技术资料

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

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis,allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk),an enzyme acting downstream of the T-cell receptor (TCR),in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor,Compound 44,created by chemical optimization of an initial high-throughput screening hit,inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition,our data reveal that human Itk,comparable to its murine homologue,is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally,silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion,Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases. View Publication

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms,however,underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite,retinoic acid (RA),as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism,we investigated the ability of HSC to function as regulatory APC. Different from previous reports,we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function,HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast,HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells,whereas they greatly inhibited the Th17 differentiation. Furthermore,the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders,and therefore,by promoting Tregs and suppressing Th17 differentiation,they might represent key players in the mechanism that drives liver-induced tolerance. View Publication

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice,N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity,cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis,spontaneous inflammatory demyelination and neurodegeneration,the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3),including genetic variants in interleukin-7 receptor-α (IL7RA*C),interleukin-2 receptor-α (IL2RA*T),MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3),optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway,namely N-glycosylation. View Publication

HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function,manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. 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

Interferon regulatory factor-1 (IRF-1) is an important transcription factor that mediates interferon-gamma (IFN-gamma)-induced cell-signaling events. In this study,we examined whether 17beta-estradiol alters IRF-1 in splenic lymphocytes,in view of the immunomodulatory effects of this natural female sex hormone including its ability to alter IFN-gamma levels. We find that IRF-1 expression is markedly downregulated in splenocytes or purified T-cells from estrogen-treated mice at all time points studied when compared with their placebo counterparts. This decrease in IRF-1 in splenocytes from estrogen-treated mice is neither due to upregulation of IRF-1-interfering proteins (nucleophosmin or signal transducer and activator of transcription (STAT)-5) nor due to alternatively spliced IRF-1 mRNA. Given that IFN-gamma is a potent inducer of IRF-1,direct addition of recombinant IFN-gamma to splenocytes from either wild-type or IFN-gamma-knockout mice,or the addition of recombinant IFN-gamma to purified T-cells,was expected to stimulate IRF-1 expression. However,robust expression of IRF-1 in cells from estrogen-treated mice was not seen,unlike what was observed in cells from placebo-treated mice. Diminished IFN-gamma induction of IRF-1 in cells from estrogen-treated mice was noticed despite comparable phosphorylated STAT-1 activation. These studies are the first to show that estrogen regulates IFN-gamma-inducible IRF-1 in lymphoid cells,a finding that may have implications to IFN-gamma-regulated immune and vascular diseases. View Publication

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period,cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets,lymphocytes and granulocytes. To improve our isolation method,a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail,recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood cells. View Publication

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD,IDS,and MPP1 genes,which together were informative in about 65% of female subjects. To increase our ability to detect clonality,we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these,all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis,whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly,interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET,and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus,these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The present study demonstrates that CD4(+)CD25(+) T cells,expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART),exhibit phenotypic,molecular,and functional characteristics of regulatory T cells. The majority of peripheral CD4(+)CD25(+) T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4(+)CD25(+) T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4(+)CD25(-) T cells,CD4(+)CD25(+) T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin,cytomegalovirus (CMV),and p24 significantly increased following depletion of CD4(+)CD25(+) T cells. Furthermore,addition of increasing numbers of CD4(+)CD25(+) T cells resulted in a dose-dependent inhibition of CD4(+)CD25(-) T-cell proliferation to tuberculin and p24. CD4(+)CD25(+) T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10),thus indicating the presence of p24-specific CD4(+) T cells among the CD4(+)CD25(+) T-cell subset. Suppressive activity was not dependent on the secretion of TGF-beta or IL-10. Taken together,our results suggest that persistence of HIV antigens might trigger the expansion of CD4(+)CD25(+) regulatory T cells,which might induce a tolerance to HIV in vivo. View Publication