gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.
Citation: Ruiss R, Jochum S, Mocikat R, Hammerschmidt W, Zeidler R (2011) EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL. PLoS ONE 6(10): e25294. https://doi.org/10.1371/journal.pone.0025294
Editor: Gabriele Multhoff, Technische Universitaet Muenchen, Germany
Received: July 27, 2011; Accepted: August 31, 2011; Published: October 10, 2011
Copyright: © 2011 Ruiss et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported by grant 107793 from the Deutsche Krebshilfe (www.krebshilfe.de) and grant DJCLS R09/17 from the Deutsche José Carreras Leukämie-Stiftung (www.carreras-stiftung.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Epstein-Barr virus (EBV) is an almost ubiquitous human gamma herpes virus that infects resting human B-lymphocytes, including B-CLL cells, with high efficacy , . EBV's B-cell tropism is mainly due to gp350, the viral envelope glycoprotein that interacts with the cellular complement receptor 2 (CR2, CD21)  on B cells. In EBV seropositive individuals, gp350 mainly elicits CD4+ T-cell responses .
Exosomes are endosome-derived membrane vesicles, which are released by cells of diverse origin including dendritic cells, cancer cells  and EBV-infected B cells . Exosomes bud from endosomal membranes and accumulate in multivesicular bodies, which eventually fuse with the cellular membrane and release the contained vesicles. Exosomes are rich in lipids and membrane proteins like MHC molecules, TNF-R and tetraspanins  but their specific composition depends on the cell of origin. Exosomes either fuse to the recipient cell membrane or are engulfed by phagocytic cells in such a way that exosome proteins are degraded and loaded onto MHC class II molecules . Obviously, exosomes can deliver proteins as cargo in a very immunogenic manner so that they efficiently reactivate specific CD4+ T cell clones . Hence, exosomes can induce strong and epitope-specific immune responses ,  and can be used as an alternative to transfer strategies using gene vectors and as promising vaccines , .
Chronic lymphocytic leukemia of B-cell origin (B-CLL) is the most common adult leukemia in the Western hemisphere. B-CLL is considered as a prototypic disease undergoing immune evasion as the malignant cells lack important accessory and co-stimulatory molecules. Thus, despite their expression of high levels of surface MHC class I and II molecules, which presumably present tumor-associated antigenic epitopes, the leukemic cells tend to induce tumor-specific T-cell anergy. Typically, activated T cells from patients show a significantly reduced expression of CD40 ligand (CD154) or are completely CD154-negative . As a consequence, T cells from B-CLL patients cannot activate cells through the CD40 receptor. This interaction, however, is essential for CD40 signaling and subsequent induction of other immune accessory molecules like CD80 and CD86, which increase the antigen-presenting capacity of normal and B-CLL cells. On the other hand, the EBV-specific cellular immunity is relatively intact in these patients . To overcome the dysfunction of potentially tumor-reactive T cells from patients with B-CLL, several approaches have been developed relying on the stimulation of B-CLL cells through the CD40 pathway, including the ectopic expression of CD154 on the leukemic cells, and aiming at the self-stimulation of these cells –. In summary, immunotherapy of B-CLL is promising and CD154 is a potential candidate molecule to improve the patients' immune status and, eventually, the clinical outcome.
The robust cellular immunity in B-CLL patients against EBV  therefore prompted us to investigate the potential of tailored exosomes to redirect this immunity to malignant B cells. We present a novel approach for the targeted transfer of functional cellular proteins to B cells via tailored gp350+ exosomes. In this approach, gp350 has a dual function: (i) it confers B-cell tropism to exosomes so that they specifically co-transfer proteins of interest and (ii) it is a viral neo-antigen for these cells so that they efficiently reactive gp350-specific T cells. As a proof of concept, we show that tailored gp350+ exosomes can co-transfer functional CD154 as immune accessory molecule to B-CLL cells, which are subsequently stimulated to express surface molecules like CD54, CD80, CD86 and CD95 and stimulate autologous tumor- and EBV-specific T cells.
EBV gp350 is packaged into exosomes, confers B-cell tropism, and reactivates specific T cells
EBV has a profound B-cell tropism that is mainly conveyed by gp350, which is the major EBV glycoprotein in the viral envelope and the ligand for cellular CD21 (CR2) on B cells. We knew from previous work that exosomes can transport ectopically expressed proteins such as green fluorescent protein (GFP), which is presumably present as a cargo in the exosomal lumen. In addition, several groups provided evidence that surface proteins are incorporated in exosome membranes . We therefore asked whether gp350 could also become an integral part of exosomes and confer B-cell tropism to these vesicles. To answer this question, we co-transfected 293 cells with expression plasmids encoding BLLF1, the gene of gp350, and gfp. Three days later, we isolated vesicles from the supernatants of transfected HEK293 cells as described in Material and Methods and analyzed them by immunoblots for the presence of gp350 and exosome markers. Gp350 was detected in vesicles that floated at a density between 1.03 and 1.08 into an OptiPrep™ gradient, corresponding to a density between 1.13 to 1.18 in a sucrose gradient and thus in the density described for exosomes. The gradient also revealed the co-sedimentation of gp350 with the exosome markers hps70, tsg101 and CD63, indicating the nature of the gp350+ vesicles as exosomes (Figure 1A). Flow cytometry of exosomes coupled to latex beads revealed that gp350 is presumably located within the exosome membrane because it could be targeted with a specific antibody (Figure 1B). To demonstrate that gp350 confers B-cell tropism also to exosomes, we incubated gp350+/gfp+ exosomes with PBMCs from a healthy donor for one day and then quantified exosome binding by measuring GFP fluorescence by flow cytometry. This assay revealed that gp350+/gfp+ exosomes had an EBV-like tropism because they bound to CD19+ B cells but not to CD19-negative cells (Figure 1C).
(A) 293 cells were transfected with a gp350 expression plasmid, vesicles in the supernatant were purified as described and fractions of the OptiPrep™ gradients were spotted onto PVDF membrane. Immunoblots demonstrated that vesicles carry gp350 and that these vesicles co-sediment with vesicle that carry the exosome markers hsp70, tsg101, CD63 and ganglioside (GM)1 (www.exocarta.org). Calnexin is present in cell lysates (CL), its absence from exosomes demonstrated the purity of the preparation. (B) Latex beads were coated with gp350+ exosomes and stained with a gp350-specific antibody. gp350 is accessible and thus probably located in the exosome membrane (isotype control is shown as grey tinted histogram). (C) gp350 confers B-cell tropism to exosomes. PBMCs from a healthy donor were incubated for 18 h with gp350+/gfp+ exosomes and then analyzed for GFP fluorescence by flow cytometry. Only CD19+ B-cells stained positive, whereas an interaction of gp350+ exosomes with CD3+ T cells and CD19– cells (T and NK cells, monocytes) could not be observed. (D) B cells from a healthy donor were incubated with serial dilutions of exosomes from 293 cells transfected either with a BNRF1 expression plasmid or co-transfected with expression plasmids for BNRF1 (293/BNRF1) and gp350 (293/BNRF1+/gp350+) for one day. Then, autologous BNRF1-specific CD4+ T cells were added and their activation was measured with an IFN-γ ELISA one day later. T cells were activated at detectable levels by cells incubated with 293/BNRF1+/gp350+ exosomes but not with 293/BNRF1+ exosomes, indicating the relevance of gp350+ as a mediator of B cell tropism. An autologous EBV-infected lymphoblastoid cell line (LCL) was enclosed as a positive control.
Phagocytic cells engulf exosomes, process their proteins in lysosomes and present epitops in association with MHC class II molecules to CD4+ T cells . To further utilize the potential of gp350+ exosomes to specifically transfer exogenous proteins to B cells, which, in turn, may activate specific T cells, we generated exosomes that carried BNRF1, the major tegument protein of EBV, either alone (BNRF1+) or together with gp350 (BNRF1+/gp350+). We then incubated purified CD19+ B cells with these exosomes overnight and used these PBMC as stimulators for an autologous BNRF1-specific CD4+ T-cell clone. As shown in Figure 1D, B cells incubated with BNRF1+/gp350+ exosomes activated the T-cell clone in a concentration-dependent manner whereas B cells incubated with BNRF1+ exosomes did not activate the T-cell clone. This result demonstrates the potential of gp350+ exosomes to transfer immunogenic foreign proteins to B cells that then can activate specific CD4+ T lymphocytes.
Exosomes transfer functional CD154 to B-CLL cells
In a next series of experiments we wanted to elucidate whether 293/gp350+ exosomes can co-transfer functional membrane proteins to B cells. As a model system but also as a potential practical application, we chose B-CLL cells that express CD21 and become immunogenic upon ectopic expression of CD154 on malignant cells. We, therefore, transfected 293 cells with expression plasmids for gp350 and CD154 and isolated exosomes as described above. Again, an immunoblot with a CD154-specific antibody demonstrated the presence of CD154 in exosome preparations and an OptiPrep™ gradient revealed co-sedimentation with gp350 (Figure 1A) indicating the presence of both proteins on exosomes (Figure 2A). In addition, flow cytometry revealed that gp350+/CD154+ exosomes specifically bound to CD19+ B cells from a B-CLL patient (Figure 2B).
(A) 293 cells were transiently transfected with an expression plasmid encoding CD154. An immunoblot with a CD154-specific antibody revealed that CD154 is highly expressed in transfected cell and that the protein is incorporated into exosomes. Shown are immunoblots of cell lysates (CL) and lysates from purified exosomes (EL) of normal and transfected 293 cells, incubated with CD154- (upper panel) and tsg101-specific antibodies (middle panel). An OptiPrep™ gradient revealed co-sedimentation of CD154- and gp350-carrying vesicles (see Figure 1A). (B) Exosomes carrying gp350+ specifically bind to CD19+ B cells from a B-CLL patient. Fresh B-CLL cells were incubated with purified exosomes from 293 cells transfected with a gp350 expression plasmid and binding of exosomes was measured by flow cytometry. (C) Exosomes carrying CD154 bind only weakly to B-CLL cells, probably through interaction with CD40, which is highly expressed on these cells. Binding of CD154-carrying exosomes is drastically enhanced by the co-expression of gp350. 293 cells were co-transfected with expression plasmids for CD154 and/or gp350. Fresh B-CLL cells were co-cultivated with CD154+/gp350– and CD154+/gp350+ exosomes for two days and binding of exosomes and thus