Professor Daisuke Kitamura of the Division of Molecular Biology, Research Institute for Biological Sciences (RIBS), Tokyo University of Science and his research group have developed a new culture system in which B cells undergo massive expansion and generate germinal centre B cells, which can then differentiate into cells responsible for immunological memory. After adoptive transfer in mice, the germinal centre B cells generated using this system had a long lifespan as memory cells and produced antibodies. This new system is expected to not only facilitate elucidation of the differentiation mechanism of germinal centre B cells into memory cells but also to lead to the development of new methods of personalized infection control and antibody therapy.
*Research group: Daisuke Kitamura et al. of the Research Institute for Biological Sciences (RIBS), Tokyo University of Science
** This study was published in a British online scientific journal, Nature Communications, on September 6, 2011.
Upon an encounter with antigens such as a virus invading the body, B cells massively proliferate and form structures called germinal centres in organs such as lymph nodes and spleen. Some B cells in the germinal centre differentiate into memory B cells or long-lived plasma cells. Memory B cells can survive for several decades and respond to the invasion of their cognate antigens to rapidly produce specific antibodies, and long-lived plasma cells are cells that continue to produce specific antibodies for extended periods of time. Immunological memory is maintained by both of these types of cells. However, the mechanism of differentiation of these cells has remained unclear because a culture method has not been available so far for the expansion of germinal centre B cells that can differentiate into memory B cells and long-lived plasma cells.
The research group has developed the first culture system for B cells in which a large number of cells equivalent to germinal centre B cells (named iGB cells) are generated from naïve B cells in the spleen and peripheral blood. They demonstrated that after adoptive transfer in mice, the iGB cells generated using this system survived for a long period of time after differentiation into memory B cells or long-lived plasma cells, depending on culture conditions. Development of this culture system is expected to remarkably facilitate studies on the differentiation mechanism of germinal centre B cells into memory B cells or long-lived plasma cells, which has so far remained unrevealed. In addition, the immunological memory against viruses may be established in humans without vaccinations by the adoptive transfer of iGB cells that are generated from human peripheral blood B cells and selected for response to certain viruses. Furthermore, long-lived plasma cells continuously producing antibodies against cancers may be generated in the body of a cancer patient by adoptive transfer of iGB cells that respond to certain cancer antigens.
Our body’s mechanism called the “immunological memory” explains why a person catches an infectious disease such as measles only once even if it is caught and how vaccinations can prevent infections of certain viruses. B cells and T cells, which are both lymphocytes, play major roles in the “immunological memory.” When activated by an encounter with antigens such as pathogens, B cells present a portion of the antigen on their cell surface and encounter helper T cells recognizing the antigen. Then, the helper T cells express the CD40 ligand on their surface and secrete cytokines such as interleukin-4 (IL-4) and interleukin-21 (IL-21) to strongly induce B cell proliferation, where B cells proliferate to form germinal centres in lymphoid tissues. Some germinal centre B cells later differentiate into memory B cells or long-lived plasma cells, although the mechanism of induction of differentiation is almost completely unknown. Memory B cells can survive for several decades and rapidly respond to the invasion of their cognate antigens to produce specific antibodies. Long-lived plasma cells continue to produce specific antibodies for an extended period of time. The immunological memory is maintained by both of these types of cells.
2. Research procedures and results
We developed a new culture system for B cells and for this purpose, we generated a fibroblast cell line 40LB that stably expresses the CD40 ligand and the B-cell-activating factor belonging to the TNF family (BAFF), which is a B cell survival factor and cytokine. When the 40LB fibroblasts were used as feeder cells for the culture of mouse splenic B cells in the presence of IL-4, a massive proliferation of B cells expressing the same surface antigens as the germinal centre B cells, which were named induced germinal center B (iGB) cells, was observed. Notably, after adoptive transfer in mice, these iGB cells differentiated into memory B cells, which prevailed for a long period of time. The memory B cells differentiated from the iGB cells, which were named induced memory B (iMB) cells, had the ability to respond to antigens and rapidly produce antibodies in the presence of helper T cells in the same way as regular memory B cells generated by immunization in mice do.
When iGB cells were cultured in the presence of IL-21 after 4-days of being cultured in the presence of IL-4, their proliferation was more robust and reached about 10,000 times the initial cell number by the eighth day. Following adoptive transfer in mice, these iGB cells differentiated into long-lived plasma cells instead of memory B cells and migrated to the bone marrow, where they produced antibodies for a long period of time. Furthermore, when iGB cells were continuously cultured after the withdrawal of IL-21, their differentiation this time after adoptive transfer in mice was into iMB cells, indicating the loss of differentiation potency into long-lived plasma cells. Thus, by changing the cytokine added during cell culture, this culture system enables in vitro induction of progenitor cells that can differentiate into either memory B cells or long-lived plasma cells. Therefore, this system enables in vitro manipulation of the differentiation fate of germinal centre B cells, greatly facilitating the dissection of induction programs for germinal centre B cell differentiation.
3. Further expectations
With the development of this culture system, remarkable progress is expected in studies of induction mechanisms for differentiation such as the identification of signaling pathways and transcription factors involved in the induction of differentiation of germinal centre B cells into memory B cells or long-lived plasma cells. In addition, since this culture system is also applicable to the culture of human peripheral blood B cells in a similar way, immunological memory against any antigen may be established in humans by adoptive transfer of in vitro-generated iGB cells into the human body. For example, memory B cells recognizing certain viruses may be generated in the human body without vaccinations by adoptive transfer of iGB cells against the specific viral antigens after their proliferation in the presence of IL-4. Alternatively, long-lived plasma cells continuously producing antibodies against cancer antigens may be generated in the human body by the adoptive transfer of iGB cells against certain cancer antigens after their proliferation in the presence of IL-4 and IL-21. Such an approach has the potential to become personalized antibody therapy for many cancer patients, as only a very limited number of antibody drugs are currently available.
[Researcher Biography: Daisuke Kitamura]
Born in Saga prefecture in 1959, he graduated from Saga Medical School (currently, Faculty of Medicine, Saga University) in 1984. When he joined the surgical department, he also entered the graduate school to start immunology research in the Department of Immunology and Serology (then headed by Professor Takeshi Watanabe). The next year he transferred to the Medical Institute of Bioregulation, Kyushu University, following the transfer of Professor Watanabe. In 1988, he became a research associate at the same institute after being awarded an MD. He then studied abroad at the Universität zu Köln (University of Cologne) in Germany (under Professor Klaus Rajewsky) from the end of that year. He worked on gene targeting in its earliest days and became the first Japanese to successfully generate knockout mice when times were rapidly changing with the death of the Japanese Emperor and the fall of the Berlin wall. He also elucidated the control mechanism of early B-lymphocyte differentiation. In 1991, he returned to Japan to his former position of research associate at the Medical Institute of Bioregulation, Kyushu University. He became an associate professor at the same institute in 1994, and has held his current position since 1995. Concurrently since 2006, he has been a professor of the Department of Medicinal and Life Science at the Faculty of Pharmaceutical Sciences, Tokyo University of Science.