Segregation of CD8⁺ T-cells into IFNγ⁺ and Pfn⁺ populations in <i>T. cruzi</i> infection.

<p>C57BL/6 mice were infected with 100 blood trypomastigotes of the Colombian strain of <i>T. cruzi</i> and the expression of IFNγ⁺ and Pfn⁺ by CD8⁺ cells in the peripheral blood and cardiac tissue was evaluated. (<b>A</b>) Representative histograms of flow cytometry analysis of peripheral blood CD8⁺ T-cells [R1 (SSCxFSC)/R2 (TCR)/R3 (CD8) gated] that were analyzed for IFNγ and Pfn expression in <i>T. cruzi</i>-infected mice (120 dpi). (<b>B</b>) Representative dot plots of flow cytometry analysis of peripheral blood CD8⁺ T-cells (R1/R2/R3 gated) expressing IFNγ and Pfn at 45 and 120 dpi. (<b>C</b>) Frequency of double-stained CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ peripheral blood T-cells cells (R1/R2/R3 gated) at 45 and 120 dpi. (<b>D</b>) Representative histograms and dot plots of flow cytometry analysis of heart infiltrating CD8⁺ cells [R1 (SSCxFSC)/R2 (CD8) gated] expressing IFNγ and Pfn at 45 and 120 dpi. (<b>E</b>) Frequency of double-stained CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ of heart infiltrating cells (R1/R2 gated) at 45 and 120 dpi. In (<b>C</b>) Bars represent the mean ± SD of five to eight mice per group. In (<b>E</b>) Bars represent the mean of two or three pools of 5 mice per group. These data represent two or three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing noninfected (NI) controls and <i>T. cruzi</i>-infected mice. ^#<i>P</i><0.05, comparing <i>T. cruzi</i>-infected mice at 45 and 120 dpi.</p

IFNγ⁺ and Pfn⁺ cells differentially invade the cardiac tissue of <i>T. cruzi</i>-infected C57BL/6 mice.

<p>Mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and the presence of IFNγ⁺ and Pfn⁺ cells in the cardiac tissue was evaluated by IHS. (<b>A</b>) Numbers of IFNγ⁺ cells infiltrating the cardiac tissue at 15, 30, 45, 60, 90 and 120 dpi. (<b>B</b>) Numbers of Pfn⁺ cells infiltrating the cardiac tissue at 15, 30, 45, 60, 90 and 120 dpi. (<b>C</b>) Relationship between IFNγ⁺ cells infiltrating the cardiac tissue and heart parasitism or CK-MB activity in serum. (<b>D</b>) Relationship between Pfn⁺ cells infiltrating the cardiac tissue and heart parasitism or CK-MB activity in the serum. (<b>E</b>) Immunohistochemistry staining of IFNγ⁺ (green arrows) and Pfn⁺ (red arrows) cells infiltrating the cardiac tissue at 60 dpi. In (<b>A</b>) and (<b>B</b>), each circle represents an individual mouse. In (<b>C</b>) and (<b>D</b>), each circle represents the mean ± SD of the studied group (5–7 animals/time point). These data represent three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing noninfected (NI) controls and <i>T. cruzi</i>-infected mice. ^#, <i>p</i><0.05, comparing <i>T. cruzi</i>-infected mice at 60 and 120 dpi.</p

Electrocardiograph parameters of <i>cd8</i>^−/− mice adoptively transferred with CD8⁺ cell from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ or <i>ifnγ</i>^+/+<i>pfn</i>^−/− and infected with the Colombian <i>T. cruzi</i> strain.

1<p><i>cd8</i>^−/− mice were: NI, non-infected; <i>T. cruzi</i>, infected with 100 bt forms of the Colombian <i>T. cruzi</i> strain 15 days after adoptive cell transfer (AdT) of CD8-enriched cells (≥98%), obtained from IFNγ-deficient (<i>ifnγ</i>^−/−<i>pfn</i>^+/+) or Pfn-deficient (<i>ifnγ</i>^+/+<i>pfn</i>^−/−) mice.</p>2<p>ECG parameters were evaluated using the following standard criteria: (i) heart rate (monitored by beats per minute (bpm), and (ii) the variation of the P wave and PR, QRS and corrected QT intervals (QTc), all measured in milliseconds (ms). ART, arrhythmia; AVB1, first-degree atrioventricular block; AVB2, second-degree atrioventricular block.</p>3<p>This table represented accumulated data from three independent experiments, with 3 to 5 mice/group in each experiment.</p>4<p>Significant differences:</p>*<p>, <i>p</i><0.05 - between the values for <i>cd8</i>^−/− noninfected and <i>T. cruzi</i>-infected mice;</p>#<p>, <i>p</i><0.05 - between the values for <i>cd8</i>^−/− non-reconstituted and <i>cd8</i>^−/− reconstituted and infected with <i>T. cruzi</i>.</p

Electrocardiograph parameters of C57BL/6 mice infected with the Colombian <i>T. cruzi</i> strain.

1<p>ECG parameters were evaluated using the following standard criteria: (i) heart rate (monitored by beats per minute (bpm), and (ii) the variation of the P wave and PR, QRS and corrected QT intervals (QTc), all measured in milliseconds (ms). ART, arrhythmia; AVB1, first-degree atrioventricular block; AVB2, second-degree atrioventricular block.</p>2<p>This data represent three independent experiments, with 7–8 infected mice/group. Three sex- and age-matched noninfected controls were analyzed in all experimental time points. The results were pooled in one representative group of 12 noninfected controls per experiment.</p>3<p>Significant differences</p>*<p>, <i>p</i><0.05;</p>**<p>, <i>p</i><0.01;</p>***<p>, <i>p</i><0.001 between the values for noninfected and <i>T. cruzi</i>-infected groups of mice.</p

Distinct migratory behavior and effector function of CD8⁺IFNγ⁺ and CD8⁺Pfn⁺ T-cells in <i>T. cruzi</i> infection.

<p>(<b>A</b>) Experimental scheme of CD8⁺ cell isolation from noninfected naïve <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors, <i>in vivo</i> reconstitution of the CD8⁺ cell compartment of naïve <i>cd8</i>^−/− mice, infection with 100 bt of the Colombian strain at 15 days after cell transfer (dact) and analysis at 30 days post-infection. Parasitemia, survival rate, cardiac parasitism and CK-MB activity levels in the serum and colonization of the cardiac tissue by IFNγ⁺ and Pfn⁺ cells were evaluated. (<b>B</b>) Parasitemia and (<b>C</b>) survival curve of <i>cd8</i>^−/− mice non-reconstituted (NR) or reconstituted with CD8⁺ cells from <i>ifnγ</i>^+/+<i>pfn</i>^−/− and <i>ifnγ</i>^−/−<i>pfn</i>^+/+ donors. In independent experiments, the animals were analyzed at 30 dpi when 100% of the mice in all the experimental groups were alive (arrow). (<b>D</b>) Number of amastigote nests in 100 microscopic fields of cardiac tissue sections. (<b>E</b>) Cardiomyocyte lesion evaluated by CK-MB activity in serum samples. The number of (<b>F</b>) IFNγ⁺ and (<b>G</b>) Pfn⁺ cells in 100 microscopic fields of cardiac tissue sections from mice non-reconstituted (NR) or reconstituted with CD8⁺ cells from <i>ifnγ</i>^+/+<i>pfn</i>^−/− and <i>ifnγ</i>^−/−<i>pfn</i>^+/+ donors, at 30 dpi. Each symbol represents an individual mouse. These data represent three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing <i>cd8</i>^−/− infected mice non-reconstituted and reconstituted with CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors.</p

CD8⁺ T-cells recognizing the VNHRFTLV ASP2 <i>T. cruzi</i> peptide are enriched in the cardiac tissue.

<p>C57BL/6 mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and the anti-parasite immune response in the spleen and cardiac tissue was assessed. (<b>A</b>) Representative spots formed after stimulation of spleen cells from noninfected (NI) and <i>T. cruzi</i>-infected mice at 45 and 120 dpi with H-2K^b-resctricted VNHRFTLV peptide. The number of CD8⁺IFNγ⁺ as determined by <i>ex vivo</i> ELISpot were analyzed and compared with parasitemia and CK-MB activity levels in the serum. (<b>B</b>) Representative histogram profiles of <i>in vivo</i> cytotoxicity assay showing the specific lysis of H-2K^b-resctricted VNHRFTLV peptide-labeled CFSE^high cells from NI controls and <i>T. cruzi</i>-infected C57BL/6 mice at 45 and 120 dpi. The frequencies of <i>in vivo</i> specific lysis of with H-2K^b -resctricted VNHRFTLV peptide-labeled target cells in <i>T. cruzi</i>-infected C57BL/6 mice at 15, 30, 45, 60, 90 and 120 dpi were analyzed and compared with parasitemia and CK-MB activity levels in the serum. The peak of parasitemia and the maximum CK-MB activity are highlighted (dotted line). Each circle and vertical lines represent the mean ± standard deviation (SD) of the studied group (5–7 animals/time point). These data represent three independent experiments. (<b>C</b>) Frequency of double-positive CD8⁺ H-2K^b /VNHRFTLV⁺ cells [R1 (SSCxFSC) gated] of spleen and heart of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 40 dpi. (<b>D</b>) Frequencies of double-positive CD8⁺ H-2K^b /VNHRFTLV⁺ cells [R1 (SSCxFSC) gated] of spleen, peripheral blood and heart of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 30, 45 and 120 dpi. Representative flow ctometry profiles and mean ± SD of two or three animals per group. Bars represent the mean of two or three pools of 5 mice per pool.</p

C57BL/6 mice infected with the Colombian strain of <i>T. cruzi</i> develop chronic cardiomyopathy.

<p>Mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and parasitological and clinical parameters were evaluated. (<b>A</b>) Kinetics of parasitemia and cardiac parasitism. (<b>B</b>) Survival rate. (<b>C</b>) CK-MB activity levels in the serum of noninfected and <i>T. cruzi</i>-infected mice. (<b>D</b>) Positive correlation between cardiac tissue parasitism and CK-MB activity levels during the acute phase (r² = 0.982) but not during the chronic phase (r² = 0.039) of infection. (<b>E</b>) Representative ECG register segments of 1200 ms of sex- and age-matched noninfected (NI) controls and <i>T. cruzi</i>-infected C57BL/6 mice at 30, 45, 60, 90 and 120 dpi, showing arrhythmia and first and second degree atrioventricular block (AVB1, AVB2; arrows) in infected mice. (<b>F</b>) Relative heart weight (mg/g) of noninfected (NI, pool of three age-matched controls per analyzed point) and <i>T. cruzi</i>-infected C57BL/6 mice at 15, 30, 45, 60, 90 and 120 dpi. (<b>G</b>) Numbers of CD4⁺ and CD8⁺ cells in the cardiac tissue during acute and chronic <i>T. cruzi</i> infection were counted after immunohistochemistry staining. Each circle represents an individual mouse. These data represent three independent experiments. ^*, <i>p</i><0.05, ^**, <i>p</i><0.01, and ^***, <i>p</i><0.001 comparing NI controls and <i>T. cruzi</i>-infected mice.</p

Number of IFNγ⁺ and Pfn⁺ cells in heart tissue sections of <i>T. cruzi</i>-infected C57BL/6 mice.

1<p>dpi, days post-infection with 100 bt of the Colombian <i>T. cruzi</i> strain.</p>2<p>The number of positive cells for each parameter was counted in 100 microscopic fields per section. Three sections were counted per each analyzed animal.</p>3<p>Ratio of IFNγ⁺/Pfn⁺ cells was determined adopting the mean number of positive cells for each parameter per 100 microscopic fields.</p

CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− infected mice differentially migrate to the cardiac tissue.

<p>(<b>A</b>) Experimental scheme showing the infection of donors and recipients mice by <i>T. cruzi</i>, isolation of CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− infected donors (20 dpi) by magnetic beads, CFSE^high labeling, adoptive transfer of CFSE⁺CD8⁺ cells to C57BL/6 and <i>cd8</i>^−/− infected recipients (20 dpi) and analysis at 3, 7 and 10 days after cell transfer (dact). The colonization of the cardiac tissue by CFSE⁺ cells, heart parasitism and CK-MB activity levels in the serum were evaluated. (<b>B</b>) Detection of CFSE⁺CD8⁺ cells in cardiac tissue sections at 3, 7 and 10 dact to <i>T. cruzi</i>-infected C57BL/6 and <i>cd8</i>^−/− recipients. (<b>C</b>) Graphics showing the confocal analysis of the intensity of fluorescence of CFSE⁺CD8⁺ cells present in the myocardium of cardiac tissue sections at 3 dact to <i>T. cruzi</i>-infected <i>cd8</i>^−/− recipients. DAPI was used to reveal the nucleus of the cells of the recipient mice. (<b>D</b>) Number of amastigote nests in 100 microscopic fields of cardiac tissue from C57BL/6 and <i>cd8</i>^−/− infected mice that were non-transferred (NT) or transferred with CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors, at 10 dact. (<b>E</b>) Cardiomyocyte lesions were evaluated at 10 dact by measuring the CK-MB activity in serum samples from C57BL/6 and <i>cd8</i>^−/− infected mice that were non-transferred (NT) or transferred with CD8⁺ cells from <i>ifnδ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors. Each symbol represents an individual mouse. These data represent three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.01; and ^***, <i>p</i><0.001, comparing C57BL/6 and <i>cd8</i>^−/− infected mice that were non-transferred and transferred with CD8⁺ cells from <i>ifnγ</i>^−/−<i>pfn</i>^+/+ and <i>ifnγ</i>^+/+<i>pfn</i>^−/− donors.</p

Differential compartmentalization of anti-<i>T. cruzi</i> CD8⁺ T-cells expressing IFNγ and Pfn.

<p>C57BL/6 mice were infected with 100 bt of the Colombian strain of <i>T. cruzi</i> and presence of CD8⁺ H-2K^b /VNHRFTLV⁺ T-cells expressing IFNγ⁺ and Pfn⁺ in the spleen, peripheral blood and in the cardiac tissue was evaluated. (<b>A</b>) Representative dot plots of double-positive CD8⁺ H-2K^b /VNHRFTLV⁺ T-cells (R1 gated) in the spleen of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 120 dpi. Frequencies of CD8⁺ H-2K^b /VNHRFTLV⁺ T-cells (R2 gated) expressing IFNγ, Pfn or coexpressing IFNγ and Pfn in the spleen, peripheral blood and cardiac tissue of NI and <i>T. cruzi</i>-infected C57BL/6 mice at 45 (<b>B</b>) and 120 dpi (<b>C</b>). Bars represent the means of two or three pools of 5 mice per group. These data represent two independent experiments.</p