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. 2005 Dec 15;106(13):4339-44.
doi: 10.1182/blood-2005-06-2341. Epub 2005 Aug 2.

Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus

Christoph Mancao  1 , Markus Altmann, Berit Jungnickel, Wolfgang Hammerschmidt
Affiliations

Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus

Christoph Mancao et al. Blood. .

Abstract

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.

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Figures

Figure 1.
Figure 1.
Cell surface expression of λ and κ light chains of primary lymphocytes and preparation of BCR- B cells. (A) Primary lymphocytes were isolated from nasal adenoids and stained for cell surface expression of the immunoglobulin light chains λ and κ. Most cells were either single λ- or κ-positive BCR+ B cells, whereas a smaller fraction was double negative for both immunoglobulin light chains. (B) The lymphocyte preparation was depleted by magnetic sorting with λ- and κ-specific antibodies indirectly coupled to magnetic beads, and the BCR- fraction was again analyzed for cell surface expression of λ and κ light chains. (C) The BCR- population consisted of a smaller CD21+ B-lymphocyte fraction, whereas CD3+ T lymphocytes dominated. CD3-/CD21- cells are probably monocytes, which were not further analyzed. (D) In a second approach primary lymphocytes from adenoids were analyzed for surface expression of CD19 and CD77, revealing the initial fraction of GC B cells. (E) The lymphocyte preparation was depleted with CD3, λ- and κ-specific antibodies, enriched for CD77 surface-positive GC B cells, and again analyzed for light chain cell surface expression. (F) Prior to EBV infection, the cells were characterized for their expression of CD77 and CD19, indicating the germinal center origin of the surface BCR- B-cell preparation. Quadrant statistics based on intact cells in the lymphocyte gate by forward and sideward scatter criteria are depicted in the top right-hand corner of each FACS plot.
Figure 2.
Figure 2.
BCR+ as well as BCR- B cells infected with EBV enter the S phase of the cell cycle and proliferate in vitro. (A) An EBV genome, which encodes GFP, was used to infect the BCR- and BCR+ cell populations (middle and bottom panels) or BCR+ cells were left uninfected (top row panels). GFP+ cells were found in BCR+ and BCR- cells, indicating that EBV can infect cells in both populations. (B) To analyze the cell cycle distribution of the uninfected and infected BCR- and BCR+ bulk populations, the cells were labeled with the nucleotide analog BrdU 4 days after infection for 2 hours. Dual parameter flow cytometry of the cells with an APC-conjugated α-BrdU antibody and with 7-AAD to reveal the cellular DNA content indicated that the EBV-infected B-cell pool entered S phase and proliferated irrespective of their BCR surface status (middle and bottom panels, gates R2 and R3). Cells in the uninfected population enter neither S phase (top panel, region R2) nor M phase (region R3), as indicated by the lack of BrdU incorporation and predominance of G1/G0 phase (gate R1). (C) Dual parameter flow cytometry of EBV-infected B cells gated for typical forward/sideward scatter lymphocyte morphology. (D) Triple parameter flow cytometry of GFP+/EBV-infected B cells (gate M1 in panel A). BrdU incorporation and 7-AAD staining showed a similar cell cycle distribution in panels B, C, and D. Since only a fraction of EBV-infected primary B cells express GFP at a detectable level, the number of GFP-positive cells in the BCR+ and BCR- populations is markedly lower than in panel C. Also in contrast to panel B, most GFP+ cells in D are not apoptotic, as indicated by a minority of cells with sub-G1 DNA content.
Figure 3.
Figure 3.
Analysis of clonal LCLs for expression of immunoglobulin chains by FACS and Western blot immunodetection. (A) Single cell clones were obtained from limiting dilution assays and tested for surface BCR expression with light chain–specific antibodies by FACS. Six LCL clones are shown as representative examples, together with a control bulk population obtained from EBV-infected BCR+ B cells (BCR+). The numbers indicate the percentile of cells in the respective quadrants. (B) Single-cell clones no. 16, 54, and 59, which all fail to express λ or κ light chains at their cell surfaces, as shown in panel A, also are negative with regard to intracellular expression of the IgG-HC (50 kDa) as revealed by Western blotting. Clones no. 15 and 26 were immunoglobulin HC positive in Western blotting, but only no. 26 expressed a surface BCR, as shown in panel A. Clone no. 12, which does not express a surface BCR, is shown to be IgG-HC negative but IgM-HC (75 kDa) positive in Western blotting. The lane labeled “Ig” is a positive control with a purified commercial human IgG preparation. Another positive control is a pool of BCR+/EBV-infected LCLs. All LCLs are EBV infected, since they express the latent viral gene product EBNA2, which also served as a loading control.
Figure 4.
Figure 4.
Sequence analysis of clonal LCLs derived from EBV-infected BCR- B cells. The sequences of 4 single-cell LCL clones are shown, which carry a crippled heavy chain allele and fail to express a functional BCR. A detailed comparison of VDJH gene regions of the 4 sequenced clones (bottom lines) to the most homologous germ line genes (top lines) is shown. Sequence identity is indicated by dashes, and nucleotide exchanges introduced by somatic hypermutation are indicated. Uppercase characters denote replacement mutations, and lowercase characters describe silent mutations. (A) In clone A54 an in-frame stop codon (marked with asterisks) in CDR1 (solid lines) abrogates HC expression. (B) In clone A16 only the variable and the joining segment could be determined, since this rearranged heavy chain allele suffers from a massive internal deletion, which is indicated by boxes. The deletion also causes a frame shift mutation such that the C segments are out of phase. The clones B5 (C) and B11 (D) are crippled due to in-frame stop codons (asterisks). The forward and backward primer binding sites (dotted lines) contain wobbled bases: M (adenosine or a cytidine), and Y (cytidine or a thymidine).
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