selection during evolution,yielding high conservation of MIM,has been combined with positive selection at key sites. Interestingly,our analysis of intra-molecular co-evolution revealed potential regulatory hotspots that coincided with reduced potentially pathogenic polymorphisms. We explored databases for the mutations and expression levels of MIM in cancer. Experimentally,we focused on chronic lymphocytic leukaemia (CLL),where MIM showed high overall expression,however,downregulation on poor prognosis samples. Finally,we propose strong conservation of MTSS1 also on the transcriptional level and predict novel transcriptional regulators. Our data highlight important targets for future studies on the role of MIM in different tissues and cancers. View Publication

Group 2 innate lymphoid cells (ILC2s) in the lung are stimulated by inhaled allergens. ILC2s do not directly recognize allergens but they are stimulated by cytokines including interleukin (IL)-33 released by damaged epithelium. In response to allergens,lung ILC2s produce T helper 2 cell type cytokines inducing T cell-independent allergic lung inflammation. Here we examined the fate of lung ILC2s upon allergen challenges. ILC2s proliferated and secreted cytokines upon initial stimulation with allergen or IL-33,and this phase was followed by a contraction phase as cytokine production ceased. Some ILC2s persisted long after the resolution of the inflammation as allergen-experienced ILC2s and responded to unrelated allergens more potently than naive ILC2s,mediating severe allergic inflammation. The allergen-experienced ILC2s exhibited a gene expression profile similar to that of memory T cells. The memory-like properties of allergen-experienced ILC2s may explain why asthma patients are often sensitized to multiple allergens. View Publication

CD46 is a glycoprotein with important functions in innate and adaptive immune responses. Functionally different isoforms are generated by alternative splicing at exons 7-9 (BC and C isoforms) and exon 13 (CYT-1 and CYT-2 isoforms) giving rise to BC1,BC2,C1 and C2. We developed a novel real-time PCR assay that allows quantitative comparisons between these isoforms. Their relative frequency in CD4(+) T cells from 100 donors revealed a distribution with high interpersonally variability. Importantly,the distribution between the isoforms was not random and although splicing favoured inclusion of exon 8 (BC isoforms),exclusion of exon 8 (C isoforms) was significantly linked to exclusion of exon 13 (CYT-2 isoforms). Despite inter-individual differences,CD4(+) and CD8(+) T cells,B cells,NK cells and monocytes expressed similar isoform profiles intra-individually. However,memory/effector CD4(+) T cells had a significantly higher frequency of CYT-2 when compared with naïve CD4(+) T cells. Likewise,in vitro activation of naïve and total CD4(+) T cells increased the expression of CYT-2. This indicates that although splicing factors determine a certain expression profile in an individual,the profile can be modulated by external stimuli. This suggests a mechanism by which alterations in CD46 isoforms may temporarily regulate the immune response. View Publication

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood (UCB) graft from an unrelated donor and allows faster count recovery,with low rates of disease recurrence and chronic graft-versus-host disease (GVHD). But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS),engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate,high,or very-high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% versus 11% p textless 0.0001) and decrease in one year progression-free (20% versus 55%,p = 0.004) and overall survival (30% versus 62%,p = 0.02). Less than 100% UCB chimerism in the CD3 lineage was associated with increase rate of disease recurrence (46% versus 12%,p = 0.007). Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% versus 15%,p = 0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful graft-versus-leukemia (GVL) effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells,but when these cells dominate,GVL-effects are limited and rates of disease recurrence are high. View Publication