技术资料

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

In early HIV-1 infection,Vdelta1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression,chemokine response,and recirculation. Herein we show that,at variance with healthy donors,in HIV-1-infected patients ex vivo-isolated Vdelta1 T cells display cytoplasmic interferon-gamma (IFN-gamma). Interestingly,these cells coexpress cytoplasmic interleukin-17 (IL-17),and bear the CD27 surface marker of the memory T-cell subset. Vdelta1 T cells,isolated from either patients or healthy donors,can proliferate and produce IFN-gamma and IL-17 in response to Candida albicans in vitro,whereas Vdelta2 T cells respond with proliferation and IFN-gamma/IL-17 production to mycobacterial or phosphate antigens. These IFN-gamma/IL-17 double-producer gammadelta T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in gammadelta T-cell transendothelial migration. Moreover,Vdelta1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory gammadelta T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections,possibly contributing to compensate for the impairment of CD4(+) T cells. View Publication

CD4+ and CD8+ T cell functions are rapidly aborted during chronic infection,preventing viral clearance. CD4+ T cell help is required throughout chronic infection so as to sustain CD8+ T cell responses; however,the necessary factor(s) provided by CD4+ T cells are currently unknown. Using a mouse model of chronic viral infection,we demonstrated that interleukin-21 (IL-21) is an essential component of CD4+ T cell help. In the absence of IL-21 signaling,despite elevated CD4+ T cell responses,CD8+ T cell responses are severely impaired. CD8+ T cells directly require IL-21 to avoid deletion,maintain immunity,and resolve persistent infection. Thus,IL-21 specifically sustains CD8+ T cell effector activity and provides a mechanism of CD4+ T cell help during chronic viral infection. View Publication

Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum,a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection,we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions,but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients,these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum. 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

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or,when activated with IFN-gamma,an APC phenotype. Herein,TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta,IL-6,IL-8/CXCL8,and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB,inducible NO synthase (iNOS),and TRAIL upon TLR activation in MSC and macrophages,but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless,TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells,as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence,TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition,IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context,a mechanism that could be applied in a cell-based vaccine. View Publication

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment,particularly during early life. To investigate this concept,we developed a model of neonatal exposure to a common pathogen-associated molecular pattern,LPS,and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE,induced at 12 wk,compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE,and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes,albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS. View Publication

Persistent viral infections are a major health concern worldwide. During persistent infection,overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection,and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously,we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However,the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers,illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen. View Publication

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences,Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore,in SmuTR(-/-) mice,deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks,particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly,when the SmuTR region is present,deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies,suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication

We have previously shown that a granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-15 (IL-15) 'fusokine' (GIFT15) exerts immune suppression via aberrant signaling through the IL-15 receptor on lymphomyeloid cells. We show here that ex vivo GIFT15 treatment of mouse splenocytes generates suppressive regulatory cells of B cell ontogeny (hereafter called GIFT15 B(reg) cells). Arising from CD19+ B cells,GIFT15 B(reg) cells express major histocompatibility complex class I (MHCI) and MHCII,surface IgM and IgD,and secrete IL-10,akin to previously described B10 and T2-MZP B(reg) cells,but lose expression of the transcription factor PAX5,coupled to upregulation of CD138 and reciprocal suppression of CD19. Mice with experimental autoimmune encephalomyelitis went into complete remission after intravenous infusion of GIFT15 B(reg) cells paralleled by suppressed neuroinflammation. The clinical effect was abolished when GIFT15 B(reg) cells were derived from mmicroMT (lacking B cells),MHCII-knockout,signal transducer and activator of transcription-6 (STAT-6)-knockout,IL-10-knockout or allogeneic splenocytes,consistent with a pivotal role for MHCII and IL-10 by sygeneic B cells for the observed therapeutic effect. We propose that autologous GIFT15 B(reg) cells may serve as a new treatment for autoimmune ailments. View Publication

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4(+) T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4(+) T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably,significant protection against FV-induced disease is afforded by FV-specific CD4(+) T cells in the absence of a virus-specific CD8(+) T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4(+) T cells required to control FV-induced disease. Furthermore,ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4(+) T-cell response. Conversely,an increased frequency or continuous supply of FV-specific CD4(+) T cells is both necessary and sufficient to effectively contain acute infection and prevent disease,even in the presence of coinfection. Thus,these results suggest that FV-specific CD4(+) T cells provide significant direct protection against acute FV infection,the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4(+) T cells. View Publication