Hepatitis C Virus-Related Lymphomagenesis in a Mouse Model

B cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis C virus (HCV) infection. The mechanism by which HCV infection leads to lymphoproliferative disorder remains unclear. Our group established HCV transgenic mice that expressed the full HCV genome in B cells (RzCD19Cre mice). We observed a 25.0% incidence of diffuse large B cell non-Hodgkin lymphomas (22.2% in male and 29.6% in female mice) within 600 days of birth. Interestingly, RzCD19Cre mice with substantially elevated serum-soluble interleukin-2 receptor α-subunit (sIL-2Rα) levels (>1000 pg/mL) developed B cell lymphomas. Another mouse model of lymphoproliferative disorder was established by persistent expression of HCV structural proteins through disruption of interferon regulatory factor-1 (irf-1_/_/CN2 mice). Irf-1_/_/CN2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders. Moreover, these mice showed increased levels of interleukin (IL)-2, IL-10, and Bcl-2 as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes

EGFR T790M Mutation as a Possible Target for Immunotherapy; Identification of HLA-A*0201-Restricted T Cell Epitopes Derived from the EGFR T790M Mutation

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non–small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p = 0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients

B-cell-intrinsic hepatitis C virus expression leads to B-cell-lymphomagenesis and induction of NF-κB signalling

Hepatitis C virus (HCV) infection leads to the development of hepatic diseases, as well as extrahepatic disorders such as B-cell non-Hodgkin's lymphoma (B-NHL). To reveal the molecular signalling pathways responsible for HCV-associated B-NHL development, we utilised transgenic (Tg) mice that express the full-length HCV genome specifically in B cells and develop non-Hodgkin type B-cell lymphomas (BCLs). The gene expression profiles in B cells from BCL-developing HCV-Tg mice, from BCL-non-developing HCV-Tg mice, and from BCL-non-developing HCV-negative mice were analysed by genome-wide microarray. In BCLs from HCV-Tg mice, the expression of various genes was modified, and for some genes, expression was influenced by the gender of the animals. Markedly modified genes such as Fos, C3, LTβR, A20, NF-κB and miR-26b in BCLs were further characterised using specific assays. We propose that activation of both canonical and alternative NF-κB signalling pathways and down-regulation of miR-26b contribute to the development of HCV-associated B-NHL

Translocase of outer mitochondrial membrane 70 induces interferon response and is impaired by hepatitis C virus NS3

Hepatitis C virus (HCV) elevated expression of the translocase of outer mitochondrial membrane 70 (Tom70). Interestingly, overexpression of Tom70 induces interferon (IFN) synthesis in hepatocytes, and it was impaired by HCV. Here, we addressed the mechanism of this impairment. The HCV NS3/4A protein induced Tom70 expression. The HCV NS3 protein interacted in cells, and cleaved the adapter protein mitochondrial anti-viral signaling (MAVS).Ectopic overexpression of Tom70 could not inhibit this cleavage. As a result, IRF-3phosphorylation was impaired and IFN-β induction was suppressed. These results indicate thatMAVS works upstream of Tom70 and the cleavage of MAVS by HCV NS3 protease suppresses signaling of IFN induction

The expression of genes involved in oncogenic pathways associated with BCL.

<p><b>A</b>: Highly modified gene signals in B cell lymphoma in RzCD19Cre mice BCL vs. B cells in RzCD19Cre male (Pair 1) or female (Pair 3) mice (left), and the genes modified by HCV expression in B cells in male (Pair 2) or female (Pair 4) (right). Red indicates the relative enhancement of the expression ratio of the processed signal (Test/Control, 532/635), and green indicates the relative reduction of expression. <b>B</b>: Quantification of Fos mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice (numbers of individual mice were indicated) by quantitative RT-PCR. Fos mRNA was normalised against 18S rRNA, and the relative ratio was calculated. Vertical bars indicate S.D. <b>C</b>: Quantification of C3 mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice. C3 mRNA was normalised against 18S rRNA, and relative ratio was calculated. Vertical bars indicate S.D. <b>D</b>: Quantification of LT βR mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice by quantitative RT-PCR. RNA copies per total RNA (μg) were indicated and vertical bars indicate S.D.</p

The expression of A20 in HCV-associated BCL.

<p><b>A</b>: Expression levels of A20 in the spleen from RzCD19Cre mice with or without BCL. Whole-tissue extracts prepared from the spleen in CD19Cre mice (control, n = 3; lanes 1–3 217–2, 2 224–2, 224–3), RzCD19Cre mice without BCL (HCV/Tumour(-), n = 5; lanes 4–8 217–3, 224–4, 232–3, 254–4, 240–2) and RzCD19Cre mice with BCL (HCV/Tumour(+), n = 5; lanes 9–13 24–1, 56–5, 69–5, 59–1, 43–4) were subjected to SDS-PAGE and were analysed by immunoblotting using anti-N terminal (A20N), anti-C terminal A20 (A20C), and anti-GAPDH antibodies. GAPDH was used as protein loading control. <b>B</b>: Quantitation of A20 (N and C), the average is indicated and statistical analysis was performed. Vertical bars indicate S.D.</p

Double immunofluorescence localisation of B220 (Green) and NF-κB p65 (Red) in HCV-Tg mice and the fractionation analysis of mouse tissues.

<p><b>A</b>: Co-localisation of NF-κB p65 immunoreactivity with B220 is indicated by arrows. (a–b) Cells double-positive for B220 and NF-κB in the control mouse (CD19cre). (c–d) Cells double-positive for B220 and NF-κB in the asymptomatic HCV-Tg mouse (RzCD19cre). (e–f) Cells double-positive for B220 and NF-κB in the lymphomatous HCV-Tg mouse (RzCD19cre). <b>B</b>: Quantitative analysis of the ratio of double-positive cells among B220-positive cells in each HCV-Tg mouse. Bar graph indicates the percentage of cells with NF-κB p65 nuclear translocation in B220-positive cells. <b>C</b>: Bar graph shows the ratio of double-positive cells within the B220-positive cells in normal, asymptomatic and lymphomatous HCV-Tg mice. Ho: Hoechst33342 Data are presented as means ± S.E., * P<0.05, ** P<0.01, *** P<0.001. <b>D</b>: Western blot analysis: tissues from the spleen of controls (224–2, 3) or HCV-Tg mice without BCL (217–3, 224–4, 232–3) or with BCL (56–5, 69–5) were fractionated into nuclear and cytoplasmic fractions. NF-κB p50 and p65 were detected by antibodies. Relative ratios of quantitation by imager are indicated. GAPDH was detected as a loading control of the cytoplasmic fraction.</p

Data from array performed once with mixed RNA samples (<b>Table 1</b>) were analysed using MetaCore software.

<p>Signals were analysed in the disease network (<b>A</b>) and in the process network (<b>B</b>) the values for the microarray data [(Feature number; yellow), (Process Signal (635); blue), (Process signal (532); red), Test/Control (532/635); green], (Process Signal (635); orange), (Process signal (532); purple)] are indicated by coloured bars. Abbreviations: BCL = B cell lymphoma. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091373#pone-0091373-t001" target="_blank">Table 1</a> for construction of pairings.</p

Mice subjected to microarray analysis.

<p>*BCL: B cell lymphoma; ⁺4EBP(+/−): heterozygous knockout of 4E-BP1 gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091373#pone.0091373-TsukiyamaKohara2" target="_blank">[73]</a>.</p

Quantification of miR-26b in BCL from HCV-positive and HCV-negative and non-tumour Tg mice.

<p>Formalin-fixed, paraffin-embedded (FFPE) splenic tissue from 24 animals (BCL HCV+, n = 8; BCL HCV-, n = 5; non-tumorous spleen HCV+/−, n = 11) was analysed for miR-26b expression by single assay stem-loop Q-RT-PCT by triplicate experiments. Data are shown as scatter dot-plots, and horizontal bar depicts the mean; y-axis: delta Ct (inverted scale) calculated in relation to endogenous control (snoRNA202). HCV-positive lymphoma tissue: filled circles; HCV-negative lymphoma tissue: filled squares; non-tumorous splenic tissue: filled triangles. P-values are shown in the graph.</p