BackgroundCD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out.Methodology/Principal FindingsSeventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor.ConclusionsChagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.

BackgroundChronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls.Methodology/Principal FindingsForty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζbright/CD3ζdim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζbright/CD3ζdim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ.ConclusionsCD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.

The K1 peptide is a CD8(+)T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein. We have previously shown that this peptide induces IFN-gamma secretion by CD8(+)T cells. The aim of this study was to characterize the frequency of K1-specific CD8(+)T cells in chagasic patients. Nineteen HLA-A2(+)individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP-PCR assays. Twelve HLA-A*0201(+)noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA-A2-K1 tetramer, showing that 15 of 19 infected patients have K1-specific CD8(+)T cells (0.09-0.34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1-specific CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)infected patients was performed. The results showed that both non-HLA-A*0201 and HLA-A*0201(+)individuals have a predominant effector memory CD8(+)T cell phenotype (CCR7-, CD62L-). Moreover, CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)individuals expressed IL-2, IFN-gamma and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA-A2 supertype molecules and is highly recognized by chagasic patients.

Monocytes are classified according to their CD14 and CD16 expression into classical (reparative), intermediate (inflammatory), and non-classical. This study assessed the frequency of monocyte and the relationship between monocyte subset percentages and the levels of blood cytokines in Colombian chagasic patients with different clinical forms. This study included chagasic patients in different clinical stages: indeterminate (IND) n = 14, chronic chagasic cardiomyopathy (CCC) n = 14, and heart transplant chagasic (HTCC) n = 9; controls with non-chagasic cardiopathy (NCC) n = 15, and healthy individuals (HI) n = 15. Peripheral blood mononuclear cells (PBMCs) were isolated, labeled for CD14, CD16, and HLA-DR, and analyzed by flow cytometry. Cytokines were measured with a bead-based immunoassay. Percentages of total CD14+ CD16+ and CD14+ HLA-DR+ monocytes were higher in patients with heart involvement (CCC, HTCC, and NCC) than controls. Percentages of intermediate monocytes increased in symptomatic chagasic patients (CCC and HTCC) compared to asymptomatic chagasic patients (IND) and controls (HI). Asymptomatic chagasic patients (IND) had higher percentages of classical monocytes, an increased production of CCL17 chemokine compared to chagasic symptomatic patients (CCC), and their levels of CCL17 was positively correlated with the percentage of classical monocyte subset. In CCC, the percentages of intermediate and classical monocytes were positively correlated with IL-6 levels, which were higher in this group compared to HI, and negatively with IL-12p40 concentration, respectively. Remarkably, there also was an important increased of classical monocytes frequency in three chronic chagasic patients who underwent cardiac transplant, of which one received anti-parasitic treatment. Our findings suggest that cardiac chagasic patients have an increased percentage of inflammatory monocytes and produce more IL-6, a biomarker of heart failure and left ventricular dysfunction, whereas asymptomatic chagasic individuals present a higher percentage of reparative monocytes and CCL17.

Objective: CD4+CD8+ double-positive T-cells (DPTs) have been classified as a separate T-cell subpopulation, with two main phenotypes: CD4 high CD8 low and CD4 low CD8 high . In recent years, the relevance of DPTs in the pathogenesis of infections, tumors, and autoimmune diseases has been recognized. Reference values among healthy individuals remain unknown. Therefore, the aim of this study is to provide a reference value for DPTs in peripheral blood from healthy donors in a blood bank in Bogotá, Colombia, and to determine the activation status using a surface marker. Materials and Methods:One hundred healthy donors were enrolled in the study. Peripheral blood cells were stained for CD3, CD4, CD8, and CD154 (CD40L), and cellular viability was assessed with 7-aminoactinomycin D and analyzed by flow cytometry. Results:The median value for DPTs was 2.6% (interquartile range=1.70%-3.67%). Women had higher percentages of DPTs than men (3.3% vs. 2.1%). The subpopulation of CD4 low CD8 high showed higher expression of CD154 than the other T-cell subpopulations. Conclusion:DPT reference values were obtained from blood bank donors. A sex difference was found, and the CD4 low CD8 high subpopulation had the highest activation marker expression.Amaç: CD4+CD8+ çift pozitif T hücreleri (ÇPT) ayrı bir T hücre alt popülasyonu olarak iki temel fenotip ile sınıflandırılmaktadır: CD4 yüksek CD8 düşük ve CD4 düşük CD8 yüksek . Son yıllarda, ÇPT'lerin enfeksiyonlar, tümörler ve otoimmün hastalıkların patogenezi ile ilişkisi tanımlanmıştır. Sağlıklı bireyler arasında referans değerleri bilinmemektedir. Bu nedenle, bu çalışmanın amacı, Kolombiya Bogota'daki bir kan bankasındaki sağlıklı vericilerden alınan periferik kandaki ÇPT'ler için bir referans değeri sağlamak ve bir yüzey belirteci kullanarak aktivasyon durumunu belirlemektir. Gereç ve Yöntemler:Çalışmaya yüz sağlıklı verici dahil edilmiştir. Periferik kan hücreleri CD3, CD4, CD8 ve CD154 (CD40L) için işaretlendi ve hücresel canlılık 7-aminoactinomycin D kullanarak akan hücre ölçer ile analiz edildi.Bulgular: ÇPT'ler için ortanca değer %2,6 (çeyrekler arası aralık=%1,70-%3,67) olarak saptandı. Kadınlarda ÇPT yüzdesi erkeklere göre daha yüksek bulundu (%3,3 karşı %2,1). CD4 düşük CD8 yüksek alt popülasyonu diğer T hücre alt popülasyonlarından daha yüksek CD154 ekspresyonu gösterdi.Sonuç: ÇPT referans değerleri kan bankası vericilerinden elde edilmiştir. Cinsiyetler arası fark ve CD4 düşük CD8 yüksek alt popülasyonunda da en yüksek aktivasyon belirteci ekspresyonu saptanmıştır.