Immunological exhaustion and functional profile of CD8+ T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas disease patients

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Immunological exhaustion and functional profile of CD8+ T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas patients ´ ´ , Adriana Egui , Mª Carmen Thomas , Elena Perez-Ant on ´ , Manuel Segovia , Manuel Carlos Lopez ´ Marina Simon PII: DOI: Reference:

S0001-706X(19)30927-1 https://doi.org/10.1016/j.actatropica.2019.105242 ACTROP 105242

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Acta Tropica

Received date: Revised date: Accepted date:

12 July 2019 21 October 2019 23 October 2019

´ ´ , Adriana Egui , Mª Carmen Thomas , Marina Simon ´ , Please cite this article as: Elena Perez-Ant on ´ Manuel Segovia , Manuel Carlos Lopez , Immunological exhaustion and functional profile of CD8+ T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas patients, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105242

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Highlights 

CD8+ T cells undergo functional exhaustion during the chronic infection by T. cruzi.

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The dysfunctional process is correlated to the progress of the pathology.

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Benznidazole treatment partially reverses the exhaustion of CD8 + T cells.

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The treatment induces an improvement of the multifunctional capacity of CD8 + T cells.

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Title: Immunological exhaustion and functional profile of CD8 + T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas patients.

Short title: Functional response of CD8+ T cells in chronic Chagas disease.

Authors: Elena Pérez-Antón1, Adriana Egui1, Mª Carmen Thomas1, Marina Simón2, Manuel Segovia2, Manuel Carlos López1.

1

Instituto de Parasitología y Biomedicina López-Neyra. Consejo Superior de

Investigaciones Científicas; Granada, Spain. 2

Unidad Regional de Medicina Tropical, Hospital Virgen de la Arrixaca; Murcia, Spain.

# Corresponding author: Manuel C. López, PhD, Instituto de Parasitología y Biomedicina “López-Neyra”, Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Granada – Spain, Parque Tecnológico de la Salud (PTS) Avenida del Conocimiento, 17; 18016. Tel.: +34 958181661, Fax: +34 958181632, e-mail: [email protected].

Abstract The lack of useful tools for detection the impact of treatment during the follow-up of chronic Chagas disease treated patients difficult the adequate care to the affected population. The objective of this study was to evaluate the functional response of CD8+ T lymphocyte population, critical for the control of Trypanosoma cruzi infection, as a possible cellular biomarker of treated Chagas disease patients. Thus, we analyzed the

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antigen-specific CD8+ T-cell response before and after benznidazole treatment in asymptomatic (indeterminate) and cardiac chronic Chagas disease patients. A marked dysfunctional process of the CD8+ T cell population was found in patients with an advanced pathology. Thus, the cardiac patients have a higher co-expression of inhibitory receptors and a lower antigen-specific multifunctional capacity compared with that of asymptomatic patients. Remarkably, benznidazole treatment partially reverses this functional exhaustion process of CD8 + T cells in both asymptomatic and cardiac Chagas patients. Thus, the co-expression of inhibitory molecules tends to be reduced after benznidazole treatment, mainly in asymptomatic patients, finding a significant drop in the expression of inhibitory receptors such as PD-1 and 2B4. In addition, the multifunctional antigen-specific response of CD8+ T cells is enhanced after treatment in chronic patients. An increase in the subset of cells with cytotoxic capacity and production of the IFN-γ cytokine was also observed in both treated asymptomatic and cardiac chronic Chagas patients. The results derived from this study show the improvement of the functional capacity of CD8+ T cells after treatment which could be have a positive effect on parasitic control. In addition, the phenotypic and functional profile of the CD8+ T cells described could serve as a tool for monitoring the impact of benznidazole treatment.

Keywords: Chagas disease; benznidazole treatment; CD8+ T cell; inhibitory receptor; cytokines; cytotoxic molecules.

Abbreviations: CCC: Cardiac chronic Chagas disease patients; cChD: Chronic Chagas disease patients; FMO: Fluorescence minus one; HD: Healthy donors; IND:

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Indeterminate chronic Chagas disease patients; MFI: Mean fluorescence intensity; Tc1: Cytotoxic T cell type 1; TcSA: Trypanosoma cruzi-soluble antigens.

1. Introduction Trypanosoma cruzi, the etiological agent of Chagas disease, is an obligate intracellular protozoan parasite causing a chronic infection in human and in a large number of other mammalian species. This disease, that affects around 8 million people worldwide (WHO, 2015), is endemic from southern California to South America. However, at the present, given the new patterns of migration, this disease is no longer restricted only to endemic regions (Gascon et al., 2010). Furthermore, it is known that this parasite invades a large range of host cell, in which it is able to maintain a replicative cycle in its intracellular form. A complex immunological response is induced by the host to control the T. cruzi infection, but normally this is not enough to prevent the spread of the parasite, which in absence of treatment leads to establishment of chronic phase of the disease (Cardoso et al., 2015). In this chronic phase, most patients remain in a clinically asymptomatic stage (indeterminate form). However, usually after decades a 40%, approximately, of patients develop cardiomyopathies associated with T. cruzi infection (Chatelain, 2017). The intracellular lifestyle of the parasite makes CD8+ T cells key players in the immune response against this infection (Acevedo et al., 2018). The critical importance of these cells, has been widely demonstrated, by the depletion of the CD8+ T-cell population with anti-CD8 antibodies (Tarleton, 1990; Tarleton et al., 1994) or studying knockout mice models (Rottenberg et al., 1993; Rottenberg et al., 1995; Tarleton et al., 1996; Tarleton et al., 1992). In human chronic T. cruzi infection a clear increase in the

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of CD3+HLA-DR+ cells, as well as a decrease in the percentage of CD45RA+CD8+ T cells has also been reported (Dutra et al., 1994), which evidence the active role of this T-cell population in the control of this infection. CD8+ T cells are characterized by their cytotoxic capacity to kill infected cells by the secretion of cytolytic molecules, through the perforin/granzyme pathway (Trapani and Smyth, 2002). In the context of T. cruzi infection, the cytotoxic capability of these cells has been related with the clearance of the parasite (Martin and Tarleton, 2004) which is essential for the infection control. The production of the cytotoxic molecules is also related with the late stage of differentiated CD8+ T cells, which are in high proportion in severe heart disease patients (Lasso et al., 2015). On the other hand, it is also known that IFN-γ secretion is critical for the protective function of CD8 + T cells against T. cruzi infection (Acevedo et al., 2018; Martin and Tarleton, 2004). Studies using IFN-γ knockout mice have reported that they failed to control parasitemia and dying faster than knockout mice for other important molecules or wild-type mice (Rodrigues et al., 2012; Tzelepis et al., 2006), demonstrating the primordial role of this cytokine to control T. cruzi infection. Also, an inverse correlation between the severity of the chronic Chagas disease patients and the frequency of IFN-γ-producing CD8+ T cells specific against T. cruzi has been reported (Albareda et al., 2006; Albareda et al., 2009; Laucella et al., 2004), being significantly lower in patients with severe heart disease in comparison to those with no symptoms or mild heart disease (Albareda et al., 2006). This fact not only identify IFN-γ as an important tool to assess the quality of CD8+ T cell response, but could also be provide a useful tool for chronic patients in predicting the disease progression (Egui et al., 2018). The co-production of IFN-γ, IL-2 and TNF-α by CD8+ T cells antigen-specific has been observed in greater proportion in

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asymptomatic chronic patients than in Chagas patients with cardiac symptomatology (Lasso et al., 2015; Mateus et al., 2015). Benznidazole, which is the primary recommended drugs for treat Chagas disease, improves the immune response against the parasite and prevents the development of cardiomyopathy as has been reported (Bustamante et al., 2007; FragataFilho et al., 2016; Garcia et al., 2005). Thus, we estimated that the dynamic of functional and phenotypic cellular profile that occurs during the progression of chronic Chagas disease from the asymptomatic to a symptomatic clinical form could be potential biomarkers for evaluating the effectiveness of the treatment. In fact, recently published data support that the treatment in asymptomatic Chagas patients induces an improvement in the multifunctional response of CD8+ T cells specific-antigen accompanied by a reduction of inhibitory molecule expression (Mateus et al., 2017). These findings support that in asymptomatic Chagas chronic patients the antiparasitic treatment enhanced in part the quality of antigen-specific CD8+ T-cell response. However, further research should be done to determine the impact of the treatment on the response of CD8+ T cells in chronic Chagas disease patients with cardiac symptomatology, as well as analyze their potential use as cellular biomarkers of therapeutic efficacy. On this base, the aim of the present study was to evaluate, prior and after benznidazole treatment, the functional profile and exhaustion phenotype of antigen-specific CD8+ T cells in chronic Chagas disease patients (asymptomatic and with cardiac symptomatology) living in a non-endemic area of T. cruzi infection.

2. Material and Methods 2.1 Study populations

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The study cohort was composed of patients mostly of Bolivian origin (95.1%) and all of them resident in non-endemic areas, Spain. All the patients evaluated were diagnosed with chronic Chagas disease; 25 of them were classified as asymptomatic patients, or also called indeterminate clinical form (IND), and 15 as symptomatic patients with cardiac alterations (CCC). These cardiac patients were distributed based on the Kuschnir classification, 9 in the stage G1, 5 in the stage G2, and 1 in the stage G3. The study also included the evaluation of 12 healthy donors (HD). The patients were recruited before having taken any antiparasitic therapy. Subsequently, they were treated with benznidazole chemotherapy, by administration of 5 mg/kg/d for 60 days per patient at the Virgen de la Arrixaca Hospital in Murcia, Spain, in which also a clinical follow-up was carried out for the entire study period after therapy. A signed informed consent was obtained from all individuals before their inclusion in the study. All the developed protocols were approved by the Ethics Committees of the Consejo Superior de Investigaciones Científicas and of the Hospital Virgen de la Arrixaca (Ref. no. 094/2016 and MTR-05/2016, respectively).

2.2 Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were obtained from thirty-milliliter blood samples collected, from each subject, in tubes with EDTA prior and after administration treatment. These cells were purified via a density gradient through centrifugation and cryopreserved in liquid nitrogen until use, diluted in inactivated fetal bovine serum supplemented with 10% dimethyl sulfoxide.

2.3 Isolation of Trypanosoma cruzi soluble antigens

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To obtain T. cruzi soluble antigens (TcSA), Rhesus Monkey Kidney Epithelial cells (LLC-MK2 line; CCL-7™, Manassas, VA) were cultured and grown until they formed a monolayer, which was infected with T. cruzi trypomastigote forms (MHOM/BR/1950/ Y isolated strain). After 96 h, the parasites were recovered in trypomastigote and amastigote forms. Parasites, in a ratio of 1 amastigote: 1 trypomastigote, were suspended at 1x106 parasites/µL in lysis buffer (50 mM Tris-HCl at pH 7.4, 50 mM NaCl, 0.005% NP-40, 1 mM PMSF, and 1 µg/mL leupeptin) and subsequently sonicated 3 times with pulses of 50-62 KHz for 40-s time intervals of 20 s. Soluble protein extracts were obtained by centrifugation at 10,000 rpm for 20 min at 4ºC. The protein concentration was determined using the micro BCA protein assay kit (Thermo Fisher Scientific).

2.4 Detection of surface and intracellular molecules by flow cytometry assays 2.4.1 Intracellular cytokine and cytotoxic molecules detection by flow cytometry assays in PBMC after stimulation with Trypanosoma cruzi soluble antigens PBMC were cultured at 1x106 cells/mL in the presence of anti-CD28 (1 µg/mL) and anti-CD49d (1 µg/mL) and stimulated with TcSA (1 µg/mL) or without TcSA (basal response). The cells, under each condition, were incubated for 10 h at 37ºC in a humidified atmosphere with 5% CO2, and the last 9 h in the presence of brefeldin A (1 µg/mL) and monensin (2 µM) (BD Bioscience). To evaluate intracellular cytokine and cytotoxic molecules production, at least 1x106 cells were stained for each condition. Before beginning the staining process, the cells were incubated with 5% of iFBS for 10 min at room temperature to block non-specific binding. Later, PBMC were stained with

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a viability marker, LIVE/DEAD Fixable Aqua (Invitrogen), for 20 min in darkness at room temperature. Subsequently, the cells were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies for 20 min in darkness at 4ºC. After washing, the cells were fixed and permeabilized with fixation/permeabilization solution (Cytofix/Cytoperm™), which it is incubated with this reagent for 20 min in darkness at 4ºC. After two wash with BD Perm/Wash™ intracellular staining was performed with anti-granzyme B, anti-IFN-, anti-IL-2, anti-TNF-α, and anti-perforin antibodies for 30 min in darkness at 4ºC. The cellular acquisition was carried out through a FACSAria III flow cytometer (BD Biosciences). At least 100,000 live lymphocytes were acquired according to FCS/SSC parameters for each condition. The data file was subsequently analyzed using FlowJo 9.3.2 software (Tree Star, Ashland, OR). The positivity for each marker was determined using the fluorescence minus one (FMO), and unstained controls.

2.4.2 Identification of inhibitory receptor expression by flow cytometry PBMC were cultured under the same conditions previously described in presence of anti-CD28, anti-CD49d and TcSA (1 µg/mL) for 10 h. After incubation cells were stained with the following cell surface antibodies: anti-CD3, anti-CD4, and anti-CD8,

anti-2B4,

anti-CD160,

anti-PD-1,

and

anti-TIM-3.

After

cell

permeabilization, intracellular staining was performed with anti-CTLA-4 antibody. The cells were acquired and analyzed as described above.

2.5 Statistical analysis Statistical analyses were performed using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA). Nonparametric tests were used to test for statistical significance. The Mann Whitney U test was used to carry out comparisons

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among study groups (patients versus healthy donors; or CCC versus IND). The Wilcoxon paired test was used for the longitudinal study performed in the follow-up of the treatment. An additional analysis was performed to compare co-expression pie charts using 10,000 permutations calculated with SPICE version 5.3 software (the National Institutes of Health, Bethesda, MD). Statistical significance was assigned to values of p<0.05.

3. Results 3.1 CD8+ T cells from a cohort of chronic Chagas disease patients undergo a cellular exhaustion process The surface expression of inhibitory receptors 2B4, CD160, PD-1, CTLA-4 and TIM-3 was measured in the CD8+ T-cell population from IND and CCC patients and healthy donors. The results show that CD8+ T cells from patients chronically infected with T. cruzi present a greater expression of inhibitory receptors compared with healthy donors (Figure 1). Thus, in Chagas disease patients was observed a higher percentage of CD8+ T cells expressing 2B4 (p<0.0001), CD160, CTLA-4, PD-1 and TIM-3 (p<0.05) (Figure 1A), as well as a greater expression of these co-inhibitory molecules, 2B4 (p<0.0001), CD160, CTLA-4 (p<0.01) and PD-1 (p<0.01), per cell (measured by the mean fluorescence intensity [MFI]) (Figure 1B).

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Figure 1: Expression of inhibitory receptors 2B4, CD160, CTLA-4, PD-1 and TIM-3 in CD8+ T cells from chronic Chagas disease patients and healthy donors. (A) Frequency of CD8+ T cells expressing each of the inhibitory receptors under study in 38 chronic Chagas disease patients (cChD) and in 12 healthy donors (HD). The mean values of the percentage of CD8+ T cells and the standard deviations (SD) for each marker (mean ± SD) are : 2B4 of HD: 11.49 ± 14.7; 2B4 of cChD: 41.96 ± 21.7; CD160 of HD: 8.19 ± 5.1; CD160 of cChD: 10.78 ± 6.1; CTLA-4 of HD: 3.58 ± 2.4; CTLA-4 of cChD: 5.72 ± 4.0; PD-1 of HD: 12.98 ± 4.7; PD-1 of cChD: 17.24 ± 8.7; TIM-3 of HD: 2.57 ± 1.2; TIM-3 of cChD: 4.81 ± 3.2. (B) Expression level of 2B4, CD160, CTLA-4, PD-1 and TIM-3 in CD8+ T cells of 27 cChD and 12 HD, measured by the mean fluorescence intensity (MFI) of each marker. MFI values and standard deviations (mean values ± SD) are : 2B4 of HD: 241.8 ± 21.8; 2B4 of cChD: 351.9 ± 59.8; CD160 of HD: 211.3 ± 16.9; CD160 of cChD: 216.1 ± 19.7; CTLA-4 of HD: 147.0 ± 15.3; CTLA-4 of cChD: 162.8 ± 15.1; PD-1 of HD: 336.3 ± 37.5; PD-1 of cChD: 409.1 ± 67.9; TIM3 of HD: 119.2 ± 7.7; TIM-3 of cChD: 122.1 ± 13.0. The range of values represented by the

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whiskers of the box-plots goes from the minimum to the maximum. The asterisk symbols show the data of p-values that follow: p<0.05 (*), p<0.01 (**) and p<0.0001 (****).

The expression of inhibitory receptor in CD8+ T cells from chronic Chagas disease patients considering the stage of the disease [indeterminate patient (IND) or cardiac patient (CCC)] was also analyzed. The obtained results showed a higher frequency of CD160, CTLA-4 (p<0.0001), PD-1 and TIM-3 in cardiac patients compared to indeterminate patients (Figure 2A). Likewise, a higher fluorescence intensity of the CD160 marker was observed in CD8+ T cells of cardiac patients versus IND patients (p<0.05) (Figure 2B). Remarkably, a substantial greater frequency of expression of 2B4 (p<0.001), CD160 (p<0.05), CTLA-4 (p<0.001), PD-1 (p<0.05) and TIM-3 (p<0.01) was detected in the CD8+ T-cell population from chronic cardiac patients with respect to healthy donors (Figure 2A). Moreover, a marked increase in the intensity of inhibitory receptor expression per cell was also detected in this cardiac patients group versus healthy subjects (Figure 2B). Thus, the most marked increments were detected for PD-1 and 2B4 molecules (p<0.0001, for both inhibitor receptors) and CTLA-4 (p<0.01) (Figure 2B). The indeterminate patients also showed a higher percentage of CD8+ T cells expressing 2B4 (p<0.0001), CTLA-4, PD-1 and TIM-3 versus HD (Figure 2A), as well as a higher intensity of expression per cell of 2B4 (p<0.001), CTLA-4 and PD-1 (p<0.05) compared to HD (Figure 2B).

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Figure 2: Expression of inhibitory receptors in CD8+ T cells of healthy donors and indeterminate and symptomatic patients infected with Trypanosoma cruzi. (A) Frequency of CD8+ T cells that express 2B4, CD160, CTLA-4, PD-1 or TIM-3 in indeterminate patients (IND, n=25) and in patients with cardiac manifestations (CCC, n=13), in comparison with those obtained in healthy donors (HD, n=12). Mean values of the percentage of CD8+ T cells and standard deviations (SD) for each marker (mean ± SD) are: 2B4 of IND: 41.23 ± 23.4; 2B4 of CCC: 43.36 ± 18.6; CD160 of IND: 9.41 ± 5.3; CD160 of CCC: 13.41 ± 6.9; CTLA-4 of IND: 4.07 ± 3.1; CTLA-4 of CCC: 9.14 ± 3.6; PD-1 of IND: 15.70 ± 7.9; PD-1 of CCC: 20.21 ± 9.6; TIM-3 of IND: 4.53 ± 3.5; TIM-3 of CCC: 5.35 ± 2.7. (B) Mean level of expression per cell of each inhibitory receptor under study in CD8+ T lymphocytes, measured using the mean fluorescence intensity (MFI) value of 14 IND, 13 CCC and 12 HD. MFI values and SD (mean values ± SD) are: 2B4 of IND: 339.8 ± 66.2; 2B4 of CCC: 364.9 ± 51.3; CD160 of IND: 207.7 ± 16.0; CD160 of CCC: 225.1 ± 19.9; CTLA-4 of IND: 158.5 ± 15.7; CTLA-4 of CCC: 167.9 ± 13.2; PD-1 of IND: 388.7 ± 70.7; PD-1 of CCC: 443.7 ± 48.2; TIM-3 of IND: 123.4 ± 15.1;

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TIM-3 of CCC: 120.6 ± 10.9. The range of values represented by the box and the whiskers includes the values from the minimum to the maximum. The p-values were represented with asterisks as follows: p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****).

The co-expression of inhibitory receptors in the same cell was also analyzed in the CD8+ T-cell population of chronic Chagas disease (IND and CCC) patients and healthy donors (Figure 3). The results showed that the co-expression of two, three, four or five inhibitory molecules in the CD8+ T-cell population was statistically higher in chronic Chagas disease patients versus healthy donors (p<0.0001, p<0.001, p<0.0001 and p<0.05; respectively) (Figure 3). Furthermore, the Chagas disease patients who manifested cardiac alterations had a statistically higher proportion of cells co-expressing three, four and five inhibitory receptors compared to indeterminate ones (p<0.05) as indication that CD8+ T cells from this patients with a more severe Chagas pathology had a greater degree of exhaustion (Figure 3).

Figure 3: Evaluation of the co-expression level of inhibitory receptors in CD8+ T cells from healthy donors and chronic Chagas disease patients. The inhibitory receptors studied were: 2B4, CD160, CTLA-4, PD-1 and TIM-3. The data shows the frequency values of CD8+ T coexpressing 2, 3, 4 or 5 inhibitory receptors in chronic Chagas disease patients (cChD) (indeterminate [IND] and with cardiac symptomology [CCC]) compared to values obtained in

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healthy donors (HD). The size of the study population was 37 cChD (25 IND and 12 CCC) and 12 HD. Mean values of the percentage of CD8+ T cells and standard deviations (SD) for each marker (mean ± SD) are: Express 2 of HD: 7.66 ± 5.7; Express 2 of cChD: 18.02 ± 8.3; Express 2 of IND: 16.09 ± 7.5; Express 2 of CCC: 22.03 ± 8.8; Express 3 of HD: 1.03 ± 0.8; Express 3 of cChD: 3.42 ± 3.1; Express 3 of IND: 2.61 ± 1.9; Express 3 of CCC: 5.10 ± 4.3; Express 4 of HD: 0.03 ± 0.03; Express 4 of cChD: 0.25 ± 0.3; Express 4 of IND: 0.17 ± 0.2; Express 4 of CCC: 0.43 ± 0.6; Express 5 of HD: 0.0005 ± 0.001; Express 5 of cChD: 0.009 ± 0.02; Express 5 of IND: 0.007 ± 0.02; Express 5 of CCC: 0.0117 ± 0.02. The box and whiskers on the graph represent all the values (from minimum to maximum). The p-values are symbolized with asterisks in the following way: p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****).

3.2 Impact of benznidazole treatment on the CD8+ T-cell exhaustion process in a cohort of chronic Chagas disease patients (indeterminate and cardiac) Surface expression of inhibitory receptors 2B4, CD160, CTLA-4, PD-1 and TIM-3 in the CD8+ T cells from chronic Chagas disease in IND and CCC was evaluated pre-treatment (T0) and at 6-12 months (T1) and from 24 to 48 months (T2) after treatment (Figure 4). The obtained results showed that after treatment occurs a decrease of the percentage of CD8+ T cells expressing 2B4 and PD-1. Thus, the percentage of CD8+ T cells expressing 2B4+ and PD-1+ decreased significantly after treatment (T0 versus T1, p<0.05; and T0 versus T2, p<0.01) and (T0 versus T2; p<0.05), respectively. Inversely, CD8+TIM-3+ T cells increased its proportion at post-treatment times (T0 versus T1, and T0 versus T2; p<0.05) (Figure 4A). When the evaluation of the chemotherapy impact was performed, separating Chagas disease patients in asymptomatic and cardiac patients (Figure 4B), marked changes in the expression of the inhibitory receptors were observed in the group of indeterminate patients. In this group of patients, the frequency of CD8+2B4+ and CD8+PD-1+ T cells decreased statistically

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after 24 to 48 months of benznidazole treatment (both p<0.01). In addition, a statistically significant decrease of CD8+2B4+ T cells was also detected after a short period after treatment (6-12 months) (p<0.05) in indeterminate patients (Figure 4B). Likewise, in the cardiac patients, the frequency of CD8+ CTLA-4+ and CD8+ PD-1+ T cells decreases after 6-12 months (T1) and 24-48 months (T2) of treatment, although without reaching statistically significant values. The statistical increase in the frequency of CD8+TIM-3+ T cells observed after treatment was only detected in the group of cardiac patients (T0 versus T2, p<0.05) (Figure 4B).

Figure 4: Impact of benznidazole on the expression of inhibitory receptors in CD8 + T cells from chronic Chagas disease patients. 2B4, CD160, CTLA-4, PD-1 and TIM-3 were the inhibitory receptors assessed. The cellular expression frequencies of each marker were evaluated in CD8 + T cell population at pre-treatment (T0) and after 6-12 (T1) and 24-48 months of treatment (T2) in cChD patients (IND and CCC). (A) Frequency of CD8+ T cells that express each inhibitory receptor under study evaluated before and after treatment in cChD. (B) Evaluation of the frequency of CD8+ T cells expressing each inhibitory receptor before the

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treatment administration (T0) and monitored after treatment (T1 and T2) in the indeterminate group of patients and in the cardiac group separately. The statistical analyses were carried out using the Wilcoxon paired test. Statistically significant differences are indicated by (*) p<0.05 and (**) p<0.01. The cChD was grouped according T0, IND (n=25) and CCC (n=13); T1, IND (n=22) and CCC (n=10); and T2, IND (n=21) and CCC (n=10).

Additionally, the frequency of CD8+ T cells that co-express several inhibitory receptors (2, 3, 4, or 5) was monitored after treatment in indeterminate and cardiac chronic Chagas disease patients (Figure 5). The obtained results showed a reduction in both group of patients, but without achieving statistically significant values.

Figure 5: Impact of benznidazole treatment on the co-expression level of inhibitory receptors in the CD8+ T-cell population, evaluated in chronic Chagas disease patients. The inhibitory receptors evaluated were: 2B4, CD160, CTLA-4, PD-1 and TIM-3. Frequency of CD8+ T cells co-expressing 2, 3, 4 or 5 inhibitory receptors evaluated before and after treatment with benznidazole, in indeterminate patients (IND, n=25) and cardiac patients (CCC, n=14). The time points evaluated were: T0, pre-treatment; T1, after 6-12 months of the treatment; and T2, after 24-48 months of treatment. The range of values represented by the box and the whiskers includes the values from minimum to maximum.

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3.4 Functional activity of CD8+ T cells in chronic Chagas disease patients before and after benznidazole treatment The multifunctional capacity of T. cruzi-specific CD8+ T cells from IND and CCC, before and after 6-12 (T1) and 24-48 (T2) months of receiving benznidazole treatment, was assessed by the simultaneous measurement of cytokines (IFN-γ, TNF-α, and IL-2) and cytotoxic molecules production (perforin and granzyme B) after in vitro antigenic stimulation using total soluble antigen of Y strain of T. cruzi (TcSA). The Figure 6 shows the proportion of CD8+ T cells antigen-–specific that produce or coproduce these immunological molecules. The obtained results, in chronic indeterminate Chagas disease patients, evidenced (Figure 6A) that at T0 the 59% out of 61% of the total antigen-specific CD8+ T-cell population co-expressed the two cytotoxic molecules under study (perforin and granzyme). Likewise, 3.9% of CD8+ T cells co-express three functional molecules and 2.2% have a Tc1 profile (IFN-γ+Perforin+Granzyme B+). At T1 period after treatment, a substantial increase of CD8+ T cells that produce three functional molecules was observed (from 3.9% at T0 to 10.0% at T1), being this mainly due to the increase of the Tc1 subset (from 2.2% at T0 to 9.0% at T2) (Figure 6A). The proportion of CD8+ T cells co-expressing four molecules also increased after treatment (from 0.2 at T0 and 0.3% at T1 to 0.6% at T2), as well as those cells that produced IL-2 or IFN-γ (Figure 6A). Moreover, the percentage of CD8+ T cells co-expressing two functions showed an increase in the course of the treatment follow-up (from 23.1% at T1 to 35.3% at T2) with a notorious increase of those cells with a cytotoxic profile (from 20% at T1 to 28% at T2). The functional profile observed in patients with cardiac symptomatology showed at T0 a high population of monofunctional antigen-specific CD8+ T cells (73.1%), being of 51.2% that expressed only perforin (Figure 6B). A clear reduction in

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the CD8+ T-cell population that produces only one of the molecules under studied (from 73.1% at pre-treatment to 57.6% at T1 and 28.0% at T2) was detected after treatment (Figure 6B). In addition, a significant increase in the multifunctional capacity of CD8 + T cells (producing 3 molecules) was observed at 12-24 month after treatment (from 4.3% of the population at T0 to 15.3% at T1). Interestingly, the most notable increment observed was that of the CD8+ T-cell subset that co-express the cytotoxic molecules (granzyme B and perforin) in conjunction with IFN-γ (from 2.9% at T0 to 4.5% at T1 and 11.0% at T2). Furthermore, the CD8+ T cell population that express 2 molecules markedly increased at T2 period after treatment (from 22.3% at T0 to 59.7% at T2), being mainly due to the observed increase in the frequency of T cells that co-express granzyme B and perforin cytotoxic molecules (from 15.2% at pre-treatment to 54.9% at T2).

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Figure 6: Multifunctional profile of antigen-specific CD8+ T cells from chronic Chagas disease patients, before and after treatment with benznidazole. (A) Indeterminate patients and (B) Chagas disease patients with cardiac symptomatology were assessed. Proportion of CD8+ T cells that produce or co-produce IFN-γ, IL-2, TNF-α, perforin and/or granzyme B functional molecules after in vitro stimulation with total antigens of T. cruzi (TcSA) is shown. The color of the portions of the pie charts depicts the number of molecules produced by CD8+ T cells in response to TcSA. The arcs of the pie charts represent the proportion of cells expressing each one of the molecules under study. The time points evaluated were a pre-treatment time (T0; IND, n=19; CCC, n=13); and two post-treatment times, after 6-12 months of therapy (T1; IND, n=19; CCC, n=11) and after 24-36 months (T2; IND, n=19; CCC, n=10).

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4. Discussion The immune system of chronic Chagas disease patients has developed a specific adaptive response against the parasite that strives to maintain the balance between infection control and immune homeostasis. Until now, the mechanisms that trigger this chronic disease to develop a manifest symptomatology or remain in an asymptomatic stage are not well known. Therefore, knowledge of the differential immune dynamics between symptomatic and asymptomatic chronic patients, as well as in comparison with the healthy condition, has been and continues to be a constant focus of study (Egui et al., 2018). In this sense, the findings obtained show that the population of CD8 + T cells, a critical subset of lymphocyte for the control of this intracellular infection, undergoes a process of functional exhaustion during chronic T. cruzi infection, which is accentuated in chronic patients with cardiac symptoms compared with asymptomatic (revised, in (Perez-Anton et al., 2019)). The so-called cell exhaustion process occurs with the expression and co-expression of inhibitory receptors on the surface membrane of antigen-specific T cells due to the persistent antigen exposure (Wherry and Kurachi, 2015). From the evaluation of this process in CD8+ T cells of patients with chronic Chagas disease, we demonstrate that a fraction of this T-cell population exhibits this dysfunctional process. Thus, more than half of the total CD8 + T-cell population express in their membranes at least one of the inhibitory receptor evaluated (2B4, CD160, PD-1, CTLA-4 and TIM-3) during the chronic infection by T. cruzi. The frequency of CD8+ T cells that express these coinhibitory molecules is higher in chronic patients with cardiac symptoms than in those who are asymptomatic. Furthermore, the inhibitory signaling pathways are not redundant, since they act by a synergistic mechanism. Thus, it has been described that the blockade of several inhibitory receptors leads in a greater improved of the functional T-cell response; and also the presence of more than one

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inhibitory route, activated in the cell membrane, causes an increase in the level of cellular inhibition and for that conduce to a less functional potential (Blackburn et al., 2009; Jin et al., 2010; Teigler et al., 2017). Therefore, the evaluation of the coexpression of these different markers has a strong potential. The results showed in this study evidence that the co-expression of inhibitory receptors in the CD8+ T cells is highly elevated in the patients under study (living in non-endemic area) with cardiac symptoms compared to asymptomatic one, as was previously reported in a cohort of patients living in endemic areas (Lasso et al., 2015). This fact suggests the association between the functional exhaustion of this critical immune population and the advance of the Chagas disease pathology. In addition, the level of expression per cell of these inhibitory pathways in turn marks the degree of cellular exhaustion. It has been described that cells with a high expression of PD-1 marker (PD-1hi) had a lower functional capacity than cells with intermediate or low expression of this molecule (PD-1mid/lo) (Kim et al., 2018). In this sense, the obtained results in this work revealed a higher expression of these inhibitory receptors per cell in the CD8+ T cells of chronic Chagas disease patients compared to healthy donors, which supports the fact that this population exhibits an exhaustion process, and these expression values were superior in patients with a more advanced degree of the pathology. This observation, reported for the first time, manages to reinforce the conclusion previously reported by Lasso et al. (Lasso et al., 2015). The quality or multifunctional capacity of antigen-specific T cells response is crucial for determining the disease outcome to infection (Seder et al., 2008). It had been demonstrated that the antiparasitic treatment induced a substantial T cells activation status and changes in T cells response related with the loss of the cytokine production capacity by T cells (Laucella et al., 2009; Sathler-Avelar et al., 2008). Other studies, 22

detected increased of the cytokine production by T cell after the trypanocidal chemotherapy such as IFN-γ, TNF-α and IL-17, in some cases, associated with a decrease of antibody serum level (Alvarez et al., 2016; Vallejo et al., 2016). The results obtained in this study, by follow-up after treatment, showed that benznidazole treatment improved the multifunctional capacity of the antigen-specific CD8+ T cells in chronic Chagas patients. These changes occurred in parallel to the decrease of inhibitory receptors co-expression, and in a short-medium time after the therapy. These findings suggested that it could be due to a partial reversion of the T-cell exhaustion process in chronic Chagas disease patients. The impact of chemotherapy seems to have a greater impact on the reinvigorate of the CD8+ T cells in asymptomatic patients than in the cells of symptomatic ones. Since, asymptomatic patients showed greater decrease in expression and co-expression of inhibitory receptors after treatment compared to symptomatic patients. This fact could be associated to the degree of severity of the exhaustion process exhibited by CD8+ T cells of patients with a cardiac pathology. In this sense, it has been reported that the severe exhaustion process (marked by a high coexpression of inhibitory receptors), in comparison with partial exhaustion (less inhibitory receptor co-expression), limits the reversal of the process (Blackburn et al., 2009; Teigler et al., 2017). Other factors may be influencing the improvement of the functional ability of CD8+ T cells after treatment. Such as the cellular state of other immune populations that activate and potentiate the action of CD8+ T cells, such as CD4+ T helper cells (Novy et al., 2007); as well as the possible reduction of parasitic load due to the trypanocidal action of the drug. In this sense, we observe that cardiac patients, although they do not present great modifications in the expression of the inhibitory receptors evaluated, greatly increase their multifunctional capacity. Thus, a significant increase in the 23

proportion of cells with antigen-specific multifunctional capacity after the antiparasitic therapy was observed in CD8+ T-cell population. Interestingly, the proportion of cytotoxic cells that express granzyme B and perforin markedly increased after treatment. In parallel, the proportion of cells that only express perforin, in cardiac patients which numbers were be so higher prior to treatment, decreased strongly after treatment. This fact is relevant since it has been described that the population that coproduces both molecules is more efficient, than the one that produces only one of the molecules to eliminate the T. cruzi parasite (Heusel et al., 1994; Waterhouse et al., 2006). Furthermore, it is considered that the single perforin population could be associated with tissue damage (Silverio et al., 2010; Silverio et al., 2012). Remarkably, we also observed the increase in the population of T. cruzi-specific CD8+ T cells that co-produce granzyme B and perforin with the joint expression of IFN-γ (CD8+Granzyme B+ Perforin+IFN-γ+) after treatment both in cardiac and indeterminate patients. These cells are known as Tc1 for their cytotoxic capacity and their Th1-like profile, for the production of the cytokine IFN-γ, have been described in the context of T. cruzi infection as an efficient population involved in antiparasitic control (Rodrigues et al., 1999; Wizel et al., 1997). The data reported in this manuscript reflect the improvement in the functional response of antigen-specific CD8+ T cells of asymptomatic and symptomatic patients after treatment with benznidazole, which could be associated with better control of the parasite. These results are in agreement with what was recently published by Mateus et al. (Mateus et al., 2017) studying a cohort of asymptomatic patients composed mainly of patients who live in endemic areas from Colombia. Furthermore, we show the improvement in the quality of response of antigen-specific CD8+ T cells, characterized by an increase in the multifunctional profile, also occurs in chronic Chagas disease 24

patients with cardiac symptomatology. This result is it considered relevant due to the controversy existing on the convenience or not of the administration of treatment in adult patients with cardiac symptomatology.

5. Conclusions A marked dysfunctional process of the CD8+ T cell population was found in Chronic Chagas disease patients with an advanced pathology. Thus, the cardiac patients have a higher co-expression of inhibitory receptors and a lower antigen-specific multifunctional capacity compared with that of asymptomatic patients. The benznidazole treatment partially reverses this functional exhaustion process of CD8 + T cells in both asymptomatic and cardiac Chagas patients. The evaluation of the phenotypic and functional profile of CD8+ T cells involved in the control of the infection could serve as a biomarker tool for monitoring the effect of treatment in chronic Chagas disease patients. This fact is critical since one of the greatest challenges on Chagas disease research is the search for efficient tools that will enable to assess pharmacological treatment efficacy. Declaration of interest None.

Acknowledgments We thank the patients and healthy volunteers who participated in this study. We also thank Dr. Bartolomé Carrilero (Hospital Virgen de la Arrixaca, Murcia) for contributions to the recruitment and medical examination of Chagas disease patients. This work was financially supported by grants SAF2016-81003-R, SAF2016-80998-R

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from the Programa Estatal I+D+i (MINECO); the Network of Tropical Diseases Research RICET (RD16/0027/0005 and RD16/0027/0016) and FEDER. This publication is part of the PhD thesis of student Elena Pérez Antón at the University of Granada in the Biomedicine Program.

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Graphical abstract

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