Minimal T cell activation following infection with wild-type or recombinant CVB3.

<p>Mice were infected with wtCVB3, rCVB3.6, or LCMV as a positive control, and the extent of T cell activation was analyzed on day 8. (A) CD44 and CD62L expression profiles of CD8⁺ or CD4⁺ splenocytes were compared for naïve and virus-infected mice. Representative histograms are gated on CD8⁺ or CD4⁺ cells, and the numbers indicate the proportion of cells that are CD44^hi or CD62L^lo. The frequency of CD44^hi (B) or CD62L^lo (C) CD4⁺ or CD8⁺ T cells in the spleen was determined for each group of mice. (D) The capacity of CD4⁺ and CD8⁺ T cells to produce IFNγ was evaluated following simulation with PMA and ionomycin and ICCS. The percentage of CD4⁺ or CD8⁺ T cells that produce IFNγ is shown. All data are representative of 2 independent experiments, and show the mean+SE for 3 mice per group. * p<0.05, ** p<0.01, *** p<0.001, compared to the naïve (uninfected) control.</p

The absence of CVB3-induced CD8⁺ T cell responses cannot be attributed to redistribution into non-lymphoid target organs.

<p>Equal numbers (10⁴) of P14 and SMARTA cells from uninfected mice were combined and transferred into uninfected recipients. Four days post transfer, mice were infected with rCVB3.6 or LCMV and, 8 days later, mononuclear cells were isolated from the spleen, heart, and pancreas. (A) P14 and SMARTA responses were analyzed by flow cytometry. The gates shown in the dot plots identify P14, SMARTA, or CD4⁺ SMARTA⁻ cells, and the numbers indicate their percentage among all mononuclear cells. (B) Frequency of P14 and SMARTA cells as a percentage of relevant cells (CD8⁺ or CD4⁺) in the spleen. (C) Total number of P14 and SMARTA cells in the spleen. Data are shown as the mean+SD of 3 or 4 mice per group. (D) The expression of CD44, CD62L, and CD127 on SMARTA cells from the spleen, heart, and pancreas was compared in rCVB3.6-infected and LCMV-infected mice. The dot plots shown are gated on CD4⁺ cells, and the numbers indicate the proportion of cells in each quadrant, as a percentage of all CD4⁺ cells. CD45.1⁺ SMARTA cells are present in the upper right and left quadrants. All data are representative of 2 independent experiments.</p

SMARTA T cell responses to rCVB3.6 provide an accurate reflection of the responses of endogenous epitope-specific CD4⁺ T cells.

<p>(A) The capacity of transgenic and endogenous CD4⁺ T cells to produce IFNγ and TNF was evaluated. Dot plots are gated on SMARTA cells (top row) or on endogenous CD4⁺ T cells (bottom row); the numbers indicate the proportion of cytokine-producing cells in each quadrant, as a percentage of total gated cells. (B) The frequency of splenic GP₆₁-specific CD4⁺ T cells was enumerated using I-A^b/GP_66–77 MHC class II tetramers. I-A^b/hCLIP tetramers were included as a negative control. (C) Differing numbers of naïve SMARTA cells [10⁴; 10³; 10²; 10¹; or none (media only)] were transferred into uninfected mice, and 4 days after transfer, the mice were infected with rCVB3.6. Eight days p.i., the SMARTA response in the spleen was analyzed by flow cytometry. The oval gates in the dot plots identify SMARTA cells, and the numbers shown are the percentage of SMARTA cells among all mononuclear cells. (D) Frequency of SMARTA cells as a percentage of CD4⁺ splenocytes. (E) Total numbers of SMARTA cells (measured by surface staining for CD4 and CD45.1) and GP_61–80-specific endogenous cells (measured by IFNγ ICCS) in the spleen. Data are shown as the mean+SD of 3 mice per group, * p<0.005. (F) The expression of CD44, CD62L, and CD127 on SMARTA cells was compared among rCVB3.6-infected mice that had received 10⁴, 10³, or 10² transgenic cells. Dot plots are gated on CD4⁺ cells, and the numbers indicate the proportion of cells in each quadrant, as a percentage of CD4⁺ cells. Data are representative of two independent experiments.</p

Kinetics of CD4⁺ and CD8⁺ transgenic T cell responses in rCVB3-infected and LCMV-infected mice.

<p>10⁴ P14 cells and 10⁴ SMARTA transgenic T cells from uninfected mice were combined and transferred into uninfected congenic recipients. One day post transfer, mice were infected with rCVB3.6 or LCMV. On the indicated days p.i., the frequencies of P14 and SMARTA cells were determined by flow cytometry, and are presented as a percentage of total PBMCs. Each symbol represents the time-course in an individual mouse. Note that the y-axes differ among the graphs; this was done to facilitate the comparison of the kinetics of T cell responses that differ substantially in their magnitudes.</p

rCVB3.6 drives the <i>in vitro</i> proliferation of CD4⁺ T cells, but not of CD8⁺ T cells.

<p>Stimulator cells: Splenocytes from wt C57BL/6 mice were left uninfected, or were infected with rCVB3.5 or rCVB3.6 (moi = 10). Some splenocytes (uninfected or rCVB3.6-infected) were pulsed with GP_33–41 peptide. Indicator cells: P14 and SMARTA splenocytes were CFSE-labeled, then mixed to generate a 1∶1 ratio of both types of transgenic T cell. Indicator cells were added to wells containing stimulator cells and, 72 hours later, wells were harvested and analyzed by flow cytometry using FloJo. The panels shown are gated on CD8⁺/Thy1.1⁺ cells (P14) or CD4⁺/CD45.1⁺/Vα2⁺ cells (SMARTA). P14 cells were not gated on Vα2 because this molecule is down-regulated on activated/dividing P14 cells (as shown in the peptide-stimulated population in panel B); had we gated on Vα2^hi cells, we would have failed to detect a large proportion of dividing cells. All analyses were done in triplicate, and the data shown are representative. The numbers are the proportion of (A) SMARTA cells or (B) P14 cells that have divided at least once, expressed as a percentage of total number of SMARTA or P14 transgenic T cells that were present in the plots.</p

CVB3 infection does not globally suppress the host CD8⁺ T cell response.

<p>(A) Groups of mice were infected with LCMV or rCVB3.3, or co-infected with these viruses as shown. Spleens were harvested 8 days after rCVB3.3 infection (group II) or 8 days after LCMV infection (all other groups), and LCMV-specific CD8⁺ and CD4⁺ T cell responses were analyzed. (B) Splenocytes were stimulated with peptide and the frequencies of GP₃₃-, NP₃₉₆-, and GP₆₁-specific IFNγ⁺ T cells were determined by flow cytometry. Representative dot plots are gated on mononuclear cells, and the numbers indicate the proportion of cells in each quadrant, as a percentage of total gated cells. (C) Percentages of epitope-specific CD8⁺ or CD4⁺ T cells that produced IFNγ; * p<0.01 compared to all other groups. (D) Total numbers of epitope-specific IFNγ-producing CD8⁺ or CD4⁺ T cells; * p<0.05, ** p<0.01 compared to all other groups. Data are shown as the mean+SE of 3 mice per group.</p

No significant change in CD69 or CD127 expression on T cells 8 days after CVB3 infection.

<p>Mice were infected with wtCVB3 or rCVB3.6, and the extent of T cell activation was analyzed on day 8. (A) CD69 and CD127 expression profiles of CD4⁺ and CD8⁺ splenocytes were compared for naïve and virus-infected mice. CD4⁺ and CD8⁺ cells were gated, and representative histograms are shown. CD69/CD127 staining is shown by solid black lines, and isotype control staining is shown in gray. The numbers indicate the percentage of gated T cells that are CD69^hi (left columns) or CD127^lo (right columns). The frequency of CD69^hi (B) or CD127^lo (C) CD4⁺ or CD8⁺ T cells in the spleen was determined for each group of mice. Data are representative of 2 independent experiments, and show the mean + SE for 3–11 mice per group; statistical analyses (ANOVA) revealed no significant differences among the groups.</p

rCVB3.6 induces memory CD4⁺ T cells that expand in number and differentiate into secondary effectors following a challenge infection.

<p>Equal numbers (1×10⁴) of P14 and SMARTA cells from uninfected mice were combined and transferred into uninfected recipients. One to five days later, these mice were infected with rCVB3.3, rCVB3.6, or LCMV. Sixty to sixty-five days after the primary infection, the mice were challenged with LCMV (or left unchallenged), and P14 and SMARTA responses were analyzed on day 4 and 7 post challenge. Frequency of (A) P14 and (B) SMARTA cells as a percentage of CD8⁺ or CD4⁺ cells, respectively, in the blood of unchallenged and LCMV-challenged mice. Data are shown as the mean+SE of 6–16 mice per group, pooled from 4 independent experiments. (C) P14 and SMARTA responses in the spleens of these mice; the numbers in the dotplots indicate the percentage of transgenic cells among all mononuclear cells. (D) Changes in CD127 and CD62L expression on SMARTA cells following LCMV challenge of mice previously infected with rCVB3.6 (squares) or LCMV (circles). The percentage of SMARTA cells that were CD127^hi or CD62L^hi are shown as the mean ± SE of 2–4 mice per time point, pooled from 2 independent experiments; * p<0.05, ** p<0.005, *** p<0.001. The capacity of the transgenic and endogenous GP₆₁-specific CD4⁺ T cells to produce IFNγ and TNF (E), or IFNγ and IL-2 (F) was evaluated. Data are shown as the mean + SE of 2–4 mice per group, combined from 2 independent experiments.</p

Recombinant CVB3 encoding CD8+ or CD4+ (MHC class I/II) epitopes.

a<p>described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000618#ppat.1000618-Slifka1" target="_blank">[11]</a>.</p>b<p>described in this paper.</p>c<p>described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000618#ppat.1000618-Kemball1" target="_blank">[12]</a>.</p

CVB3 infection induces the <i>in vivo</i> proliferation of virus-specific CD4⁺, but not CD8⁺, transgenic T cells.

<p>(A) Equal numbers (6×10⁵) of P14 and SMARTA cells from uninfected mice were labeled with CFSE, combined, and transferred into uninfected recipients. One day post transfer, mice were infected with rCVB3.6 or LCMV, or were not infected. Eight days later, P14 and SMARTA responses in the spleen were analyzed by flow cytometry. The square gates in the dot plots identify P14 or SMARTA cells, and the numbers shown are the percentages of transgenic cells among mononuclear cells. The histograms of CFSE fluorescence are gated on P14 or SMARTA cells. Data are representative of 3 mice per group. (B) Mice were inoculated with rCVB3.2, 3.3, 3.4, or 3.5. Two days after infection, naïve P14 and SMARTA cells were labeled with CFSE, and 9×10⁵ transgenic T cells (P14 or SMARTA) were injected i.v. into the infected mice. Eight days post transfer (10 days p.i.) the P14 or SMARTA cells were identified by flow cytometry (ovals within dot plots). The histograms show the level of CFSE fluorescence by the donor cells. As a positive control, the CFSE-labeled transgenic T cells were given to uninfected mice; the following day these mice were infected with LCMV, and 7 days later these cells were identified (bottom row, dot plots) and their near-total loss of CFSE-fluorescence was revealed (bottom row, histograms).</p