Different input TRBV13-2/TRBV13-1 ratios affect the magnitude of TRBV13-2 skewing.

<p>10⁶ or 10⁴ cells were transferred into mice at input ratios of 2:1, 1:1, and 1:2 of TRBV13-2:TRBV13-1 and mice were infected with RSV one day later. (A) Flow cytometry plots showing the input frequency for TRBV13-2 and TRBV13-1 at different ratios for the 10⁶ cell number transfer. (B and C) TRBV13-2/TRBV13-1 ratio on day 6 post-RSV infection in the lungs and MLN of mice who received different TRBV13-2/TRBV13-1 ratios. Dotted line indicates input ratio of TRBV13-2/TRBV13-1.</p

Proliferation is dependent on transfer number, but does not differ between TRBV 13–1 and TRBV13-2.

<p>(A and B) 1:10 dilutions of a 1:1 ratio of TRBV13-1:TRBV13-2 cells were transferred 1 day prior to RSV infection. Four days post-infection, the percentage of proliferating cells in the lung (A) and MLN (B) were identified by <i>in vivo</i> incorporation of BrdU during a two-hour incubation. <b>(</b>C and D) violet labeled TRBV13-1 and TRBV13-2 were co-transferred into CB6F1 mice prior to infection with RSV. Proliferation profiles of TRBV13-1and TRBV13-2 (C) in the MLN were evaluated at day 4 post-infection and percentage of dividing cells was determined (D).</p

Cell transfer number affects the Tg T cell phenotype.

<p>Different numbers of a 1:1 mixture of TRBV13-2:TRBV13-1 cells were transferred 1 day prior to RSV infection. On day 6 post-infection, we assessed the phenotype of the transferred Tg T cells based on expression of CD62L, CD127, and KLRG1. The percentage of Tg CD8+T cells of each phenotype in the 10⁴ transfer group are in the lungs (A) and MLN (B) are shown. The CD127^lo, CD62L^lo; KLRG1^lo to CD127^hi, CD62L^lo; KLRG-1 ^lo ratio in the lungs (C) and MLN (D) was determined. Bars represent mean±SEM.</p

Transfer of TRBV 13–2 leads to increased levels of proinflammatory cytokines in the lungs following RSV infection.

<p>10⁶ of TRBV13-1, TRBV13-2 cells, or a 1:1 mixture of both (mix) were transferred into CB6F1 mice one day prior to infection with RSV. On days 4, 5, and 6 post-infection, lungs were removed, homogenized, and analyzed for IFNγ, TNFα, IL-6 and MIP-1α by multiplex ELISA. Bars represent mean ± SEM from 4 mice/group.</p

Transfer of TRBV13-1, TRBV13-2, and 1:1 mix leads to increased specific lysis, decreased viral loads, and increased morbidity after RSV infection.

<p>(A-D) CB6F1 mice received 10⁶ of either TRBV13-1 or TRBV13-2 cells or a 1:1 mixture of both (mix) one day prior to infection with RSV. On day 5 (A) and 6 (B) post-infection lungs were removed and processed to determine viral titer in the lungs by plaque assay. (C) CFSE labeled target cells were transferred by IN instillation into TRBV13-1 and TRBV13-2 recipient mice to evaluate cytolysis in the lung on day 4 post-infection. (D) Morbidity of mice in the different groups was evaluated by calculation of weight loss. * indicates days in which TRBV13-1 and Mix were significantly different than TRBV13-2.</p

Effects of transfer cell number on TRBV13-2/TRBV13-1 ratio and endogenous CD8+T cell response.

<p>The indicted number of a 1:1 mix of TRBV13-1 and TRBV13-2 were transferred into CB6F1 mice one day prior to RSV infection. On <b>(A-C)</b> day 4 and <b>(D-F)</b> day 6, lungs and mediastinal lymph node (MLN) were collected and the percentages of transferred TRBV13-1, TRBV13-2, and endogenous M2-specific CD8+T cells were determined. <b>(C, F)</b> TRBV13-2/TRBV13-1. Dotted line indicates input ratio of TRBV13-2/TRBV13-1. NA indicates that there were not enough events to calculate TRBV13-2/13-1.</p

Uptake and processing of soluble antigen is age-dependent.

<p>Soluble ovalbumin-FITC protein (A and B) or ovalbumin-DQ (C and D) were co-administered at the time of RSV infection and the percent of the CD103+ and CD11b+ DC populations positive for FITC expression (A or B) or shown to be processing antigen by unquenching of ova-DQ were measured in the MLN at days 1, 2, and 3 post-infection. Data are combined from two independent experiments using pooled lymph node samples (3–7 mice/pool depending on age and availability) and error bars indicate the SEM. * p≤0.05, **p≤0.01, ***p≤0.001, **** p≤0.0001 by two-way ANOVA and Tukey's multiple comparisons test.</p

Preferential seeding of TRBV13-2 in the MLN and TRBV13-1 in the lung.

<p>10⁶ of a 1:1 Mix of TRBV13-1 and TRBV13-2 Tg cells were transferred into mice. Seeding of transferred, Thy1.1+ T cells was determined one day later in the lung and MLN. A) Expression of CD62L on TRBV13-2 (blue) and TRBV13-1 (red) before transfer. B) Expression of CD44 on TRBV13-2 and TRBV13-1 prior to transfer. C) Ratio of TRBV13-2/TRBV13-1 in the lung and MLN one day after transfer. Dotted line indicates input ratio of TRBV13-2/TRBV13-1. D) CD62L expression on TRBV13-1 and TRBV13-2 Tg cells in the lung or MLN one day after transfer. Bars represent mean±SEM of five samples of 2-pooled mice each. **p<0.01 by paired t test.</p

RSV infection of lung migratory dendritic cell populations in the lung and draining mediastinal lymph node.

<p>Percent of infected (GFP+) dendritic cell populations in the lung (A and B) and the posterior mediastinal lymph node (MLN, C and D) at days 1–3 post-infection. Lung and MLN DC populations were gated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003934#ppat.1003934.s001" target="_blank">Figure S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003934#ppat.1003934.s002" target="_blank">S2</a>, respectively. Percent GFP+ of CD103+ DCs (A and C) and CD11b+ DCs (B and D) was determined by comparison to mice infected at the same time with wtRSV. Data is representative of two experiments done with two pooled samples of 3–7 mice each and the error bars represent the SEM. * p≤0.05, **p≤0.01, ***p≤0.001, ns is no significance by two-way ANOVA and Sidak's multiple comparisons test.</p

Modulating CD28-mediated costimulatory signals differentially affects K^dM2_82–90 and D^bM_187–195-specific responses.

<p>A) Mice were infected with RSV on day 0 and subsequently given IP injections of 20 µg each of antibodies against CD80 and CD86 at the indicated day post-infection. Epitope-specific CD8+ T cell responses were measured by surface and tetramer staining at 7 days post-infection. All groups were compared using a two-way ANOVA and Tukey's multiple comparisons tests, and there were no significant differences in the D^bM_187–195 response. Differences between the K^dM2_82–90 response on Day 2 and other groups are indicated below the figure (* p≤0.05, *** p≤0.001, **** p≤0.0001). B) The response ratio/immunodominance profile of CD8+ T cell responses was obtained for each mouse by dividing the K^dM2_82–90 response by the response to D^bM_187–195. * indicates p≤0.05 following one-way ANOVA and Dunnett's multiple comparisons test. C) Day 7 CD8+ T cell responses of mice injected with the indicated dose of anti-CD80 and CD86 at day 2 post-infection or isotype control antibodies (either 50 µg or 20 µg). All groups were compared using a two-way ANOVA and Tukey's multiple comparisons tests. Differences in the K^dM2_82–90 response between the 50 and 25 µg groups and all other groups are indicated below the figure (ns not significant, ** p≤0.01, and **** p≤0.0001). The D^bM_187–195 responses are significantly different between both the 50 and the 25 µg groups and isotype (p≤0.01) only. D) CD8+ T cell response ratios of mice treated with each dose of anti-CD80 and CD86 antibodies. Groups were compared with a one-way ANOVA and Tukey's multiple comparisons test (** p≤0.01, *** p≤0.001, **** p≤0.0001), and all error bars represent the SEM.</p