技术资料

DNA immunization with in vivo electroporation is an efficient alternative protocol for the production of monoclonal antibodies (mAb). Generation of mAb by DNA immunization is a novel approach to circumvent the following technical hurdles associated with problematic antigens: low abundance and protein instability and use of recombinant proteins that lack posttranslational modifications. This chapter describes the use of a DNA-based immunization protocol for the production of mAb against a house dust mite allergen,designated as Blo t 11,which is a paramyosin homologue found in Blomia tropicalis mites. The Blo t 11 cDNA fused at the N terminus to the sequence of a signal peptide was cloned into the pCI mammalian expression vector. The DNA construct was injected intramuscularly with in vivo electroporation into mice,and the specific antibody production in mice was analyzed by enzyme-linked immunosorbent assay (ELISA). Hybridomas were generated by fusing mouse splenocytes with myeloma cells using the ClonaCell-HY Hybridoma Cloning Kit. Six hybridoma clones secreting Blo t 11 mAb were successfully generated,and these mAb are useful reagents for immunoaffinity purification and immunoassays. View Publication

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis,type 1 diabetes mellitus,and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5),containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin,is a potent and selective blocker of these channels. However,a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability,ShK-192,contains a nonhydrolyzable phosphotyrosine surrogate,a methionine isostere,and a C-terminal amide. ShK-192 shows the same overall fold as ShK,and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg,approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24,48,and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells. 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

BACKGROUND: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD),little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming,or whether allogeneic Treg are safe to use clinically. METHODS: To address these questions,we used an in vitro human skin explant GVHD model,which mimics the physiopathology of GVHD. First,donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase)� View Publication

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation,successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy,we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition,mice in remission after CpG ODN treatment were protected from leukemia rechallenge,and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge,this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL,suggesting this treatment may have clinical utility in patients with minimal residual disease. View Publication

IL-33,a new member of the IL-1 family,has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study,we demonstrate that murine basophils sorted directly from the bone marrow,without prior exposure to IL-3 or Fc(epsilon)R cross-linking,respond to IL-33 alone by producing substantial amounts of histamine,IL-4,and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo,IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3,GM-CSF,and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice,which implies that it is not the unique growth-promoting mediator in this setup,but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor,which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore,GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells,whereas IL-5 that is also generated in vivo,affects neither their functions nor their growth in vitro or in vivo. In conclusion,our data provide the first evidence that IL-33 not only activates unprimed basophils directly,but also promotes their expansion in vivo through induction of GM-CSF and IL-3. View Publication

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells,the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung,gut and liver. In the liver,these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus,T(H)-17 cells,like T(reg) cells,can be selected by self antigens in the thymus. View Publication

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1,-2,and -3) signals through the same heterodimeric receptor complex,which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution,the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells,IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition,IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13,IFN-gamma,and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa,which,through their ability to synthesize retinoic acid (RA),appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC,CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs,but not CX3CR1-expressing cells,migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover,CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs,whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens. View Publication

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor,including ULBP-1,-2,and -3,but not MICA or MICB,in infected cells both in vitro and in vivo. However,the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest,conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly,Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly,Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells,suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall,these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction. View Publication