Evaluation of CD 4 + CD 25 + T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction: CD4+CD25+ T lymphocytes have been implicated in the regulation of host infl ammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods: Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results: It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions: Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

The clinical course of chronic Chagas disease (ChD) varies from absence of symptoms (indeterminate form -IND) to severe manifestations with cardiovascular (cardiac form -CARD) and/ or digestive (digestive form) impairment 1 .It has been widely accepted in the scientifi c community that symptomatic forms of chronic ChD are associated with the establishment of an exacerbated cytotoxic cellular immune response that leads to infl ammatory tissue damage and development of symptoms 2,3 .Nevertheless, there is evidence of a specifi c CD4 + T lymphocyte lineage, characterized by expression of CD25 surface markers a nd the FoxP3 transcription factor, that is involved in the regulation of the cytotoxic response in chronic ChD [4][5][6] .Furthermore, developmental or functional disturbances in these cells have been linked to autoimmune and infl ammatory diseases in humans and animals 7 .Higher percentages of CD4 + CD25 + T lymphocytes are observed in peripheral blood from patients with IND forms of ChD [4][5][6] , which indicates that these cells have a role in controlling development of the morbidities common to human ChD.These lymphocytes produce cellular factors such as interleukin 10 (IL10) and transforming growth factor beta (TGF-β), which are among the host immunosuppression mechanisms employed during the host immune response to Trypanosoma cruzi 4 .While the role of these cytokines in immune modulation guided by CD4 + CD25 + FoxP3 + T cells has been reported 4,8,9 , there is still no consensus about their mechanisms of action.
The development of specifi c recombinant antigens of T. cruzi represents an important step in better understanding the role of the cellular responses in ChD 10 .Our group has been evaluating the use of the recombinant antigens CRA (cytoplasmic repetitive antigen) and FRA (fl agellar repetitive antigen) as immunological markers of the clinical evolution of ChD [11][12][13] , demonstrating signifi cant results for the differentiation of distinct clinical manifestations of the chronic phase of ChD.
This study aimed to evaluate detection of CD4 + CD25 + FoxP3 + , CD4 + CD25 + IL10 + T cells, and TGF-β in patients with chronic ChD at different cell culture time points after stimulation with CRA and FRA antigens.Eleven patients from the Ambulatório de Doença de Chagas e Insufi ciência Cardíaca, PROCAPE-Universidade de Pernambuco/UPE who were diagnosed with chronic ChD (IND=06/CARD=05) were selected.Forty milliliters of blood were collected in heparin tubes (Vacuette) to obtain the peripheral blood mononuclear cells (PBMC) through a concentration gradient technique (Ficoll-Paque TM Plus -Amersham Biosciences).Approximately 5 × 10 6 cells/mL were cultured (RPMI 1640 medium/Sigma with 10% of fetal bovine serum/Sigma) in the presence of Phytohemagglutinin (PHA -GIBCO TM /Invitrogen Corporation) (5µg⁄mL) (positive control), CRA, or FRA (2µg/mL), or with no stimulatory agent (negative control).Culture fl asks were then incubated Braz SCM et al -CD4 + CD25 + T cell response time kinetics in chronic Chagas disease at 37°C with 5% CO 2 for 1, 3, and 5 days.Four hours before the end of the respective incubation period, Brefeldin A (Sigma) (10µg⁄mL) was added to the cultures.Cells were washed by centrifugation (300 × g/5 min) with PBS-Wash (Phosphate-Buffered Saline with 0.5% bovine serum albumin/Sigma and 0.1% sodium azide/Sigma) and incubated for 30 min with surface antibodies (anti-CD4-fluorescein isothiocyanate/ Caltag; anti-CD25-allophycocyanin/Invitrogen).The steps that followed were cell fi xation (Human FoxP3 Buffer Set/BD Biosciences®; BD Cytofi x ™ /BD Biosciences®), centrifugation (300 × g/5 min), and permeabilization (Human FoxP3 Buffer Set/BD Biosciences®; PBS-Wash add 0.2% of saponin/Sigma).Samples were washed again with PBS-Wash and incubated for 30 min with intracellular antibodies (FoxP3-PE/BD Pharmingen; IL10-PE/Caltag).After another round of wash by centrifugation (300 × g/5 min), cells were fi xed (BD Cytofi x ™ ) and 30,000 events/sample were acquired in a FACScalibur ® fl ow cytometer (Becton Dickinson) and analyzed using the CellQuest Pro Software.Detection of TGF-β in supernatants from days 1 and 5 was performed using the Human TGF-β1 Immunoassay Kit -Quantikine ® (R&D Systems © ).Comparison of different culture time points within each group (IND and CARD) was performed though Friedman test followed by Wilcoxon test.To compare groups at each time point, data normality was analyzed by Student's t-test.When presumption of homogeneity was not confi rmed, Mann-Whitney test was used.A signifi cance level of 5% was considered in all analyses.The software utilized was Excel 2007, GraphPad Prism 5.0, and Statistical R 2.9.0.The procedures described here were approved by the Ethics Committee at Centro de Pesquisas Aggeu Magalhães (CPqAM)/ Fundação Oswaldo Cruz (FIOCRUZ)(Nº: 157/08).
Analysis of CD4 + CD25 + T lymphocyte population in each group (CARD and IND) showed that there was no statistical difference among cell culture time points (1, 3, and 5 culture days) after stimulation with either recombinant antigen (Figures 1A  and 1B).However, an increase in the mean number of CD4 + CD25 + T cells in IND patients was observed over the course of the cultures, mainly after FRA stimulation, while in the CARD group, there was a decrease in the mean number of CD4 + CD25 + T cells from the fi rst to the fi fth day of culture.The means of CD4 + CD25 + T cell counts between the 2 groups (CARD versus IND) for each culture time point (culture day 1, 3, or 5) were also compared.Differences between CARD and IND were statistically signifi cant at days 3 (p = 0.014) and 5 (p = 0.010) after in vitro stimulation with FRA (Figure 1B).However, no differences were observed after stimulation with CRA (Figure 1A).
In regard to IL10-producing CD4 + CD25 + T lymphocytes, the mean values of CD4 + CD25 + IL10 + T cells in the IND group, in the presence of FRA, were statistically different between days 1 and 3 (p = 0.031) and between days 1 and 5 (p = 0.031).Comparison of the CARD and IND forms showed that the mean number of CD4 + CD25 + IL10 + T cells was higher in the IND group than in the CARD patients after stimulation with CRA and FRA, however, this difference was non-signifi cant (Figures 1C  and 1D).With regard to the presence of CD4 + CD25 + FoxP3 total and CD4 + CD25 + FoxP3 high T cells, there was no statistical difference observed between the groups or among the cell culture time points within each group (Figure 2).

The presence of TGF-β in supernatants was not signifi cantly different between patients with CARD and IND forms of chronic
ChD.Nevertheless, a reduction in TGF-β production after 5 days was observed upon stimulation with CRA (p = 0.043) (Figure 3).Thus, in the present study we found that in vitro culture of PBMC for 3 and 5 days allowed differentiation between CARD or IND forms of ChD through evaluation of CD4 + CD25 + T lymphocyte counts after antigenic stimulation.Previous studies using whole blood (ex vivo) 5,6 and after 22 hours of culture in the presence of epimastigote antigens 7 were also able to differentiate CARD and IND forms by analyzing the percentage of these cells through fl ow cytometry.Because CD4 + CD25 + T cells represent a small percentage of total circulating CD4 + T lymphocytes 7 , we believe that longer periods of cell culture may improve studies regarding the possible roles of these cells in the clinical course of ChD.In regard to IL10 production by CD4 + CD25 + T lymphocytes in chronic ChD, our data corroborated with those presented by Araújo et al. 6 , in that no difference was found between patients with CARD and IND clinical forms.However, the increased numbers of CD4 + CD25 + IL10 + T lymphocytes throughout the cell culture period reinforced the importance of considering longer periods of cultivation.

A B
The observation that there were no differences in TGF-β production among patients with CARD and IND forms of ChD may have been due to the lower sample size evaluated.Moreover, with the chosen method (capture ELISA), the source of cytokine production is not identifi ed.With regard to the reports citing TGF-β as a pathway of immunoregulation by CD4 + CD25 + FoxP3 + T lymphocytes 7,8 , the technique reported here did not clarify whether CD4 + CD25 + FoxP3 + T cells were responsible for TGF-β production.Thus, the evolution of these growth factors under the regulation of CD4 + CD25 + FoxP3 + T cells in individuals with chronic ChD will only be confi rmed upon further study.
We conclude that CD4 + CD25 + T lymphocyte cultures derived from serum of patients chronic ChD that are maintained for longer periods have the potential to be used in prospective studies aimed at understanding the clinical evolution of the disease.Moreover, this approach may also aid in the identifi cation of cellular patterns that can serve as biological markers to monitor patients.Further, it is important to mention that a greater number of individuals should be evaluated to prove the importance of this cell population in immunoregulation in individuals with the IND form of ChD.

FIGURE 1 -
FIGURE 1 -Detection of CD4 + CD25 + (A and B) and CD4 + CD25 + IL10 + (C and D) T lymphocytes in peripheral blood mononuclear cells of patients with chronic Chagas disease after in vitro stimulation with CRA and FRA.Signifi cant differences are indicated in the fi gures with the respective p-values.An "a" indicates signifi cant differences in comparison with day 1.CRA: cytoplasmic repetitive antigen; FRA: fl agellar repetitive antigen; CARD: patients with cardiac clinical form of chronic Chagas disease; IND: patients with indeterminate clinical form of chronic Chagas disease; CD: cluster of differentiation; IL10: interleukin 10.

FIGURE 2 -FIGURE 3 -
FIGURE 2 -Detection of CD4 + CD25 + FoxP3 total (A and B) and CD4 + CD25 + FoxP3 high (C and D) T lymphocytes in peripheral blood mononuclear cells of patients with chronic Chagas disease after in vitro stimulation with CRA and FRA.Signifi cant differences are indicated in the fi gures with the respective p-values.CRA: cytoplasmic repetitive antigen; FRA: fl agellar repetitive antigen; CARD: patients with cardiac clinical form of chronic Chagas disease; IND: patients with indeterminate clinical form of chronic Chagas disease; CD: cluster of differentiation.