title: Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells.
abstract: OBJECTIVE
  Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation.
  
  
  METHODS
  Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81.
  
  
  RESULTS
  Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity.
  
  
  CONCLUSIONS
  Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.
authors: Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Morten P
authors: MP
authors: Oksvold
authors: Life Technologies AS, Oslo, Norway.
authors: Anette
authors: A
authors: Kullmann
authors: Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Lise
authors: L
authors: Forfang
authors: Life Technologies AS, Oslo, Norway.
authors: Bente
authors: B
authors: Kierulf
authors: Life Technologies AS, Oslo, Norway.
authors: Mu
authors: M
authors: Li
authors: Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Andreas
authors: A
authors: Brech
authors: Life Technologies AS, Oslo, Norway.
authors: Alexander V
authors: AV
authors: Vlassov
authors: Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Erlend B
authors: EB
authors: Smeland
authors: Life Technologies AS, Oslo, Norway.
authors: Axl
authors: A
authors: Neurauter
authors: Life Technologies AS, Oslo, Norway. Electronic address: Ketil.pedersen@lifetech.com.
authors: Ketil W
authors: KW
authors: Pedersen
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D000954:Antigens, Surface; D001402:B-Lymphocytes / Q000276:immunology*; D015415:Biomarkers; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D006684:HLA-DR Antigens; D006801:Humans; D016393:Lymphoma, B-Cell / Q000378:metabolism*; D008854:Microscopy, Electron; D060225:Tetraspanin 28 / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000954:Antigens, Surface; D015415:Biomarkers; D006684:HLA-DR Antigens; D060225:Tetraspanin 28
keywords: B-cell lymphoma; B-cell surface antigens; exosomes; flow cytometry; immunotherapy; magnetic beads