TLR agonist and Epstein Barr Virus

Source: Iskra S, Kalla M, Delecluse HJ, Hammerschmidt W, Moosmann A. Toll-like

receptor agonists synergistically increase proliferation and activation of B

cells by epstein-barr virus. J Virol. 2010 Apr;84(7):3612-23. Epub 2010 Jan 20.

PubMed PMID: 20089650; PubMed Central PMCID: PMC2838115.

 

EBV maintains itself in its host in latent state for prolonged times. The most preferred host cell type for EBV is B cell. Four modes of latent infection have been described. Mode 0, I, and II are characterized by resting B cell phenotype and expression of very few of EBV proteins. In contrast, mode III involves expression of 12 EBV proteins. The combined action of these proteins induces B cell activation and proliferation, alter receptor expression and enhance antigen presentation.

Three categories of exogenous physiological signals that lead to B cell activation have been recognized. These are signal 1, the stimulation of B cell receptor by antigen binding, signal 2, stimulation of CD40 by CD40L and signal 3, the stimulation of pattern recognition receptors, TLRs, by microbe associated molecular patterns (MAMP). It is known that combination of all three signals is required for maximal proliferation of naïve B cells. Studies have also reported that stimulation with TLR ligands alone causes transient activation of B cells.

Earlier studies have established that EBV's latent membrane proteins LMP2A and LMP1 mimic signaling by the BCR and CD40, respectively. However, in case of primary B cell infection by EBV, whether and how a potential signal 3 is generated is not known. Studies have reported that EBV infection of naïve B cells elevates expression of TLR7, thus, TLR7 pathway might play a role in generation of signal 3.

It is also known from previous studies that other microbial agents present at the site of EBV infection might influence EBV infection, B cell transformation and release of virus. Thus, in the present study, the authors investigate the effects of CpG DNA and other exogenous TLR ligands on EBV driven B cell proliferation, clonal outgrowth and induction of EBV associated cellular receptors and cytokines.

RESULTS:

• Controlled analysis of infection and transformation by EBV: EBV particles or their components might trigger TLR mediated activation of B cells. Thus, it was necessary to control for such effects by using virus like particles alongside transformation competent EBV. The investigators used a recombinant EBV system that expresses enhanced GFP. To produce transforming, GFP-encoding EBV, EBV lyric cycle was induced in human epithelial 293 cells that stably carry genomes of this recombinant EBV as episomes. Cells which carry a mutant version of EBV genome (lacking terminal repeats, 293/TR- cells), release virus like particle with empty capsids without DNA. These virus like particles are able to bind to B cells and are also taken up by B cells. When B cells are infected with EBV/TR- (pseudoinfection) there is sufficient uptake of virus particles including GFP and there is also recognition of B cells by CD4+ T cells specific for EBV-proteins. The authors next analyzed virus-associated GFP expression and de novo GFP expression in cells infected with EBV and EBV/TR-. GFP expression was detected in both set of cells on day 1 of post-infection. In contrast, a high increase in fluorescence was observed in cells infected with EBV and not with EBV/TR- on day 3 post-infection. These results indicate de novo expression of GFP from the viral genome. Later on, this GFP-high subpopulation of cells increased in number and dominated the cultures by day 7. These results are consistent with the EBV-mediated B cell transformation.
  
• Rapid activation and proliferation of B cells in response to EBV: When B cells are infected with EBV, their phenotype changes from resting to lymphoblastoid one. This phenotype is characterized by larger cell size, enlarged cytoplasm, expression of activation markers and proliferation. To reassess the timing of this process, the investigators infected primary B cells with EBV or EBV/TR- and analyzed cells' scatter characteristics by flow cytometry. On day 3 after infection with EBV and not with EBV/TR-, a distinct population of lymphoblastoid cells with high forward and side scatter levels emerged and continuously expanded thereafter. The authors also looked for surface expression of co-stimulatory surface molecule CD86 on these B cells and found that there was no expression of CD86 on B cells infected with EBV/TR-. In contrast, in cells infected with EBV, CD86 was detected at day 3, highly expressed at day 7 and remained highly expressed thereafter.
  
• CpG DNA synergistically enhances B cell transformation by EBV: The authors next infected primary B cells with EBV or EBV/TR- in the presence or absence of CpG DNA. CpG DNA is bacterial or viral DNA containing unmethylated CpG dinucleotides and it is a ligand to TLR9, and it causes activation and proliferation of B cells. On day 7 after infection, infection with EBV alone caused growth of two lymphoblastoid cell lines from resting B cells. EBV/TR- alone produced no lymphoblastoid cells and presence of CpG DNA along with EBV/TR- led to only week production. When CpG DNA was present along with EBV, several lymphoblastoid cell lines were produced. In addition, the immediate presence of CpG DNA was critical for production of lymphoblastoid cells. To further test whether other immune cells could affect the EBV-CpG synergism, the investigators repeated the same experiments with total PBMCs in the presence or absence of Cyclosporin (inhibits T cell activation). They observed that effect of CpG DNA on EBV-mediated production of lymphoblastoid cells from PBMCs and from B cells were similar. They also observed that cyclosporine had no effect. These results indicated that EBV-CpG synergism was mediated by direct effects of EBV and CpG DNA on B cells. The authors also tested whether lymphoblastoid cells detected after EBV infection in the presence of CpG DNA are transformed permanently or not. Their results showed that presence of CpG DNA at the time of EBV infection led to a long term increase in number of transformed B cell clones. The authors further determine live/dead cell ratios in EBV-CpG DNA treated versus EBV-infected B cell cultures. They observed that addition of CpG DNA together with EBV in early infection led to a six fold higher survival rate on the day when lymphoblastoid cell growth was initiated. Combined together, all these results indicate that the presence of a TLR ligand at the site of EBV infection increases the efficiency of infection and transformation of B cells.
  
• CpG DNA increases activation of B cells by EBV: To determine whether CpG modifies the expression of CD80 or CD86 molecules on the surface of B cells, the authors infected peripheral B cells with EBV or EBV/TR- in the presence or absence of CpG DNA and analyzed induction of CD80 by flow cytometry. They observed that CD80 induction was highest and fastest after EBV infection in presence of CpG DNA. The authors further looked for secretion of cytokines (IL-6 and IL-10) in these cells. IL-6 was rapidly induced by combined action of EBV and CpG DNA. In contrast, IL-10 secretion was delayed. However, when blocking antibodies against IL-6 or IL-10 were added, no effect on lymphoblastoid cell outgrowth was observed in the early days of infection. Thus, the authors concluded that enhanced IL-6 or IL-10 release is not a major factor in the rapid CpG-mediated increase of outgrowth in the days after EBV infection.
  
• Susceptibility of naïve and memory B-cell subsets to EBV transformation in the presence of CpG DNA: The authors isolated peripheral B cells from blood and separated them into naïve (CD27-) and memory (CD27+) B cells. These cells were then infected with EBV or EBV/TR- in the presence or absence of CpG DNA. The results obtained suggested that the synergistic effect of EBV-CpG is similar on both naïve and memory B cells while CpG alone was effective on memory cells only.
  
• Effect of CpG DNA on early activation of EBV latency genes: The authors infected primary B cells with EBV in the presence or absence of CpG DNA and analyzed expression levels of EBNA1, EBNA2 and LMP1 genes of EBV at different time points. The authors could see the induction of these genes on infection with EBV, but presence or absence of CpG DNA had little effect.
  
• Effect of CpG DNA on the induction of EBV's lytic cycle: The investigators cultivated lymphoblastoid cell lines in the presence or absence of CpG DNA and determined expression levels of five genes from EBV's immediate, early and late lytic stages by quantitative real-time PCR. They observed that most of these genes were downregulated by CpG DNA except BALF4. When the authors tested supernatants of these cells to determine whether they contain EBV that can infect and transform sensitive B cells, they found that supernatants from CpG DNA treated cells had this ability while those from untreated cells did not have the ability to transform B cells. These results suggested that CpG DNA might favor EBV release by latently infected B cells without up-regulation of lytic genes.
  
• Effects of various TLR ligands or bacteria on EBV transformation of tonsillar or peripheral B cells: The authors next used various other TLR liagnds e.g., Pam3CSK4 (TLR-2ligand), imiqiomod (TLR7 ligand), LPS (TLR4 lignad), LTA (TLR2 lignad) and whole fixed Staphylococcus aureus bacteria. The authors infected B cells with EBV in presence of these ligands or CpG DNA at various concentrations and analyzed lymphoblastoid cells growth 7 days after infection. The authors observed that several TLR ligands favored the outgrowth of EBV-infected B cells. Of these, CpG DNA had the strongest effect, followed by S. aureus and TLR2 ligand Pam3CSK4 and LTA. Only high concentrations of imiquimod were effective while LPS had no effect. In the presence of EBV TR-, effects of all TLR ligands were small. Not much difference was observed in tonsillar or peripheral B cells. These results suggest that not only agonists of TLR9 but also those of other TLRs, such as TLR2, support EBV-driven B cell activation and early transformation.