DOCSLIB.ORG

Human Tumor-Specific Antigen Dendritic Cell Immunogens

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Human Tumor-Specific Antigen Dendritic Cell Immunogens The Journal of Immunology Enhanced Effector and Memory CTL Responses Generated by Incorporation of Receptor Activator of NF-␬B (RANK)/RANK Ligand Costimulatory Molecules into Dendritic Cell Immunogens Expressing a Human Tumor-Specific Antigen1,2 Carsten Wiethe,3*†‡ Kurt Dittmar,* Tracy Doan,†‡ Werner Lindenmaier,* and Robert Tindle4†‡ The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-␬B (RANK)/ RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8؉ T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7- expressing DCs augmented E7-specific IFN-␥-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma. The Journal of Immunology, 2003, 171: 4121–4130. nitiation of T cell immunity depends on the Ag-presenting DCs present Ags to T cells via the TCR/MHC/epitope synapse capacity of mature dendritic cells (DCs).5 DCs occur in tis- and, in so doing, signal T cells for activation. This event requires I sues as immature cells and are specialized to capture and a second signal, also known as a costimulatory signal, which is process foreign or tumor Ag. After Ag capture, they mature in classically provided by ligation of CD28 on T cells with CD80 response to inflammatory stimuli characterized by up-regulation of (B7-1) or CD86 (B7-2) on DCs, but is now known to be provided MHC and costimulatory molecules (1), and migrate to the T cell by a family of B7 molecules, by the TNF superfamily, and by areas of draining lymph nodes where processed Ag is presented to cytokines (3, 4). ϩ T cells. DC-induced stimulation of CD8 CTL directed to tumor The interaction between DCs and T cells results in up-regulation epitopes is a vital component of the immune response to tumors (2). of receptor-ligand pairs of the TNF superfamily, including 4-1BB ligand (4-1BBL) (CD137L), FasL (CD95L), CD27, CD30, CD154 (CD40L), receptor activator of NF␬B (RANK)L (or TNF-related *Gesellschaft fu¨r Biotechnologische Forschung, Department of Molecular Biotech- activation-induced cytokine (TRANCE)), lymphotoxin, TNF-re- nology, Braunschweig, Germany; and †Sir Albert Sakzewski Virus Research Centre, lated apoptosis-inducing ligand, and members of the TNFR super- ‡ Royal Children’s Hospital, and Clinical Medical Virology Centre, University of family including 4-1BB (CD137), RANK, and CD40 (5, 6). Co- Queensland, Brisbane, Australia ligation of these receptor-ligand pairs induces downstream Received for publication April 10, 2003. Accepted for publication August 6, 2003. signaling events via TNFR-associated factor adaptor molecules. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance This signaling up-regulates adhesion and costimulatory molecules with 18 U.S.C. Section 1734 solely to indicate this fact. (7), enhances stable cellular interactions between DCs and T cells, 1 This work was supported by the National Health and Medical Research Council regulates survival of either the APC or the T cell (8), and leads to (Australia). C.W. was supported by a grant from the Helmholtz-Gemeinschaft Fors- production of T cell-stimulatory cytokines (e.g., IL-12, IL-1, and chungszentren-Strategiefond I Infektionsabwehr und Krebspra¨vention and by a short- term Ph.D. fellowship from the Deutscher Akademischer Austauschdienst. IL-6) (5, 8–10) and/or down-regulation of T cell-inhibitory cyto- 2 This paper is contribution number 192 of the Sir Albert Sakzewski Virus Research kines (e.g., IL-10) (9). A result is amplification and sustenance of Center. the ensuing immune response. 3 Current address: Dermatologische Klinik, Universitat Erlangen, Hartmannstrasse Manipulation of DCs ex vivo to display tumor epitopes before 14, D-19052 Erlangen, Germany. reinfusion is an attractive approach to tumor immunotherapy, but 4 Address correspondence and reprint requests to Dr. Robert Tindle, Sir Albert Sak- the approach is limited by the short life span of fully differentiated zewski Virus Research Centre, Royal Children’s Hospital, Herston Road, Herston, QLD 4029, Australia. E-mail address: [email protected] or mature DCs (11). This short life span is related to their rapid 5 Abbreviations used in this paper: DC, dendritic cell; RANK, receptor activator of apoptosis, but this can be relieved at least in tissue culture by NF-␬B; L, ligand; TRANCE, TNF-related activation-induced cytokine; HPV, human treatment of DCs with several costimulatory molecules, including papillomavirus; eGFP, enhanced green fluorescent protein; MOI, multiplicity of in- fection; Ad, adenovirus; CMVie, CMV immediate early; TAA, tumor-associated Ag; RANKL and CD40L (12). Furthermore, pretreatment of Ag-pulsed IRES, internal ribosome entry site. mature DCs with soluble RANKL in vitro enhances the number Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 4122 RANK/RANKL ENHANCES DC-TUMOR Ag IMMUNOGENICITY and persistence of Ag-presenting DCs in draining lymph nodes in fragment into the pCR-Blunt-II-TOPO (Invitrogen) resulting in pCRII-CD40 vivo (13). In addition, RANKL treatment increased Ag-specificT and pCRII-CD40L, respectively, we sequenced the inserts. In comparison to cell responses (13). GenBank sequences, we found in all our CD40 clones 1 aa substitution at position 227 (Met3Ile), and in the CD40L clones 1 aa substitution at position In the present study, we used novel adenovirus (Ad) vectors to 198 (Ile3Ser). This mutation in CD40L has been described (GenBank se- express a model tumor Ag (the E7 oncoprotein of human papillo- quence S21738) and does not affect biological function (18). The mutation in mavirus type 16 (HPV16)) and costimulatory molecules in DCs. CD40 has also been described (GenBank sequence X65453). To create the We investigated whether the immune response induced by immu- bicistronic expression cassette for either the murine CD40 or CD40L, the mouse ecotropic retrovirus receptor gene Rec-1 from the bicistronic construct nization with DCs expressing the tumor Ag would be enhanced by pGEMRec-1IRESegfp was replaced by the murine CD40 or CD40L gene from coprovision of RANK/RANKL or CD40/CD40L DC/T cell recep- pCRII-CD40 or pCRII-CD40L, resulting in pGEMCD40IRESegfp and tor-ligand pairs to the DCs. We reasoned that ligation of these pGEMCD40LIRESegfp, respectively. costimulatory molecules and their respective receptors within DCs Construction of Ad cosmids may effect an autocrine activation of individual DCs and/or allow reciprocal activation of interacting DCs. This would augment the rAds derived from human Ad type 5 were constructed using a cosmid activating signals received by DCs from T cells that themselves cloning procedure that allows direct assembly of rAds by cloning in Esch- erichia coli (W. Lindenmaier, unpublished data). Briefly, Ad cosmid pAd- express RANKL and CD40L. We demonstrate that coexpression cos45 was digested by XbaI and ClaIorSwaI and ClaI for CD40L, de- RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs in- phosphorylated, and ligated to the Psp1406 I/XbaI- or SwaI/XbaI-excised creased E7-directed effector and memory CTL responses. Simi- mono- or bicistronic expression cassettes including CMVie promoter and larly, we demonstrate that coexpression of the T cell costimulatory 3Ј regulatory sequences from pGEMegfp, pGEME7mutIRESegfp, pGEM- molecules (CD40L and RANKL) or DC costimulatory molecules RANKIRESegfp, pGEMRANKLIRESegfp, pGEMCD40IRESegfp, and pGEMCD40LIRESegfp, respectively. Packaging in vitro and transduction (CD40 and RANK) in E7-expressing DCs also increased E7-directed into E. coli DH5␣ yielded Ad cosmids with the corresponding expression effector and memory CTL responses. We show that augmentation of cassettes integrated into the E1 region. T cell responses correlated with up-regulation of CD80 and CD86 in rAds DCs by these costimulatory molecule combinations. rAds were propagated, purified, and titrated as described (19) with minor modifications. For production of rAd, cosmid DNA was transfected into Materials and Methods ϩ Cell lines and cell culture 293LP cells. The formation of rAds was confirmed by monitoring eGFP adenoviral plaques, and virus particles were harvested. Restriction analysis 293LP cells, an Ad-transformed human embryonic kidney cell line that of viral DNA was performed using DNA extracted from benzonase provides phenotypic complementation of the E1 genes, were purchased (Merck, West Point, PA)-digested lysates of infected cells. Purified virus from Microbix Biosystems (Toronto, Ontario, Canada). A549 cells were a was isolated by two rounds of CsCl density gradient centrifugation, and gift from T. Adrian (Medizinischen Hochschule Hannover, Hannover, Ger- extensively dialyzed. The titers of the dialyzed stocks were determined by many). The E7-expressing EL4.E7 cell line was derived from EL4 cells plaque assay on the 293LP cells. (H-2b thymoma) transfected to stably express the full-length HPV16 E7 gene (14). The EL4.A2 cell line has been described (13).
Load more

Recommended publications
  • Induction of Antitumor Immunity by Transduction of CD40 Ligand Gene
    D2001 Nature Publishing Group 0929-1903/01/$17.00/+0 www.nature.com/cgt Induction of antitumor immunity by transduction of CD40 ligand gene and interferon- gene into lung cancer Masahiro Noguchi,1 Kazuyoshi Imaizumi,1 Tsutomu Kawabe,1 Hisashi Wakayama,1 Yoshitsugu Horio,1 Yoshitaka Sekido,2 Toru Hara,1 Naozumi Hashimoto,1 Masahide Takahashi,3 Kaoru Shimokata,2 and Yoshinori Hasegawa1 1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan; Departments of 2Clinical Preventive Medicine and 3Pathology, Nagoya University School of Medicine, Nagoya, Japan. CD40±CD40 ligand (CD40L) interaction is an important costimulatory signaling pathway in the crosstalk between T cells and antigen-presenting cells. This receptor±ligand system is known to be essential in eliciting strong cellular immunity. Here we demonstrate that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Immunohistochemical study showed that inflammatory cells, including CD4+, CD8+ T cells and NK cells, infiltrated into the inoculated 3LLSA-CD40L tumor tissue. Inoculation of 3LLSA-CD40L cells into mice resulted in the induction of 3LLSA-specific cytotoxic T-cell immunity, and the growth of parental 3LLSA tumors was inhibited when 3LLSA cells were inoculated into C57BL/6 mice mixed with 3LLSA-CD40L cells or when they were rechallenged 4 weeks after 3LLSA-CD40L cells were rejected. Furthermore, co-inoculation of interferon (IFN)- ± transduced cells (3LLSA-IFN ) with 3LLSA-CD40L cells enhanced the antitumor immunity efficiently in vivo. These results indicate that the in vivo priming with CD40L- and IFN- gene±transduced lung cancer cells is a promising strategy for inducing antitumor immunity in the treatment of lung cancer.
    [Show full text]
  • BD Pharmingen™ FITC Mouse Anti-Rat CD134
    BD Pharmingen™ Technical Data Sheet FITC Mouse Anti-Rat CD134 Product Information Material Number: 554848 Alternate Name: OX-40 Antigen Size: 0.5 mg Concentration: 0.5 mg/ml Clone: OX-40 Immunogen: Activated rat lymph node cells Isotype: Mouse (BALB/c) IgG2b, κ Reactivity: QC Testing: Rat Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide. Description The OX-40 antibody reacts with the 50-kDa OX-40 Antigen (CD134), also known as OX-40 Receptor, on CD4+ T lymphocytes activated in vitro and in vivo. The antigen is a member of the NGFR/TNFR superfamily, which includes low-affinity nerve growth factor receptor, TNF receptors, the Fas antigen, CD137 (4-1BB), CD27, CD30, and CD40. CD134 supplies costimulatory signals for T-cell proliferation and effector functions. While OX-40 mAb is not mitogenic, it does augment some in vitro T-cell responses. It is also reported to block binding of OX-40 Ligand to OX-40 Antigen. Preparation and Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4° C and protected from prolonged exposure to light. Do not freeze. Application Notes Application Flow cytometry Routinely Tested Suggested Companion Products Catalog Number Name Size Clone 559532 FITC Mouse IgG2b, κ Isotype Control 0.25 mg MPC-11 Product Notices 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results. 2. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
    [Show full text]
  • Innovating Antibodies, Improving Lives
    Innovating Antibodies, Improving Lives H.C. Wainwright & Co. Global Life Sciences Conference April 9, 2019 Forward Looking Statement This presentation contains forward looking statements. The words “believe”, “expect”, “anticipate”, “intend” and “plan” and similar expressions identify forward looking statements. All statements other than statements of historical facts included in this presentation, including, without limitation, those regarding our financial position, business strategy, plans and objectives of management for future operations (including development plans and objectives relating to our products), are forward looking statements. Such forward looking statements involve known and unknown risks, uncertainties and other factors which may cause our actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by such forward looking statements. Such forward looking statements are based on numerous assumptions regarding our present and future business strategies and the environment in which we will operate in the future. The important factors that could cause our actual results, performance or achievements to differ materially from those in the forward looking statements include, among others, risks associated with product discovery and development, uncertainties related to the outcome of clinical trials, slower than expected rates of patient recruitment, unforeseen safety issues resulting from the administration of our products in patients, uncertainties related to product manufacturing, the lack of market acceptance of our products, our inability to manage growth, the competitive environment in relation to our business area and markets, our inability to attract and retain suitably qualified personnel, the unenforceability or lack of protection of our patents and proprietary rights, our relationships with affiliated entities, changes and developments in technology which may render our products obsolete, and other factors.
    [Show full text]
  • Induces Antigen Presentation in B Cells Cell-Activating Factor of The
    B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells This information is current as of September 27, 2021. Min Yang, Hidenori Hase, Diana Legarda-Addison, Leena Varughese, Brian Seed and Adrian T. Ting J Immunol 2005; 175:2814-2824; ; doi: 10.4049/jimmunol.175.5.2814 http://www.jimmunol.org/content/175/5/2814 Downloaded from References This article cites 54 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/175/5/2814.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells1 Min Yang,* Hidenori Hase,* Diana Legarda-Addison,* Leena Varughese,* Brian Seed,† and Adrian T.
    [Show full text]
  • Production by OK-432 Via the CD40/CD40 Ligand Pathway
    cancers Article The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway Go Ohe 1,2,* , Yasusei Kudo 3 , Kumiko Kamada 1, Yasuhiro Mouri 3, Natsumi Takamaru 1, Keiko Kudoh 1, Naito Kurio 1 and Youji Miyamoto 1 1 Department of Oral Surgery, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan; [email protected] (K.K.); [email protected] (N.T.); [email protected] (K.K.); [email protected] (N.K.); [email protected] (Y.M.) 2 Dentistry and Oral Surgery, Takamatsu Municipal Hospital, 847-1 Ko Busshozan-cho, Takamatsu 761-8538, Japan 3 Department of Oral Bioscience, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan; [email protected] (Y.K.); [email protected] (Y.M.) * Correspondence: [email protected] Simple Summary: OK-432 is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously reported that OK-432 treatment can induce the production of high levels of IFN-γ from peripheral blood mononuclear cells (PBMCs). Moreover, the IFN-γ production from PBMCs by OK-432 is impaired by conditioned media (CM) from oral cancer cells. To determine the inhibitory mechanism of IFN-γ production by CM, the genes involved in IFN-γ production was Citation: Ohe, G.; Kudo, Y.; Kamada, retrieved by cDNA microarray analysis. We found that CD40 played a key role in IFN-γ production K.; Mouri, Y.; Takamaru, N.; Kudoh, via IL-12 production.
    [Show full text]
  • Absence of Igd-CD27(+) Memory B Cell Population in X-Linked Hyper-Igm Syndrome
    Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome. K Agematsu, … , H D Ochs, A Komiyama J Clin Invest. 1998;102(4):853-860. https://doi.org/10.1172/JCI3409. Research Article The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti- CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL- 10 in vitro. Find the latest version: https://jci.me/3409/pdf Absence of IgD2CD271 Memory B Cell Population in X-linked Hyper-IgM Syndrome Kazunaga Agematsu,* Haruo Nagumo,* Koji Shinozaki,* Sho Hokibara,* Kozo Yasui,* Kihei Terada,‡ Naohisa Kawamura,§ Tsuyoshi Toba,i Shigeaki Nonoyama,¶ Hans D.
    [Show full text]
  • Lipid Rafts Are Important for the Association of RANK and TRAF6
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 4, 279-284, August 2003 Lipid rafts are important for the association of RANK and TRAF6 Hyunil Ha1,3, Han Bok Kwak1,2,3, Introduction Soo Woong Lee1,2,3, Hong-Hee Kim2,4, 1,2,3,5 Osteoclasts are multinucleated giant cells responsible and Zang Hee Lee for bone resorption. These cells are differentiated from 1 hematopoietic myeloid precursors of the monocyte/ National Research Laboratory for Bone Metabolism macrophage lineage (Suda et al., 1992). For the dif- 2Research Center for Proteineous Materials 3 ferentiation of osteoclast precursors into mature osteo- School of Dentistry clasts, a cell-to-cell interaction between osteoclast Chosun University, Gwangju 501-759, Korea 4 precursors and osteoblasts/stromal cells are required Department of Cell and Developmental Biology (Udagawa et al., 1990). Recently, many studies have College of Dentistry, Seoul National University provided ample evidences that the TNF family mem- Seoul 110-749, Korea κ 5 ber RANKL (receptor activator of NF- B ligand; also Corresponding author: Tel, 82-62-230-6872; known as ODF, OPGL, and TRANCE) is expressed Fax, 82-62-227-6589; E-mail, [email protected] on the surface of osteoblasts/stromal cells and es- sential for osteoclast differentiation (Anderson et al., Accepted 19 June 2003 1997; Yasuda et al., 1998; Takahashi et al., 1999). When its receptor RANK was stimulated by RANKL, Abbreviations: MAPK, mitogen-activated protein kinase; MCD, several TNF receptor-associated factors (TRAFs), methyl-β-cyclodextrin; RANK, receptor activator of NF-κB; TLR, especially TRAF6, can be directly recruited into RANK Toll-like receptor; TNFR, TNF receptor; TRAF, TNF receptor- cytoplasmic domains and may trigger downstream associated factor signaling molecules for the activation of NF-κB and mitogen activated protein kinases (MAPKs) (Darnay et al., 1998; Wong et al., 1998; Kim et al., 1999).
    [Show full text]
  • B-Cell Development, Activation, and Differentiation
    B-Cell Development, Activation, and Differentiation Sarah Holstein, MD, PhD Nov 13, 2014 Lymphoid tissues • Primary – Bone marrow – Thymus • Secondary – Lymph nodes – Spleen – Tonsils – Lymphoid tissue within GI and respiratory tracts Overview of B cell development • B cells are generated in the bone marrow • Takes 1-2 weeks to develop from hematopoietic stem cells to mature B cells • Sequence of expression of cell surface receptor and adhesion molecules which allows for differentiation of B cells, proliferation at various stages, and movement within the bone marrow microenvironment • Immature B cell leaves the bone marrow and undergoes further differentiation • Immune system must create a repertoire of receptors capable of recognizing a large array of antigens while at the same time eliminating self-reactive B cells Overview of B cell development • Early B cell development constitutes the steps that lead to B cell commitment and expression of surface immunoglobulin, production of mature B cells • Mature B cells leave the bone marrow and migrate to secondary lymphoid tissues • B cells then interact with exogenous antigen and/or T helper cells = antigen- dependent phase Overview of B cells Hematopoiesis • Hematopoietic stem cells (HSCs) source of all blood cells • Blood-forming cells first found in the yolk sac (primarily primitive rbc production) • HSCs arise in distal aorta ~3-4 weeks • HSCs migrate to the liver (primary site of hematopoiesis after 6 wks gestation) • Bone marrow hematopoiesis starts ~5 months of gestation Role of bone
    [Show full text]
  • IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells
    cells Article IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells Fridolin Franke 1,2, Greg A. Kirchenbaum 1 , Stefanie Kuerten 2 and Paul V. Lehmann 1,* 1 Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA; [email protected] (F.F.); [email protected] (G.A.K.) 2 Institute of Anatomy and Cell Biology, Friedrich-Alexander University Erlangen-Nürnberg, 91054 Erlangen, Germany; [email protected] * Correspondence: [email protected]; Tel.: +1-216-965-6311 Received: 3 December 2019; Accepted: 12 February 2020; Published: 13 February 2020 Abstract: Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help. Herein, we report that the combination of agonistic anti-CD40, IL-4 and IL-21 affords polyclonal B cell stimulation that was comparable to R848 and IL-2 for detection of influenza-specific memory B cells. An additional advantage of anti-CD40, IL-4 and IL-21 stimulation is the selective activation of IgM+ memory B cells, as well as the elicitation of IgE+ ASC, which the former fails to do. Thereby, we introduce a protocol that mimics physiological B cell activation through helper T cells, including induction of all Ig classes, for immune monitoring of antigen-specific B cell memory.
    [Show full text]
  • Cooperation of Multiple Signaling Pathways in CD40-Regulated Gene Expression in B Lymphocytes
    Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes Hajir Dadgostar*†, Brian Zarnegar*, Alexander Hoffmann‡, Xiao-Feng Qin‡, Uyen Truong§, Govinda Rao§, David Baltimore‡, and Genhong Cheng*¶ *Molecular Biology Institute and †Medical Scientist Training Program, School of Medicine, University of California, Los Angeles, CA 90095; ‡California Institute of Technology, Pasadena, CA 91125; and §Affymetrix, Incorporated, Santa Clara, CA 95051 Contributed by David Baltimore, December 12, 2001 CD40͞CD40L interaction is essential for multiple biological events factors, and hence it is generally believed that CD40 achieves in T dependent humoral immune responses, including B cell sur- many of its complex effects on B cells through alterations in gene vival and proliferation, germinal center and memory B cell forma- expression. CD40 up-regulates expression of numerous genes, tion, and antibody isotype switching and affinity maturation. By including CD23, intercellular adhesion molecule-1 (ICAM-1), using high-density microarrays, we examined gene expression in Fas, B7.1, B7.2, MHC II, LT-␣,c-myc, Bcl-x, Bfl-1, A20, CDK4, primary mouse B lymphocytes after multiple time points of CD40L CDK6, IgC␥, and IgC␧ (2, 14–21). From the standpoint of stimulation. In addition to genes involved in cell survival and signaling specificity, it is not presently clear which (if any) growth, which are also induced by other mitogens such as lipo- CD40-mediated gene expression responses are specific to CD40, polysaccharide, CD40L specifically activated genes involved in as a B cell costimulus, and which are less specific manifestations germinal center formation and T cell costimulatory molecules that of mitogenic B cell activation.
    [Show full text]
  • Co-Stimulatory Versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia
    cancers Article Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia Raquel Delgado 1,2 , Karoline Kielbassa 1,3,4,5, Johanna ter Burg 1, Christian Klein 6 , Christine Trumpfheller 6, Koen de Heer 7, Arnon P. Kater 3,4,5,8 and Eric Eldering 1,3,4,5,* 1 Department of Experimental Immunology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands; [email protected] (R.D.); [email protected] (K.K.); [email protected] (J.t.B.) 2 Chronic Diseases Research Center, NOVA Medical School, 1150-082 Lisbon, Portugal 3 Cancer Center Amsterdam, Amsterdam University, 1081 HV Amsterdam, The Netherlands; [email protected] 4 Amsterdam Institute for Infection and Immunity, 1081 HV Amsterdam, The Netherlands 5 LYMMCARE (Lymphoma and Myeloma Center Amsterdam), 1105 AZ Amsterdam, The Netherlands 6 Cancer Immunotherapy Discovery, Roche Innovation Centre Zurich, 8952 Schlieren, Switzerland; [email protected] (C.K.); [email protected] (C.T.) 7 Department of Hematology, Flevoziekenhuis, 1315 RA Almere, The Netherlands; [email protected] 8 Department of Hematology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands * Correspondence: [email protected]; Tel.: +31-205667018 Citation: Delgado, R.; Kielbassa, K.; Simple Summary: Previous observations have shown that CD40 activation of CLL cells via coculture ter Burg, J.; Klein, C.; Trumpfheller, with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and C.; de Heer, K.; Kater, A.P.; Eldering, obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in E.
    [Show full text]
  • Characterization of a 23-Kda Protein Associated with CD40 TOMOHIRO MORIO, SILVA HANISSIAN, and RAIF S
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11633-11636, December 1995 Immunology Characterization of a 23-kDa protein associated with CD40 TOMOHIRO MORIO, SILVA HANISSIAN, AND RAIF S. GEHA* Division of Immunology, Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115 Communicated by Mary Ellen Avery, Harvard Medical School, Boston, MA, August 30, 1995 (received for review May 1, 1995) ABSTRACT CD40 is a 45-kDa glycoprotein member ofthe MATERIALS AND METHODS tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carci- Cells. The human B-cell lines Raji, BJAB, and Ramos and the human bladder carcinoma cell line T24 were obtained from noma cells. The unique capacity of CD40 to trigger immuno- American Type Culture Collection. The human B-cell line globulin isotype switching is dependent on the activation of Jijoye which expresses TNFR p80 (CD120b) was a kind gift protein-tyrosine kinases, yet CD40 possesses no kinase do- from G. Mosialos and E. Kieff (Harvard Medical School). main and no known consensus sequences for binding to Human tonsil B cells were purified as described (13). protein-tyrosine kinases. Recently, an intracellular protein Monoclonal Antibodies (mAbs) and Reagents. Mouse anti- (CD40bp/LAP-1/CRAF-1) which belongs to the family of human CD40 mAb 626.1 (IgGl) was a gift from S. M. Fu TNFR-associated proteins was reported to associate with (University of Virginia, Richmond). Murine anti-major histo- CD40. We describe a 23-kDa cell surface protein (p23) which compatibility complex (MHC) class I mAb W6/32 (IgGl), and is specifically associated with CD40 on B cells and on urinary anti-HLA-DR mAb L243 (IgG2a) were obtained through bladder transitional carcinoma cells.
    [Show full text]