Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Co-infection of Cytomegalovirus and Epstein-Barr Virus Diminishes the Frequency of CD56dimNKG2A+KIR- NK Cells and Contributes to Suboptimal Control of EBV in Immunosuppressed Children With Post-transplant Lymphoproliferative Disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a rare but potentially life-threatening complication, frequently associated with Epstein-Barr virus (EBV), which develops after solid organ or stem cell transplantation. Immunosuppression received by transplant recipients has a significant impact on the development of PTLD by suppressing the function of T cells. The preferential proliferation of NKG2A-positive natural killer (NK) cells during primary symptomatic EBV infection known as infectious mononucleosis (IM) and their reactivity toward EBV-infected B cells point to a role of NK cell in the immune control of EBV. However, NK cell-mediated immune response to EBV in immunosuppressed transplant recipients who develop PTLD remains unclear. In this study, we longitudinally analyzed the phenotype and function of different NK cell subsets in a cohort of pediatric liver transplant patients who develop PTLD and compared them to those of children with IM. We found persistently elevated plasma EBV DNA levels in the PTLD patients indicating suboptimal anti-viral immune control. PTLD patients had markedly decreased frequency of CD56dimNKG2A+Killer Immunoglobulin-like receptor (KIR)- NK cells from the time of diagnosis through remission compared to those of IM patients. Whilst the proliferation of CD56dimNKG2A+KIR- NK cells was diminished in PTLD patients, this NK cell subset maintained its ability to potently degranulate against EBV-infected B cells. Compared to cytomegalovirus (CMV)-seropositive and -negative IM patients, PTLD patients co-infected with CMV and EBV had significantly higher levels of a CMV-associated CD56dimNKG2ChiCD57+NKG2A-KIR+ NK cell subset accumulating at the expense of NKG2A+KIR- NK cells. Taken together, our data indicate that co-infection of CMV and EBV diminishes the frequency of CD56dimNKG2A+KIR- NK cells and contributes to suboptimal control of EBV in immunosuppressed children with PTLD.

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection.

Long term carriers were shown to generate robust polyfunctional T cell (PFC) responses against lytic and latent antigens of Epstein-Barr virus (EBV). However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM]) and asymptomatic (AS) EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs) as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions) concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6-12 months. In conclusion, EBV antigen-specific CD4+ and CD8+ PFC responses emerge during the first year of primary EBV infection, with greatest responses toward immunodominant epitopes in both lytic and latent proteins, correlating to steady decline in PBMC and plasma viral loads.