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In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model
Published on: November 28, 2017
Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific
Takashi Inagaki1, Tatsunari Yoshimi, Satoshi Kobayashi
1Innovative Antibody Engineering Laboratory, Advance Co. Ltd., 5-7 Kobunacho, Chuo-ku, Tokyo, Japan.
Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained.
Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained.
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