title: Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.
abstract: Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: xenografts@hanmail.net.
authors.forename: Jung-Sik
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, South Korea; BK21Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: nr.byun@gmail.com.
authors.forename: Nari
authors.initials: N
authors.lastname: Byun
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, South Korea; BK21Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: ramzawoo@snu.ac.kr.
authors.forename: Hyunwoo
authors.initials: H
authors.lastname: Chung
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, South Korea; BK21Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: tte9801@hanmail.net.
authors.forename: Hyun-Je
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: vinaka00@cbnu.ac.kr.
authors.forename: Jong-Min
authors.initials: JM
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, South Korea. Electronic address: tchun@korea.ac.kr.
authors.forename: Taehoon
authors.initials: T
authors.lastname: Chun
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, South Korea; BK21Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: wonwoolee@snu.ac.kr.
authors.forename: Won-Woo
authors.initials: WW
authors.lastname: Lee
authors.affiliation: Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Biomedical Research Institute, Seoul National University College of Medicine, Seoul, 110-799, South Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, South Korea; BK21Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, 110-799, South Korea. Electronic address: chgpark@snu.ac.kr.
authors.forename: Chung-Gyu
authors.initials: CG
authors.lastname: Park
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology; D015703:Antigens, CD / Q000378:metabolism; D018951:Antigens, CD20 / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology*; D051940:B7-2 Antigen / Q000378:metabolism; D019013:CD40 Antigens / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008213:Lymphocyte Activation; D008253:Macaca mulatta; D008562:Membrane Glycoproteins / Q000378:metabolism; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D018951:Antigens, CD20; D051940:B7-2 Antigen; D019013:CD40 Antigens; D023201:CD40 Ligand; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D000097802:CD83 Antigen
keywords: B cell; CD40-CD40L signals; Ex-vivo expansion; Monkeys