GENETIC MODIFICATION OF PRIMARY HUMAN B CELLS TO MODEL THE PROCESS OF B CELL DEVELOPMENT IN GERMINAL CENTERS
- Authors: Byazrova M.G.1,2, Sukhova M.M.1,3, Mikhailov A.A.1,3, Prilipov A.G.1, Filatov A.1,3
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Affiliations:
- National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation
- Peoples’ Friendship University of Russia of the Ministry of Science and Higher Education of the Russian Federation, 117198, Moscow, Russian Federation
- Lomonosov Moscow State University, 119991, Moscow, Russian Federation
- Section: Joint Immunology Forum 2024
- URL: https://rusimmun.ru/jour/article/view/16622
- DOI: https://doi.org/10.46235/1028-7221-16622-GMO
- ID: 16622
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Abstract
Abstract
The main stages of maturation of antigen-specific B cells occur in the germinal centers of the lymph nodes. During the process of differentiation, a decision is made on which path the B cells will take to develop further. They will either turn into short-lived plasmablasts or memory B cells or plasma cells. The relationship between these processes is very important for the development of a productive humoral immune response. The goal of the work was to create a system that is capable of simulating ex vivo processes occurring in germinal centers. We used primary B cells from human peripheral blood as starting material. B lymphocytes were stimulated in vitro using feeder cells carrying CD40L molecules and recombinant IL-21. Upon IL-21/CD40L stimulation, B lymphocytes changed their morphology, surface phenotype, and functional activity. After active expansion for 10 days, further cell growth stopped, and after some time they died. To generate stably proliferating B cells, we used lentiviral transduction of IL-21/CD40L stimulated IgM+ B cells. For this purpose, lentivirus preparations were obtained that carried a cassette consisting of the BCL6 and BCL2L1 genes, separated by a sequence encoding the self-cutting peptide P2A, as well as a GFP reporter gene separated from the target genes by an IRES element. The cassette used ensured the synthesis of the Bcl-6 transcription factor and the Bcl-XL protein in target cells. The Bcl-6 repressor prevented B cells from undergoing terminal differentiation and becoming plasma cells, and the Bcl-XL protein had an anti-apoptotic effect. Transduced B cells proliferated for more than a month and maintained a plasmablast phenotype. 42 days after the start of stimulation, transduced B cells remained GFP-positive, coexpressed CD27 and CD38 antigens, carried surface CD20 and IgM, intracellular Bcl-6, Bcl-XL and IgM, retained IgM secretion, but remained negative for surface and IgM. intracellular IgG. The proven stimulation system will allow us to simulate key aspects of B cell development in germinal centers to study the formation of B cell memory, which will ultimately facilitate the development of effective vaccines.
Keywords
About the authors
Maria Georgievna Byazrova
National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation;Peoples’ Friendship University of Russia of the Ministry of Science and Higher Education of the Russian Federation, 117198, Moscow, Russian Federation
Email: mbyazrova@list.ru
without degree, research fellow
Russian Federation, 115522, Russian Federation Moscow, Kashirskoye Shosse, 24Maria Mikhailovna Sukhova
National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation;Lomonosov Moscow State University, 119991, Moscow, Russian Federation
Email: mary.sukhova13@gmail.com
without degree, junior researcher
Russian Federation, 115522, Russian Federation Moscow, Kashirskoye Shosse, 24Artem Andreevich Mikhailov
National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation;Lomonosov Moscow State University, 119991, Moscow, Russian Federation
Email: artem.mihaylov.2001@mail.ru
without degree, laboratory assistant
Russian Federation, 115522, Russian Federation Moscow, Kashirskoye Shosse, 24Alexey Gennadievich Prilipov
National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation
Email: a_prilipov@mail.ru
ORCID iD: 0000-0001-8755-1419
Doctor of Biological Sciences, senior researcher, Laboratory of Immunochemistry
Russian Federation, 115522, Russian Federation Moscow, Kashirskoye Shosse, 24Alexander Filatov
National Research Center – Institute of Immunology of the Federal Medical-Biological Agency, 115522, Moscow, Russian Federation;Lomonosov Moscow State University, 119991, Moscow, Russian Federation
Author for correspondence.
Email: avfilat@yandex.ru
ORCID iD: 0000-0002-6460-9427
SPIN-code: 4229-3397
Scopus Author ID: 264609427
ResearcherId: AAH-2126-2021
professor, head of the Laboratory of Immunochemistry
Russian Federation, 115522, Russian Federation Moscow, Kashirskoye Shosse, 24References
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