技术资料

Preferential activation of regulatory T (Treg) cells limits autoimmune tissue damage during chronic immune responses but can also facilitate tumor growth. Here,we show that tissue-produced inflammatory mediators prime maturing dendritic cells (DC) for the differential ability of attracting anti-inflammatory Treg cells. Our data show that prostaglandin E(2) (PGE(2)),a factor overproduced in chronic inflammation and cancer,induces stable Treg-attracting properties in maturing DC,mediated by CCL22. The elevated production of CCL22 by PGE(2)-matured DC persists after the removal of PGE(2) and is further elevated after secondary stimulation of DC in a neutral environment. This PGE(2)-induced overproduction of CCL22 and the resulting attraction of FOXP3(+) Tregs are counteracted by IFN alpha,a mediator of acute inflammation,which also restores the ability of the PGE(2)-exposed DC to secrete the Th1-attracting chemokines: CXCL9,CXCL10,CXCL11,and CCL5. In accordance with these observations,different DCs clinically used as cancer vaccines show different Treg-recruiting abilities,with PGE(2)-matured DC,but not type 1-polarized DC,generated in the presence of type I and type II IFNs,showing high Treg-attracting activity. The current data,showing that the ability of mature DC to interact with Treg cells is predetermined at the stage of DC maturation,pave the way to preferentially target the regulatory versus proinflammatory T cells in autoimmunity and transplantation,as opposed to intracellular infections and cancer. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication

The dynamic interplay between dendritic cells (DCs) and human immunodeficiency virus type-1 (HIV-1) is thought to result in viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin receptor (CLR) DC-SIGN was responsible for the trans-infection function of the virus,subsequent studies demonstrated that trans-infection of CD4(+) T cells with HIV-1 can also occur through DC-SIGN-independent mechanisms. We demonstrate that a cell surface molecule designated DCIR (for DC immunoreceptor),a member of a recently described family of DC-expressing CLRs,can participate in the capture of HIV-1 and promote infection in trans and in cis of autologous CD4(+) T cells from human immature monocyte-derived DCs. The contribution of DCIR to these processes was revealed using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data from transfection experiments indicated that DCIR acts as a ligand for HIV-1 and is involved in events leading to productive virus infection. Finally,we show that the neck domain of DCIR is important for the DCIR-mediated effect on virus binding and infection. These results point to a possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation. View Publication

Experimental autoimmune encephalomyelitis (EAE) models,in animals,many characteristics of multiple sclerosis,for which there is no adequate therapy. We investigated whether lithium,an inhibitor of glycogen synthase kinase-3 (GSK3),can ameliorate EAE in mice. Pretreatment with lithium markedly suppressed the clinical symptoms of EAE induced in mice by myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization and greatly reduced demyelination,microglia activation,and leukocyte infiltration in the spinal cord. Lithium administered postimmunization,after disease onset,reduced disease severity and facilitated partial recovery. Conversely,in knock-in mice expressing constitutively active GSK3,EAE developed more rapidly and was more severe. In vivo lithium therapy suppressed MOG35-55-reactive effector T cell differentiation,greatly reducing in vitro MOG35-55- stimulated proliferation of mononuclear cells from draining lymph nodes and spleens,and MOG35-55-induced IFN-gamma,IL-6,and IL-17 production by splenocytes isolated from MOG35-55-immunized mice. In relapsing/remitting EAE induced with proteolipid protein peptide139-151,lithium administered after the first clinical episode maintained long-term (90 days after immunization) protection,and after lithium withdrawal the disease rapidly relapsed. These results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of multiple sclerosis and other autoimmune and inflammatory diseases afflicting the CNS. View Publication

The intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naive T(reg) cells with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of T(reg) impairments in multiple sclerosis (MS) is still contradictory,we analyzed naive PD1- and PD1+ T(reg) cells in peripheral blood and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS) patients and of healthy control subjects. Results showed that 1) CSF PD1- T(reg) cells were significantly augmented in MS patients; 2) PD1- T(reg) cells were significantly increased in the peripheral blood of patients with stable disease (SMS) compared to those with acute (AMS) disease,and in patients responding to glatiramer acetate (COPA) compared to AMS- and COPA-unresponsive patients; and 3) PD1+ T(reg) cells were similar in CSF and peripheral blood of all groups analyzed. PD1- T(reg) cells were not increased in the peripheral blood of interferon-beta (IFNbeta) -responsive patients,but the suppressive ability of T(reg) cells was significantly higher in SMS and in COPA- or IFNbeta-responsive compared to AMS- and COPA-unresponsive individuals. The data herein suggest that PD1- T(reg) cells play a pivotal role in MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNbeta. View Publication

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15,a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients,significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues,and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors,we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore,STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM,53BP1,and MDC1. Furthermore,protein levels of these DNA damage response molecules are reduced by IL-15,as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally,pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

Immune dysfunction develops in patients with many cancer types and may contribute to tumor progression and failure of immunotherapy. Mechanisms underlying cancer-associated immune dysfunction are not fully understood. Efficient IFN signaling is critical to lymphocyte function; animals rendered deficient in IFN signaling develop cancer at higher rates. We hypothesized that altered IFN signaling may be a key mechanism of immune dysfunction common to cancer. To address this,we assessed the functional responses to IFN in peripheral blood lymphocytes from patients with 3 major cancers: breast cancer,melanoma,and gastrointestinal cancer. Type-I IFN (IFN-alpha)-induced signaling was reduced in T cells and B cells from all 3 cancer-patient groups compared to healthy controls. Type-II IFN (IFN-gamma)-induced signaling was reduced in B cells from all 3 cancer patient groups,but not in T cells or natural killer cells. Impaired-IFN signaling was equally evident in stage II,III,and IV breast cancer patients,and downstream functional defects in T cell activation were identified. Taken together,these findings indicate that defects in lymphocyte IFN signaling arise in patients with breast cancer,melanoma,and gastrointestinal cancer,and these defects may represent a common cancer-associated mechanism of immune dysfunction. View Publication

BACKGROUND: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step,using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes,whereas single cell suspensions from tonsils contain relatively few red blood cells. RESULTS: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however,the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells,but not sheep red blood cells,reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. CONCLUSION: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods. View Publication