Phenotypic characterization of specific CD8⁺ T cells of infected and/or AdASP-2 immunized A/Sn mice.

<p>A/Sn mice were infected or immunized as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002699#ppat-1002699-g003" target="_blank">Figure 3A</a>. Splenic cells were collected nineteen days after infection/immunization. The histograms show FACS analysis on CD8⁺ cells (Gr. 1) and H-2K^k-TEWETGQI⁺ CD8⁺ cells (Gr. 2, 3, and 4) and the indicated marker (blue). Control cells were from naive mice (red lines). Results of CD44, KLRG1 and CD183 staining are presented as MFI and frequencies of the CD44^High, KLRG1^High or CD183^High cells, respectively. On the other hand, results of CD27 and CD62L staining are presented as MFI and frequencies of the CD27^Low or CD62L^Low cells. Representative analyses are shown from pools of cells from 3 mice. Stainings were performed 2 or more times with identical results.</p

Schematic representation of the ASP-2 antigen and its expression in intra-cellular amastigotes of <i>T. cruzi</i>.

<p><b>A</b>) The protein is a prototypical member of the <i>trans</i>-sialidase family of <i>T. cruzi</i> surface antigens containing a putative signal peptide at the N-terminal region, 2 ASP-box sequences, and a VTV-box at the C-terminal domain. The protein is attached to the membrane through a glycophosphatidylinositol anchor. <b>B</b>) ASP-2 expression in intra-cellular amastigotes was determined by immunofluorescence with the specific MAb K22. HeLa cells were infected for 48 h with trypomastigotes of the Y strain. After fixation, indirect immunofluorescence and DAPI staining were performed and imaged under fluorescence microscopy. Bar, 14 µM. Photomicrography kindly provided by Dr. Clara Claser (Singapore Immunology Network -SIgN, Singapore).</p

Frequency, phenotypic characterization and proliferative capacity of specific CD8⁺ T cells.

<p>The protocol of the experiment was as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002699#ppat-1002699-g003" target="_blank">Figure 3A</a>. <b>A</b>) Nineteen days after the immunization/challenge, we estimated the frequency of splenic H2K^k-TEWETGQI⁺ CD8⁺ cells. FACS charts are for a representative mouse (median) from 3 mice. Numbers represent frequencies of splenic cells. <b>B</b>) Splenic cells were stained for CD8, H2K^k-TEWETGQI and CD95 prior to analysis by FACS (blue lines). Control cells were from naive mice (red lines). <b>C</b>) The histograms show FACS analysis on CD8⁺ cells (Gr. 1) and H-2K^k-TEWETGQI⁺ CD8⁺ cells (Gr. 2, 3, and 4) stained for CD95 and annexin V ligand. Numbers represent frequencies of CD8⁺ cells (Gr.1) or H2K^k-TEWETGQI CD8⁺ cells (Gr. 2, 3, and 4). <b>D</b>) Mice were treated with BrdU for 4 days prior to euthanasia and splenic cells were stained for CD8, H-2K^k-TEWETGQI, CD95 and BrdU prior to FACS analysis. The histograms show analysis on CD8⁺ cells (Gr. 1) or H2K^k-TEWETGQI⁺ CD8⁺ cells (Gr. 2, 3, and 4) stained for CD95 and incorporated BrdU. Numbers represent frequencies of CD8⁺ cells (Gr.1) or H2K^k-TEWETGQI CD8⁺ cells (Gr. 2, 3, and 4). Mean numbers represents MFI for CD95 staining. Representative analyses (medians) are shown for 3 mice per experiment. The experiment was performed two or more times with similar results.</p

Parasitemia and mortality in A/Sn mice immunized with AdASP-2 vaccine and challenged with trypomastigotes.

<p><b>A</b>) A/Sn mice were immunized i.m. with the indicated doses (pfu) of AdASP-2 vaccine or control Adβ-gal. Seven days later, mice were challenged s.c. with 150 bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Parasitemia was followed daily from days 9 to 13 after challenge. The results represent the mean ± SD values for 6 mice. At the peak parasitemia (day 11), values of mice immunized with each different dose were compared by one-way ANOVA and Tukey HSD tests and the results were as follows: <b>i)</b> Groups of mice that received doses of 10⁸ or 10⁷ pfu of AdASP-2 vaccine had values lower than the group of mice injected with Adβ-gal (<i>P</i><0.01). <b>B</b>) Kaplan–Meier curves for survival of the different groups were compared using the logrank test. The results of the comparisons were as follows: <b>i)</b> Groups of mice injected with 10⁸ or 10⁷ pfu of AdASP-2 vaccine survived longer than the group injected with Adβ-gal (<i>P</i><0.01). <b>C</b>) A/Sn mice were immunized i.m. on the indicated days before or after infection with AdASP-2 vaccine or control Adβ-gal (2×10⁸ pfu/mouse). All mice were challenged s.c. as described above. Although the day of the peak parasitemia was different for each group, we used the maximal values for statistical comparison. The results of the comparisons were as follows: <b>i)</b> Mice vaccinated on day −7 (red) with AdASP-2 vaccine had values lower than those of any other group. <b>ii)</b> Mice vaccinated on days 0 (green) or +4 (yellow) with AdASP-2 vaccine had values lower than the group of mice injected with Adβ-gal (black) or AdASP-2 on day +7 (blue, <i>P</i><0.01). <b>D</b>) Kaplan–Meier curves for survival of the different groups were compared and the results were as follows: <b>i)</b> Groups of mice vaccinated with AdASP-2 vaccine on days −7 (red) or 0 (green) survived longer than the groups of mice injected with Adβ-gal (black) or AdASP-2 vaccine on days +4 (yellow) or +7 (blue) (<i>P</i><0.01 in all cases). <b>E</b>) A/Sn mice were immunized with AdASP-2 vaccine or Adβ-gal (2×10⁸ pfu/mouse) by the indicated routes (i.m., s.c., or i.p.). All mice were challenged s.c. on the same day as the immunization, as described above. The parasitemia data for each mouse group are represented in terms of mean ± SD (n = 6). Although the day of the peak parasitemia was different for each group, we used the maximal values for statistical comparison. The results of the comparisons were that mice vaccinated i.m. or s.c. with the AdASP-2 vaccine had values lower than those of any other groups (<i>P</i><0.01). <b>F</b>) Kaplan–Meier curves for survival of the different groups were compared and the results were as follows: <b>i)</b> Mice vaccinated i.m. or s.c. with the AdASP-2 vaccine survived longer than any other groups (<i>P</i><0.01). <b>ii)</b> Mice vaccinated i.p. with AdASP-2 vaccine survived longer than mice that received Adβ-gal (<i>P</i><0.05). <b>G</b>) A/Sn mice were immunized i.m. with AdASP-2 vaccine or control Adβ-gal (2×10⁸ pfu/mouse). Seven days later, mice were challenged s.c. as described above. Before and after challenge, mice were treated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002699#s4" target="_blank">Methods</a> section with rat IgG (control) or anti-CD8 MAb. The parasitemia for each mouse group is represented as mean ± SD (n = 6). Although the day of the peak parasitemia was different for each group, we used the maximal values for statistical comparison. AdASP-2-immunized mice treated with rat IgG had a significantly lower parasitemia (<i>P</i><0.01) when compared to those of non-immune animals or AdASP-2-vaccinated mice treated with anti-CD8 MAb. <b>H</b>) Kaplan–Meier curves for survival of the different groups were compared and the results showed that AdASP-2-immunized mice treated with rat IgG survived significantly longer (<i>P</i><0.01) than non-immune animals or AdASP-2-immunized mice treated with anti-CD8 MAb. The results described above are representative of 2 or more independent experiments.</p