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

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.


INTRODUCTION
The production of antibodies that prevent or limit infections is central to humoral immunity. Recognition of pathogens by B cells and their activation is achieved through B cell receptor (BCR) signaling. In addition, efficient activation and differentiation of B cells into effector PCs requires either engagement of Toll-like receptors (TLRs) that are able to sense pathogen-derived molecules, or ligation of costimulatory receptors that enable crosstalk with other immune cells. Additionally, cytokines interacting with their receptors expressed on B cells can enhance and/or modulate the response. A hallmark of B cell responses is the generation of germinal centers (GCs) in draining lymph nodes and the spleen. GCs are microenvironments where activated B cells receive signals from T follicular helper (Tfh) cells to undergo BCR affinity maturation and selection (1,2). Tfh cells support B cell activation and differentiation through CD40L-CD40, CD28-CD80/86, and ICOS-ICOSL interactions, and through the production of cytokines, such as IL-2 and IL-4 that increase B cell proliferation, and IL-10 and IL-21, that enhance PC differentiation (3)(4)(5)(6)(7)(8). Activated B cells may differentiate into either antibody-producing PCs or memory B cells, where the latter are programmed to rapidly differentiate into PCs after antigen re-exposure (9,10).
Most of our understanding of PC differentiation and regulation has been acquired from mouse models, in which genetic defects or engineered reporter systems have provided detailed insight in the regulation of the PC lineage. The process of differentiation and fate commitment is an intricately regulated process that involves the coordinated actions of the transcription factors Pax5, IRF4 and Blimp-1, and occurs in a cell divisionlinked manner (11)(12)(13)(14). When B cells acquire increasing levels of Blimp-1 during the differentiation process, they extinguish the B cell programme through downregulation of Pax5 (15)(16)(17)(18), arrest cell cycling through repression of c-myc (19) and remodel the sub-cellular organization to support high antibody production through induction of XBP-1 (18,20). Terminally differentiated PCs require continued expression of IRF4 for survival, as well as Blimp-1, to maintain their ability to produce and secrete antibodies (20).
Alterations in immune responses due to mutations affecting the development or function of immune cells often results in increased susceptibility to infectious diseases. Patients diagnosed with common variable immune deficiency (CVID) typically suffer from recurrent infections, with the respiratory and gastrointestinal tracts commonly affected (21). Diagnosis of CVID is predominantly based on hypogammaglobulinemia, and most patients present in the clinic with a reduced memory B cell pool. This aside, the clinical phenotype of patients diagnosed with primary antibody deficiencies with late B cell defects, such as CVID, is widely heterogeneous and the genetic causes of most cases remain undefined (22)(23)(24)(25). However, mutations in the genes encoding Transmembrane Activator and CAML Interactor (TACI), Inducible T cell Costimulator (ICOS) and B cell Activating Factor Receptor (BAFFR) are commonly found, emphasizing the importance of intact B cell activation and signaling (26)(27)(28). The recent developments of next-generation sequencing (NGS) methods, such as whole-exome sequencing (WES) and whole-genome sequencing (WGS), have led to the discovery of mutations in many other genes associated with primary immunodeficiencies (25,29,30). For example, mutations in several components of the NF-κB and the PI3K signaling pathways, which are activated after BCR signaling, were identified (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Recently, mutations in IRF4 causing complete deficiency or haploinsufficiency have been linked to severe immune defects and predisposition to develop Whipple's disease as a result of infection with Tropheryma whipplei bacteria (42,43). Impaired plasma cell differentiation has been linked to CVID (44), but so far, only a few mutations have been identified as genetic causes for diminished plasma cell generation in PID patients, such as those resulting in IRF4 deficiency and constitutive NF-κB signaling caused by gain-of-function CARD11 mutations (42,45). Thus, the functional relevance of newly identified mutations and the extent to which they contribute to the clinical phenotype of the patient remains poorly understood.
Here, we established an approach to analyse PC differentiation from primary human B cells in response to in vitro stimulation. Firstly, we evaluated different stimulation conditions to determine which was most efficient at inducing PC differentiation. We then developed panels for intracellular staining of transcription factors known to regulate the PC differentiation process (Pax5, IRF4, Blimp-1) and methods to monitor cell proliferation (CellTrace and Ki67) to allow in-depth flow cytometric analyses of the PC differentiation state. We found that, differentiation of human B cells in vitro generated IRF4 hi Pax5 lo CD38 + cells, representing cells committed to PC differentiation. We also found that cryopreservation, which is required for the storage of patient samples, did not markedly affect the ability of the B cells to differentiate into PCs using the stimulation conditions presented here. Thus, this study offers an approach that is applicable to assess B cell function in patient samples for identification of potential plasma cell differentiation defects. We propose that applying such functional assays to CVID cases may allow the stratification of CVID patients into subgroups that do, or do not, display PC differentiation defects, which may help explain how different genetic alterations associate with clinical phenotypes.

Cell Preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Ficoll Paque Plus and washed twice in PBS. Cell suspensions were treated with red blood cell lysis buffer, after which cells were washed twice with PBS, and resuspended in complete medium. Cell suspensions were passed through a 70 µm cell strainer to remove debris. PBMCs were frozen in FBS supplemented with 10% DMSO and stored at −80 • C. Frozen PBMCs were thawed at 37 • C in a water bath, washed twice in complete medium, and passed through a 70 µm cell strainer. Cell count and viability was performed using Trypan Blue and the automated cell counter Countess II. Total peripheral human B cells were isolated using the EasySep Human B Cell Enrichment Kit (STEMCELL Technologies) according to the manufacturer's protocol. Human memory B cells and naïve B cells were both isolated using the EasySep Human Memory B cell isolation kit (STEMCELL Technologies) according to the manufacturer's protocol. Purity of the total human B cell isolation based on CD20 expression was at least 97% (Supplementary Figures 5A,B).

Flow Cytometry
To track cellular divisions, CellTrace Violet (CTV) (Invitrogen) was used at a concentration of 0.25 µM in PBS to label 1 × 10 6 cells prior to culturing. To block non-specific binding to Fc receptors, cells were incubated with human Fc block reagent (BD). Cells were then stained with different panels of fluorochrome conjugated monoclonal antibodies (Supplementary Table 1) in FACS buffer. For intracellular staining of transcription factors, cells were fixed, permeabilised and stained for 6 h using the transcription factor buffer set (BD) according to the manufacturer's protocol. For intracellular IgG staining, purified IgG was included in the surface panel to block binding to surface IgG. Samples were acquired on a BD FACSCelesta cytometer and data were analyzed by FlowJo software v9.6.4 (Tree Star, Ashland, OR).

ELISA
ELISA plates (Nunc) were coated with a 1:1,000 dilution of goat anti-human unlabelled IgG (Southern Biotech) in 100 µL PBS per well. After overnight incubation at 4 • C, plates were washed six times with washing buffer (PBS+0.05% Tween20) and blocked for 1 h with PBS containing 2% dry milk. Fifty microlitre of culture supernatant was added in a total volume of 150 µL, followed by 3-fold serial dilutions in blocking buffer, and incubated for 2 h at room temperature (RT). Plates were washed six times, and HRP-coupled goat anti-human IgG (Southern Biotech) was added in 100 µL PBS per well in a 1:1,000 dilution, followed by incubation for 1.5 h at RT. After six washes, plates were developed with TMB substrate (KPL) (Life Technologies), and the reaction was stopped with 1 M H 2 SO 4 . The OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd.).

Efficient in vitro B Cell Differentiation to PCs by BCR Ligation and Stimulation via TLR9 and CD40
To establish an efficient protocol for human PC differentiation, we cultured primary human B cells in four different stimulation cocktails that were adapted from conditions described previously (7,46,47). For each condition, we evaluated antibody production by ELISA and PC generation by flow cytometry (Figure 1A). Stimulation I induced TLR9 signaling combined with a stimuli mimicking CD4 + T cell help (46). Stimulation II contained a mixture of superantigens known to induce polyclonal B cell activation through non-specific ligation of BCRs and engagement of TLR receptors (46). Stimulation III ligated the BCR and co-stimulatory molecules and provided additional cytokine stimulation via IL-21, which is an important inducer of Blimp-1 expression, particularly in combination with CD40 ligation (47). Stimulation IV utilized a 2-step approach where, at day 4, the activation medium was replaced with medium supplemented with IL-2, IL-4, and IL-10 to assist cells at the terminal stage of differentiation (7). The optimal concentration of IL-2 for enhancing PC differentiation was determined after titration (Supplementary Figure 1). When assessing IgG immunoglobulin secretion after 6 days of culture we found that the highest IgG levels were achieved by stimulation III (Figure 1B).
To assess proliferation, we labeled B cells with CellTrace Violet (CTV) prior to culturing. Proliferation was observed in all cultures at day 3.5 but was most notable in the cultures treated with stimulation III at day 6 ( Figure 1C and Supplementary Figure 2). Cells that upregulated CD38 whilst proliferating were described to represent a population of (pre)plasma blasts and plasma cells (13,48). At day 3.5, stimulation III and IV generated similar frequencies of CD38 + cells with lower frequencies observed for stimulation I and II.
At day 6, we observed the highest frequency of CD38 + cells for stimulation IV (Figures 1D,E). During differentiation, B cells downregulate surface CD20 expression on CD38 + cells (48). Again, at day 3.5 we observed no differences between stimulation III and IV while at day 6, most CD20 − CD38 + cells were obtained for stimulation IV (Figures 1F,G). Thus, stimulation III and IV were most efficient at generating CD38 + cells and were both suitable for assessing PC differentiation in vitro, with stimulation III resulting in higher levels of IgG secretion.

In vitro Stimulation of Human B Cells Generated Populations With Distinct IRF4 and Pax5 Expression Levels
To evaluate PC differentiation in vitro at the transcription factor level, we next assessed the expression of IRF4 and Pax5. We identified three populations present at both 3.5 and 6 days of stimulation; IRF4 lo Pax5 hi , IRF4 hi Pax5 lo , and IRF4 int Pax5 lo referred to hereafter as P1, P2, and P3, respectively (Figure 2A). Between day 3.5 and day 6, we observed a decrease in the frequency of population P1, and an increase in the frequency of population P3 (Figures 2A,B). Both populations P2 and P3 contained CD27 + CD38 + cells suggesting that these two populations represent plasma blasts and plasma cells ( Figure 2C and Supplementary Figure 3A). In support of this finding, we detected intracellular IgG in 28.58 ± 11.65% of P2 cells and 29.75 ± 10.73% of P3 cells at day 3.5, which increased to ca. 40% for both populations at day 6 (Supplementary Figure 3B). Interestingly, at day 6, the majority of P3 cells exhibited lower expression of Blimp-1 and Ki67, indicating that these cells were no longer actively cycling. In contrast, population P2 had high expression of both Blimp-1 and Ki67 and thus resembled plasma cells phenotypically and transcriptionally (Figure 2C and Supplementary Figure 4). These results indicate that human plasma cell differentiation in vitro can be characterized by three stages defined by Pax5 upregulation (P1), IRF4 upregulation and Pax5 repression (P2), and finally, decrease in IRF4 expression (P3). Of these three populations, P2 exhibits a phenotype most characteristic of plasma cells as evidenced by high expression of both IRF4 and Blimp-1.

Naïve and Memory B Cells Exhibited Distinct Differentiation Kinetics
As memory B cells have the ability to respond to cognate antigen and differentiate into PCs with faster kinetics compared to naïve B cells (9,10,49), the ratio between memory and naïve B cells in the starting PBMC sample may affect the efficiency of PC differentiation in stimulated B cell cultures. Since the frequencies of memory B cells may vary between donors and, particularly for CVID patients, may be lower than those observed in healthy donors, we compared the differentiation kinetics of cultures enriched for memory B cells or naïve B cells. Based on CD27 expression, purity of the enriched populations was 91.7 ± 6.5% and 96.2 ± 1.4% for memory B cells and naïve B cells, respectively (Supplementary Figures 5A,B). The B cell enrichment methods could potentially result in the inclusion of plasma cells. However, we found very few CD20 − CD38 + cells and no distinguishable P1, P2, and P3 populations in enriched memory and naïve B cells (Supplementary Figures 5C,D). As expected, memory B cells were most efficient at producing IgG and generating CD38 + cells (Figures 3A-C). In addition, they generated higher frequencies of the P2 population already at day 3.5 (Figures 3D,E). Even though naïve B cells generated lower frequencies of CD38 + cells and populations P2 and P3 compared to total B cells at day 3.5, at day 6 the frequencies of CD38 + cells and population P2 were similar to total B cell cultures (Figures 3B,F). Taken together, these results demonstrate that the differentiation conditions used here induced PC differentiation from both memory and naïve B cells and are thus applicable to patient samples with low memory B cell frequencies.

The Efficiency of in vitro B Cell Differentiation to PCs Was Retained in Cryopreserved PBMCs
As collection of patient materials often involves freezing samples, we assessed whether the process of freezing and thawing affected the differentiation potential of B cells into PCs. To this end, B cells isolated from freshly obtained donor blood were compared to B cells isolated from frozen PBMCs. B cell purity based on CD20 expression was 98.6 ± 1.5% from fresh samples, and 98.4 ± 1.7% from frozen samples (Supplementary Figures 6A,B). The prevalence of naïve and memory B cells obtained from fresh and frozen PBMC samples after B cell isolation was similar; 65.1 ± 9.3% from fresh samples and 62.5 ± 12.6% from frozen samples for naïve B cells, and 24.7 ± 8.3% from fresh samples and 21.3 ± 5.9% from frozen samples for memory B cells (Supplementary Figures 6C,D). After 6 days of culture with stimulation III, similar levels of IgG were detected from the supernatant from frozen and fresh samples (Figure 4A). B cells isolated from frozen PBMCs proliferated to a similar extent as those from fresh PBMCs but exhibited a tendency to generate less CD38 + cells (Figures 4B,C). Frequencies of populations P1, P2, and P3 and their phenotypes were also similar between fresh and frozen samples (Figures 4D-F). Hence, cryopreservation of PBMC samples did not appear to affect the ability of the B cells to differentiate into PCs and produce antibodies after in vitro culturing using the stimulation III conditions described here.

DISCUSSION
CVID comprises a family of primary immunodeficiencies with patients suffering from increased susceptibility to infections due to hypogammaglobulinemia and impaired antibody responses (21). Most underlying genetic causes have been described in the receptors and ligands required for B cell signaling or B-T cell interactions, thus interfering with B cell activation and differentiation (28). The association between mutations in certain genes and the development of CVID is only well established for 2-10% of cases (21). Whole genome or exome sequencing efforts have resulted in the discovery of novel mutations in genes that are associated with CVID (29,30). However, how most of these mutations impact B cell differentiation and function remains poorly understood. Immunophenotypic characterization of immunodeficient patients is useful for defining subcategories as patients can present with variable B cell numbers, and reduced frequencies of isotype switched memory B cells and plasma cells (50,51). However, immunophenotyping does not address potential causes of B cell abnormalities. These studies include analysis of transcription factors involved in PC differentiation and B cell proliferation. Based on these parameters, and in contrast to standard analysis of PCs (CD38 + CD27 − IgM − ), our assay may be able to further differentiate CVID patients into subgroups displaying more specific defects. In this study, we established flow cytometry-based protocols for the analysis of human plasma cell differentiation of primary B cells, which are suitable for the analysis of CVID patient samples ( Figure 5).
Our results showed that in vitro PC differentiation was most efficiently induced by combining BCR and TLR9 ligation with stimuli mimicking T cell help, which induced the production of IRF4 hi Pax5 lo CD38 + cells, a phenotype typical of B cells that have committed to PC differentiation. The next best stimulation conditions based on our evaluation was a two-step approach (stimulation IV), which also efficiently generated PC differentiation. However, this protocol was less practical, and the washing and reseeding steps resulted in a lower yield of secreted antibodies due to the need to change medium on day 4. Since the frequency of memory B cells present in some CVID patients may be lower than that observed in healthy donors, we considered it important to investigate the relative contribution of naïve vs. memory B cells to plasma cell differentiation under the conditions used. In the present study, the frequency of memory B cells was 24.7 ± 8.3% and the frequency of naïve B cells was 65.1 ± 9.3% based on expression of CD27 in the different donors (Supplementary Figures 5A,B). Despite this variation, plasma cell differentiation in naïve B cell cultures was readily detectable as CD38 + IRF4 hi Pax5 lo cells were generated, albeit at a lower pace compared to total B cell cultures, suggesting that the method described is applicable also to donors with low memory B cell frequencies. In addition, B cells from cryopreserved PBMCs retained the ability to generate CD38 + IRF4 hi Pax5 lo cells, though at day 3.5 we observed a tendency toward lower frequencies of CD38 + cells and IRF4 int Pax5 lo cells compared to those from fresh samples.
IRF4 is required for initiating plasma blast differentiation, whereas Blimp-1 is critical for converting the gene expression state of the cell to being PC-specific. Blimp-1 is absolutely indispensable for antibody production and secretion through its regulation of both the unfolded protein response (UPR) and cellular metabolism (18,20). During differentiation, the B cell-specific gene program is extinguished, which is reflected by decreased Pax5 levels (15,16). Absence or reduced frequencies of CD38 + cells, altered or reduced expression of IRF4 or Blimp-1, or failed downregulation of Pax5 after in vitro stimulation compared to healthy donor controls could help identify defects in plasma cell differentiation in patients. Additionally, accelerated or premature upregulation of Blimp-1 could indicate impaired survival of differentiating cells committed to PC fate (52).
Identifying and characterizing mutations in genes involved in PC differentiation might help explain alterations in memory and PC frequencies, hypogammaglobulinemia, and the increased susceptibility of infections observed in CVID patients. In summary, establishing and applying functional PC differentiation assays to patient samples may be used to stratify patients suffering from primary antibody deficiencies with late B cell defects into subgroups to increase our understanding of disease phenotypes.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
SK and GKH conceived and directed the study. SK performed experiments, data collection, analysis and interpretation with help of MÁ. All authors discussed the results. SK wrote the manuscript with critical feedback from MÁ, GP, and GKH.   Figure 1A, and analyzed at day 3.5 and day 6 by flow cytometry. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. Representative histogram overlays show cellular divisions from five donors based on CTV dilution. Data are representative of three independent repeats with 5 donors per experiment.
Supplementary Figure 3 | Population P2 and P3 contain cells with a plasma blast/plasma cell phenotype. Total B cells were isolated from healthy donor blood, cultured with stimulation III, and analyzed at day 3.5 and day 6 by flow cytometry. Cells were pre-gated on FSC-A/FSC-W to identify singlets, on FSC-A/SSC-A to identify lymphocytes, on Live/Dead Green − to exclude dead cells, and on IRF4 and Pax5 expression to identify populations P1, P2, and P3.  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, on FSC-A/SSC-A to identify lymphocytes, and on IRF4/ Pax5 expression to identify the P1, P2, and P3 populations. Representative plots showing CD38, Blimp-1 and Ki67 expression for the IRF4 low Pax5 hi (P1), IRF4 hi Pax5 lo (P2), and IRF4 int Pax5 lo (P3) populations.
Supplementary Figure 5 | Purity of naïve and memory B cell populations before and after enrichment. (A) Representative plots showing gating strategy for CD20 + CD38 + CD27 − naïve and CD20 + CD38 + CD27 + memory B cell populations from fresh PBMCs and enriched populations. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. Numbers adjacent to gates indicate cell frequencies. (B) Frequencies of CD20 + CD38 + CD27 − naïve and CD20 + CD38 + CD27 + memory B cell populations before and after enrichment. Data are representative of three independent repeats with 4-5 donors per experiment. (C) Representative plots showing gating strategy for CD20 − CD38 + cells and populations P1, P2, and P3 based on IRF4 and Pax5 expression from total, memory, and naïve B cells immediately after enrichment. Cells cultured for 3.5 days with stimulation III are included as gating control. Cells were pre-gated on FSC-A/FSC-W to identify singlets, on FSC-A/SSC-A to identify lymphocytes, and on Live/Dead Green − to exclude dead cells. Numbers adjacent to gates indicate cell frequencies. (D) Frequencies of CD20 − CD38 + cells. Data are representative of two independent repeats with 3 donors per experiment.
Supplementary Figure 6 | Naïve and memory B cell populations in pre-and post-isolation samples from fresh PBMCs and frozen PBMCs. Cells were pre-gated on FSC-A/FSC-W to identify singlets and on FSC-A/SSC-A to identify lymphocytes. (A) Representative histogram showing gating strategy for the CD20 + population before and after isolation. (B) Frequencies of CD20 + B cells before and after isolation are shown for four samples from fresh PBMCs and four samples from frozen PBMCs. (C) Representative plots showing gating strategy for CD20 + CD38 + CD27 − naïve and CD20 + CD38 + CD27 + memory B cell populations from fresh PBMCs (left panel) and frozen PBMCs (right panel). Numbers adjacent to gates represent the frequencies of cells gated on. (D) Graph showing frequencies of CD20 + CD38 + CD27 − naïve and CD20 + CD38 + CD27 + memory B cell populations before and after isolation. Data are representative of three independent repeats with 4-5 donors per experiment.