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. 2020 Sep 29:11:571321.
doi: 10.3389/fimmu.2020.571321. eCollection 2020.

Flow Cytometry-Based Protocols for the Analysis of Human Plasma Cell Differentiation

Sharesta Khoenkhoen  1 , Monika Ádori  1 , Gabriel K Pedersen  1 , Gunilla B Karlsson Hedestam  1
Affiliations

Flow Cytometry-Based Protocols for the Analysis of Human Plasma Cell Differentiation

Sharesta Khoenkhoen et al. Front Immunol. .

Abstract

Humoral immunity is established after differentiation of antigen-specific B cells into plasma cells (PCs) that produce antibodies of relevant specificities. Defects in the development, activation, or differentiation of B cells severely compromises the immune response. Primary immunodeficiencies are often characterized by hypogammaglobulinemia and the inability to mount effective antigen-specific antibody responses, resulting in increased susceptibility to infections. After IgA deficiency, which is most often asymptomatic, common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency, but in most cases the underlying genetic causes are unknown or their roles in disease pathogenesis are poorly understood. In this study, we developed a protocol for in vitro stimulation of primary human B cells for subsequent analyses of PC differentiation and antibody production. With this approach, we were able to detect a population of CD38+ IRF4+ Blimp-1+ cells committed to PC fate and IgG production, including when starting from cryopreserved samples. The application of functional assays to characterize PC differentiation and possible defects therein in B cells from patients suffering from primary antibody deficiencies with late B cell defects could increase our understanding of the disease pathophysiology and underlying mechanisms.

Keywords: B cell proliferation; Blimp-1/PRDM1; CVID; IRF4; Pax5; common variable immunodeficiency; plasma cell differentiation.

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Figures

Figure 1
Figure 1
Antibody production and phenotype of primary human B cells after in vitro stimulations and culturing. (A) Schematic representation of the experimental approach. Total human B cells were isolated from healthy donor blood, stimulated under the indicated conditions and analyzed at day 3.5 and day 6 by flow cytometry to evaluate PC generation. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. The supernatant was collected at day 6 to evaluate IgG production by ELISA. This overview was created using images from Servier Medical Art, which are licensed under a Creative Commons Attribution 3.0 Unported License (http://smart.servier.com). (B) Total IgG production from supernatant of cultured cells at day 6 was evaluated by ELISA. Each line and error bar indicate mean ± s.d. (C) Cells were labeled with CTV prior to stimulation. Representative histogram plots show an overlay of CTV dilution indicating proliferation in response to the four stimulations. (D) Representative plots showing gating strategy for CD38+ cell population. Numbers in gates represent cell frequencies. (E) Frequencies of CD38+ cells are summarized in a box plot with whiskers indicating the minimum and maximum values. Data are pooled from three independent repeats with 5 donors per experiment. Statistical significance was determined by Mann-Whitney U-test. (F) Representative plots showing gating strategy for CD20− CD38+ plasma cell population. Numbers in or adjacent to gates represent cell frequencies. (G) Frequencies of CD20− CD38+ plasma cell population are summarized in a box plot with whiskers indicating the minimum and maximum values. Data are pooled from three independent repeats with 5 donors per experiment. Statistical significance was determined by Mann-Whitney U-test.
Figure 2
Figure 2
Flow cytometric analysis of transcription factor expression in human primary B cells after in vitro differentiation. Total human B cells from healthy donors were stimulated under conditions described in Figure 1A and analyzed by flow cytometry at days 3.5 and 6. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. (A) Representative plots showing gating strategy for IRF4lo Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Numbers in or adjacent to gates indicate cell frequencies. (B) Frequencies of IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) cells. Bars and error bars indicate mean ± s.d. Data are pooled from three independent repeats with 5 donors per experiment. (C) Representative plot overlays showing CD38, Blimp-1 and Ki67 expression for the IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations.
Figure 3
Figure 3
Differentiation of memory B cells and naïve B cells in vitro. Memory B cells, naïve B cells, and total B cells were isolated from healthy donors, cultured with stimulation III, and analyzed by flow cytometry at day 3.5 and day 6. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. (A) Total IgG production from supernatant of cultured cells at day 6 was evaluated by ELISA. Each line and error bar indicate mean ± s.d. (B) Representative plots showing gating strategy for CD38+ population. Numbers adjacent to gates indicate cell frequencies. (C) Frequencies of CD38+ cells with symbols and error bars indicating mean ± s.d. (D) Representative plots showing gating strategy for IRF4lo Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Numbers in or adjacent to gates represent cell frequencies. (E) Frequencies of IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Bars and error bars indicate mean ± s.d. (F) Plot overlays showing CD38, Blimp-1 and Ki67 expression for IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Data are representative of three independent repeats with 3–5 donors per experiment.
Figure 4
Figure 4
Comparison of PC differentiation of B cells isolated freshly from PBMCs or from cryopreserved PBMCs. Total human B cells were isolated from fresh PBMCs and cryopreserved PBMCs. IgG production and PC differentiation were assessed in response to stimulation III. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. (A) Total IgG production from supernatant of cultured cells at day 6 was evaluated by ELISA. Each line and error bar indicate mean ± s.d. (B) Representative plots showing gating strategy for CD38+ population. Numbers adjacent to gates indicate cell frequencies. (C) Frequencies of CD38+ cells summarized with bars and error bars indicating mean ± s.d. (D) Representative plots showing gating strategy for IRF4lo Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Numbers in or adjacent to gates represent cell frequencies. (E) Frequencies of IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Bars and error bars indicate mean ± s.d. (F) Plot overlays showing CD38, Blimp-1 and Ki67 expression for IRF4low Pax5hi (P1), IRF4hi Pax5lo (P2), and IRF4int Pax5lo (P3) populations. Data are representative of three independent repeats with 4–5 donors per experiment.
Figure 5
Figure 5
Schematic representation of the optimized protocol for the in vitro induction and analysis of plasma cell differentiation from human primary B cells. Proliferation (CTV), CD38, CD20, Pax5, IRF4, Blimp-1, and Ki67 expression can be analyzed simultaneously and requires 2.5 × 105 isolated B cells per time point. This overview was created using images from Servier Medical Art, which are licensed under a Creative Commons Attribution 3.0 Unported License (http://smart.servier.com).
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