Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a B cell specific process required for the generation of specific and high affinity antibodies during the maturation of the immune response against foreign antigens. This process depends on the activity of both activation-induced cytidine deaminase (AID) and several DNA repair factors. AID-dependent SHM creates the full spectrum of mutations in Ig variable (V) regions equally distributed at G/C and A/T bases. In most mammalian cells, deamination of deoxycytidine into uracil during S phase induces targeted G/C mutagenesis using either direct replication of uracils or TLS mediated bypass, however only the machinery of activated B lymphocytes can generate A/T mutagenesis around AID-created uracils. The molecular mechanism behind the latter remains incompletely understood to date. However, the lack of a cellular model that reproduces both G/C and A/T mutation spectra constitutes the major hurdle to elucidating it. The few available B cell lines used thus far to study Ig SHM indeed undergo mainly G/C mutations, that make them inappropriate or of limited use. In this report, we show that in the Ramos cell line that undergoes constitutive G/C-biased SHM in culture, the low rate of A/T mutations is due to an imbalance in the ubiquitination/deubiquitination reaction of PCNA, with the deubiquitination reaction being predominant. The inhibition of the deubiquitinase complex USP1-UAF1 or the expression of constitutive fusion of ubiquitin to PCNA provides the missing clue required for DNA polymerase η recruitment and thereafter the introduction of A/T base pair (bp) mutations during the process of IgV gene diversification. This study reports the establishment of the first modified human B cell line that recapitulates the mechanism of SHM of Ig genes in vitro.

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“…Human tonsils were obtained as discarded material from routine tonsillectomies. B cell isolation was performed as described in (26).…”

Section: Cell Lines and Culture Conditionsmentioning

confidence: 99%

Expression of Constitutive Fusion of Ubiquitin to PCNA Restores the Level of Immunoglobulin A/T Mutations During Somatic Hypermutation in the Ramos Cell Line

et al. 2022

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“…No replicates were tested since the number of primary cells from a given donor is, regardless of the expansion step, limited, while cells from different donors must be expected to vary in their experimental response [39]. We limited our testing to N/P ratios corresponding to polymer concentrations ≤40 µg mL −1 for nano-stars and ≤4 µg mL −1 for No replicates were tested since the number of primary cells from a given donor is, regardless of the expansion step, limited, while cells from different donors must be expected to vary in their experimental response [39]. We limited our testing to N/P ratios corresponding to polymer concentrations ≤40 µg mL −1 for nano-stars and ≤4 µg mL −1 for l-PEI to avoid possible cytotoxic effects.…”

Section: Comparison Of Pdmaema-nano-stars To L-pei For the Transfection Of Primary Human B Lymphocytesmentioning

confidence: 99%

“…The tonsillar B cells used in this study consisted of several heterogeneously represented subpopulations distinguishable by their CD markers [57,58] (i.e., CD19, CD20, CD27, CD38) [39]. Moreover, a set of mitogenic factors was used to activate the B cells prior to transfection.…”

Section: Possible Effects Of B Cell Polyclonality During Transfectionmentioning

confidence: 99%

“…B cells were isolated as previously described [39]. Briefly, after removal by surgery, tonsillar tissue was immediately transferred into an ice-cold buffer (HBSS containing 100 U mL −1 penicillin, 100 µg mL −1 streptomycin, 2.5 µg mL −1 amphotericin B, 2mM Ethylenediaminetetraacetic acid (EDTA), and 0.5% (w/v) bovine serum albumin (BSA)) and placed on ice for the transport.…”

Section: Cell Culturementioning

confidence: 99%

“…In particular, the polymer density (µg per 10 6 cells), the polymer concentration (µg per mL), the cell density during the transfection, as well as the contact time between DNA/polycation polyplexes and cells, are critical for the transfection outcome (transfection efficiency, cell viability) [36]. Moreover, preparations of tonsillar B cell pools contain several B cell subpopulations that further differentiate upon cultivation in the presence of the mitogenic agents [39]. Compared to transfection of cell lines, the non-clonality of the primary B cells introduces an additional parameter that needs to be considered during protocol development.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Keim

Gollner

et al. 2021

IJMS

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Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current “gold standard”, namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.

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Cultivation of Encapsulated Primary Human B Lymphocytes: A First Step toward a Bioartificial Germinal Center

Helm

Huang

Gollner

et al. 2022

Macromolecular Bioscience

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Polyelectrolyte microcapsules based on sodium cellulose sulfate (SCS) and poly‐diallyl‐dimethyl‐ammonium chloride (PDADMAC) have previously been proposed as a suitable ex vivo microenvironment for the cultivation and differentiation of primary human T lymphocytes. Here, the same system is investigated for the cultivation of human primary B cells derived from adult tonsillar tissue. Proliferation and differentiation into subtypes are followed and compared to suspension cultures of B cells from the same pool performed in parallel. Total cell expansion is somewhat lower in the capsules than in the suspension cultures. More importantly, however, the differentiation of the initially mainly memory B cells into various subtypes, in particular into plasma cell (PC), shows significant differences. Clearly, the microenvironment provided by the microcapsules is beneficial for an accelerated induction of a germinal center‐like B cell phenotype and afterward supports the long‐term survival of the PC cells. Then, varying the encapsulation conditions (i.e., presence of human serum and dedicated cytokines in the capsule core) provides a tool for finetuning the B cell response. Hence, this methodology is suggested to pave the way toward ex vivo development of human immune organoids.

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Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application

Cited by 4 publications

References 66 publications

Expression of Constitutive Fusion of Ubiquitin to PCNA Restores the Level of Immunoglobulin A/T Mutations During Somatic Hypermutation in the Ramos Cell Line

Expression of Constitutive Fusion of Ubiquitin to PCNA Restores the Level of Immunoglobulin A/T Mutations During Somatic Hypermutation in the Ramos Cell Line

Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Cultivation of Encapsulated Primary Human B Lymphocytes: A First Step toward a Bioartificial Germinal Center

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