Additional file 2: Table S2. of Inhibition of emotional needs and emotional wellbeing predict disease progression of chronic hepatitis C patients: an 8-year prospective study

Baseline associations between the FACIT scales and physical factors. (DOCX 16 kb

Infection of Adult Thymus with Murine Retrovirus Induces Virus-Specific Central Tolerance That Prevents Functional Memory CD8⁺ T Cell Differentiation

<div><p>In chronic viral infections, persistent antigen presentation causes progressive exhaustion of virus-specific CD8⁺ T cells. It has become clear, however, that virus-specific naïve CD8⁺ T cells newly generated from the thymus can be primed with persisting antigens. In the setting of low antigen density and resolved inflammation, newly primed CD8⁺ T cells are preferentially recruited into the functional memory pool. Thus, continual recruitment of naïve CD8⁺ T cells from the thymus is important for preserving the population of functional memory CD8⁺ T cells in chronically infected animals. Friend virus (FV) is the pathogenic murine retrovirus that establishes chronic infection in adult mice, which is bolstered by the profound exhaustion of virus-specific CD8⁺ T cells induced during the early phase of infection. Here we show an additional evasion strategy in which FV disseminates efficiently into the thymus, ultimately leading to clonal deletion of thymocytes that are reactive to FV antigens. Owing to the resultant lack of virus-specific recent thymic emigrants, along with the above exhaustion of antigen-experienced peripheral CD8⁺ T cells, mice chronically infected with FV fail to establish a functional virus-specific CD8⁺ T cell pool, and are highly susceptible to challenge with tumor cells expressing FV-encoded antigen. However, FV-specific naïve CD8⁺ T cells generated in uninfected mice can be primed and differentiate into functional memory CD8⁺ T cells upon their transfer into chronically infected animals. These findings indicate that virus-induced central tolerance that develops during the chronic phase of infection accelerates the accumulation of dysfunctional memory CD8⁺ T cells.</p></div

FV-specific CD8⁺ T cells can differentiate into functional memory CD8⁺ T cells if recruited during the chronic phase of infection.

<p>(A–D) B6AF₁ mice were infected with either FV or FV-OVA. Six weeks later, FACS-sorted naïve (CD44^lo) CD8⁺ T cells (1–2×10⁷) from (OT-1-Thy1.1× A/WySnJ)F₁ mice were transferred i.v. Splenocytes and BM cells were isolated at 28 days post transfer and stained with the indicated Abs and OVA_257–264/K^b tetramer. (B) Percentages of CD44^hi cells among OT-1 cells or endogenous OVA-specific CD8⁺ T cells, and actual numbers of CD44^hi OT-1 cells recovered from FV-OVA- or FV-infected mice. Averages between groups in the left panel were compared by one-way ANOVA with Tukey's multiple comparison test: *, <i>p</i><0.001; †, <i>p</i><0.05. Averages between FV-OVA and FV groups were compared by Welch's <i>t</i>-test: *, <i>p</i> = 0.047. (C) Representative staining patterns for PD-1 and CD69 among OT-1 cells or endogenous OVA-specific CD8⁺ T cells in the BM and spleen. (D) Expression of CD69 and PD-1 on CD44^hi OVA-specific CD8⁺ T cells in the spleen. Averages were compared for the two parameters between the endogenous and OT-1 cells: *, <i>p</i><0.00001<α₂ (0.05) = 0.0253 by student's <i>t</i> test with Bonferroni's correction for multiple comparisons. (E–F) A group of mice received 5×10³ OT-1 cells 1 day prior to FV-OVA infection as a control for CD8⁺ T cell responses that were primed by initial infection. Splenocytes were stimulated in vitro with OVA_257–264/K^b peptide. Shown are intracellular expression levels of IFN-γ and IL-2 of CD44^hi OT-1 cells primed at the initial infection or during the chronic phase of infection. Averages were compared for the two parameters between the preinfection transfer and chronic phase groups: *, <i>p</i> = 0.012<α₂ (0.05) = 0.0253 for IFN-γ and <i>p</i> = 0.013<α₂ (0.05) = 0.0253 by Welch's <i>t</i>-test with Bonferroni's correction. (G) B6AF₁ mice were infected with 5,000 focus-forming units of F-MuLV-OVA plus 2,000 SFFU of FV and naïve OT-1 cells (1×10⁷) were transferred 28 days after infection. Four weeks after transfer of naïve OT-1 cells, spleen weights were measured, and splenocytes were cocultured with <i>M. dunni</i> cells to enumerate F-MuLV infectious centers. Each symbol represents an individual mouse. *, significantly smaller in the numbers of infectious centers in comparison with those in non-transferred mice; <i>p</i> = 0.0357 by Mann-Whitney test for non-Gaussian distributions.</p

Identification of FV-infected cells in the thymus.

<p>Mice were infected with 1,000 SFFU of FV. (A) At day 14 post infection, cells in the thymus were isolated and stained with the indicated Abs. Shown are frequencies of F-MuLV gp70⁺ cells among indicated populations as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003937#ppat-1003937-g002" target="_blank">Figure 2</a>. Differences in means between uninfected and FV-infected groups were analyzed by two-way ANOVA with Bonferroni's corrections for multiple comparisons: *, <i>p</i><0.0001; †, <i>p</i><0.001; ‡, <i>p</i><0.05. (B) Shown are representative staining patterns for cell surface gp70 and p15<i>^gag</i> of each thymocyte population. (C) Representative frozen sections of the thymus from FV-infected mice (14 days post infection) were stained for CD11c and F-MuLV gag p30 (top), cTEC-specific ER-TR4 and F-MuLV gag p30 (middle), or mTEC-specific ER-TR5 and F-MuLV gag p30 (bottom). Arrowheads indicate cells double positive for the indicated cell surface marker and the viral antigen. A larger view field of the sections shown here can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003937#ppat.1003937.s003" target="_blank">Figure S3</a>. (D) Mice were infected with 1,000 SFFU of FV. At day 14 after infection, cells in the thymus were isolated, stained with the indicated Abs, and FACS sorted into the indicated populations as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003937#ppat-1003937-g002" target="_blank">Figure 2</a>. Cells were cocultured with <i>M. dunni</i> cells to enumerate F-MuLV infectious centers. Each symbol represents cells from an individual mouse. Dashed lines indicate the detectable limits (e.g. maximum available numbers of DCs and TECs used in this experiment were 1×10⁵). Data are representative of two independent experiments with essentially equivalent results.</p

Thymic DCs and TECs are the major deleters of FV-specific thymocytes.

<p>(A) Thymic stromal cells of B6AF₁ mice were prepared from E15.5 fetal thymus lobes. OT-1 DP thymocytes were sorted from adult (OT-1-Thy1.1× A/WySnJ)F₁ mice. Thymocytes, thymic DCs and TECs from FV-OVA infected mice (21 days post infection) were sorted into each population as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003937#ppat-1003937-g002" target="_blank">Figure 2</a>. Cells were mixed and cultured as a reassembled organ for 4 days. (B) Shown are relative percentages of CD8 SP cells as compared to the control. The percentage of the control value was calculated by [(% of CD8 SP cells in the experimental culture)/(% of CD8 SP cells in the control culture)]×100 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003937#ppat.1003937.s005" target="_blank">Figure S5</a>). Averages were compared between FV-OVA- and FV-infected groups for each third cell population by two-way ANOVA with Bonferroni's correction for multiple comparisons, and statistically significant differentiations are indicated: *, <i>p</i><0.01; †, <i>p</i><0.05.</p

Viral antigen expression in each cell population in the thymus after FV infection.

<p>Mice were infected with 1,000 SFFU of FV. At day 14 after infection, cells in the thymus were isolated and stained with indicated Abs. Shown are representative staining patterns and gating protocols of thymocytes (A), thymic DCs (B), and TEC populations (C). (B) Cells purified from the thymus were incubated with microbeads-labeled anti-CD90.2 antibody, and antibody-negative populations were further stained with fluorescent-labeled anti-CD11b, anti-CD11c, anti-B220, and anti-gp70. CD11c⁺ cells were separated into B220⁺ plasmacytoid DCs, B220⁻CD11b⁻ DCs of intrathymic origin, and CD11b⁺ DCs of extrathymic origin. (C) CD90.2⁻ populations were stained with fluorescent-labeled anti-EpCAM, anti-CD80, anti-Ly51, and anti-gp70. EpCAM⁺ cells were separated into CD80⁺ medullary TECs (mTECs), and Ly51⁺ cortical TECs (cTECs). Nonspecific binding of the biotinylated anti-gp70 mAb 720 especially onto DN thymocytes, DCs and TECs was inevitable even in the presence of anti-Fc receptor Abs, as evidenced by the background staining with the isotype control IgG. Thus, the percentages of “gp70⁺” cells shown here include some background values.</p

FV disseminates to and persists in the thymus following infection.

<p>Mice were infected with 1,000 SFFU of FV. (A) Cells in the BM, spleen and thymus were isolated at indicated time-points, and were cocultured with <i>M. dunni</i> cells to enumerate F-MuLV infectious centers. Each symbol represents an individual mouse. Data are representative of two independent experiments with essentially equivalent results. *, <i>p</i><0.0001 in comparison with the numbers of FV-producing cells in the BM at the same time-point; †, <i>p</i><0.0001 in comparison with those in the spleen, by two-way ANOVA with Bonferroni's correction for multiple comparisons. (B) Total RNA was purified from the BM, spleen and thymus of FV-infected mice at day 14. Expression levels of F-MuLV and SFFV mRNA were analyzed by quantitative real-time PCR assays. Shown are copy numbers of viral DNA fragments amplified from 1 µg of total cDNA.</p

Infection of thymus with FV leads to clonal deletion of FV-specific thymocytes.

<p>(A) To make a T cell-free microenvironment in B6AF₁ mice, day 5 neonatal pups were thymectomized, and 5 weeks later were injected intraperitoneally with depleting Abs for both CD4 and CD8. Two weeks later, a thymic lobe from either FV-infected (2–3 weeks post infection) or age-matched uninfected mice was grafted under the kidney capsule. (B) At day 40 after transplantation, splenocytes were isolated and stained with the indicated Abs. Shown are representative staining patterns for CD8 and CD19. (C) Six weeks after transplantation, when peripheral CD8 T cells were reconstituted, mice were challenged with either FV-antigen bearing FBL3 tumor cells or influenza virus ×31. Splenocytes were isolated at 10–14 days post challenge, and stained with the indicated Abs and either F-MuLV gag_75–83/D^b or Flu NP_366–374/D^b tetramer. Shown are representative staining patterns for CD8 and each tetramer among CD8⁺ T cells. (D) Shown are frequencies of tetramer⁺ cells among CD8⁺ T cells (left panels), and actual numbers of tetramer⁺ CD8⁺ T cells (right panels). Averages of % and actual numbers of tetramer⁺ cells were compared between uninfected and FV-infected groups by two-tailed Welch's <i>t</i>-test, as variances in both cases were not regarded as equal. *, <i>p</i><0.021; NS, not significant.</p