Inhibitory Receptors Are Expressed by Trypanosoma cruzi-Specific Effector T Cells and in Hearts of Subjects with Chronic Chagas Disease

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4+ T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4+CTAL-4+ T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4+LIR-1+ among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3+ T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease

CTLA-4 and CD57 expression in the heart of chronic Chagas disease patients with severe cardiomyopathy.

<p>CTLA-4 and CD57 expression was assessed by immunohistochemistry in explanted heart tissue sections from chronic Chagas disease recipients. Two representative staining where CTLA-4 expression (arrows) was detected. Original magnification, 400× (A and B inset magnification, 1000×). Giant cell myocarditis infiltrate showing a typical giant cell (arrowhead), severe diffuse infíltrate and CTLA-4 expression (arrows). Original magnification 400× (C). No CTLA4⁺ cells were observed in idiopathic dilated cardiomyopathy heart tissues. Original magnification, 400× (D). CD57 expression in heart tissues from a <i>T. cruzi</i>-infected subject (E) and in lymph node tissues (F). Original magnification, 400×.</p

Effect of cross-linking of CTLA-4 and LIR-1 on T cell responses against <i>T. cruzi</i>-antigens.

<p>IFN-γ ELISPOT responses of PBMCs from <i>T. cruzi</i>-infected subjects stimulated with <i>T. cruzi</i> lysate or media alone were measured in the presence of a plate bound isotype control, anti-CTLA-4 or anti-LIR-1 antibodies. The data represent the mean spot number/10⁶ PBMCs for individual subjects with positive IFN-γ ELISPOT responses prior to cross-linking assays. (*) Indicates significant differences in <i>T. cruzi</i>-specific IFN-γ ELISPOT responses (SFCs in media subtracted) between previous and post cross-linking assays, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035966#s2" target="_blank">Material and Methods</a>. The data represent the mean SFCs number/1×10⁶ PBMCs. ^(a) LIR-1 cross-linking was not performed. The clinical status of each subject is indicated between brackets.</p

CTLA-4 and LIR-1 are expressed by <i>T. cruzi</i> antigen-responsive CD4⁺ T cells.

<p>PBMCs were stimulated for 18 hours in the presence of an amastigote <i>T. cruzi</i> lysate (middle panel), a mix of tetanus/diphtheria toxoids (low panel) or media alone (top panel). Cells were stained with anti-CD4 and anti-LIR-1 monoclonal antibodies followed by fixation and permeabilization for the intracellular staining with anti-IFN-γ and anti-CTLA-4 monoclonal antibodies. (A) Representative dot plot from a G0 <i>T. cruzi</i>-infected subject. Lymphocytes were gated by forward and side light scatter and subsequently analyzed by IFN-γ vs. CD4. The right graphs show CTLA-4 and LIR-1 staining of R3 gated cells. The figures indicate the percentage of CD4⁺IFN-γ⁺CTLA-4⁺ (lower right quadrant), CD4⁺IFN-γ⁺LIR-1⁺ (upper left quadrant), and double CD4⁺IFN-γ⁺CTLA-4⁺ LIR-1⁺ T cells (upper right quadrant). (B) Frequencies of CTLA-4⁺ and LIR-1⁺ T cells in the CD4⁺IFN-γ⁺ T cell compartment from 18 chronically infected subjects without cardiac symptoms (closed symbols, n = 10) or with severe cardiomyopathy (open symbols, n = 8). Median values are indicated by the horizontal lines. (C) MFI of IFN-γ on CTLA-4⁺ and CTLA-4⁻ CD4⁺ T cells specific for <i>T. cruzi</i> in asymptomatic and symptomatic <i>T. cruzi</i>-infected subjects. (D) MFI of IFN-γ on LIR-1⁺ and LIR-1⁻ CD4⁺ T cells specific for <i>T. cruzi</i> in asymptomatic and symptomatic <i>T. cruzi</i>-infected subjects.</p

Increased frequencies of CD4⁺CTLA-4⁺ T cells following polyclonal activation with anti-CD3 antibodies.

<p>PBMCs were stimulated with anti-CD3 for 18 h or media alone. Cells were surface stained with anti-CD4 monoclonal antibody followed by fixation and permeabilization and intracellular staining with anti-CTLA-4 monoclonal antibody. Lymphocytes were gated by forward and side light scatter. From this population single color CD4 staining histogram was made and CD4⁺ T cells were selected and analyzed for CD4 vs. CTLA-4 dot plot. (A) Representative dot plots from an uninfected control, an asymptomatic subject (G0) and a patient with severe cardiomyopathy (G3). The numbers in the quadrants represent percent cells in each out of total CD4⁺ T cells. (B) The frequency of induced CD4⁺CTLA-4⁺ was calculated by subtracting the percentage of CD4⁺CTLA-4⁺ T cells in unstimulated cultures from the percentage of CD4⁺CTLA-4⁺ T cells responding to anti-CD3 stimulation. Values from individual uninfected controls, G0 or G3 subjects are depicted as separate points and median values are indicated by the horizontal lines. Kruskal-Wallis test with pairwise comparison was used to compare differences between subject groups.</p

CTLA-4 is expressed by infiltrating T lymphocytes in the heart of chronic Chagas disease patients.

<p>Double immunofluorescence staining with CD3 and CTLA-4 antibodies was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035966#s2" target="_blank">Material and Methods</a>. From total CD3-expressing T cells present in the inflammatory infiltrate (A) a small proportion showed CTLA-4 expression (B). Nuclei staining with DAPI. The arrowheads point the nuclei of CTLA4⁺ cells (C). Composite of figures A, B and C showing the double stained cells (arrowheads) and a CD3⁺CTLA-4⁻ single stained cell (large arrow)(D). Original Magnification 400×.</p

CD4⁺ T cells expressing LIR-1 following treatment with benznidazole in subjects with increased CD4⁺LIR-1⁺ T cells at baseline.

<p>PBMCs from <i>T. cruzi</i>-infected subjects were taken prior and at different time points following treatment with benznidazole and stained with anti-CD4 and anti-LIR-1 monoclonal antibodies. Plots show representative data for single subjects from 10 chronically infected subjects. Significant changes in the levels of CD4⁺LIR-1⁺ T cells, as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035966#s2" target="_blank">Materials and Methods</a> are depicted with dotted lines. <i>Horizontal line</i>, cut-off CD4⁺LIR-1⁺ T cell levels in the normal range, as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035966#s2" target="_blank">Material and Methods</a>.</p