Large-Scale In Vitro Expansion of Polyclonal Human Switched-Memory B Lymphocytes
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{"title"=>"Large-Scale In Vitro Expansion of Polyclonal Human Switched-Memory B Lymphocytes", "type"=>"journal", "authors"=>[{"first_name"=>"Sonia", "last_name"=>"Néron", "scopus_author_id"=>"6603069945"}, {"first_name"=>"Annie", "last_name"=>"Roy", "scopus_author_id"=>"7402705278"}, {"first_name"=>"Nellie", "last_name"=>"Dumont", "scopus_author_id"=>"16136674700"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"366298621", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0051946", "scopus"=>"2-s2.0-84871314693", "pmid"=>"23284827", "sgr"=>"84871314693"}, "id"=>"ea654593-1fad-3412-9a6d-3f04c23ea126", "abstract"=>"Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients' accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10(6)-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG(1), IgG(2), IgG(3) and IgG(4) is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.", "link"=>"http://www.mendeley.com/research/largescale-vitro-expansion-polyclonal-human-switchedmemory-b-lymphocytes", "reader_count"=>40, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>14, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>7, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>14, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>7, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>25, "Medicine and Dentistry"=>4, "Immunology and Microbiology"=>6}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>6}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"Canada"=>2, "United States"=>1, "Brazil"=>1, "United Kingdom"=>1, "France"=>1, "Spain"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/524178"], "description"=>"<p>In all experiments, purified CD19<sup>+</sup> B lymphocytes (A) were depleted for IgD<sup>+</sup>IgM<sup>+</sup> and IgM+ cells in a two-step selection process (B). Analysis of the resulting cell populations showed a relatively similar proportion of cells with surface IgA (C) and IgG (D).</p>", "links"=>[], "tags"=>["switched-memory"], "article_id"=>194672, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Selection_of_switched_memory_B_lymphocytes_/194672", "title"=>"Selection of switched-memory B lymphocytes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 18:36:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/524375"], "description"=>"<p>(<b>A</b>) Ten samples of switched-memory B lymphocytes were cultured for 35 to 65 days in the presence of IL-2, IL-4, IL-10 and CD154<sup>+</sup> cells (L4.5 cells) at a ratio of five B cells per L4.5 cell. Expansion factors for the ten independent samples were plotted as a function of time (days) in culture. (<b>B</b>) Regression analysis of the ten exponential growth curves presented in (A) resulted in a correlation coefficient of 0.9965 corresponding to the equation y = 10 (0.1344x+0.5415). (<b>C</b>) The proportion of viable cells during long-term culture was monitored on a regular basis for each culture. (<b>D</b>) The mean value and standard deviation of viability (%) showed a similar evolution for each samples.</p>", "links"=>[], "tags"=>["switched-memory"], "article_id"=>194871, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Long_term_expansion_of_switched_memory_B_lymphocytes_/194871", "title"=>"Long-term expansion of switched-memory B lymphocytes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 18:37:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/524515"], "description"=>"<p>Cells from the same ten independent experiments shown in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051946#pone-0051946-g002\" target=\"_blank\">Fig. 2</a>, were collected during the exponential phase, namely on days 28 (e, f), 33 (b, c, d) 35 (a), and 37 (g, h, i, j), and seeded in fresh IMDM at 1–2×10<sup>6</sup> cells/mL for 20–22 h. For each experiment, secretion rates were determined for IgA, IgG and IgM (A, B) and IgG<sub>1</sub>, IgG<sub>2</sub>, IgG<sub>3</sub> and IgG<sub>4</sub> (C, D). Data are presented as the mean ± SD.</p>", "links"=>[], "tags"=>["lymphocytes", "secrete"], "article_id"=>195011, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Switched_memory_B_lymphocytes_secrete_mostly_IgG_/195011", "title"=>"Switched-memory B lymphocytes secrete mostly IgG.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 18:37:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/524630"], "description"=>"<p>IgG’s polyclonality was determined by IEF for experiments a to j. A representative pattern is presented in panel A. (A) Briefly, IEF standard (Std), IVIg, a human monoclonal IgG (mAb) and culture supernatants from experiment a sampled on days 16, 21, 30, 41 and 49 are shown. This polyclonal IEF pattern is similar for the ten independent cultures presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051946#pone-0051946-g001\" target=\"_blank\">Fig. 1</a>. (B) IEF pattern was determined for the cumulated supernatants of the three experiments presented in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051946#pone-0051946-g005\" target=\"_blank\">Fig. 5</a> as well as the pooled supernatants of 13 representative experiments. As mentioned above, Std, IVIg and mAb were used as controls and a sample from a healthy human serum was also used for the same purpose. For all samples in (A) and (B), analyses were done on 100 ng IgG per well.</p>", "links"=>[], "tags"=>["lymphocytes", "secrete", "polyclonal"], "article_id"=>195130, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Switched_memory_B_lymphocytes_secrete_polyclonal_IgG_/195130", "title"=>"Switched-memory B lymphocytes secrete polyclonal IgG.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 18:38:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/524770"], "description"=>"<p>Three switched-memory B lymphocyte samples were cultured as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051946#pone-0051946-g001\" target=\"_blank\">Fig. 1</a> and transferred in petri dishes to test the feasibility of increasing the culture volume up to 500 mL. (<b>A</b>) Expansion factors were similar to those obtained in 6-well plates (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051946#pone-0051946-g001\" target=\"_blank\">Fig. 1</a>). (<b>B</b>) Culture volumes are shown as a function of time. (C) IgA, IgG and IgM concentrations were determined in supernatants of the three independent samples at the end of the culture. (D) Flow cytometry analyses for kappa and lambda chain expression was similar for all three independent samples.</p>", "links"=>[], "tags"=>["immunology", "microbiology", "hematology"], "article_id"=>195270, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_expansion_during_long_term_culture_/195270", "title"=>"Validation of expansion during long-term culture.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 18:39:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/524937"], "description"=>"1<p>Data present the IgG signal (1.0 µg/mL; threshold >1000) corresponding to background subtracted pixel intensity value (RFU), the CV associated with the duplicate spots for each protein and the fold increase between IgG and negative control signals.</p>2<p>(*) Indicated significant interaction when IgG was adjusted to 0.1 µg/ml concentration (Threshold >500 RFU).</p>3<p>(#) Indicated significant interaction with a commercial intravenous immunoglobulin preparation (IVIg) adjusted to 1.0 µg/mL (Threshold >1000 RFU).</p>", "links"=>[], "tags"=>["polyclonal", "igg", "interacting", "proteins"], "article_id"=>195430, "categories"=>["Hematology", "Microbiology", "Immunology"], "users"=>["Sonia Néron", "Annie Roy", "Nellie Dumont"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0051946.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Human_polyclonal_IgG_interacting_proteins_using_a_protein_array_/195430", "title"=>"Human polyclonal IgG interacting proteins using a protein array.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-19 18:40:05"}

PMC Usage Stats | Further Information

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Relative Metric

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