IL-21/CD40L stimulation of human B-lymphocytes in vitro and their characteristics

Abstract

Introduction. Cell lines and cultures are indispensable tools in cell physiology research. Cultures of B lymphocytes are essential for the study of B-cell response, antibody generation, as well as for the creation of therapeutic monoclonal antibodies. At the same time, the activation of B-lymphocytes and their long-term cultivation in vitro continues to be an unsolved problem.

The aim of the study - development of a protocol for the stimulation of human B lymphocytes in vitro, characterization of the phenotype and functional characteristics of stimulated B cells for the subsequent use of the protocol for determining the number of memory B cells, sequencing of Ig genes, as well as obtaining immortalized clones of B lymphocytes.

Material and methods. Cells stably transfected with the CD40L gene were generated by lentiviral transduction. B lymphocytes were isolated from human peripheral blood by centrifugation in a density gradient of ficoll-verografin with further enrichment by negative selection using magnetic beads. B lymphocytes were stimulated in vitro with feeder cells carrying surface CD40L in the presence of exogenous recombinant interleukin(IL)-21. The number and phenotype of stimulated B lymphocytes were determined using multicolor flow cytometry. The secretion of IgM and IgG in cultures of B lymphocytes was assessed using enzyme-linked immunosorbent assay.

Results. Using lentiviral transduction, HEK293, K562 and A549 cells stably transfected with the CD40L gene were obtained. Based on the expression of the CD40L molecule, as well as growth properties, the transfected A549 cell line was selected for further research as a feeder. When feeder cells and B lymphocytes were co-cultured in the presence of exogenous IL-21, stimulation of B lymphocytes was observed. B lymphocytes began to proliferate and after 7 days their number increased by an average of 8 times. The stimulated B lymphocytes changed their morphology. They acquired an irregular shape with pseudopodia, grouped around feeder cells, and began to move actively. Upon IL-21/CD40L stimulation, B lymphocytes changed their surface phenotype and by day 10, about 40 % of B cells had differentiated into plasmablasts (CD19+CD27+CD38+). At the same time, the proportion of CD20+ cells decreased to 20 %. The percentage of cells with surface Ig expression also decreased. In contrast, the secretion of IgM and IgG increased during stimulation. By the 12th day, the proliferative potential of B cells was exhausted, and they died.

Сonlusion. In vitro cultivation of B lymphocytes in the presence of exogenous IL-21, as well as feeder cells expressing the CD40L molecule, is a convenient system for obtaining activated B lymphocytes that can be used in different applications.

Keywords:cell culture; B lymphocytes; plasmablasts; IL-21; CD40L

For citation: Byazrova M.G., Astakhova E.A., Spiridonova A.B., Vasileva Yu.V., Prilipov A.G., Filatov A.V IL-21/CD40L stimulation of human B-lymphocytes in vitro and their characteristics. Immunologiya. 2020; 41 (6): 501-10. DOI: https://doi.org/10.33029/0206-4952-2020-41-6-501-510. (in Russian)

Funding. The study was supported by the grant of Russian Science Foundation No 19-15-00331.

Conflict of interests. The authors declare no conflict of interests.

References

1. Gearhart P.J., Mock B.A., Casellas R., Cancro M.P. The reign of antibodies: A celebration of and tribute to Michael Potter and his homogeneous immunoglobulin workshops. J. Immunol. 2018; 200: 23–6. DOI: https://www.doi.org/10.4049/jimmunol.1701516.

2. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256: 495–7. DOI: https://www.doi.org/10.1038/256495a0.

3. Küppers R. B cells under influence: transformation of B cells by Epstein–Barr virus. Nat. Rev. Immunol. 2003; 3: 801–2. DOI: https://www.doi.org/10.1038/nri1201.

4. Nilsson K. Human B-lymphoid cell lines. Hum. Cell. 1992; 5: 25–41. PMID: 1329931.

5. Crain M.J., Sanders S.K., Butler J.L., Cooper M.D. Epstein-Barr virus preferentially induces proliferation of primed B cells. J. Immunol. 1989; 143: 1543–8. PMID: 2547870.

6. Laffly E., Sodoyer R. Monoclonal and recombinant antibodies, 30 years after. Hum. Antibodies. 2005; 14: 33–55. PMID: 16424599.

7. O’Nions J., Allday M.J. Proliferation and differentiation in isogenic populations of peripheral B cells activated by Epstein–Barr virus or T cell-derived mitogens. J. Gen. Virol. 2004; 85: 881–95. DOI: https://www.doi.org/10.1099/vir.0.19704-0.

8. Steinitz M. Production of human monoclonal antibodies by the Epstein–Barr virus method. In: Steinitz M. (ed). Methods Mol. Biol. Humana Press: Totowa, N.J. 2014; 1060: 111–22. DOI: https://www.doi.org/10.1007/978-1-62703-586-6_6.

9. Mossalayi M.D., Lecron J.C., Tanzer J., Goube de Laforest P. Relationship between IL-2 and human T cell colony formation. Clin. Exp. Immunol. 1986; 66: 532–8. PMID: 3494553.

10. Rohaan M.W., van den Berg J.H., Kvistborg P., Haanen J.B.A.G. Adoptive transfer of tumor-infiltrating lymphocytes in melanoma: a viable treatment option. J. Immunother. Cancer. 2018; 6: 102. DOI: https://www.doi.org/10.1186/s40425-018-0391-1.

11. Auladell M., Nguyen T.H., Garcillán B., Mackay F., Kedzierska K., Fox A. Distinguishing naïve-from memory-derived human B cells during acute responses. Clin. Transl. Immunol. 2019; 8. DOI: https://www.doi.org/10.1002/cti2.1090.

12. Banchereau J., Bazan F., Blanchard D., Briè F., Galizzi J.P., van Kooten C., Liu Y.J., Rousset F., Saeland S. The CD40 antigen and its ligand. Annu. Rev. Immunol. 1994; 12: 881–926. DOI: https://www.doi.org/10.1146/annurev.iy.12.040194.004313.

13. Huggins J., Pellegrin T., Felgar R.E., Wei C., Brown M., Zheng B., Milner E.C.B., Bernstein S.H., Sanz I., Zand M.S. CpG DNA activation and plasma-cell differentiation of CD27− naive human B cells. Blood. 2007; 109: 1611–9. DOI: https://www.doi.org/10.1182/blood-2006-03-008441.

14. Wiesner M., Zentz C., Mayr C., Wimmer R., Hammerschmidt W., Zeidler R., Moosmann A. Conditional immortalization of human B cells by CD40 ligation. PLoS ONE. 2008; 3: e1464. DOI: https://www.doi.org/10.1371/journal.pone.0001464.

15. Filatov A.V., Lavrova O.I., Mazurov D.V., Chevalier A.F. Expression of Lymphocyte Phosphatase-Associated Protein on human lymphoid lines. Immunologiya. 2011; 32 (1): 30–4. (in Russian)

16. Filatov A.V., Krotov G.I., Zgoda V.G., Volkov Y. Fluorescent immunoprecipitation analysis of cell surface proteins: A methodology compatible with mass-spectrometry. J. Immunol. Meth. 2007; 319: 21–33. DOI: https://www.doi.org/10.1016/j.jim.2006.09.014.

17. Khvastunova A.N., Kuznetsova S.A., Al-Radi L.S., Vylegzhanina A.V., Zakirova A.O., Fedyanina O.S., Filatov A.V., Vorobjev I.A., Ataullakhanov F. Anti-CD antibody microarray for human leukocyte morphology examination allows analyzing rare cell populations and suggesting preliminary diagnosis in leukemia. Sci. Rep. 2015; 5: 12573. DOI: https://www.doi.org/10.1038/srep12573.

18. Berg E.A., Fishman J.B. Labeling antibodies with Cy5-phycoerythrin. Cold Spring Harb. Protoc. 2019; pdb.prot099317. DOI: https://www.doi.org/10.1101/pdb.prot099317.

19. Vira S., Mekhedov E., Humphrey G., Blank P.S. Fluorescent-labeled antibodies: Balancing functionality and degree of labeling. Anal. Biochem. 2010; 402: 146–50. DOI: https://www.doi.org/10.1016/j.ab.2010.03.036.

20. Lushova A.A., Zheremyan E.A., Astakhova E.A., Spiridonova A.B., Byazrova M.G., Filatov A.V. B-lymphocyte subsets: functions and molecular markers. Immunologiya. 2019; 40 (6): 63–76. DOI: https://www.doi.org/10.24411/0206-4952-2019-16009. (in Russian)

21. Kwakkenbos M.J., Bakker A.Q., van Helden P.M., Wagner K., Yasuda E., Spits H., Beaumont T. Genetic manipulation of B cells for the isolation of rare therapeutic antibodies from the human repertoire. Methods. 2014; 65: 38–43. DOI: https://www.doi.org/10.1016/j.ymeth.2013.07.002.

22. Kwakkenbos M.J., Diehl S.A., Yasuda E., Bakker A.Q., van Geelen C.M.M., Lukens M.V. van Bleek G.M., Widjojoatmodjo M.N., Bogers W.M.J.M., Mei H., Radbruch A., Scheeren F.A., Spits H., Beaumont T. Generation of stable monoclonal antibody–producing B cell receptor–positive human memory B cells by genetic programming. Nat. Med. 2010; 16: 123–8. DOI: https://www.doi.org/10.1038/nm.2071.

23. Kwakkenbos M.J., Helden P.M., Beaumont T., Spits H. Stable longterm cultures of selfrenewing B cells and their applications. Immunol. Rev. 2016; 270: 65–77. DOI: https://www.doi.org/10.1111/imr.12395.

24. Boldyreva M.N. SARS-CoV-2 virus and other epidemic coronaviruses: pathogenetic and genetic factors for the development of infections. Immunologiya. 2020; 41 (3): 197–205. DOI: https://www.doi.org/10.33029/0206-4952-2020-41-3-197-205. (in Russian)

All articles in our journal are distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0 license)


JOURNALS of «GEOTAR-Media»