Low Frequency of Circulating CD8⁺ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease

<div><p>Background</p><p>CD8+ T cells have been shown to play a crucial role in <i>Trypanosoma cruzi</i> infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control <i>T. cruzi</i> in the chronic phase of the infection are unknown.</p><p>Methodology/Principal Findings</p><p>To characterise the CD8+ T cell subsets that could be participating in the control of <i>T. cruzi</i> infection, in this study, we compared total and <i>T. cruzi</i>-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells.</p><p>Conclusions/Significance</p><p>These findings suggest that CD8+ TSCM cells may be associated with the immune response to <i>T. cruzi</i> and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.</p></div

Distribution of total CD8⁺ T cell subsets from CCPs and HDs.

<p>(<b>A</b>) Representative contour plot of the ex vivo selection of CD8⁺ T cell subsets based on the differential expression of CD45RA, CCR7, CD28, CD27, CD95 and CD127. (<b>B</b>) Frequency of CD8⁺ T_SCM, T_CM, T_TM, T_EM and T_TE cells from CCPs with different degrees of disease severity and HDs. Box and whiskers indicate the median frequency and range of the total CD8⁺ T cell subsets (25–75 percentile). The <i>p</i> values were calculated using a one-way ANOVA non-parametric Kruskal–Wallis test (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001). (<b>C</b>) The correlations between the frequencies of CD8⁺ T_SCM cells and CD8⁺ T_TE cells from CCPs were calculated with Spearman's rank correlation coefficient. CCPs were grouped according to the disease severity as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a> (A = 10, B = 8, C = 9 and D = 5). Additionally, nine HDs were included.</p

Distribution of <i>T. cruzi</i>-specific CD8⁺ T cell subsets from CCPs.

<p>(<b>A</b>) Representative density plot of the selection of <i>T. cruzi</i>-specific CD8⁺ T cells producing IFN-γ, TNF-α or IL-2 following cultivation with parasite lysate. A positive cytokine response was defined as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a>. (<b>B</b>) The proportions of <i>T. cruzi</i>-specific CD8⁺ T_SCM, T_CM, T_EM and T_TE cells were expressed as percentages of total cytokine-producing CD8⁺ T cells. The <i>p</i> values were calculated using a one-way ANOVA non-parametric Kruskal–Wallis test (**<i>p</i><0.01, ****<i>p</i><0.0001). (<b>C</b>) Frequencies of antigen-specific CD8⁺ T_SCM, T_CM, T_EM and T_TE cells among CCPs with differing degrees of disease severity. The <i>p</i> values were calculated using the Mann-Whitney test (***<i>p</i><0.001). Box and whiskers indicate the median frequency and range of the <i>T. cruzi</i>-specific CD8⁺ T cell subsets (25–75 percentile). CCPs were grouped according to their disease severity as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a> (A = 8, B = 6, C = 6 and D = 4).</p

Proportions of total and <i>T. cruzi</i>-specific CD8⁺ T cell subsets from CCPs.

<p>Representation of the proportions of CD8⁺ T_SCM, T_CM, T_TM+T_EM and T_TE cells from CCPs with different degrees of disease severity. The black bars represent the functional activities evaluated for parasite lysate-stimulated cells from CCPs for antigen-specific CD8⁺ T cell subsets.</p

Proportions of functional activity profiles for <i>T. cruzi</i>-specific CD8⁺ T cell subsets from CCPs.

<p>Functional profiles of CD8⁺ T cell subsets stimulated with Staphylococcal enterotoxin B (SEB) and parasite lysate are color-coded according to the number of functions and summarised in the pie charts. Each pie slice corresponds to the mean production of one (dark grey), two (light grey) and three (red) functions. The white pie corresponds to the absence of a response when IFN-γ, TNF-α and IL-2 production were observed. CCPs were grouped according to their disease severity as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a> (A = 8, B = 6, C = 6 and D = 4).</p

Cytokine combinations produced by <i>T. cruzi</i>-specific CD8⁺ T cells from CCPs.

<p>Frequencies of CD8⁺ T cells with one and two functions among CD8⁺ T cells stimulated with parasite lysate. Box and whiskers indicate the median frequency and range of the <i>T. cruzi</i>-specific CD8⁺ T cell subsets (25–75 percentile). CCPs were grouped according to their disease severity as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a> (A = 8, B = 6, C = 6 and D = 4).</p

Characteristics of study participants.

<p>CCPs, chronic chagasic patients; HDs, healthy donors; LVEF, left ventricular ejection fraction.</p>#<p>Clinical characteristics of CCPs are described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a>.</p>+<p>Mann-Whitney test, CCP svs. HDs.</p>++<p>One-way ANOVA non-parametric Kruskal–Wallis test with Dunn's test.</p><p>*<i>p</i><0.05 (A vs. C, B vs. C),</p><p>**<i>p</i><0.001 (A vs. D, B vs. D).</p>###<p><i>p</i><0.0001 (A vs. C),</p>#<p><i>p</i><0.05 (B vs. C).</p><p>Characteristics of study participants.</p

Functional activity profiles of <i>T. cruzi</i>-specific CD8⁺ T cell subsets from CCPs with different degrees of disease severity.

<p>(<b>A</b>) Frequencies of CD8⁺ T_SCM, T_CM, T_EM and T_TE cells with one, two and three functions among CD8⁺ T cells stimulated with Staphylococcal enterotoxin B (SEB). (<b>B</b>) Frequencies of CD8⁺ T_SCM, T_CM, T_EM and T_TE cells with one, two and three functions among CD8⁺ T cells stimulated with parasite lysate. Box and whiskers indicate the median frequency and range of the CD8⁺ T cell subsets (25–75 percentile). CCPs were grouped according to their disease severity as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003432#s2" target="_blank">Materials and Methods</a> (A = 8, B = 6, C = 6 and D = 4). Additionally, nine HDs were included.</p

Analytical sensitivity of LAMP assay.

<p>Top panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from <i>CL</i> Brener stock (DTU VI). Bottom panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from Sylvio X10 stock.The aliquots were expressed in fg/μL. A: 0; B: 1.0 x 10⁻³; C: 1.0 x 10⁻²; D: 1.0 x 10⁻¹, E: 1. NC: Non template control.</p

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p