Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B+ regulatory phenotype

Abstract

Introduction: The infusion of ex-vivo-generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease.

Methods: Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (GraB cells). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases.

Results: In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into GraB cells. The percentage of GraB cells after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR.

Discussion: Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for ex-vivo manufacturing of GraB cells.

Keywords: 12-O-tetradecanoylphorbol-13-acetate; GraB cells; GvHD; PMA; TPA; granzyme B; regulatory B cells.

Figure 1

Differentiation of pre-activated B cells into either plasma cells or GrB⁺ regulatory GraB cells and concept of generation and application of GrB⁺ regulatory B cells for the treatment of hyper-inflammatory diseases. (A) Regular CD4⁺ T cell activation includes stimulation of both the T cell receptor (TCR) via MHC/peptide complexes and CD28 via B7. Such fully activated T cells secrete IL-21 and express high levels of CD40 ligand (CD40L), enabling them to induce plasma cell differentiation in B cells, when they receive antigen-specific signals via their B cell receptor (BCR) in parallel. In contrast, in certain pathological situations such as during an HIV infection, the TCR of CD4⁺ T cells can be directly stimulated (e.g. via the protein Nef during HIV infection), without simultaneous costimulation of CD28. Such incompletely activated T cells secrete IL-21, but barely express CD40L, resulting in the induction of GrB-secreting GraB cells instead of plasma cells. Experimentally, exogenous addition of CD40L multimers can substitute for incomplete B cell/T cell interactions, resulting in enhanced plasma cell differentiation in vitro. (B) GrB⁺ regulatory B cells (GraB cells) can be generated from either autologous or allogeneic, HLA-matched donor blood. After autologous or allogeneic leukapheresis B cells are enriched, for example by magnetic depletion of CD3⁺ T cells, followed by positive magnetic selection of CD19⁺ cells using MACS technology. Subsequently, B cells are cultured in a clean room environment (A-in-B clean room environment) in the presence of IL-21, the key cytokine for the generation of GraB cells. In order to achieve robust stimulation of alloreactive pre-activated B cells, a second stimulus is required, which can either directly target the B cell receptor or subordinate signaling pathways downstream of the B cell receptor as discussed later. After an incubation period of 48 hours, alloreactive pre-activated B cells express GrB and can be harvested from the incubation flasks. After several washing steps to remove remnants of stimulatory agents in the medium, cell suspensions are quality-tested to ensure purity, absence of endotoxin and microbiological contamination including mycoplasma. Then, the final cell product can be released and either cryopreserved or directly transfused to the patient. Potential indications are hyper-inflammatory diseases including autoimmune or graft-versus-host diseases. Anti-BCR, antibodies directed against the constant part of the B Cell Receptor; B7, Costimulatory molecules CD80 and CD86; BCR, B Cell Receptor; CD, Cluster of Differentiation; CD40L, CD40 Ligand; GvHD, Graft-versus-Host Disease; HLA, Human Leukocyte Antigen; IL-21, Interleukin 21; IL-21R, IL-21 Receptor; MACS, Magnetic Activated Cell Sorting; MHC, Major Histocompatibility Complex; PMA, Phorbol Myristate Acetate; TCR, T Cell Receptor.

Figure 2

IL-21 induces and antibodies to the B cell receptor strongly enhance differentiation of pre-activated B cells into GrB⁺ GraB cells. B cells from healthy donors were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of increasing concentrations of IL-21 and anti-BCR as indicated. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Dot plots show percentages of GrB⁺ GraB cells from one representative experiment out of n > 10. (B, C) Box plots show average percentages of GrB⁺ GraB cells from n > 10 individual experiments as indicated, each dot indicates a result from one individual experiment. Box central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance levels were *** p < 0.0005, ** p < 0.005 and * p < 0.05. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; GrB, Granzyme B; IgG, Immunoglobulin G; IL-21, Interleukin 21; IQR, interquartile ranges; ns, not significant; PBMC, Peripheral Blood Mononuclear Cells; PE, Phycoerythrin; SSC, Side Scatter.

Figure 3

Optimal doses of PMA differentiate pre-activated B cells into GrB⁺ GraB cells and improve their viability ex vivo. B cells from at least ten healthy donors were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of increasing concentrations of IL-21 and PMA as indicated. Anti-BCR at 12 µg/ml and IL-21 at 100 ng/ml served as positive control. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Line graphs show mean percentages of GrB⁺ GraB cells from n > 10 individual experiments as indicated. (B) Line graphs show mean viability of GraB cells at the end of the cell culture period based on morphological criteria. Error bars indicate SEM. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; B21, Anti-BCR + IL-21; GrB, Granzyme B; IL-21, Interleukin 21; PMA, Phorbol Myristate Acetate; SEM, Standard Error of the Mean.

Figure 4

PMA may replace activating antibodies against the B cell receptor to enhance IL-21-dependent ex-vivo differentiation of pre-activated B cells into GrB⁺ GraB cells. B cells were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of different combinations of IL-21 at 100 ng/ml, anti-BCR at 12 µg/ml and PMA at 50 ng/ml as indicated. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Dot plots show percentages of GrB⁺ GraB cells from one representative experiment out of n > 10. (B) Box plots show average percentages of GrB⁺ GraB cells from n > 10 individual experiments as indicated. Box central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance level was * p < 0.05. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; B21, Anti-BCR + IL-21; GrB, Granzyme B; IL-21, Interleukin 21; IQR, interquartile ranges; ns, not significant; P21, PMA + IL-21; PBMC, Peripheral Blood Mononuclear Cells; PE, Phycoerythrin; PMA, Phorbol Myristate Acetate; SSC, Side Scatter.