Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells

Abstract

Exosomes, nano-sized membrane vesicles, are released by various cells and are found in many human body fluids. They are active players in intercellular communication and have immune-suppressive, immune-regulatory, and immune-stimulatory functions. EBV is a ubiquitous human herpesvirus that is associated with various lymphoid and epithelial malignancies. EBV infection of B cells in vitro induces the release of exosomes that harbor the viral latent membrane protein 1 (LMP1). LMP1 per se mimics CD40 signaling and induces proliferation of B lymphocytes and T cell-independent class-switch recombination. Constitutive LMP1 signaling within B cells is blunted through the shedding of LMP1 via exosomes. In this study, we investigated the functional effect of exosomes derived from the DG75 Burkitt's lymphoma cell line and its sublines (LMP1 transfected and EBV infected), with the hypothesis that they might mimic exosomes released during EBV-associated diseases. We show that exosomes released during primary EBV infection of B cells harbored LMP1, and similar levels were detected in exosomes from LMP1-transfected DG75 cells. DG75 exosomes efficiently bound to human B cells within PBMCs and were internalized by isolated B cells. In turn, this led to proliferation, induction of activation-induced cytidine deaminase, and the production of circle and germline transcripts for IgG1 in B cells. Finally, exosomes harboring LMP1 enhanced proliferation and drove B cell differentiation toward a plasmablast-like phenotype. In conclusion, our results suggest that exosomes released from EBV-infected B cells have a stimulatory capacity and interfere with the fate of human B cells.

FIGURE 1

DG75-LMP1ex contain physiological levels of LMP1, as found on exosomes released during primary EBV infection. (A) Peripheral B cells from two donors were either left untreated or infected with EBV and cultured for 3 d. The corresponding exosomes, PBex and PB-EBVex, were isolated and analyzed for LMP1 and HLA-DR expression by immunoblot. Expression levels of 10 µg PBex and PB-EBVex from donor 1 and 20 µg of PBex and PB-EBVex from donor 2 were compared with 10 µg each of BJABex and LCL1ex. (B) Exosomes (10 µg) isolated from the Burkitt’s lymphoma cell lines BJAB (BJABex), DG75 (DG75-COex), LMP1 transfected (DG75-LMP1ex), and EBV transformed (DG75-EBVex), as well as from a lymphoblastoid cell line (LCL1ex), were analyzed by immunoblot for their expression levels of LMP1, HLA-DR, CD81, and β-actin. One representative immunoblot of four is shown.

FIGURE 2

DG75 exosomes harbor phenotypic differences that reflect the phenotype of their B cell line. (A) Schematic diagram of direct phenotypic characterization of different DG75 B cell lines and indirect phenotyping of their exosomes after binding onto anti–MHC class II Dynabeads by flow cytometry. (B) The expression pattern on cells and exosomes of HLA-ABC, HLA-DR, CD19, CD40, CD80, and CD86 and (C) of the tetraspanins CD63 and CD81 on exosomes shown as mean fluorescence intensity (MFI) ratio divided by the isotype control. Horizontal lines indicate mean values from 3–5 independent experiments for DG75 B cells (DG75-CO, DG75-LMP1, and DG75-EBV) and from 5–20 independent experiments for the exosomes (DG75-COex, DG75-LMP1ex, and DG75-EBVex). (D) Nanoparticle tracking analysis (NanoSight). The mean particle size distribution from three different exosome batches for DG75-COex, DG75-LMP1ex, and DG75-EBVex. *p ≤ 0.05, ***p ≤ 0.001, Mann–Whitney U test. MVB, multivesicular body.

FIGURE 3

DG75 exosomes bind with similar efficiency to B cells in PBMCs and are internalized by B cells. (A) Gating strategy used to assess binding of PKH67-labeled exosomes, after 4 h, to human B cells and monocytes within PBMCs, showing representative contour plots. (B) Binding pattern of PKH67-labeled DG75-COex, DG75-LMP1ex, and DG75-EBVex, after 1, 2, or 4 h, to B cells and monocytes. Results from three (1 h) and five (2 or 4 h) independent experiments are shown. Horizontal lines represent the mean. (C) Human primary B cells were incubated for 24 or 48 h with no exosomes (−), BJABex, or LCL1ex, and cell lysates were assessed by immunoblot analysis for expression of LMP1 and β-actin. One representative experiment of three is shown. (D) Confocal microscopy analysis of anti-CD19⁺ (red) B cells incubated with PKH67-labeled (green) DG75 exosomes for 4 h at 37°C. Areas in the white boxes are shown in the Merge column. Arrows point to intracellular (green) localization of exosomes within B cells. Scale bars, 30 µm (left and middle panels), 7.5 µm (right panels). One representative experiment of three is shown.

FIGURE 4

DG75 exosomes do not prevent apoptosis but induce B cell proliferation in PBMCs. Purified B cells (1.8 × 10⁵ cells/200 µl) were either left unstimulated (co) or stimulated with IL-21 + CD40L or with 15 µg/well DG75-COex, DG75-LMP1ex, or DG75-EBVex. After 24 h, the induction of apoptosis was assessed by flow cytometry (A), and morphological changes were documented (B). (A) Representative contour plot showing differentiation between alive (Annexin V⁻ and PI⁻), early apoptotic (Annexin V⁺, PI⁻), or late apoptotic/necrotic (Annexin V⁺ and PI⁺) B cells. Results are shown from four independent experiments (mean ± SEM). (B) Cultures of purified B cells were stimulated, as indicated, and visualized using a light microscope. Arrows indicate the formation of clumps. One representative experiment out of two is shown. Scale bars, 200 µm. (C) PBMCs were labeled with CFSE and either left unstimulated (co) or stimulated with PHA or DG75-COex, DG75-LMP1ex, or DG75-EBVex. After 5 d, proliferation of CD3⁺ T cells and CD19⁺ B cells was assessed by flow cytometry. One representative experiment of two is shown.

FIGURE 5

DG75 exosomes induce a dose-dependent proliferative response in B cells. B cells (1.5 × 10⁵ cells/200 µl) were labeled with CFSE and either left unstimulated (co) or stimulated with IL-21, CD40L, or IL-21 + CD40L or with 5 or 25 µg exosomes/well, with or without IL-21. After 5 d of culture, proliferation was assessed by flow cytometry. (A) Representative plots from one donor. The percentage of B cells for each quadrant is shown. (B) Combined results from four blood donors (mean ± SEM). Data are normalized to proliferation rates of IL-21 + CD40L–stimulated B cells (= 100%). *p ≤ 0.05, Mann–Whitney U test.

FIGURE 6

DG75-LMP1ex induces differentiation into a CD38^highCD20^low plasmablast-like B cell population. CFSE-labeled B cells (1.5 × 10⁵ cells/200 µl) were either left unstimulated (co) or stimulated with IL-21, CD40L, or IL-21 + CD40L or with 5 or 25 µg DG75 exosomes/well, with or without IL-21. After 5 d of culture, B cells were stained for CD19, CD20, and CD38 and assessed by flow cytometry. (A) Representative plots from one donor are shown, and the percentage of CD38^highCD20^low B cells is indicated. (B) Combined results from four blood donors (mean ± SEM). *p ≤ 0.05, Mann–Whitney U test.

FIGURE 7

DG75 exosomes induce CSR in human IgD⁺ B cells. IgD⁺ B cells were either left unstimulated (co) or stimulated with IL-21, CD40L, or IL-21 + CD40L or with DG75 exosomes, with or without IL-21, for 3 d. (A) Quantitative real-time PCR of AICDA transcripts relative to unstimulated (co) B cells. AICDA was normalized to EF-1α and GAPDH mRNA levels. Data from one donor of two is shown. Southern blot analysis of (B) circle transcripts for Iμ-Cμ and Iγ1/2-Cμ and (C) for Iμ-Cμ and Iγ1/2-Cγ1 germline transcripts. Data in (B) and (C) are from two donors.