Efficient Expansion of Human Granzyme B–Expressing B Cells with Potent Regulatory Properties

Mélanie Chesneau, Hoa Le Mai, Richard Danger, Sabine Le Bot, Thi-Van-Ha Nguyen, Josselin Bernard, Cyrielle Poullaouec, Pierrick Guerrif, Sophie Conchon, Magali Giral, Béatrice Charreau, Nicolas Degauque and Sophie Brouard
J Immunol November 1, 2020, 205 (9) 2391-2401; DOI: https://doi.org/10.4049/jimmunol.2000335

Key Points

  • Circulating plasmablasts are a major B cell subpopulation that expresses GZMB.

  • GZMB+ B cells can be efficiently expanded ex vivo from total B cells.

  • GZMB+ B cells strongly suppress both autologous and allogenic T cell proliferation.

Abstract

Granzyme B–expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25− effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.

This article is featured in Top Reads, p.2325

Introduction

B cells are mainly known as effectors of the immune response because of their ability to secrete Abs and to induce T cell activation by Ag presentation (1–4). Nevertheless, evidence for B cells with suppressive properties has also been found in different situations. These B cells, also called regulatory B cells (Bregs), were first described in mice for their ability to regulate inflammation in murine models of colitis, experimental autoimmune encephalomyelitis, and arthritis (2–5). In humans, the regulatory functions of these B cells have also been demonstrated in various clinical settings, including cancer, autoimmunity, and kidney allograft tolerance (6–8). Bregs perform suppressive functions that affect different cell types and act through different mechanisms (5, 8). To our knowledge, the recognition of the roles of Bregs in controlling immune responses opens a new avenue for adoptive cell therapy. However, several obstacles currently prevent their clinical use. First, the nature, phenotypes, and suppressive features of Bregs remain poorly described and depend on the immunological context. Second, the mechanisms of action of Bregs may differ between humans and mice (6, 7). Finally, because Bregs, unlike regulatory T cells, are difficult to expand, it is challenging to obtain a sufficient quantity of Bregs to perform adoptive cell therapy.

In the last decade, a B cell population secreting GZMB was identified as a new suppressive B cell population involved in a growing number of pathological conditions, such as B cell chronic lymphocytic leukemia (9), solid tumor infiltration (10), autoimmune diseases (11), and infections (12). We previously showed that renal allograft-tolerant patients had a higher number of GZMB+ B cells than stable kidney transplant recipients and healthy volunteers, and these GZMB+ B cells were able to inhibit effector T cell proliferation (13). In this report, we showed that in the peripheral blood of healthy donors, plasmablasts were the main B cell subset that expressed GZMB. Starting with total human B cells, we were able to induce GZMB expression in more than 90% of the B cells after 3 d of culture. By further characterizing these cells, we suggest that GZMB+ B cells could be a candidate for future Breg-based adoptive cell therapies.

Materials and Methods

Identification of GZMB+ B cells in human peripheral blood

Fresh human PBMCs were isolated from the whole blood of healthy donors using Ficoll gradient centrifugation. The PBMCs were stained for CD19, CD24, CD38, CD138, and surface Ig D (see Supplemental Table I for the list of Abs and cell markers used for flow cytometry), followed by a 30-min incubation with a fixation/permeabilization solution (eBioscience), staining for intracellular GZMB, and then analysis by flow cytometry (LSRII; BD Biosciences).

B and T cell isolation and culture

B cells and CD4+ T cells were negatively selected from human PBMCs by magnetic separation using a human B cell isolation kit II and a human CD4+ T cell isolation kit, respectively. CD25+ T cells were then depleted from the CD4+ T cells using CD25 microbeads to obtain CD4+CD25− effector T cells. CD8+ T cells were negatively selected using human CD8+ T cell isolation kit (all kits and beads were from Miltenyi Biotec). Cell purities were confirmed by flow cytometry to be at least 90%. B cells were isolated from PBMCs immediately after Ficoll gradient centrifugation; for T cells, some PBMCs from the same donor were incubated overnight in culture medium at 4°C, and CD4+ T cell isolation was performed the next morning because we prestimulated B cells overnight before coculturing them with autologous T cells (see below). All cell cultures were carried out in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FCS, l-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a 5% CO2 atmosphere.

In vitro plasmablast differentiation and Ig production

B cells were stimulated to differentiate into plasmablasts using a two-phase cell culture protocol as previously described (13, 14). Briefly, B cells were activated for 4 d with 50 ng/ml soluble CD40L (R&D Systems), 2.5 μg/ml CpG oligodeoxynucleotide (ODN) 2006 (InvivoGen), 2 μg/ml F(ab′)2 goat anti-human IgG/IgM/IgA (H + L) polyclonal Abs (anti-BCR) (Jackson ImmunoResearch Laboratories), and 50 IU/ml human IL-2 (Novartis). The cells were then washed and cultured for three additional days in fresh medium containing 50 IU/ml human IL-2, 10 μg/ml human IL-4 (R&D Systems), and 10 μg/ml human IL-10 (R&D Systems) to induce B cell differentiation into plasmablasts. Flow cytometry was performed at the end of each phase, specifically on days 4 and 7, to assess GZMB expression and Ig production. Cells were stained with a LIVE/DEAD fixable yellow dye and an anti-human CD19 APC–Vio 770–conjugated Ab. Surface Ig was blocked with an anti-human IgM FITC-conjugated, anti-human IgG FITC-conjugated, or anti-human IgA APC–Vio 770–conjugated Ab. After permeabilization and fixation, intracellular staining for cytoplasmic Ig and GZMB was performed with an anti-human IgM APC-conjugated, anti-human IgG VioBlue-conjugated or anti-human IgA VioBright FITC-conjugated Ab and an anti-GZMB AF700-conjugated Ab (Supplemental Table I).

GZMB+ B cell expansion and characterization

B cells were cultured at 1 × 106 cells/ml for 1, 2, or 3 d in the presence of different combinations of IL-21 (10 ng/ml), F(ab′)2 anti-BCR Abs (5 μg/ml), CpG ODN 2006 (1 μg/ml), CD40L (50 ng/ml), and IL-2 (50 IU/ml) as indicated. At each time point, specifically days 1, 2, and 3, cells were stained with Fixable Viability Dye (FVD) eFluor 450 to identify dead cells, followed by staining for CD19 and intracellular GZMB to evaluate B cell GZMB expression. In some experiments, cell apoptosis was also evaluated by annexin V staining or intracellular active caspase-3 staining. To quantify B cell proliferation, B cells were labeled with Cell Proliferation Dye (CPD) eFluor 450 and stimulated for 3 d by the combination of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN 2006, CD40L, and IL-2 at the aforementioned concentrations (referred to as the expansion mixture) with or without a GZMB inhibitor (GZMBinh) (Calbiochem Research Biochemicals) or ERK1/2 inhibitor (Sigma-Aldrich). B cells cultured for 3 d in the presence of the expansion mixture or medium alone were also stained for different extracellular markers associated with B cell activation, differentiation, and regulation, including CD21, CD25, CD27, CD38, CD72, CD258, CD307b, FasL, LAG-3, PD-1, PD-L1, and PD-L2, and then analyzed by flow cytometry for phenotypic characterization (Supplemental Table I). After exclusion of dead cells and doublets, B lymphocytes were identified according to their morphological features and positive CD19 expression. For each donor and each staining combination, CD19 cells from stimulated and unstimulated cell cultures were then downsampled and concatenated to generate new flow cytometry standard files with equal numbers of CD19 cells. Supervised analysis of the expression of different target molecules by stimulated (i.e., GZMB+) B cells or unstimulated (i.e., GZMB−) B cells was performed using FlowJo software to identify the optimal association of markers that enabled the identification of GZMB+ B cells.

B cell protein phosphorylation assays

B cells were incubated for 30 min in culture medium containing the expansion mixture with or without the GZMBinh. Medium alone and 5 ng/ml PMA were used as negative and positive controls, respectively. An equal volume of prewarmed Cytofix Buffer (BD Biosciences) was added to each well and incubated for 10 min at 37°C to stop activation. The cells were then washed, permeabilized with Phosflow Perm Buffer III (BD Biosciences) for 30 min on ice and stained with different anti-phosphoprotein Abs, including anti–P-ERK1/2, anti–P-BAD, anti–P-STAT3, anti–P-STAT5, anti–P-P38, anti-P–MEK-1, and anti–P-EGFR.

Western blot analysis

B cells were cultured at 1 × 106 cells/ml in 12-well plates for 2 d in culture medium containing the expansion mixture with or without the GZMBinh. The cells were lysed in radioimmunoprecipitation assay buffer on day 2. Samples were separated by SDS-PAGE, and immunoblotting for Notch1 (1:1000) (52627; Abcam) and GAPDH (1:1000) (Sigma-Aldrich) was performed.

T cell suppression assays

To evaluate the capacity of B cells to suppress effector T cell proliferation, we prestimulated B cells (1 × 106 cells/ml) overnight (for ∼20 h) with different combinations of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 as indicated. The B cells were then washed, resuspended in fresh culture medium (without adding any cytokines or Abs) and cocultured with autologous CPD-labeled CD4+CD25− effector T cells for 3 d in the presence of CD3/CD28 Dynabeads (Invitrogen) at a T cell/bead ratio of 1:1. A total of 1 × 105 B cells were cocultured with 0.5 × 105 autologous T cells in a total volume of 150 μl/well in a 96-well, U-bottom plate. In some experiments, allogenic T cells were used instead of autologous T cells. T cell proliferation was evaluated as the percentage of CPD-low T cells on day 3. T cell apoptosis was assessed by annexin V staining. To study the importance of cell contact in the suppressive capacity of B cells, B and T cell coculture was also performed in the presence of a Transwell polycarbonate insert (0.4 μm; Corning). Finally, to study the roles of different signaling pathways in the suppressive effect of B cells, different inhibitors, including IL-10 inhibitor (BD Biosciences), LAG-3 inhibitor (AdipoGen Life Sciences), FasL inhibitor (R&D Systems), PD-L1 inhibitor (BioLegend), and GZMBinh (Calbiochem Research Biochemicals), were added into the B and T cell coculture system. All inhibitors were used at 10 μg/ml.

GZMB-based cytotoxicity assays

To detect the GZMB-mediated cytotoxic activity of expanded GZMB+ B cells against target CD4+CD25− T cells, we used a PanToxilux kit (OncoImmunin) according to the manufacturer’s instructions. B cells were stimulated for 24 h with the expansion mixture (expanded GZMB+ B cells). Autologous CD4+CD25− T cells were incubated for 45 min at 37°C with the target-labeling reagent TFL4 diluted 1:1000 in PBS and then washed twice. The target CD4+CD25− T cells and expanded GZMB+ B cells (T/B cells = 1:2) were then incubated together for 2 h at 37°C in 75 μl PanToxilux substrate using a 96-well, V-bottom plate, resuspended in 200 μl wash buffer, and analyzed by flow cytometry. When active GZMB was transferred from the GZMB+ B cells into the target T cells, the GZMB cleaved the PanToxilux substrate inside the T cells and released the quencher, leading to the emission of green fluorescence, indicating GZMB-mediated cytotoxicity (15).

Statistical analyses

Statistical analyses were performed using GraphPad Prism software V.5. Continuous variables were compared using Mann–Whitney U test or t test when n > 30. Statistical significance was set at p < 0.05.

Results

Circulating human GZMB+ B cells are mainly found in the plasmablast subset

To determine which B cell subsets in normal human peripheral blood express GZMB, PBMCs from more than 30 healthy donors were isolated by Ficoll gradient centrifugation and stained for B cell–surface markers and intracellular GZMB. Four B cell subsets were defined based on the expression of CD24 and CD38: transitional (CD24+CD38+), naive (CD24−CD38−), and memory (CD24+CD38−) B cells and plasmablasts (CD24−CD38hi) (Fig. 1A). We found that the differentiated B cell subsets such as memory B cells and especially plasmablasts contained higher percentages of GZMB+ cells (Fig. 1B) and expressed higher levels of GZMB (Fig. 1C, 1D) than the naive B cell subset. Approximately 25% of the circulating plasmablasts expressed GZMB, and conversely, ∼50% of the circulating GZMB+ B cells were indeed plasmablasts (Supplemental Fig. 1). In addition, more than half of the GZMB+ B cells had a mature IgDlo/– phenotype, whereas the majority of the GZMB− B cells were IgD+ (Fig. 1E). Concordantly, the GZMB+ B cells expressed lower levels of IgD than the GZMB− cells (Fig. 1F). Although differentiated plasma cells (CD19+CD138+) are a rare population in the peripheral blood (Supplemental Fig. 2A), we were able to show that higher percentage of CD19+CD138+ cells expressed GZMB compared with total B cells (Supplemental Fig. 2B). Accordingly, compared with GZMB− B cells, GZMB+ B cells expressed higher levels of CD138 as determined by mean fluorescence intensity (MFI) (Supplemental Fig. 2C) and contained higher percentage CD138+ cells (Supplemental Fig. 2D).

FIGURE 1.
FIGURE 1.

Circulating human GZMB+ B cells are mainly found in the plasmablast subset. Fresh human PBMCs from healthy donors were isolated by Ficoll gradient centrifugation, stained for B cell surface markers and intracellular GZMB, and then analyzed by flow cytometry. (A) Dot plots showing gating for transitional (CD24+CD38+), naive (CD24−CD38−), and memory (CD24+CD38−) B cells and plasmablasts (CD24−CD38hi). (B) Frequency of GZMB+ cells in different B cell subsets (n = 52). (C) Histogram representing the expression of GZMB in transitional, naive, and memory B cells and plasmablasts (n = 52). (D) MFI of GZMB in different B cell subsets. (E) Histogram representing surface IgD expression on GZMB+ and GZMB− B cells. (F) Expression level (MFI) of surface IgD on GZMB+ and GZMB− B cells. Bars indicate the mean ± SEM. **p < 0.01, ***p < 0.001 (n = 34).

In vitro–differentiated plasmablasts do not highly express GZMB

Because circulating plasmablasts are the main GZMB-expressing B cells in normal human peripheral blood, we wondered whether in vitro–differentiated plasmablasts also express GZMB. To answer this question, we isolated B cells from PBMCs and performed B cell culture to induce plasmablast differentiation using a two-phase protocol (14, 16). We found that at the end of the activation phase (day 4), ∼20% of the B cells expressed GZMB, but at the end of the differentiation phase (day 7), when a proportion of the B cells had differentiated into plasmablasts (CD20lo CD38hi), <2% of the B cells still expressed GZMB (Fig. 2A, 2B). We also compared the capacities of GZMB+ and GZMB− B cells to produce Igs and found that on day 7, a higher percentage of GZMB+ B cells contained cytoplasmic IgM (Fig. 2C, Supplemental Fig. 3), and these cells also expressed higher levels of cytoplasmic IgM as measured by MFI (Fig. 2D). In contrast, the percentages and expression levels of cytoplasmic IgG and IgA were not different between the GZMB+ and GZMB− B cells.

FIGURE 2.
FIGURE 2.

In vitro–differentiated plasmablasts do not highly express GZMB. Purified B cells were cultured in vitro to induce plasma cell differentiation using a two-phase culture protocol as described in Materials and Methods. Cells harvested at the end of the activation (day 4) or differentiation phase (day 7) were stained for B cell–surface markers, intracellular GZMB, and cytoplasmic IgA, IgM, and IgG and then analyzed by flow cytometry. (A) Dot plot showing the gating strategies for GZMB+ B cells and CD20loCD38hi plasmablasts. (B) Percentages of GZMB+ B cells on days 4 and 7 (n = 5). (C) Percentages of GZMB− and GZMB+ B cells expressing cytoplasmic IgA, IgM, and IgG on day 7. (D) The corresponding MFIs on day 7. Bars indicate the mean ± SEM. **p < 0.01 (n = 5).

The combination of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 promotes ex vivo expansion of GZMB+ B cells

Because in healthy individuals under steady-state conditions, <3% of circulating B cells express GZMB, it is important to identify an optimal culture protocol to effectively expand GZMB+ B cells for preparing cellular therapies in the future. To achieve this goal, we cultured purified B cells for 1, 2, or 3 d in the presence of different combinations of reagents known to be important for B cell activation, namely IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2. We found that the combination of all five aforementioned reagents (the expansion mixture) was necessary to ensure both strong expression of GZMB (Supplemental Fig. 4A) and high cell survival (Supplemental Fig. 4B). On day 3 of culture with the expansion mixture, the GZMB expression level as measured by MFI was increased by more than 30-fold (Fig. 3A) and more than 90% of B cells expressed GZMB (Fig. 3B). However, beyond day 3, the percentage of GZMB+ B cells was decreased, and <60% of B cells expressed GZMB on day 6 of culture (Fig. 3B).

FIGURE 3.
FIGURE 3.

GZMB+ B cells can be efficiently expanded in vitro by a combination of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2. Purified B cells from healthy blood donors were cultured for up to 6 d in the presence of the expansion mixture, which contained IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2. Cells were harvested on days 1, 2, 3, and 6; stained with an FVD, annexin V, B cell–surface markers, intracellular GZMB, and intracellular active caspase-3; and then analyzed by flow cytometry. GZMB expression levels as measured by the MFI (A), percentage of GZMB+ B cells (B) (n = 5). (C) Percentage of live B cells (FVD−annexin V−) at different time points. (D) Representative histogram of active caspase-3 staining. (E) Percentages of cells expressing active caspase-3 in GZMB+ and GZMB− B cells evaluated on day 3. Bars indicate the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5).

GZMB+ B cells are relatively sensitive to apoptosis

We found that the percentage of viable B cells as defined by the FVD−annexin V− phenotype decreased from 90% on day 3 to <80% on day 6 (Fig. 3C), suggesting that B cells tend to undergo apoptosis with prolonged stimulation. Because one of the functions of GZMB is to cleave procaspase-3 into the active form to initiate the caspase-dependent apoptotic pathway, we asked whether high expression of GZMB leads to caspase-3 activation within B cells. Therefore, we stained B cells for intracellular active caspase-3 and found that the GZMB+ B cell population expressed a higher level of active caspase-3 (Fig. 3D) and contained a higher percentage of active caspase-3–positive cells (Fig. 3E) than the GZMB− B cell population, at least partially explaining the apoptotic tendency of GZMB+ B cells.

Expanded GZMB+ B cells show a regulatory phenotype and are enriched in the CD307bhi, CD258hiCD72hi, and CD21loPD1hi subpopulations

To further characterize the phenotype of expanded GMZB+ B cells, we performed flow cytometry staining for different extracellular molecules known or reported to be associated with B cell activation, differentiation, or regulation including CD21, CD25, CD27, CD38, CD72, CD258, CD307b, FasL, LAG-3, PD-1, PD-L1, and PD-L2 (Fig. 4A). We found that B cells cultured for 3 d in the presence of the expansion mixture expressed relatively high levels of CD25 and CD38, consistent with previous reports (17–19). Interestingly, expanded GMZB+ B cells upregulated the expression of molecules with immunomodulatory properties such as PD-1, PD-L1, PD-L2, CD72, CD307b (FcRL2), and CD258 (TNFSF14) (20). In contrast, unlike in mice, in which LAG3 has been shown to be a key marker of Bregs (21), expanded GZMB+ B cells did not upregulate LAG3. Finally, to further define the phenotype of expanded GZMB+ B cells, we interrogated our flow cytometry data to test whether expanded GZMB+ B cells can be identified using solely cell-surface markers (Fig. 4B–E, and see Materials and Methods). For each marker panel, equal randomized numbers of expanded and nonexpanded cells were mixed before supervised gating. Altogether, our results show that expanded GMZB+ B cells display enrichment (80–99.9% of GZMB+ cells) in the CD307bhi, CD258hiCD72hi, and CD21loPD1hi B cell subpopulations (Fig. 4B–E), suggesting a regulatory phenotype.

FIGURE 4.
FIGURE 4.

GZMB+ B cells acquire a regulatory phenotype. Purified B cells were cultured for 3 d in the presence of the expansion mixture containing IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 or in medium alone; harvested; stained with an FVD, anti-CD19, and various cell-surface markers associated with B cell activation, differentiation, or regulation; and then analyzed by flow cytometry. Cells were gated as FVD−CD19+. (A) Expression levels of various cell-surface markers measured by the MFI. (B–E) Identification of GZMB+ B cells by cell-surface markers using supervised analysis. For each marker panel, CD19+ cells from expanded and nonexpanded cells were concatenated in equal randomized cell numbers before supervised clustering based on selected cell-surface markers. Alternative gating strategies enabled the identification of expanded GZMB+ B cells with up to 99.9% purity. A representative example is shown before gating (B) and after gating (C–E). No stim, no stimulation (medium alone); Stim, stimulation with the expansion mixture. Bars indicate the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5).

GZMB per se induces B cell proliferation in an ERK1/2-dependent manner

To study the effect of stimulation on B cell proliferation, we labeled purified B cells with a CPD and then cultured them for 3 d in the presence of the expansion mixture. We found that the B cells strongly proliferated. Surprisingly, B cell proliferation was reduced by half when GZMBinh was added (Fig. 5A), suggesting that GZMB itself may promote the proliferation of B cells. Because GZMB has been shown to be involved in the Notch-1 and ERK1/2 signaling pathways (22, 23), we further investigated these two pathways. We confirmed that Notch1, a substrate of GZMB, was expressed by B cells, but we found no difference in Notch1 expression in expanded GZMB+ B cells with or without GZMBinh, suggesting that Notch1 expression by GZMB+ B cells is not dependent on GZMB or involved in GZMB-dependent B cell proliferation (Fig. 5B). In contrast, B cell proliferation was associated with strong ERK1/2 phosphorylation, and the addition of GZMBinh significantly reduced ERK1/2 phosphorylation (Fig. 5C, 5D). Consistent with these data, inhibiting ERK1/2 significantly prevented B cell proliferation (Fig. 5E, 5F). No difference was observed in the phosphorylation of molecules in the JNK and p38 MAPK pathways (data not shown). Altogether, our results suggest that GZMB+ B cells expand through phosphorylation of ERK1/2 and under the control of GZMB produced by the GZMB+ B cells.

FIGURE 5.
FIGURE 5.

GZMB induces B cell proliferation in a Notch1-independent and ERK1/2-dependent manner. (A) Purified B cells were labeled with a CPD and cultured for 3 d in the presence of a combination of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 (referred to as the expansion mixture) with or without GZMBinh. The cells were harvested, stained with an FVD and anti-CD19 Ab, and then analyzed by flow cytometry. Shown are percentages of proliferating B cells (n = 9). (B) B cells were cultured for 2 d in the presence of the expansion mixture with or without GZMBinh and then lysed for Western blot analysis. Notch1 protein expression was quantified as a ratio to the loading control GAPDH (n = 7). (C and D) B cells were stimulated for 30 min with PMA (positive control) or the expansion mixture with or without GZMBinh. The cells were then washed, stained for intracellular P-ERK1/2, and analyzed by flow cytometry. Representative FACS plots (C) and statistical graph (D) show the percentage of B cell ERK1/2 phosphorylation under different culture conditions (n = 6). (E) Histogram representing proliferation of B cells by CPD staining. (F) Percentages of B cell proliferation analyzed after 3 d of culture in the presence of the expansion mixture with or without ERK1/2 inhibitor (n = 5). Bars indicate the mean ± SEM. *p < 0.05.

Expanded GZMB+ B cells efficiently suppress autologous and allogenic T cell proliferation

Next, we assessed the capacity of B cells stimulated by different combinations of reagents to inhibit effector T cell proliferation to determine the combination that leads to the strongest suppressive effect. To this end, we prestimulated purified B cells overnight with different combinations of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 and then cocultured them with autologous purified CD4+CD25− effector T cells in the presence of CD3/CD28 microbeads for 3 d. We found that the B cells prestimulated by a combination of all five reagents (the expansion mixture) most effectively suppressed CD4+CD25− T cell proliferation (Supplemental Fig. 5). The suppressive effect of expanded GZMB+ B cells on effector T cells was partially reversed by the addition of GZMBinh (Fig. 6A, 6B), indicating that this effect is at least partially GZMB dependent. In contrast, blockade of IL-10, LAG3, FASL, or PD-L1 did not alter the suppressive properties of expanded GZMB+ B cells (Supplemental Fig. 6). Expanded GZMB+ B cells completely lost their ability to inhibit T cell proliferation when coculture was performed using a Transwell system (Fig. 6C), demonstrating that the suppressive effect of GZMB+ B cells on effector T cell proliferation is contact dependent. Interestingly, expanded GZMB+ B cells also inhibited the proliferation of allogeneic CD4+CD25− effector T cells with the same efficiency as that seen with autologous CD4+CD25− effector T cells (Fig. 6D). In addition, GZMB+ B cells were also capable of suppressing the proliferation of CD8+ T cells (Supplemental Fig. 7A, 7B). In contrast, they did not have any effect on the polarization of monocyte-derived macrophages into M1 or M2 macrophages (Supplemental Fig. 7C–E).

FIGURE 6.
FIGURE 6.

Expanded GZMB+ B cells suppress effector CD4+CD25− T cell proliferation. Purified B cells were cultured overnight with either a combination of IL-21, F(ab′)2 anti-BCR Abs, CpG ODN, CD40L, and IL-2 to induce GZMB expression (GZMB+ B cells), or medium alone (B cells), washed, and then cocultured for three additional days with autologous, CPD-labeled CD4+CD25− effector T cells in the presence of CD3/CD28 beads (T cells/beads = 1:1). The cells were then harvested, stained for CD4 and CD19, and analyzed by flow cytometry. Representative histograms from one experiment (A) and a statistical graph (B) showing the percentages of T cell proliferation in different culture conditions (n = 24). (C) Percentage inhibition of CD4+CD25− T cell proliferation with or without a Transwell (TW) insert (n = 5). (D) Percentage inhibition of autologous or allogenic CD4+CD25− T cell proliferation in the context of coculture with B cells or GZMB+ B cells (n = 9). (E) Percentages of apoptotic T cells (annexin V+) in different culture conditions (n = 16). (F) Percentage of CD4+CD25− T cells experiencing GZMB+ B cell–mediated cytotoxicity, as measured with the PanToxilux kit (see Materials and Methods) (n = 5). Bars indicate the mean ± SEM. *p < 0.05, **p < 0.01.

The suppressive activity of expanded GZMB+ B cells does not rely on CD4+CD25− T cell apoptosis and/or the uptake of GZMB by CD4+CD25− T cells

To evaluate the effect of GZMB+ B cells on T cell apoptosis, we stained CD4+CD25− T cells with annexin V after 3 d of coculture with expanded GZMB+ B cells. We found that the percentage of annexin V+ T cells was unchanged (Fig. 6E), suggesting that the expanded GZMB+ B cells did not induce T cell apoptosis. Finally, we assessed whether the regulatory properties of GZMB+ B cells rely on the direct transfer of active GZMB from expanded GZMB+ B cells to target CD4+CD25− T cells using a PanToxilux kit. We found that the percentage of T cells experiencing GZMB-mediated cytotoxicity did not significantly differ regardless of whether unstimulated B cells, expanded GZMB+ B cells, or expanded GZMB+ B cells plus GZMBinh were incubated with the T cells (Fig. 6F), suggesting that the regulatory function of GZMB+ B cells did not rely on the uptake of GZMB by CD4+CD25− T cells (Figs. 6F, 7).

FIGURE 7.
FIGURE 7.

Proposed role of GZMB+ Bregs in inflammation. During inflammation or infection, the presence of bacterial or viral Ags (BCR recognition) and DNAs (TLR stimulation); inflammatory cytokines, especially IL-21 and IL-2; and T cell costimulatory signals (CD40L) induces B cell secretion of GZMB. GZMB boosts the proliferation of B cells via an ERK-dependent pathway. GZMB+ B cells regulate the T cell response by inhibiting effector T cell proliferation in a GZMB− and contact-dependent manner. Finally, GZMB+ B cells are relatively susceptible to apoptosis, enabling negative feedback control.

Discussion

B cell subsets with suppressive properties play important roles in inflammation (24), autoimmunity (25), and transplantation (26), as well as in physiological conditions, by regulating cell homeostasis (27). The importance of Bregs is well illustrated by the deleterious consequences of B cell depletion in several pathological conditions (28–30) and by the fact that adoptive transfer of these B cell subpopulations into animal models of autoimmune diseases improves disease symptoms (31–33). There are different subpopulations of Bregs that exert their suppressive effects via different pathways, including the IL-10 (27, 34–36), IL-35 (37), TGF-β (38, 39), and PDL-1 pathways (40). Apart from well-known Bregs, GZMB-expressing B cells have been shown, to our knowleged, to be a new Breg subset capable of suppressing T cells (10, 17). GZMB+ B cells have been detected in solid tumor infiltrates (10) and the peripheral blood of patients with autoimmune disease (11) or HIV infection (12). Recently, we reported that this GZMB+ B cell population could prevent autologous T cell proliferation (17).

Given the importance of this Breg subset, we first determined its frequency in freshly isolated human PBMCs without any stimulation. We found that GZMB+ B cells accounted for ∼2% of the total B cells in the peripheral blood of healthy donors under steady-state conditions. We showed that ∼25% of circulating CD24−CD38hi plasmablasts expressed GZMB and that plasmablasts accounted for approximately half of the GZMB+ B cells in the peripheral blood. Although plasma cells present in the human tonsils (41) and intestinal mucosa (42) are known to express GZMB, this is the first time, to our knowledge, that circulating plasmablasts have been shown to be the major B cell subset that expresses GZMB in human peripheral blood. We also found that GZMB+ cells were mature B cells expressing low levels of IgD. Interestingly, a recent report showed that some populations of mature IgD-low B cells could exert their regulatory activity by promoting regulatory T cell expansion (29). Taken together, these data suggest that mature B cells may comprise different Breg subsets. This is reinforced by the fact that expanded GMZB+ B cells upregulate the expression of molecules with immunomodulatory properties such as PD-1, PD-L1, PD-L2, CD72 (43) CD307b (FcRL2) (44), and CD258 (TNFSF14) (20), suggesting a regulatory phenotype.

Because plasmablasts normally account for only ∼1% of the B cells in the peripheral blood, it is difficult to isolate a sufficient number of these cells to be used as a source of GZMB+ Bregs. Therefore, we investigated whether an in vitro plasmablast differentiation protocol can effectively produce GZMB+ plasmablasts with regulatory properties. However, <2% of the plasmablasts obtained with a 7-d in vitro B cell differentiation protocol expressed GZMB (Fig. 2A, 2B), excluding the use of this protocol as a method to expand GZMB+ B cells. Other GZMB+ B cell expansion protocols have been described. Lindner et al. (10) reported that B cells cultured for 48 h in the presence of IL-21 and F(ab′)2 anti-BCR Abs expressed GZMB in ∼30% of the cells and inhibited CD4+ T cell proliferation. Similarly, we previously showed that another B cell culture protocol using CD40L and CpG ODN induced GZMB expression in ∼20% of B cells, and those B cells were capable of inhibiting CD4+CD25− effector T cell proliferation (17). Such levels of GZMB+ B cell expansion, although permitting biological and functional characterization, are not sufficient to envision the use of GZMB+ B cells as an adoptive cell therapy. In the current study, using an expansion mixture combining five reagents, CD40L, CpG ODN, IL-21, F(ab′)2 anti-BCR Abs, and IL-2, we were able to induce GZMB expression in more than 90% of B cells after 3 d of culture. More importantly, we showed that B cells prestimulated with this combination efficiently suppressed both autologous and allogenic effector T cell proliferation. We also showed that the suppressive activity of these Bregs was partially GZMB dependent and totally contact dependent because it was abrogated by the Transwell culture. In the Transwell system, we used the same proportion of T cells, B cells, and medium volume as that used in the normal culture, but there was no suppression of T cell proliferation, proving that competition for nutrients and cytokines is unlikely the cause of T cell suppression. The precise molecular pathway underlying the contact-dependent suppressive effects of GZMB+ Bregs remains to be elucidated. Although GMZB+ B cells presented upregulated expression of several immunoregulatory molecules, especially molecules in the PD-1 signaling pathway, PD-L1 blockade did not affect their suppressive function, suggesting that the mechanisms of action of GZMB+ Bregs are different from those of recently described PD-L1+ Bregs (40, 45, 46). Likewise, inhibition of the Fas/FasL pathway did not affect the suppressive function of our Bregs.

Although the classic function of GZMB is to induce apoptosis in target cells, in the current study, we found that the presence of expanded GZMB+ B cells in cocultures did not lead to a significant increase in the frequency of annexin V–positive T cells, a result consistent with the findings of a previous report (10). GZMB has other concomitant or alternative functions (47). GZMB is a serine protease that can digest the extracellular matrix and cell-surface proteins (48, 49). Moreover, GZMB can act directly on T cells by altering their migration (50) or inducing their proliferation (9). However, to our knowledge, no direct effect on B cells has been described to date. In this study, we showed that GZMB per se stimulates B cell proliferation in an ERK1/2-dependent manner, suggesting that GZMB+ B cells may control their own proliferation and expansion. However, this process is counteracted by the relatively high susceptibility of GZMB+ B cells to apoptosis, which may indeed serve as a negative feedback control to avoid uncontrolled proliferation.

Although human Bregs are best known to exert their suppressive effects through the secretion of IL-10, the identification of IL-10–secreting B cells always requires ex vivo stimulation. Moreover, even after robust stimulation with PMA and ionomycin, either alone or combined with other stimuli, fewer than 20% of B cells produce IL-10 (6), making it difficult to use this Breg subset for future cell therapy. In this study, we described an ex vivo expansion protocol allowing us to obtain up to 99% human GZMB+ B cells, which strongly suppress effector T cell proliferation, suggesting that ex vivo–expanded GZMB+ B cells could be a candidate for future adoptive Breg-based therapies. Further studies on the roles of human GZMB+ Bregs in humanized mouse disease models are necessary to provide proof-of-concept data on the efficacy of this approach in vivo before envisaging human clinical trials.

Finally, based on our in vitro experimental results, we propose a scheme for the role of GZMB+ Bregs in vivo (Fig. 7). In the presence of different stimulatory signals during infection and inflammation, such as bacterial or viral proteins and DNA (which serve as BCR and TLR-9 stimulators, respectively), inflammatory cytokines (especially IL-21 and IL-2), and T cell costimulatory signals (CD40L), a proportion of B cells express GZMB, which, in turn, directly boosts B cell proliferation via an ERK-dependent pathway. GZMB+ B cells downregulate inflammation by preventing effector T cell proliferation in a GZMB-dependent and contact-dependent manner. However, GZMB+ B cells display a relatively high susceptibility to apoptosis, which may serve as a negative feedback control to decrease their numbers to avoid uncontrolled B cell proliferation. The relatively short life of GZMB+ B cells may be a challenge in considering their use in adoptive cell therapy. Nevertheless, we are convinced that this short-acting therapy might be appropriate for disease conditions such as acute allograft rejection or exacerbation of autoimmune diseases in which a temporary intervention is preferable to avoid prolonged overimmunosuppression.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank clinical research assistants Astrid Fleury and Irène Guihard.

Footnotes

  • ↵1 M.C. and H.L.M. equally contributed and are listed by alphabetic order.

  • This work was supported by Agence Nationale de la Recherche BIKET Project Grant ANR-17-CE17-0008, Grant ANR-11-JSV1-0008-01, and Investment into the Future Program Grant ANR-10-IBHU-005 (which provided French government financial support to the IHU-Cesti project, of which this study is a part), Grant ANR-17-RHUS-0010, and Grant ANR-11-LABX-0016-01 in the context of the PRELUD Project (ANR-18-CE17-0019). This work was supported in part by the RTRS Fondation de Coopération Scientifique CENTAURE. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. This work was also supported by the FP7 VISICORT Project, which has received funding from the European Union’s Seventh Framework Programme for Research (Grant Agreement 602470). This work was performed as a part of the Labex IGO - Immonothérapies Grand Ouest.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    Breg
    regulatory B cell
    CPD
    Cell Proliferation Dye
    expansion mixture the combination of IL-21 F(ab′)2 anti-BCR Abs CpG ODN 2006 CD40L
    and IL-2
    FVD
    Fixable Viability Dye
    GZMBinh
    GZMB inhibitor
    MFI
    mean fluorescence intensity
    ODN
    oligodeoxynucleotide.

  • Received March 31, 2020.
  • Accepted August 10, 2020.

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