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Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells

Abstract

Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR–Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.

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Fig. 1: Schematic overview of the expansion, immortalization and use of human GC B cells cultured on FDC-like feeder cells.
Fig. 2: Summary of the GC B-cell purification strategy.
Fig. 3: Example of successful GC B-cell enrichment.
Fig. 4: Examples of the use of YK6-CD40Lg-IL21 and modified feeder systems.
Fig. 5: Gene knockout using virally transduced Cas9 and gRNA.
Fig. 6: Gene knockout using transient nucleofection of RNP complexes.
Fig. 7: Typical histological and immunohistochemical appearances of an example ‘synthetic’ human tumor.

Data availability

All raw data underlying the figures are provided as source data files. Other examples of data can be made available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

D.H. was personally supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). Research in the Hodson laboratory is supported by the Kay Kendall Leukaemia Fund, The Addenbrooke’s Charitable Trust and the Evelyn Trust. The Hodson laboratory receives core funding from Wellcome (203151/Z/16/Z) and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and from CRUK to the CRUK Cambridge Centre (A25117). We thank J. Beswick, A. Mitchel and N. Jonas from the ENT Department at Addenbrooke’s Hospital, Cambridge for their assistance in the collection of primary tonsil tissue. We are grateful to K. Elston and J. Baxter from the Cambridge Blood and Stem Cell Bank for collection and storage of primary tonsils tissue and to the staff of the Central Biomedical Services for animal housing and care. We thank A. Weiss for expert technical advice. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub and we wish to thank all members of the flow cytometry core for their advice and support in flow cytometry.

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Contributions

R.C., J.G., M.D.R., C.G. and D.H. contributed to the development and optimization of the protocols described in this manuscript. R.C. and D.H. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Daniel J. Hodson.

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Competing interests

R.C.: consultancy for Karus Therapeutics. D.H.: research funding from Gilead Sciences. The remaining authors declare no competing interests.

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Peer review information Nature Protocols thanks Wolfgang Hammerschmidt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Caeser, R. et al. Nat. Commun. 10, 4543 (2019): https://doi.org/10.1038/s41467-019-12494-x

Sommermann, T. et al. Proc. Natl Acad. Sci. USA 117, 14421–14432 (2020): https://doi.org/10.1073/pnas.1921139117

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Caeser, R., Gao, J., Di Re, M. et al. Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells. Nat Protoc 16, 2499–2519 (2021). https://doi.org/10.1038/s41596-021-00506-4

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