Fewer WNV-specific CD4⁺ T cells are present in the draining lymph node of old WNV-infected mice compared to adult.

<p>C57Bl/6 mice were infected with 1000 PFU 385–99 WNV, sc (foot pad). ndLN, dLN and the spleen were harvested on day 8 post-infection for lymphocyte analysis by flow cytometry (FCM). Representative gating strategy for CD4⁺ tetramer⁺ T cells via live, forward and side scatter, CD3⁺ T cells, CD8α^- CD4⁺ T cells and E641:I-A^b+ CD4⁺ T cells harvested from (A) ndLN, (B) dLN, and (C) spleen are shown on day 8 post-WNV infection. Absolute numbers of E641:I-A^b+ CD4⁺ T cells in adult and old C57BL/6 and DEREG mice enumerated for (D) ndLN, (E) dLN, and (F) spleen are shown as labeled. (G) Percent CD4⁺ INF-γ⁺ T cells in dLN of adult and old C57BL/6 and DEREG mice. Shown are the aggregate results of two experiments with (A-F) 6–9 mice/group and (G) 5–8 mice/group. Each dot represents a single mouse (*p<0.05; **p<0.01; Mann-Whitney).</p

Disconnect between precursor number and response magnitude after immunization.

<p>Adult and old C57Bl/6 mice were immunized subcutaneously with 20μg peptide and 10μg LPS on both sides of the base of the tail and mice were kept on BrdU drinking water during the course of the challenge. Day 6 post immunization, pooled spleen and lymph node cells were incubated with E641:I-A^b, OVA:I-A^b and MCC:I-E^k tetramers, enriched using anti-His magnetic beads, and enumerated by flow cytometry after antibody staining and gating on: lymphocytes via forward and side scatter; then CD3⁺ CD19⁻, CD11c⁻, F4/80⁻, CD8⁻ T-cells; then CD3⁺ CD4⁺ T-cells; and finally E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− T-cells in the tetramer enrichment bound fraction from adult and old mice [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198354#pone.0198354.ref015" target="_blank">15</a>]. (A) Absolute number of E641:I-A^b+, OVA:I-A^b−, MCC:I-E^k− cells from adult and old mice after immunization with E641+LPS. Bars represent median values (ns p>0.05; Mann-Whitney). The average number per group (±SEM) is also shown below the respective label. (B) Concatenated contour plots showing BrdU incorporation by E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− cells in adult and old mice (gated as above). E641:I-A^b+ cells are divided into BrdU negative (neg), intermediate (int) and high (hi) subsets. Numbers indicate percent (±SEM) E641:I-A^b+ CD4⁺ T cells in the respective gates. Shown are the aggregate results of two experiments with 4 mice/group.</p

Larger precursor population does not equate to a larger response upon <i>Lm</i> infection.

<p>Adult and old C57Bl/6 mice were infected i.v. with 10⁷ CFU <i>Lm</i>E641-OVA in 100μl PBS. On day 6 post-infection, pooled spleen plus lymph node cells were incubated with E641:I-A^b, OVA:I-A^b and MCC:I-E^k tetramers, enriched, and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198354#pone.0198354.g001" target="_blank">Fig 1</a>. (A) Absolute number of responding E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− CD4⁺ T-cells in adult and old mice (ns p>0.05; Mann-Whitney). The average number of cells (±SEM) is shown below the respective label. (B) Representative concatenated contour plot showing BrdU incorporation by E641:I-A^b only cells in adult and old mice. Numbers indicate percent (±SEM) E641:I-A^b+ CD4⁺ T cells in the respective gates. Shown are the aggregate results of two experiments with 3–4 mice/group.</p

Relative and absolute counts of naïve, CM and EM cells in blood, spleen and LN of MCMV, VACV or MOCK-infected mice.

<p>3 month-old BALB/c mice were injected with 2×10⁵ PFU of MCMV, 10⁶ PFU of VACV or 200 µl PBS (mock infected). 6 months later blood, spleen and inguinal LN CD8⁺ cells were gated on CD11a, CD44, CD62L and CD27 (for a representative gating strategy, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat.1002849.s003" target="_blank">Fig. S3</a>) to define (A) the relative representation and (B) the absolute count of naïve, CM and EM subsets in each compartment. Each dot represents data from a single mouse, horizontal lines show medians.</p

CD8 T-cell responses to superinfection upon MCMV infection.

<p>(A) groups of 3 (young) and 12 (old) month old mice were primed with 10⁵ PFU of MCMV, 10⁶ PFU of VACV-WR or PBS (MOCK), and challenged 5 months later with 300 EID of influenza virus, PR/8 strain, intranasally (i.n.). 7 days post challenge blood lymphocytes were tested by ICCS for IFNγ responses to a 9 h in vitro stimulation with the NP³⁶⁶ peptide in the presence of BrefeldinA. Mean IFNγ responses in animal groups (n = 5/group)+SD is shown on the y axis. (B) 12 month-old mice were primed as indicated and challenged 5 months later with Influenza. The frequency of peptide specific cells in the blood CD8 pool was defined by peptide restimulation and ICCS. Group average (+SD) values for either peptide are indicated on the y axis. (C) Mice were primed with 10⁵ PFU of MCMV or 10⁶ PFU of VACV-IE1 at 2 months of age, and i.p. challenged with 50 PFU of West Nile Virus at 8 months of age. 7 days later, blood lymphocytes were in vitro stimulated with a pool of two immunodominant H^2d restricted WNV peptides and analyzed by ICCS. The frequency of IFNγ expressing T-cells in the CD8 pool of individual mice is displayed on the y axis. Horizontal lines indicate medians. (D) C57BL6/DBA2 F1 mice were primed with MCMV or VACV-IE1 at 3 months of age, challenged with HSV-1 at 8 months of age and frequencies of HSV-1 specific responses were determined 7 months later by pMHC tetramer staining and FCM. Each mouse is indicated with a symbol, horizontal lines are means, the dashed line shows the detection threshold. The p value for Mann-Whitney analysis is shown. (E, F) Mice were primed with MCMV or VACV-IE1 at 6, 12, 16 or 20 months of age, and assayed for responses to WNV challenge at 22 months of age (16, 10, 6 or 2 months post prime, as indicated below axis) using the protocol as in panel C. Each mouse is indicated with a symbol, horizontal lines are means. (E) % of CD8 cells responding to peptide stimulation in ICCS. (F) Leukocytes were in parallel assayed by polyclonal stimulation with anti-CD3 antibodies, and the peptide specific response was normalized to the CD3 (Max) response. Values in panels A, C, E and F were compared by 1-way ANOVA followed by Bonferroni comparison of individual columns, and significance is indicated (n.s. - p>0.05, *- p<0.05, ** - p<0.01, *** - p<0.001).</p

Poor CD8 response to WNV in latently infected mice is not caused by viral immune evasive genes.

<p>(A) Mice were primed with 10⁵ PFU of MCMV, 5×10⁵ PFU of ΔMCMV or 10⁶ PFU of VACV-IE1 at 6–8 months of age and challenged with 50 PFU of WNV at 22 months of age. Peptide stimulation with WNV peptides was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat-1002849-g006" target="_blank">figure 6A</a>, and group averages+SD of IFNγ responses are indicated by histograms. Statistical comparison was performed by ANOVA followed by Bonferroni analysis of individual groups (*** p<0.0001, n.s. p>0.05). (B, C) IFNγ responses upon WNV challenge were correlated to the frequency of (B) EM (CD62L⁻) or (C) naïve (CD11a⁻CD44⁻) CD8 T-cells in individual MCMV (red dots) VACV-IE1 (green dots) or MOCK (black dots) infected mice. Trend indicates the linear correlation, Pearson r and significance (p) are indicated, where the p value indicates the probability that the trend deviates from a horizontal line.</p

Vβ family analysis of CD8 pools upon MCMV infection.

<p>(A,B,C) BALB/cxDBA/2 F1 Mice infected at 6 months of age with MCMV or VACV-IE1 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat-1002849-g001" target="_blank">Fig. 1A</a> and 1B were bled at 14 months following infection and analyzed for frequency of Vβ8, Vβ9, Vβ10, Vβ13 and Vβ14 populations. Representative gating for Vβ 8 and Vβ10 is shown (A). Frequencies of CD8 cells belonging to Vβ families were analyzed in individual mice and a representative analysis is shown for Vβ14 (B), where each mouse is displayed by a symbol, and group medians by horizontal lines. The cohort variability of Vβ population frequencies were defined in groups of MCMV or VACV infected mice (C), and are indicated as SD on the x axis. Variance F-tests were performed to identify differences in group variabilities and p values are indicated in the chart.</p

Cytomegalovirus infection irreversibly perturbs the CD8 T-cell pool.

<p>(A, B) 6 months old mice were infected with 10⁵ PFU of MCMV or 10⁶ PFU of VACV-IE1 and compared to untreated controls. Blood leukocytes were stained for CD3, CD4, CD8, CD11a, CD62L and YPHFMPTNL-L(d) tetramers and analyzed by flow cytometry. (A) CD8 T-cells were gated on tetramer⁺ CD11a⁺ gate as indicated in the representative plot on the left and group means (+/− SD) at 7, 14 28, 60, 90 180, 270 and 420 days post infection are connected. (B) The same cells as in panel A were gated on a CD62L⁻CD11a⁺ gate to identify EM cells (plot on the left) and group means (+/− SD) for indicated days are connected. (C) At 9 months post infection, we gated CD8⁺CD4⁻ lymphocytes and compared the surface TCR expression, defined as the mean fluorescence index (MFI) of CD3, in the MCMV, VACV and mock infected mice. Symbols indicate individual mice, horizontal lines are medians. n.s. – p>0.05; *** - p<0.001 according to Bonferroni statistical comparison. (D) At 9 months post infection, the frequency of activated (Ly6C⁺) cells in the CD8 EM lymphocytes of MCMV, VACV or mock infected mice was compared by Bonferroni statistical comparison. Symbols indicate individual mice, horizontal lines are medians. n.s. – p>0.05; *** - p<0.001.</p

Long-term maintenance of effector and decrease of naïve CD8 subsets upon MCMV infection.

<p>Mouse littermates were infected at 6, 12 or 16 months of age with MCMV (red symbols) or VACV-IE1 (green symbols) and compared at 20 months of age (4, 8 or 14 months post infection, as indicated below x axes) to uninfected controls (black symbols). (A) CD8⁺ cells were gated on CD11a⁺CD44⁺, then on a CD62L⁻ gate, and frequencies of CD11a⁺CD44⁺CD62L⁻ cells in the CD8 pool were calculated. Each symbol represents a mouse, horizontal lines indicate medians. (B) CD8⁺ cells from mice shown in panel A were gated on a KLRG1⁺ gate, and their frequency in individual mice is shown. Each symbol represents a mouse, horizontal lines indicate medians. Significance was assessed by ANOVA followed by Bonferroni post-analysis for indicated columns (* - p<0.05, *** - p<0.001). (C) Naïve cells were defined by progressive gating on a CD11a⁻CD44⁻, and then on a CD127⁺CD62L⁺ gate (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat.1002849.s002" target="_blank">Fig. S2</a>). Each symbol represents a mouse, horizontal lines indicate medians. Significance was assessed by ANOVA followed by Bonferroni post-analysis for indicated columns (ns – p>0.05, * - p<0.05, ** - p<0.01, *** - p<0.001).</p

Blocking TGFβ prevents acute CHIKV-induced disease in O mice.

<p>A and O B6 mice were inoculated and treated with 100ug of anti-TGFβ antibody or isotype control and swelling was measured daily as described in Methods. Data are mean ± SEM (n = 8 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. Red stars indicate reduction of swelling in O mice from αIgG1 to αTGFβ treated; black stars indicate reduction of swelling in A mice from αIgG1 to αTGFβ treated.</p