Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study

Abstract

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient's peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19⁺ cells from the patient's peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo.

Keywords: Breg induction ex vivo; adoptive Breg transfer; immune response regulation; immunosuppression; mBregs; primary B cell activation; regulatory B cells; tBregs.

Figure 1

Differential activation of primary CD19⁺ lymphocytes alter the Breg subsets ratio, their viability, and proliferation rates ex vivo. (A) Total live cell count of mBregs and tBregs after various stimulation (50,000 events were acquired). Untreated control values were measured on day 0 and day 7. Mean values ± SEM of five independent experiments for each condition are shown. (B) Heatmap showing average B cell viability percentages after various stimulation. The mean values of five independent experiments for each stimulation are shown. (C) The ratio of mBregs and tBregs among live B cells after activation with various stimuli for 7 days. Untreated control values were measured on day 0 and day 7. Mean values of five independent experiments for each condition are shown. (D) Heatmap showing the average percentage of divided mBregs and tBregs induced by differential activation of primary B cells. The mean values of five independent experiments for each stimulation are shown.

Figure 2

Immunoregulatory molecular profile of differentially activated B cells. (A) Heatmap showing EBI3, CD274, IL10, and TNF normalized average expression levels in differentially activated B cells. (B) EBI3 and CD274 relative expression levels were determined by RT-PCR using total RNA extracted from differentially activated B cells on day 7 of cultivation. (C) IL10 and TNF levels were determined by ELISA in the medium from differentially activated B cells on day 7 of cultivation. Mean values ± SEM of at least four independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated control, as calculated by ANOVA.

Figure 3

Differentially activated B cells suppress the activity of effector cells and mediate CD4⁺ T cells differentiation into Tregs (CD4⁺CD25^hi) with simultaneous induction of IL10 secretion. (A) Tregs proliferation induced by co-cultivation of differentially activated B cells and CD4⁺ T cells. The percentage of divided Tregs was determined by flow cytometry using CellTrace Violet dye. (B) IL10 level was determined in the medium from CD4⁺ T cells co-cultivated with differentially activated B cells on day 5 of cultivation using ELISA. Mean values ± SEM of four independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated control, as calculated by ANOVA. (C) Activated B cells influence the proliferation rate of T helpers, T killers, and NK cells from CD19-depleted PBMCs after 5 days of co-cultivation. (D) IFNγ, TNF, IL1β, IL6, IL10 normalized levels were determined using ELISA in the medium from B cell-depleted PBMCs after co-cultivation. The average values of four independent experiments are shown. (E) Activated B cells reduce NK-mediated killing of cancer cell line MCF7. Mean values ± SD of three independent experiments is shown.

Figure 4

Radar graph representing the regulatory phenotype of B cells induced by various activation stimuli. Axes display immunosuppressive parameters of activated B lymphocytes assessed in the study. All data from Figures 1 - 3 used for immunogram construction were proportionally normalized to a single scale from 0 to 10. The values of each axis have been joined to form the central polygon area, which represents the general regulatory phenotype induced by various activation cocktails.

Figure 5

Potential mechanisms of induction of a suppressive phenotype in B lymphocytes by synergistic activation by different ligands. Created with BioRender.com.