Abstract
The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B cell culture is the capacity to support mature B cell proliferation. We developed a culture method to support the efficient activation and proliferation of naive and memory human B cells. This culture supports extensive B cell proliferation, with ∼103-fold increases following 8 d in culture and 106-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naive B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved and, when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHC class II, CD80, and CD86. CD B cells act as APCs and present alloantigens and microbial Ags to T cells. We are able to activate and expand Ag-specific memory B cells; these cultured cells are highly effective in presenting Ag to T cells. We characterized the TCR repertoire of rare Ag-specific CD4+ T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4+ T cells differs from resting and unspecifically activated CD4+ T cells. Moreover, we found that TT-specific TCR Vβ usage by CD4+ T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.
Footnotes
This work was supported in part by Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant AI100645-02 and Autoimmunity Center of Excellence Grant AI56363.
The TCRβ sequence data sets presented in this article have been submitted to the immunoSEQ Analyzer database (https://clients.adaptivebiotech.com/pub/9816555b-5673-4316-85e2-244acb293f0b) under accession numbers 14_d0_CD4naive-2016881253, 14_d7_activatedCD4-2016881253, 14_d7_TTspecificCD4-2016881253, 6_d0_CD4naive-2016881253, 6_d7_activatedCD4-2016881253, and 6_d7_TTspecificCD4-2016881253.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- 7-AAD
- 7-aminoactinomycin D
- CD
- culture-derived
- DC
- dendritic cell
- FSC
- forward scatter
- HA
- recombinant influenza hemagglutinin
- MFI
- mean fluorescent intensity
- MHCII
- MHC class II
- rPA
- recombinant Bacillus anthracis protective Ag
- Td
- tetanus–diphtheria toxoid
- TT
- tetanus toxoid
- TT-PE
- TT conjugated with PE.
- Received October 19, 2015.
- Accepted August 30, 2016.
- Copyright © 2016 by The American Association of Immunologists, Inc.