CD8+ and CD4+ T cell recognition of B cells 24hrs post-EBV infection.

<p>(A) Representative plots showing CD8+ (left panels, black) and CD4+ (right panels, grey) T cell recognition of primary B cells 24hrs post-EBV infection (B + EBV) by effectors specific for EBNA2 (latent, top), BMLF1 (E lytic, middle) and gp110 (L lytic structural, bottom). Uninfected B cells (B) and epitope-peptide loaded B cells (B + pep) are included as negative and positive controls respectively. (B) Summary of T cell recognition data for all epitopes included in the study and derived from “first wave” (EBNA2, BHRF1) or IE (BZLF1, BRLF1) E (BMLF1) and L (gp85, gp110, gp350) lytic cycle antigens. Epitopes recognised by CD8+ and CD4+ effectors at the 24hr time-point are highlighted in black or grey respectively; epitopes on a white background are not recognised by CD8+ or CD4+ effectors within 24hrs post-EBV infection.</p

Identification of EBNA2-, EBNA-LP- and BHRF1-specific CD4+ T cell responses.

<p>(A) EBNA2-specific responses. Top panel: In vitro expanded CD4-selected polyclonal T cells from Donor 14 were screened for reactivity against overlapping peptides spanning the complete unique sequence of EBNA2. Results are expressed as the mean IFNγ concentration +/- SD for duplicate wells. Lower panel: Individual component peptides from adjacent pools 15 and 16 were screened for their ability to induce IFNγ production by the CD4-selected T cell population. The sequence of the 15mer peptide common to pools 15.5 and 16.1 is given below the figure. (B) EBNA-LP-specific responses. Top panel: In vitro expanded total polyclonal T cells from Donor 5 were screened for recognition of peptide pools spanning the complete unique sequence of EBNA-LP. Bottom panel: Individual component peptides from pool 2 were screened for their ability to mediate IFNγ production by the total T cell population; the sequence of the active peptide, 2.3, is given. Note that this response was abolished following CD4-depletion. (C) BHRF1-specific responses. Top panel: In vitro expanded CD4-selected polyclonal T cells from Donor 3 were screened for reactivity against overlapping peptides spanning BHRF1. Lower panel: Individual peptides from pools 3 and 5 were screened for their ability to induce IFNγ production by the CD4-selected T cell population. The sequence of the two active peptides (3.1 and 5.1) is given.</p

Identification of EBNA2-, EBNA-LP- and BHRF1-specific CD8+ T cell responses.

<p>(A) EBNA2-specific responses. Top left panel: In vitro expanded, CD8-enriched polyclonal T cells from Donor 17 were screened against overlapping 20mer peptides spanning the complete unique sequence of EBNA2 (sub-divided into 18 pools). T cell recognition was assessed by IFNγ production, measured in an ELISA. Results are expressed as the mean IFNγ concentration +/- SD for duplicate wells. Top right panel: Individual component peptides from pool 6 were screened for their ability to mediate IFNγ production by the CD8-enriched T cell population. Middle panel: To identify the HLA restriction, LCLs sharing one or more class I alleles with Donor 17 (class I type in bold) were pre-loaded with peptides 6.4/6.5 (1μg/ml) and co-cultured overnight with a specific T cell clone. Results are expressed as the mean IFNγ concentration +/- SD for triplicate wells. Table: Peptides 6.4, 6.5 and the predicted minimal epitope were screened for their ability to induce IFNγ production by the CD8-enriched polyclonal T cell population. (B) EBNA-LP-specific responses. Left panel: In vitro expanded CD8-enriched polyclonal T cells from Donor 20 were screened against overlapping 15mer peptides spanning the complete unique sequence of EBNA-LP (sub-divided into 4 pools); T cell recognition was assessed by IFNγ production. Right panel: Individual component peptides from pool 3 were screened for their ability to induce IFNγ production by the CD8-enriched T cell population. Sequences and T cell recognition data for peptide 3.6 and the minimal epitope are shown in the table. (C) BHRF1-specific responses. Top left panel: In vitro expanded CD8-enriched polyclonal T cells from Donor 8 were screened against overlapping 15mer peptides spanning the complete unique sequence of BHRF1 (sub-divided into 6 pools). Top right panel: Individual component peptides from pool 1 were screened for their ability to mediate IFNγ production by the total polyclonal T cell population. Middle panel: To identify the HLA restriction, LCLs sharing one or more class I alleles with Donor 8 (class I type in bold) were pre-loaded with peptides 1.3/1.4 (1μg/ml) and co-cultured overnight with a specific T cell clone. Results are expressed as the mean IFNγ concentration +/- SD for triplicate wells. Table: Peptides 1.3/1.4 and the predicted minimal epitope were screened for their ability to mediate IFNγ production by the CD8-enriched T cell population.</p

Analysis of EBNA2 expression and T cell recognition following EBV infection of B cells in vitro.

<p>(A) Analysis of gene expression using a 48:48 Dynamic Array QPCR assay to measure EBNA2, EBNA3B and LMP2 mRNA transcripts at time points between 6hrs and 10 days following infection of primary B cells with EBV (wt2089). Results are normalised to cellular PGK1 transcript levels. (B) Immunoblotting to detect expression of EBNA2, EBNA3B and LMP2 at time points between 0.5 and 6 days post EBV-infection; uninfected B cells (UI) and an established LCL were included as negative and positive controls respectively. (C) CD8+ T cell recognition of newly infected B cells. Left panels: Primary B cells (HLA-A11-, B*3801-positive) were infected with EBV (B95.8 supernatant) then co-cultured with latent antigen-specific (EBNA2: YHL/B*3801, EBNA3B: IVT/A11 LMP2: SSC/A11) T cell clones (40,000 B cells + 5000 T cells/well). Culture supernatant was harvested at the specified time points and the IFNγ concentration measured by ELISA; results are the mean of triplicate wells +/- SD. Right panels: T cell recognition of established HLA-B38 or -A11 matched LCLs -/+ cognate epitope peptide shown for reference. (D) Comparison of EBNA2-specific CD8+ versus CD4+ T cell recognition of newly infected B cells. Primary B cells (HLA-B*3801, DR7-positive) were infected with purified virus (wt2089), co-cultured with EBNA2-specific T cell clones (35,000 B cells + 2500 T cells/well) and assayed as in (C). Results for T cell co-culture assays are representative of ≥3 independent experiments using multiple effectors of different specificities.</p

CD4+ T cell epitopes.

<p>CD4+ T cell epitopes.</p

CD8 and CD4 epitope maps of EBNA-2, EBNA-LP and BHRF1.

<p>These epitope maps denote all responses identified in the present study and all previously characterised epitopes. Each protein is represented according to its relative size (B95.8 sequence; EBNA2: 487aa, EBNA-LP: (66 x 7) + 44 = 506aa, BHRF1: 191aa). The polyproline repeat domain of EBNA2 is shown as a hatched box and the seven amino terminal repeat domains of EBNA-LP as separate boxes. Epitopes (CD8 top, CD4 bottom) are illustrated as vertical bars and are identified by the first three amino acids of their sequence. CD8 responses for which the minimal epitope is not defined are shown in italics. Full details of all CD8 and CD4 epitopes relevant to the present study are given in Tables <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005549#ppat.1005549.t001" target="_blank">1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005549#ppat.1005549.t002" target="_blank">2</a>.</p

Analysis of BHRF1 expression and CD8+ T cell recognition following EBV infection of B cells in vitro.

<p>(A) Analysis of gene expression using a 48:48 Dynamic Array QPCR assay to measure BHRF (Y2-HF, latent) and EBNA2 transcripts at time points 0.5 to 14 days post-infection with EBV (wt2089). Results are expressed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005549#ppat.1005549.g005" target="_blank">Fig 5A</a>. (B) Immunoblotting to detect expression of BHRF1 at time points between 0.5 and 7 days post EBV-infection; uninfected B cells (UI), established wild-type (WT) and BZLF1-KO LCLs and a BL cell line (Sal) were included as controls. (C) CD8+ T cell recognition of newly infected B cells. Primary B cells (HLA-A68, B*5501-positive) were infected with EBV (wt2089) then co-cultured with BHRF1 (ETF/A68) and EBNA2 (RPT/B*5501)-specific CD8+ T cell clones (40,000 B cells + 2500 T cells/well). Supernatant was harvested at the specified time points and the IFNγ concentration measured by ELISA; results are the mean of triplicate wells +/- SD. (D) BHRF1 expression during latency. BHRF1 (ETF)-specific CD8+ T cell clones were screened against a panel of HLA-matched (A68-positive) and mismatched LCL pairs; each LCL pair included the WT and BZLF1-KO LCL. IFNγ ELISA, results are shown for one representative clone. Results for T cell assays are representative of ≥2 independent assays using multiple effectors of each specificity.</p

CD8+ T cell inhibition of B cell transformation and LCL outgrowth.

<p>(A) Representative results from three experiments where primary B cells were infected with B95.8 supernatant, either neat (bottom panels) or diluted 1:100 (middle panels) and co-cultured with EBNA2-, EBNA3B/C- or LMP2-specific CD8+ T cell clones at effector to target ratios between 2:1 and 0:1. Results (shown for one effector population of each specificity/experiment) are expressed as the minimum T cell seeding required to completely inhibit B cell transformation and LCL outgrowth, scored visually at ~4 weeks. T cell recognition of an established LCL from the B cell donor is included for reference (top panels). (B) Combined results (mean +/- SD) for all EBNA2 (YHL/RPT), EBNA3 (RPP/AVF/IVT/EGG) and LMP2 (TYG)-specific effectors from six independent experiments.</p

EBV-specific CD8+ T cell response and phenotype of PBMCs from children seronegative at visit one who at visit four had been very recently infected (IgM+ IgG+/-).

<p>PBMC samples from two donors that were EBV non-infected at visit one and became VCA IgM+ six months later were analysed for EBV-specific responses using appropriate MHC class I tetramers. Epitope-specific CD8+ T cells were further analysed for activation status by measuring CD38 HLA DR co-expression, cell cycle status by measuring Ki-67 status and Bcl-2 status. Flow plots and gating are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004746#ppat.1004746.g005" target="_blank">Fig. 5</a>.</p

Prospective analysis of EBV-specific CD8+ T cell response and phenotype of PBMCs from a child very recently infected (IgM+ IgG−) at visit one.

<p>Serial PBMC samples from an HLA B*0801 donor found to be EBV VCA IgM+ at visit one were analysed for EBV-specific responses using the B*0801 RAK-specific MHC class I tetramer. A sample from visit three was available in addition to the one from visit four for this donor. The epitope-specific CD8+ T cells were further analysed for activation status by measuring CD38 HLA DR co-expression, cell cycle status by measuring Ki-67 status and Bcl-2 status. Flow plots and gating are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004746#ppat.1004746.g005" target="_blank">Fig. 5</a>.</p