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Christian P Larsen,
Eric T Elwood,
Diane Z Alexander,
Shannon C Ritchie,
Rose Hendrix,
Carol Tucker-Burden,
Hong Rae Cho, Alejandro Aruffo,
Diane Hollenbaugh,
Peter S Linsley,
Kevin J Winn,
Thomas C Pearson
The Journal of Immunology 03/2011; 186(5):2693-7. · 5.79 Impact Factor
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Immunological Reviews 04/2006; 138(1):23 - 37. · 11.15 Impact Factor
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ABSTRACT: This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell-surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 06/2003; Chapter 6:Unit 6.11.
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Alejandro Aruffo
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ABSTRACT: This unit describes the use of COS cells to efficiently produce a desired protein in a short period of time. These cells express high levels of the SV40 large tumor (T) antigen, which is necessary to initiate viral DNA replication at the SV40 origin. Each COS cell transfected with DNA encoding a cell-surface antigen (in the appropriate vector) or cytoplasmic protein will express several thousand to several hundred thousand copies of the protein 72 hr posttransfection. If the transfected DNA encodes a secreted protein, up to 10 mg of protein can be recovered from the supernatant of the transfected COS cells 1 week posttransfection. COS cell transient expression systems have also been used to screen cDNA libraries, to isolate cDNAs encoding cell-surface proteins, secreted proteins, and DNA binding proteins, and to test protein expression vectors rapidly prior to the preparation of stable cell lines.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 12/2002; Chapter 16:Unit 16.12.
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Thomas C Pearson,
Joel Trambley,
Kris Odom,
Daniel C Anderson,
Shannon Cowan,
Robert Bray,
Angello Lin,
Diane Hollenbaugh, Alejandro Aruffo,
Anthony W Siadak,
Elizabeth Strobert,
Randall Hennigar,
Christian P Larsen
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ABSTRACT: Organ transplant recipients currently require lifetime immunosuppressive therapy, with its accompanying side effects. Biological agents that block T-cell costimulatory pathways are important components of strategies being developed to induce transplantation tolerance. The aim of this study was to test the effect of a novel chimeric anti-human CD40 monoclonal antibody (Chi 220), either alone or in combination with CTLA4-Ig, on the survival of renal allografts in a nonhuman primate model.
Captive-bred adolescent male rhesus monkeys (Macaca mulatta) (4-10 kg) were used as recipients and donors. Four treatment protocols were tested: Chi220 monotherapy, CTLA4-Ig monotherapy, Chi220 combined with CTLA4-Ig, and H106 (anti-CD40L) combined with CTLA4-Ig. Control animals received human albumin. Recipients were followed for survival, renal allograft function as determined by measurement of serum blood urea nitrogen (BUN) and creatinine, chemistries (sodium, potassium, chloride, and bicarbonate), complete blood cell count (CBC) with differential, and the development of donor-specific alloantibody.
Treatment with Chi220 for 14 days prolonged renal allograft survival (MST 38.5 vs. 7 days in untreated controls). Notably, simultaneous blockade of the CD28/B7 pathway did not further augment graft survival but did suppress the development of donor-specific antibodies, an effect not achieved with Chi220 alone, despite peripheral B cell depletion. Finally, treatment with Chi220 suppressed the primary immune response to cytomegalovirus, resulting in severe systemic manifestations.
Blockade of the CD40 pathway with anti-CD40 mAb is immunosuppressive in a large animal, preclinical renal transplant model. The potential effect of this therapy on viral immune responses will be important to consider for the design of safe clinical trials.
Transplantation 11/2002; 74(7):933-40. · 4.00 Impact Factor
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ABSTRACT: Recombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 06/2002; Chapter 10:Unit 10.19A.
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04/2002;
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Eric T. Elwood,
Christian P. Larsen,
Hong Rae Cho,
Matthias Corbascio,
Shannon C. Ritchie,
Diane Z. Alexander,
Carol Tucker-Burden,
Peter S. Linsley, Alejandro Aruffo,
Diane Hollenbaugh,
Kevin J. Winn,
Thomas C. Pearson
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ABSTRACT: Background. The prompt and vigorous immune response to xenogenic tissue remains a significant barrier to clinical xenotransplantation. Simultaneous blockade of the CD28 and CD40 costimulatory pathways has been shown to dramatically inhibit the immune response to alloantigen.
Methods. In this study, we investigated the ability of simultaneous blockade of the CD28 and CD40 pathways to inhibit the immune response to xenoantigen in the rat-to-mouse and pig-to-mouse models.
Results. Simultaneous blockade of the CD28 and CD40 pathways produced marked inhibition of the cellular response to xenoantigen in vivo and produced long-term acceptance of xenogeneic cardiac and skin grafts (rat-to-mouse), and markedly suppressed an evoked antibody response to xenoantigen. In addition, this strategy significantly prolonged the survival of pig skin on recipient mice.
Conclusions. Long-term hyporesponsiveness to xenoantigen across both a concordant and discordant species barrier, measured by the stringent criterion of skin grafting, can be achieved using a noncytoablative treatment regimen.
Transplantation 06/1998; 65(11):1422-1428. · 4.00 Impact Factor
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Walter W. Shuford,
Kerry Klussman,
Douglas D. Tritchler,
Deryk T. Loo,
Jan Chalupny,
Anthony W. Siadak,
T. Joseph Brown,
John Emswiler,
Hong Raecho,
Christian P. Larsen,
Thomas C. Pearson,
Jeffrey A. Ledbetter, Alejandro Aruffo,
Robert S. Mittler
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ABSTRACT: The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily
that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated
T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by
demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and
4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through
4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T
cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d–specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac
allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-γ production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB
receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.
Journal of Experimental Medicine 07/1997; 186(1):47-55. · 13.85 Impact Factor
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Journal of Computer-Aided Molecular Design. 01/1997; 11:3-8.
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Christian P. Larsen,
Eric T. Elwood,
Diane Z. Alexander,
Shannon C. Ritchie,
Rose Hendrix,
Carol Tucker-Burden,
Hong Rae Cho, Alejandro Aruffo,
Diane Hollenbaugh,
Peter S. Linsley,
Kevin J. Winn,
Thomas C. Pearson
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ABSTRACT: THE receptor–ligand pairs CD28–B7 and CD40–gp39 are essential for the initiation and amplification of T-cell-dependent immune responses1,2. CD28–B7 interactions provide 'second signals' necessary for optimal T-cell activation and IL-2 production3–5, whereas CD40–gp39 signals co-stimulate B-cell, macrophage, endothelial cell and T-cell activation6–12. Nonetheless, blockade of either of these pathways alone is not sufficient to permit engraftment of highly immunogenic allografts13–15. Here we report that simultaneous but not independent blockade of the CD28 and CD40 pathways effectively aborts T-cell clonal expansion in vitro and in vivo, promotes long-term survival of fully allogeneic skin grafts, and inhibits the development of chronic vascular rejection of primarily vascularized cardiac allografts. The requirement for simultaneous blockade of these pathways for effective inhibition of alloimmunity indicates that, although they are interrelated, the CD28 and CD40 pathways are critical independent regulators of T-cell-dependent immune responses.
05/1996; 381(6581):434-438.
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ABSTRACT: Interactions between T and B cells are dynamic and regulated by interacting receptor: co-receptors. Interactions between CD40 and its ligand, gp39, and the CD28/CTLA-4 and B7 family members play a decisive role in regulating the progression of cognate interactions. The interdependence of gp39-CD40 and CD28/CTLA-B7 expression and function was studied in vitro during an antigeninduced immune response using T cells from mice expressing a transgenic T cell receptor (TCR). gp39 was induced on pigeon cytochrome c (PCC)-transgenic T cells in the presence of antigen and antigen-presenting cells. The antigen-induced expression of gp39 on transgenic T cells was inhibited by antibodies to class II major histocompatibility complex, CD4 and LFA-1, but not by CTLA-4 Ig, anti-B7-1 or anti-B7-2. These data established that the antigen-induced expression of gp39 was not dependent on co-stimulation via CD28/CTLA-4. The addition of PCC also resulted in the modest expression of B7-1 and a more robust expression of B7-2 on the cognate B cells. The addition of anti-gp39 blocked the up-regulated expression of B7-1 and partially blocked the up-regulated expression of B7-2. The addition of anti-gp39 and anti-interleukin-4 inhibited antigen-induced expression of B7-2 on B cells to near background levels. Studies on the up-regulation of B7-1 and B7-2 on resting B cells showed that soluble gp39 up-regulated B7-1 and B7-2 expression on B cells. In addition, interleukin-4 and interferon-γ up-regulated B7-2 expression on B cells. Taken together, these data demonstrate that the antigen-induced expression of gp39 is dependent on TCR-derived signals, yet independent of CD28/CTLA-4 co-stimulatory signals. Cognate interactions also resulted in the modest enhancement of B7-1 expression and a more profound expression of B7-2 which were completely or partially dependent on gp39-CD40 interactions.
European Journal of Immunology 01/1995; 25(2):596 - 603. · 5.10 Impact Factor
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ABSTRACT: The interaction between the CD40 ligand (gp39), expressed by activated T cells, and CD40, constitutively expressed by B cells, is critical for an effective antibody response to T cell dependent antigens. Patients with X-linked hyper IgM (HIM) syndrome fail to express a functional CD40 ligand due to a mutation within the gene for gp39. As a direct consequence, HIM patients, when immunized with T dependent antigens, produce only small amounts of IgM antibody without the development of immunologic memory, amplification and switch from IgM to IgG. Mutations affecting the gene for the HIM syndrome are localized throughout the coding region of gp39 and consist predominantly of point mutations. The resulting amino acid substitutions interfere directly with the receptor binding site or lead to stop codons or deletions secondary to splice site mutations. Expression of gp39 by activated T cells from patients with common variable immunodeficiency (CVI) is low in approximately half of the patients and is associated with depressed expression of IL-2. These findings suggest that inefficient signaling via CD40 may be responsible in part for failure of B cell differentiation in CVI.
Seminars in Immunology.
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ABSTRACT: Many humanized antibodies and fusion proteins targeting T-cell co-stimulatory molecules are now in late-stage clinical development (phase II, phase III) or have recently completed phase III clinical trials. Both Amevive, an LFA-3–Ig fusion protein targeting CD2, and Xanelim, a humanized anti-CD11a antibody, have shown efficacy in pivotal phase III trials in patients with plaque psoriasis. These new medicines are poised to enter clinical use in 2002.
Current Opinion in Immunology.
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ABSTRACT: Ligation of CD40 using anti-CD40 or soluble CD40-ligand activates numerous intracellular kinases which transduce signals to the nucleus. The nature whereby these signaling events are coupled to distal functional events in B cells is poorly understood. In this study, using anti-CD40 monoclonal antibodies which recognize different epitopes on CD40, we compare the ability to activate the stress-activated protein kinases (SAPK) such as c-Jun NH2 terminal kinase and p38 in human B cells with CD40 function. Activation of the SAPK pathway correlated with levels of activation of Rel/NF-κB transcription factors, but did not appear to be associated with rescue from anti-IgM induced apoptosis by suppressing caspase (CPP32) activity. Somewhat surprisingly, in the presence of IL-4, those antibodies to CD40 which failed to activate SAPK were most active in IgE production. IgE production was augmented in the presence of wortmannin. These studies suggest that rescue from apoptosis and IgE production mediated via CD40 may be independent of SAPK activation, induction of Rel/NF-κB, or suppression of CPP32 and that IgE production is, at least in part, regulated by signaling pathways that are dependent on phosphatidylinositol 3-kinase.
Cellular Immunology.
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ABSTRACT: A novel approach to the study of proteins is the construction of chimeras consisting of a target protein fused to an immunoglobulin constant domain. These recombinant globulins have been used in the identification of ligand-receptor pairs, determination of functional consequences of receptor engagement, the elucidation of structural domains necessary for ligand binding, and as potential therapeutic agents.
Current Opinion in Immunology.
