Optimized Protocols for In-Vitro T-Cell-Dependent and T-Cell-Independent Activation for B-Cell Differentiation Studies Using Limited Cells

Abstract

Background/methods: For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19⁺ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared.

Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B-cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples.

Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.

Keywords: B-cell activation; B-cell differentiation; CD40L; CpG; IL-2; IL-21; plasma cells.

Figure 1

Proliferation, differentiation, and antibody production after T-cell-dependent in-vitro stimulation and culturing of low numbers of primary human CD19⁺ B cells. (A) Schematic overview of the T-cell-dependent (TD) culture system to induce B-cell differentiation. A total of 25,000, 2,500, or 250 CD19⁺ human B cells (n = 4) were stimulated with a human CD40L-expressing 3T3 feeder layer and recombinant IL-21 (50 ng/ml) enabling conditions I (dark blue), II (cobalt blue), and III (light blue). Cells were analyzed on day 6 and day 9 by flow cytometry to evaluate the (B) number of live CD19⁺ events, (C) amount of proliferation by CTY dilution, and frequency of (D) plasmablast (CD27⁺CD38⁺ B cells) and (E) plasma cell (CD27⁺CD38⁺CD138⁺ B cells). A cutoff of 1,000 events was used to proceed with further analysis. (F) The supernatant was collected on day 6 and day 9 to evaluate IgG, IgA, and IgM production by ELISA (n = 4). Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using two-way ANOVA with Sidak’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 2

The addition of anti-BCR stimuli in a T-cell-dependent stimulation results in increased B-cell differentiation, while IL-4 severely inhibits antibody production. Human primary B cells obtained from healthy donors were stimulated under conditions described in Figure 1A (condition II) and Supplementary Figure 2A (condition II.2, PBMC cultures) with or without anti-BCR [anti-Ig F(ab)₂ mix (5 µg/ml) targeting IgM, IgG, and IgA] and/or recombinant IL-4 (25 ng/ml). Frequencies of CD27⁺CD38⁺ B cells on day 6 and day 9 in (A) condition II and (B) condition II.2 (n = 4–6). (C, D) Total secretion of IgG, IgA, and IgM measured in culture supernatants of eligible conditions after 6 and 9 days (C) without PBMCs (condition II) and (D) within PBMC cultures (condition II.2). Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using one-way ANOVA with Dunnett’s multiple comparison test (A, B) or two-way ANOVA with Sidak’s multiple comparison test (C, D). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant.

Figure 3

Proliferation, differentiation, and antibody production after T-cell-independent in-vitro stimulation and culturing of low numbers of primary human CD19⁺ B cells. (A) Schematic overview of the T-cell-independent (TI) culture system to induce B-cell differentiation. A total of 25,000, 2,500, or 250 CD19⁺ human B cells (n = 4) were stimulated with CpG (1 µM) and IL-2 (50 ng/ml) enabling conditions IV (dark blue), V (cobalt blue), and VI (light blue). Cells were analyzed on day 6 and day 9 by flow cytometry to evaluate the (B) number of live CD19⁺ events, (C) amount of proliferation by CTY dilution, and frequency of (D) plasmablast (CD27⁺CD38⁺) and (E) plasma cell (CD27⁺CD38⁺CD138⁺) generation. A cutoff of 1,000 events was used to proceed with further analysis. (F) The supernatant was collected on day 6 and day 9 to evaluate IgG, IgA, and IgM production by ELISA (n = 4). Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using two-way ANOVA with Sidak’s multiple comparison test (B) or unpaired t-test (D–F). **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4

The addition of BAFF in a T-cell-independent stimulation results in increased IgG and IgA production in isolated B-cell cultures. Human primary B cells obtained from healthy donors were stimulated under conditions described in Figure 3A (condition IV) and Supplementary Figure 4A (condition IV.2, PBMC cultures) with or without anti-BCR [anti-Ig F(ab)₂ mix (5 µg/ml) targeting IgM, IgG, and IgA] and/or BAFF (100 ng/ml). Frequencies of CD27⁺CD38⁺ B cells on day 6 and day 9 in (A) condition IV and (B) condition IV.2 (n = 3–5). (C, D) Total secretion of IgG, IgA, and IgM measured in culture supernatants of eligible conditions after 6 and 9 days (C) without PBMCs (condition IV) and (D) as PBMC culture (condition IV.2). Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using one-way ANOVA with Dunnett’s multiple comparison test (A, B) or two-way ANOVA with Sidak’s multiple comparison test (C, D). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant.

Figure 5

Cryopreserved B cells respond similarly to freshly isolated cells in T-cell-dependent and T-cell-independent assays. Total human B cells were isolated from fresh PBMCs (indicated in white) or frozen PBMCs (indicated in gray) and cultured for 6 and 9 days (n = 4). (A, B) Using T-cell-dependent (TD) stimuli (CD40L and IL-21 with/without IL-4), 2,500 B cells (fresh and frozen) were cultured under conditions described previously (A) without PBMCs (condition II) and (B) as PBMC culture (condition II.2). Frequencies of CD27⁺CD38⁺ B cells (left panel) and IgG production (right panel) on day 6 (upper graphs) and day 9 (lower graphs) are shown. (C, D) Using T-cell-independent (TI) stimuli (CpG and IL-2 with/without BAFF), 25,000 B cells (fresh and frozen) were cultured under conditions described previously (C) without PBMCs (condition IV) and (D) with PBMCs (condition IV.2). Frequencies of CD27⁺CD38⁺ B cells (left panel) and IgG production (right panel) on day 6 (upper graphs) and day 9 (lower graphs) are shown. Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using two-way ANOVA with Sidak’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01. ns, not significant.