Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients

Journal Pre-proof Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients

Adriana Egui, Manuel Carlos López, Inmaculada Gómez, Marina Simón, Manuel Segovia, M. Carmen Thomas PII:

S0925-4439(19)30357-6

DOI:

https://doi.org/10.1016/j.bbadis.2019.165629

Reference:

BBADIS 165629

To appear in:

BBA - Molecular Basis of Disease

Received date:

31 July 2019

Revised date:

18 October 2019

Accepted date:

28 October 2019

Please cite this article as: A. Egui, M.C. López, I. Gómez, et al., Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/j.bbadis.2019.165629

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof Title: Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients. Authors: Adriana Egui1, Manuel Carlos López1,#, Inmaculada Gómez1, Marina Simón2, Manuel Segovia2, M. Carmen Thomas1,# 1

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas; Granada, Spain. Unidad Regional de Medicina Tropical, Hospital Virgen de la Arrixaca; Murcia, Spain.

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Corresponding author: M. Carmen Thomas, PhD (Phone: +34 958181662, e-mail: [email protected]) and Manuel C. López, PhD (Phone: +34 958181661, e-mail: [email protected]).

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Short title: Cellular biomarkers in benznidazole-treated chronic Chagas patients

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Abstract One of the greatest challenges in Chagas disease research is the search for tools that will enable the assessment of pharmacological treatment efficacy. A recently described set of serological biomarkers composed of four parasite antigens and established criteria of treatment efficacy allowed the evaluation of the impact of benznidazole treatment a short/medium time after the treatment. In addition, cellular immunological parameters have also been described as potential indicators of the treatment response. The cytotoxic CD8+ T cells specific to five epitopes in the PFR2, PFR3, TcCA-2 and KMP11 antigens have been analysed, and these epitopes have been shown to be recognized, processed and presented in the context of a natural T. cruzi infection. In the present manuscript, we characterized these antigen-specific CD8+ T cells in indeterminate chronic Chagas disease patients both before and after (from 11 to 28 months) benznidazole treatment. The results indicate that there is a differential memory CD8+ T cell profile depending on the antigenic epitope and that the benznidazole treatment modulates the memory, differentiation and senescence phenotypes of the epitope-specific CD8+ T cells. Moreover, in these patients, the reactivity of sera against the referred set of biomarkers was evaluated. The data obtained show that the patients who met the established therapeutic efficacy criteria presented a differential phenotypic profile of the antigen-specific CD8+ T cells even prior to treatment compared to the patients who did not meet the therapeutic efficacy criteria, and this behaviour is associated with a better functionality of these CD8+ T cells. Keywords: Chagas disease, epitope-specific CD8+ T cells, benznidazole treatment, biomarkers, memory phenotype, cytotoxic molecules, cytokines, humoral response.

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1. Introduction Chagas disease is caused by the protozoan Trypanosoma cruzi and is one of the 20 neglected tropical diseases (NTDs) listed by the World Health Organization. It affects approximately 6 million people worldwide, causing 7 thousand deaths annually [1, 2]. Although this pathogen is considered to be endemic from southern California to South America, it is no longer restricted to endemic regions given that new patterns of migration have spread the disease to non-endemic countries [3]. This parasitic infection triggers a strong activation of the immune system, characterized by high levels of plasma cytokines, activation of B and T lymphocytes, and inflammatory reactions in the infected tissues [4]. However, this immune response is not enough to limit parasite dissemination, which, in the absence of treatment, leads to the establishment of a chronic phase of the disease [5]. A high percentage of patients remain in a clinically silent asymptomatic chronic stage also called indeterminate phase (IND). However, 10 to 40% of infected patients develop cardiomyopathies and/or digestive tract pathologies [6]. Benznidazole and nifurtimox are the currently recommended drugs for the treatment of Chagas disease at the acute and early chronic phases. Although the efficiency of these treatments in the chronic phase is controversial, their administration has been shown to reduce the risk of the development of electrocardiographic or intestinal alterations and to prevent congenital transmission [2, 7-9]. Moreover, the modulation of parasite-specific T-cell response in association with a significant decrease in T. cruzi-specific antibodies after benznidazole treatment has been described in chronic Chagas disease patients [10, 11]. Previous laboratory studies have also shown that treatment leads to an improvement in the quality of the antigen-specific responses of CD8+ T cells in IND chronic Chagas disease patients, evidenced by a higher percentage of CD8+ T cells that exhibit a multifunctional profile (IFN-γ+IL2+Perforin+Granzyme B+), an increase in the frequency of central and terminal effector memory CD8+ T cells and a decrease in the co-expression of inhibitory receptors [12]. These CD8+ T cells are essential for the control of parasite, and it has been reported that the persistent presence of the parasite can lead to a proportion of the CD8+ T cell population to become exhausted, which in turn might favour disease persistence [13, 14]. However, the main limitation for assessing treatment efficacy in the chronic phase of Chagas disease is the lack of a marker that allows to define treatment success and cure. Currently, the gold standard for evaluating treatment efficacy is based on seroconversion, which may take years or decades to be detected precluding its use in clinical trials [15, 16]. In this regard, a global effort is being made to identify useful markers that allow the evaluation of short-term treatment efficacy. Some biomarkers have been shown to be potentially useful for assessing the response to antiparasitic treatment in Chagas disease patients. These include, among others, markers for detecting specific antibodies against host antigens, markers for detecting antibodies generated against parasite antigens, markers for measuring the expression patterns of certain cytokines and markers for quantification of the response of particular cellular immune populations [6, 17-20]. In this context, previous laboratory studies have shown

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that KMP11, PFR2, HSP70 and 3973 T. cruzi antigens are recognized with high specificity and sensitivity by sera from patients at the chronic stage of the disease. It was observed that the reactivity against these antigens decreases in the short term after treatment (6 to 9 months). Furthermore, this drop in reactivity continued during the post-treatment follow-up period (24 to 48 months) [18, 21]. The evaluation of the reactivity dynamics of Chagas disease patient sera against these four antigens, used as a set of biomarkers, allowed us to establish a standard criteria of therapeutic efficacy (STEC) which may be associated with a drastic decline in the parasite load induced by the drug and could thus be connected with therapeutic efficacy [22, 23]. These criteria implied a continuous decrease in the reactivity of patient sera against the four antigens together with a significant drop in reactivity for at least two of the antigens after treatment [22]. Interestingly, the multifunctional and cytotoxic capacity of antigenspecific CD8+ T cells from patients who met the therapeutic efficacy criteria improved after treatment [22]. This is particularly relevant because the establishment of an anti-T. cruzi CD8+ immune response focused on the parasite’s immunodominant epitopes is crucial for parasitemia control and for the course of infection [24, 25]. Many of the epitopes capable of inducing a strong specific response are restricted to the HLAA*0201 allele, and approximately 50% of humans express MHC class I alleles that belong to the HLA-A2 supertype and share similar binding profiles [26]. In this regard, in our laboratory, 14 new HLA*A2:01-restricted epitopes were identified that were derived from the kinetoplastid membrane protein-11 kDa (K1) [27, 28], heat shock protein-70 (HSP70210-218, HSP70255-63, HSP70316-24 and HSP70345-53) [29], paraflagellar rod proteins (PFR219-28, PFR2156-163, PFR2449-457, PFR3428-436, PFR3475-482 and PFR3481489) [30] and TcCA-2 (TcCA-2273-281, TcCA-2442-451 and TcCA-2607-615) [31]. These peptides are recognized by CD8+ T cells from Chagas disease patients and induce the secretion of pro-inflammatory cytokines (IFN-γ and TNF-α) and cytotoxic molecules (Granzyme B) [29-31]. Characterization of the phenotype of CD8+ T cells specific to two of the epitopes contained in TcCA-2 (TcCA-2442-451, TcCA-2607-615) revealed a differential profile in cells from IND compared to chronic Chagas patients with cardiac symptomatology (CCC). Thus, IND Chagas patients presented a significantly higher percentage of TcCA-2442-451 and TcCA-2607-615 epitope-specific CD8+ T cells with a TNAIVE phenotype than patients in the cardiac phase, where an effector memory (TEM) or a terminally differentiated effector CD8+ T cell (TEMRA) phenotype was predominant. In addition, a significantly higher percentage of antigen-specific CD8+ T cells with senescent features was observed in the CCC patients [31]. Considering that the phenotypic and functional profile of antigen-specific CD8+ T-cells could be a potential early indicator of treatment effects, even in cases where cure is not achieved, the aim of this study was to evaluate the functional capacity and phenotype of antigen-experienced CD8+ T cells specific to the most immunogenic peptides in the PFR2 (PFR2449-457), PFR3 (PFR3428-436), TcCA-2 (TcCA-2442-451, TcCA2607-615) and KMP11 (K1) T. cruzi proteins in IND Chagas disease patients before and after treatment. The relationship between this cellular response and the humoral response against this set of biomarkers was also assessed in treated patients, taking into account the established criteria of therapeutic efficacy.

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2. Materials and Methods

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2.1. Study populations Twenty HLA-A*02:01 adult chronic Chagas disease patients originally from Bolivia and residents of Spain were included in this study. The patients were recruited, diagnosed and clinically evaluated at the Virgen de la Arrixaca Hospital in Murcia, Spain. Following WHO criteria [32], Chagas disease diagnosis was carried out using two conventional serological tests, an ELISA (Bioelisa Chagas Biokit, Spain) and an indirect immunofluorescence assay (Inmunofluor Chagas, Biocientífica, Argentina). All patients enrolled in this study were characterized as indeterminate (IND) patients due to the absence of clinical signs or symptoms of the disease and received 5 mg/kg/day of benznidazole for 60 days as treatment. HLA-A genotyping was carried out using the RELITM SSO HLA-A Typing kit (Invitrogen, California, USA), and the HLA-A*02:01 patients were randomly selected among the diagnosed IND Chagas disease patients. Peripheral blood mononuclear cells (PBMCs) were obtained from thirtymilliliter blood samples collected in EDTA-containing tubes both before and after treatment (from 11 to 28 months). PBMCs were purified by Ficoll density gradient centrifugation and cryopreserved into inactivated foetal bovine serum (iFBS) with 10% dimethyl sulfoxide in liquid nitrogen until use. In addition, serum samples from the IND patients included in the study were obtained from peripheral blood collected before and at nine and twenty-four months after treatment. The patients were included in the experimental trials according to the availability of PBMCs isolated from each subject. Thus, for phenotypic characterization assays, 12 (PFR3428-436, TcCA-2442-451 and TcCA-2607-615) and 13 patients (PFR2449-457, and K1) were evaluated. Analyses of the cytokine profiles against each antigen were carried out in 13 (K1), 12 (PFR2449-457), 10 (TcCA-2442-451) and 9 (PFR3428-436 and TcCA-2607-615) patients. The ability to secrete cytotoxic molecules (Granzyme B) was evaluated in 9 (PFR2449-457, and K1), 8 (TcCA-2442-451) and 7 (PFR3428-436 and TcCA-2607-615) patients. 2.2. Ethical considerations Signed informed consent was obtained from all individuals before their inclusion in the study. All the protocols were approved by the Ethics Committees of the Consejo Superior de Investigaciones Científicas (no. 094/2016) and of the Hospital Virgen de la Arrixaca (no. MTR-05/2016). 2.3. Synthetic peptides Previous laboratory studies have described the recognition of HLA-A*02:01restricted epitopes in the PFRs, TcCA-2 and KMP11 T. cruzi antigens by PBMCs from patients with Chagas disease. Therefore, this study was based on the immunological responses associated with PFR2449-457, PFR3428-436, TcCA-2442-451, TcCA-2607-615 and K1 peptides located in the above-mentioned antigens. The peptides bearing the HLAA*02:01 binding motifs were synthesized by simultaneous multiple-peptide solid-phase methods and were assembled and verified as previously described [31]. Once

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Journal Pre-proof synthesized, the peptides were dissolved to a 1 mM final concentration in water containing 10% DMSO and stored at −20ºC.

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2.4. CD8+ T cell peptide-specific phenotypic characterization Antigen-specific CD8+ T cells were characterized using HLA-A*02:01 allophycocyanin (APC)- and phycoerythrin (PE)-labelled dextramers loaded with PFR2449-457 (PE), PFR3428-436 (PE), K1 (PE), TcCA-2442-451 (APC) and TcCA-2607-615 (APC) peptides (Immudex, Copenhagen, Denmark). A total of 1x106 PBMCs were incubated with 10 µL of each dextramer in 40 µL of 5% iFBS in phosphate buffered saline (PBS) for 10 min at RT in the dark. After incubation, the PBMCs were stained with different cocktails of surface antibodies purchased from BD PharmingenTM (California, USA): anti-CD8-V500 (clone RPA-T8), anti-CD8-PerCP-Cy5.5 (clone RPA-T8), anti-CD45RA-APC-H7 (clone HI100), anti-CCR7-V450 (clone 150503), anti-CD27-FITC (clone M-T271), anti-CD127-PerCP-Cy5 (clone HIL-7R-M21), antiCD57-FITC (clone NK-1) and anti-CD44RA-APC-H7 (clone G44-26) for 20 min at 4 ºC. The labelled cells were washed twice with PBS-5% iFBS and resuspended in 400 μL of 1X PBS. At least 100,000 PBMCs were acquired according to FSC (Forware Scatter)/SSC (Side SCatter) parameters in a FACSAria III flow cytometer (BD Biosciences, California, USA). The data were analysed using Flowjo 7.6.5 software (Tree Star, Ashland, USA). Positive staining for each marker was determined using fluorescence minus one (FMO) and isotype staining controls. To determine the cut-off point for the peptide-MHC dextramer, both the negative control (unstained cells) and PBMCs stained with all the antibodies to be evaluated except the corresponding dextramer were used as a reference. The gating strategies used in the flow cytometry analysis were described previously [31].

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2.5. Detection of specific IgG antibodies against T. cruzi antigens ELISA assays were performed in triplicate at different dilutions as previously described [18]. Plates were coated with 0.5 µg of each antigen diluted in carbonate buffer (pH 9.6) for the recombinant proteins and in phosphate buffer (pH 7.4) for 3973d and stored in a dry atmosphere at -20°C until use. Positive and negative serum controls were included in all plates. Sera were assayed at dilutions ranging from 1:200 to 1:800 for PFR2 and HSP70, 1:100 to 1:400 for KMP11 and 1:100 to 1:3200 for 3973d before treatment. After treatment, each patient sera was analysed at two dilutions, which were selected based on the reactivity observed at T0. The optical density (O.D.) values of the chosen dilutions had to be between 0.45 and 2, which allowed us to evaluate a decrease or increase in the reactivity as a consequence of the treatment. 2.6. Evaluation of functional cellular responses The frequency of Granzyme B (GzB)-producing cells was evaluated by ELISPOT assays using cryopreserved PBMCs from IND Chagas disease patients as described before [29]. Briefly, plates were coated with 0.5 μg/well anti-GzB monoclonal antibody (Mabtech, Cincinnati, USA) and incubated overnight at 4 °C. Afterwards, the plates were washed and incubated with 200 μL/well blocking solution

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(RPMI-1640 with 10% iFBS) at room temperature for 30 minutes. Then, 5 × 104 PBMCs/well were added and incubated with 1 μM of each peptide for 30 h at 37 °C with 5% CO2. Both negative and positive controls were included for each patient. PBMCs were incubated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, SigmaAldrich, St Louis, USA) and 500 ng/mL ionomycin (Sigma-Aldrich, St Louis, USA), or 10 μg/mL phytohemagglutinin (PHA, Sigma) (positive control), or culture medium (negative control or basal response). Following incubation, 0.1 μg/well biotinylated GzB-specific antibody (Mabtech, Cincinnati, USA) diluted in PBS with 0.5% iFBS was added and incubated for 2 h at room temperature. After extensive washing with PBS, the wells were incubated with 100 μL/well streptavidin-alkaline phosphatase (SigmaAldrich, St Louis, USA) at a dilution of 1:6000 for 1 h at room temperature. Then, 50 μL/well substrate solution (NBT-BCIP substrate; Sigma-Aldrich, St Louis, USA) was added and incubated for 20–30 min at room temperature in the dark. The reaction was stopped by rinsing the plates with cold tap water. The spots were visualized using an AXIO PLAN 2 Imagine microscope and quantified using KS ELISPOT software. The results are expressed as the number of peptide-induced spot-forming cells (SFC) x 106 PBMCs after subtracting the number of spots of peptide un-stimulated PBMCs (basal response). The secretion of IL-6, IFN-γ, and TNF-α cytokines was determined in the supernatants of PBMCs from IND Chagas disease patients after in vitro stimulation with 1 μM of each peptide for 30 h at 37 °C in RPMI supplemented with 10% iFBS. As a positive control, PBMCs from IND Chagas disease patients were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin (SigmaAldrich, St Louis, USA). The concentration of each cytokine after stimulation was determined by using a bead-based multiplex immunoassay system (Bio-Plex; Bio-Rad Laboratories, California, USA) following the manufacturer's instructions and quantified by using Bio-Plex Manager software 4.1 (Bio-Rad Laboratories, California, USA). The basal level (un-stimulated PBMCs) for each of the cytokines was determined for each patient and was subtracted from the samples stimulated with each peptide. 2.7. Statistical analysis To determine whether the differences observed among groups were statistically significant, Mann-Whitney U, paired T-test, RM one-way ANOVA, Wilcoxon and Kruskal–Wallis with Dunn correction tests were applied according to the type of comparison using GraphPad Prism® v6.0 (GraphPad Software Inc., California, USA). Statistical significance was assigned a p-value ≤ 0.05.

3. Results 3.1. Phenotypic characterization of epitope-specific cytotoxic CD8+ T lymphocytes before and after treatment with benznidazole

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Journal Pre-proof The aim of the study was to determine the presence and phenotype of activated CD8 T cells specific to PFRs-, TcCA-2- and KMP11-derived previously described epitopes [29-31] and to evaluate whether benznidazole induced any phenotypic modifications in these cells. PBMCs from IND Chagas disease patients were first incubated with HLA-A*02:01 labelled dextramers loaded with the potentially most immunogenic epitopes in the antigens PFR2 (PFR2449-457), PFR3 (PFR3428-436), TcCA-2 (TcCA-2442-451, TcCA-2607-615) and KMP11 (K1) of T. cruzi and then with antibodies against the surface molecules CD8, CD45RA, CD27, CCR7, CD127, CD44 and CD57, as previously described [31]. This strategy allowed us to define the following populations within epitope-specific CD8+ T cells: terminal effector memory RA+ (TEMRA, CD45RA+CD27-CCR7-), effector memory (TEM, CD45RA-CD27-CCR7-), central memory (TCM, CD45RA-CD27+CCR7+), early differentiation stage (TED, CD45RA-CD127+), advanced differentiation stage (TTD, CD8+CD45RA+CD127-), senescent memory (CD44+CD57+) and non-senescent memory (CD44+CD57-), which were analysed by flow cytometry in each IND patient. The obtained results showed that all patients had antigen-specific CD8+ T cells specific for PFR2449-457 (ranging from 0.68 to 1.85%, median 1.13%), PFR3428-436 (ranging from 0.91 to 2.62%, median 1.19%), TcCA-2442-451 (ranging from 0.86 to 2.22%, median 1.40%), TcCA-2607-615 (ranging from 1.02 to 2.31%, median 1.33%) and K1 (ranging from 0.48 to 2.75%, median 1.15%) (Fig. 1, T0 in A to C). Analysis of the specific CD8+ T cell populations after benznidazole treatment showed a slight decrease in the percentage of CD8+ T cells specific for PFR2449-457 (ranging from 0.71 to 2.12%, median 1.07%), PFR3428-436 (ranging from 0.60 to 2.26%, median 0.95%), TcCA-2442451 (ranging from 0.67 to 2.92%, median 1.27%) and K1 (ranging from 0.54 to 1.31%, median 0.93%) (Fig. 1A1-2, B1 and C1-2, Post-Tt). Likewise, a slight increase in the percentage of CD8+ T cells specific for TcCA-2607-615 (ranging from 0.87 to 2.53%, median 1.44%) was observed (Fig. 1B2, Post-Tt).

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Fig. 1. Percentage of peptide-specific cells from total CD8+ T cells. Cell subpopulations were determined by multi-parametric flow cytometry. The results correspond to the percentage of cells specific to each peptide for 13 (PFR2449-457 (A1)), 12 (PFR3428-436 (A2)), 12 (TcCA-2442-451 (B1)), 12 (TcCA-2607-615 (B2)) and 13 (K1 (C1)) IND Chagas disease patients. T0 and Post-Tt correspond to measurements made before and after treatment with benznidazole, respectively. Median values are represented by horizontal lines.

Analysis of the phenotype of the CD8+ T cells specific for PFR2449-457, PFR3428+ 436, TcCA-2442-451, TcCA-2607-615 and K1 revealed a different memory CD8 T cell pattern depending on the antigen under study. Thus, the PFR2449-457-specific CD8+ T cells had a higher proportion of cells with an effector memory phenotype (TEMRA and TEM) than a central memory (TCM) phenotype (Fig. 2A1). Conversely, the percentage of TCM cells was higher than that of TEMRA and TEM in PFR3428-436-specific CD8+ T cells (Fig. 2B1). In the case of TcCA-2 (TcCA-2442-451 and TcCA-2607-615) antigen-specific T cells, a higher percentage of cells expressing the TEMRA phenotype was detected (Fig. 2C1 and D1). However, a similar proportion of effector (TEMRA and TEM) and central memory K1-specific CD8+ T cells were identified (Fig. 2E1). When the phenotypic profiles of PBMCs from treated patients were evaluated, drug-induced changes were

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observed in the proportion of antigen-specific CD8+ T cells that expressed an effector or central memory phenotype. The changes in PFR-specific CD8+ T cells (PFR2449-457 and PFR3428-436) were evidenced by the following observations: a) a decrease in the percentage of effector memory CD8+ T cells (statistically significant for PFR2449-457, ρ<0.05) and b) a non-significant increase in central memory CD8+ T cells for both peptides (Fig. 2A1 and B1). In addition, in the case of PFR3428-436-specific CD8+ T cells, a slight increase in the percentage of TEMRA was observed after treatment (Fig. 2B1). In the case of TcCA-2442-451-specific CD8+ T cells, the drug induced an increase (p<0.05) in the percentage of the effector memory subpopulations (TEM), while in TcCA-2607-615specific CD8+ T cells a decrease in the percentage of TEM and central (TCM) memory subpopulations was observed (Fig. 2C1 and D1). The percentage of cells expressing a TEMRA phenotype was higher after benznidazole treatment in the TcCA-2607-615-specific CD8+ T cells (Fig. 2D1). When the phenotype of K1-specific CD8+ T cells was evaluated after treatment, a drop in the percentage of effector memory T cells (TEMRA and TEM) was detected (Fig. 2E1). The degree of cellular differentiation of the CD8+ T cells specific to all the peptides under study was analysed using the combination of CD45RA and CD127 surface markers. The results showed that for all the peptides, there was a statistically significant higher percentage of antigen–specific CD8+ T cells at an advanced stage of differentiation (TTD, CD45RA+CD127-) than at an early stage of differentiation (TED, CD45RA-CD127+) (p≤0.0001) (Fig. 2A2 to D2) both before and after treatment. Moreover, in the case of TcCA-2607-615-specific CD8+ T cells, a statistically significant decrease in the percentage of TED and, consequently, an increase in that of TTD (p≤0.05) after treatment was observed (p≤0.05) (Fig. 2D2). Analysis of the proportion of antigenspecific CD8+ T cells expressing CD127 after treatment differed depending on the assayed peptide. Thus, the proportion of cells expressing CD127 after 24 months of treatment was lower in CD8+ T cells specific for PFR2449-457, PFR3428-436, TcCA-2442-451 and TcCA-2607-615 (p≤0.05) than in cells obtained before treatment (Fig. 2A2, 2B2, C2 and D2) and higher for K1-specific cells (Fig. 2E2). Analysis of the CD57 and CD44 surface markers, which are associated with clonal senescence [33] and antigen experience [34], resulted in the detection of a significantly higher percentage of senescent (CD44+CD57+) CD8+ T cells specific for PFR2449-457 (p≤0.05), PFR3428-436 and K1 (p≤0.01) epitopes versus non-senescent memory cells (CD44+CD57-) (Fig. 2A3, B3 and E3). However, after treatment, a significant increase in the percentage of TcCA-2442-451- and K1-specific CD8+ T cells with a senescent phenotype (CD44+CD57+) (p≤0.05 and p≤0.01, respectively) was observed. Conversely, a predominant phenotype of non-senescent TcCA-2442-451- and TcCA-2607-615-specific CD8+ memory cells (p≤0.0001 and p≤0.01, respectively) (Fig. 2C3, and D3) was observed before and after treatment.

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Fig. 2. Phenotypic characterization of antigen-specific CD8+ T cells. Peptidespecific CD8+ T cells were divided into different subpopulations according to the combination of antibodies used. The panels on the left (A1 to E1) represent the memory subpopulations TEMRA (CD8+CD45RA+CD27-CCR7-), TEM (CD8+CD45RA-CD27-

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Journal Pre-proof CCR7-) and TCM (CD8+CD45RA-CD27+CCR7+). The centre panels (A2 to E2) show the differentiation status of the TED (CD8+CD45RA-CD127+) and TTD + + + (CD8 CD45RA CD127 ) cells and the antigen-independent CD127 cells. The right panels (A3 to E3) represent the expression of the senescence marker CD57 in antigenexperienced CD8+ T cells. These results correspond to 13 (PFR2449-457 (A)), 12 (PFR3428-436 (B)), 12 (TcCA-2442-451 (C)), 12 (TcCA-2607-615 (D)) and 13 (K1 (E)) IND Chagas disease patients. T0 and Post-Tt correspond to measurements made before and after treatment with benznidazole, respectively. Median values are represented by horizontal lines. Significant differences are indicated (* p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001).

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3.2. Recognition of KMP11, HSP70, PFR2, and 3973d by sera from chronic Chagas disease patients treated with benznidazole As previously reported, the T. cruzi KMP11, PFR2, HSP70, and 3973 antigens were recognized with high specificity and sensitivity by sera from chronic Chagas disease patients [18, 21]. It was also shown that reactivity against these antigens decreased substantially early after treatment with benznidazole [18, 21]. Studying the dynamics of the reactivity against these 4 antigens allowed us to suggest criteria for monitoring the impact of the treatment and to design bioinformatics software accordingly. The reactivity against the aforementioned antigens was evaluated in 20 Bolivian asymptomatic chronic Chagas disease patients living in Spain before (T0) and after 9 (T9) and 24 (T24) months of benznidazole treatment. As observed by ELISA, all patients presented high IgG levels against the four antigens before benznidazole treatment (Fig. 3). When the reactivity against the four biomarkers (BMKs) was analysed after treatment, a continuous decrease in the antibody level was detected over time. A substantial drop in reactivity was observed at 9 (p<0.01 for the four antigens) and 24 (p<0.0001 for PFR2, p<0.01 for 3973d, p<0.001 for KMP11 and p<0.05 for HSP70) months post treatment (Fig. 3A). Next, we analysed whether the drug-induced modifications in the reactivity against the four antigens met the previously proposed therapeutic efficacy criteria (STEC) [22]. The results showed that 7 out of 20 (35%) IND Chagas disease patients met the STEC (Fig. 3B). That is, after treatment, they presented a continuous drop in the reactivity against each of the four biomarkers (T9
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Fig. 3. Reactivity of sera from asymptomatic (IND) Chagas disease patients against T. cruzi antigens before and after benznidazole treatment. A) The levels of antibodies (IgG) against PFR2, 3973d, KMP11 and HSP70 were measured by ELISA in sera from 20 Bolivian indeterminate (IND) patients before benznidazole treatment (T0) and at 9 (T9) and 24 (T24) months after treatment. The sera were always tested in triplicate and at 1/100, 1/200, 1/400, 1/800, 1/1600 and 1/3200 dilutions for each of the four antigens at T0. For post-treatment follow up, the selection of the dilution was based on the reactivity observed at T0 and at which a decrease or an increase in the reactivity could be observed as a consequence of the impact of the treatment [optical density (O.D.) values were between 2 and 0.45]. Thus, each of the 20 serum samples from the aforementioned patients was tested at the following dilutions against each antigen: 1/100 (2 patients tested for 3973d and 2 for KMP11), 1/200 (9 patients tested for PFR2, 2 for KMP11 and 5 for HSP70), 1/400 (3 patients tested for 3973d, 6 for KMP11 and 4 for HSP70), 1/800 (1 patient tested for PFR2, 1 for 3973d and for HSP70), 1/1600 (3 patients tested for 3973d) and 1/3200 (1 patient tested for 3973d). Data in both graphs were expressed as the O.D., measured at 492 nm. The whiskers of the box-plot diagrams represent the percentiles from 10th to 90th. Outliers are represented as dots. The p values were obtained using the Wilcoxon matched pair test, and significant differences are indicated (* p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001). B) Chronic Chagas disease patients who met the therapeutic efficacy criteria following the

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3.3. Functional capacity of antigen-specific CD8+ T lymphocytes from chronic Chagas disease patients prior to treatment The functional capacity of antigen-specific CD8+ T cells was analysed in an indeterminate chronic Chagas disease patients previously treated with benznidazole. Patients were separated into two groups taking into consideration their humoral response against the four antigens used as biomarkers after treatment and if they met STEC or did not. Consequently, the cytokine secretion profile (IL-6, IFN-γ and TNF-α) and Granzyme B (GzB) production were determined in PBMCs from patients following stimulation with each one of the tested peptides. The results in Fig. 4 showed that following stimulation with PFR2449-457, PFR3428-436, TcCA-2442-451, TcCA-2607-615 and K1 peptides, the CD8+ T cells from patients who met STEC (TE) when they were subsequently treated presented a higher level of secretion of pro-inflammatory cytokines such as IFN-γ and TNFα compared to CD8+ T cells from patients who did not meet STEC (TF) (Fig. 4 A1 to E1). Next, the cytotoxic activity of the CD8+ T cells specific to the five epitopes was analysed. Secretion of GzB was evaluated by ELISPOT assays in untreated patients who will be subsequently treated and will meet the STEC and those who will not. As shown in Fig. 4, following stimulation with the PFR3428-436, TcCA-2442-451 and K1 peptides located in PFRs, TcCA-2 and KMP11 proteins, respectively, a greater number of GzB-secreting cells was observed in the untreated patients who will meet STEC after treatment versus those who will not (Fig. 4 B2, C2 and E2).

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Fig. 4. Functional profile of antigen-specific CD8+ T cells from untreated Chagas disease patients associated with the therapeutic effect. The cytokine secretion (IL-6, IFN-γ and TNF-α) and Granzyme B response to T. cruzi epitopes by PBMCs from HLA-A*02:01 IND patients were analysed. The results correspond to 9 (PFR2449-457 (A), PFR3428-436 (B) and K1(E) antigens) and 8 (TcCA-2442-451 (C) and TcCA-2607-615 (E) antigens) patients who did not meet the therapeutic efficacy criteria (TF) and 4 (PFR2449-457 (A), TcCA-2442-451 (C), TcCA-2607-615 (D) and K1 (E)) and 3 PFR3428-436 (B) patients who met the therapeutic efficacy criteria (TE). Mean values are represented by horizontal lines.

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3.4. The phenotype of the antigen-specific CD8+ T cells from patients who met the STEC differs from that of patients who did not To analyse whether the differential percentage and phenotype of the antigenspecific CD8+ T cells observed in the Chagas chronic patients before and after treatment could be related to a particular response to the treatment, analyses were performed that took into consideration the reactivity of the patient sera against the 4 molecules and if they met the STEC or not 24 months after treatment. The results represented in Fig. 5 and Fig. 6 correspond to 9 (PFR2449-457, PFR3428-436 and K1 antigens) and 8 (TcCA-2442451 and TcCA-2607-615) patients who did not meet the therapeutic efficacy criteria (TF) and 4 (PFR2449-457, TcCA-2442-451, TcCA-2607-615 and K1) and 3 (PFR3428-436) patients who met the therapeutic efficacy criteria (TE). The obtained results showed that before treatment, the patients who met the STEC had a higher percentage of CD8+ T cells specific for TcCA-2442-451, TcCA-2607-615 and K1 than the patients who did not meet the STEC (Fig. 5 C1, D1 and E1), with statistical significance in the case of TcCA-2607-615-specific CD8+ T cells (p≤0.05) (Fig. 5 D1). After treatment, a higher percentage of CD8+ T cells specific to TcCA-2442-451, TcCA-2607-615 and K1 was also detected in patients who met STEC versus those who did not (Fig. 5 C1, D1 and E1). Analysis of the phenotype of antigen-specific CD8+ T cells indicated that the percentage of PFRs (PFR2449-457 and PFR3428-436) and TcCA-2 (TcCA2442-451, TcCA-2607-615) antigen-specific CD8+ T cells with a terminal effector memory phenotype (TEMRA) was reduced following treatment in those patients who met the STEC (Fig. 5 A2 to D2). However, before treatment, the frequency of CD8+ TEM cells specific for PFR2449-457 and TcCA-2607-615 was lower in patients who did not meet the STEC. In contrast, the percentage of CD8+ T cells specific for PFR3428-436, TcCA-2442451 and K1 with a TEM phenotype was higher in treated patients who met the STEC (Fig. 5 B2, C2 and E2). Before treatment, the percentage of CD8+ T lymphocytes specific for PFR3428436, TcCA-2442-451, TcCA-2607-615 and K1 with a TCM phenotype was lower in patients who met STEC than in those who did not and in most patients the percentage was decreased after treatment (Fig. 5 C2, D2 and E2). Before treatment, the percentage of PFR2449-457- (p≤0.05), PFR3428-436- and K1-specific CD8+ T lymphocytes expressing IL7 receptor α (CD127) was higher in patients who met the STEC (Fig. 5 A2, B2 and E2). In contrast, the percentage of TcCA-2607-615-specific CD8+ T cells expressing CD127 before treatment was lower in patients who met the STEC (p<0.01) compared with patients who did not (Fig. 5 D2). We also detected a lower frequency of CD8+ T cells specific for PFR2449-457, PFR3428-436, and K1 with an advanced differentiation stage memory phenotype in patients who met the STEC before and after treatment than in those who did not (Fig. 6 A1, B1, and E1). Interestingly, the patients who met the STEC presented a considerably lower percentage of CD8+ T cells specific for all the tested peptides (PFR2449-457, PFR3428-436, TcCA-2442-451, TcCA-2607-615 and K1) with senescent features (CD44+CD57+) (Fig. 6 A2 to E2). This lower percentage of antigen-specific CD8+ T cells was observed both before and after benznidazole treatment and was statistically significant before treatment for the T cells specific for TcCA-2607-615 and

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after treatment in the CD8+ T cells specific for PFR3428-436 (p≤0.05), TcCA-2442-451 (p≤0.05), TcCA-2607-615 (p≤0.05) and K1 (p≤0.05) (Fig. 6 B2 to E2).

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Fig. 5. Phenotypic characterization of antigen-specific CD8+ T cells from Chagas disease patients before and after benznidazole treatment (from 11 to 28 months) associated with the therapeutic effect. Peptide-specific CD8+ T cells were classified in different memory subpopulations according to the combination of antibodies used. The left panels (A1 to E1) show the peptide-specific cells from total CD8+ T cells. The right panels (A2 to E2) represent the following memory subpopulations: TEMRA (CD8+CD45RA+CD27-CCR7-), TEM (CD8+CD45RA-CD27CCR7-) and TCM (CD8+CD45RA-CD27+CCR7+). The results correspond to 9 (PFR2449457 (A), PFR3428-436 (B) and K1(E) antigens) and 8 (TcCA-2442-451 (C) and TcCA-2607-615 (E)) patients who did not meet the therapeutic efficacy criteria (TF) and 4 (PFR2449-457 (A), TcCA-2442-451 (C), TcCA-2607-615 (D) and K1 (E)) and 3 (PFR3428-436 (B)) patients who met the therapeutic efficacy criteria (TE). T0 and Post-Tt correspond to measurements made prior and after treatment with benznidazole, respectively. Median values are represented by horizontal lines. The whiskers of the box-plot diagrams represent the percentiles from 5th to 95th. Significant differences are indicated (* p<0.05 and ** p<0.01).

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Fig. 6. Differentiation and expression of senescence makers on antigenspecific CD8+ T cells from Chagas disease patients before and after benznidazole treatment (from 11 to 28 months) associated with the therapeutic effect. CD8 peptide-specific cells were classified according to the combination of antibodies used. The left panels (A1 to E1) represent the differentiation status, namely, TED (CD8+CD45RA-CD127+), TTD (CD8+CD45RA+CD127-), and the antigen-specific CD8+ T cells expressing CD127+. The right panels (A2 to E2) show the expression of the senescence marker CD57 in antigen-experienced CD8+ T cells. The results correspond to 9 (PFR2449-457 (A), PFR3428-436 (B) and K1(E) antigens) and 8 (TcCA-2442-451 (C) and TcCA-2607-615 (E)) patients who did not meet the therapeutic efficacy criteria (TF) and 4 (PFR2449-457 (A), TcCA-2442-451 (C), TcCA-2607-615 (D) and K1 (E)) and 3 (PFR3428-436 (B)) patients who met the therapeutic efficacy criteria (TE). T0 and Post-Tt correspond to determinations made before and after treatment with benznidazole, respectively. Median values are represented by horizontal lines. The whiskers of the box-plot diagrams represent the percentiles from 5th to 95th. Significant differences are indicated (*p<0.05 and **p<0.01).

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4. Discussion

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One of the greatest challenges that still must be addressed in chronic Chagas disease research is the search for efficient tools that will enable the assessment of shortterm pharmacological treatment efficacy. Conversion to negative serology accompanied by a favourable clinical course after treatment is currently considered an indication of parasitological cure. However, seronegativization can take more than 10 years [36]. This makes the monitoring reactivity with conventional serological tests an inadequate evaluation criterion [6, 36]. The reactivity of patient sera was evaluated before and at 9 and 24 months after treatment, against the previously described biomarker set [22] and a established criteria of therapeutic efficacy (STEC) applied [22]. The results indicated that at 24 months after treatment, 35% of patients met the STEC, as they showed a continuous decrease in the antibody level against the four biomarkers and a significant decrease in the reactivity against at least two of the biomarkers. The observed decrease in reactivity within a short period of time (up to 24 months) would be considered acceptable for the evaluation of anti-parasitic treatment according to the reported target product profile (TPP), [23]. Pharmacological treatments not only induce changes at the humoral level but also modulate the cellular immune response. Thus, an improvement in the quality of the antigen-specific CD8+ T cell response after benznidazole treatment has been observed [12]. The functional capacity and phenotype of the antigen-experienced CD8+ T cells specific for epitopes in the PFRs, KMP11 and TcCA-2 antigens were also evaluated, as these epitopes have been shown to be recognized, processed and presented in the context of a natural T. cruzi infection [29-31]. The phenotypic characterization of the antigen-specific CD8+ T cells was carried out before and after benznidazole treatment. Moreover, the data were analysed according to the STEC to determine if there was a

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relationship between the patients that met STEC and specific phenotypes of the antigenspecific CD8+ T cells. For this purpose, the pattern of antigen-specific memory T cells, effector (TEM) and central memory (TCM), were analysed before and after treatment. The results showed that there was a differential memory CD8+ T cell profile depending on the antigen that was being evaluated. The percentages of effector T cells (TEMRA and TEM) epitope-specific was higher than that detected by central memory T cells (TCM). A exception was observed by the K1-specific CD8+ T cells, in which the proportion of specific CD8+ T cells with TEMRA, TEM and TCM phenotypes was similar, and of the PFR3-specific cells, in which there was a higher percentage of TCM antigen-specific CD8+ T cells. The percentage of the TEMRA cells was particularly elevated in the CD8+ T cells specific for TcCA-2442-451 and TcCA-2607-615. Variations in the phenotype of the CD8+ T cells specific to the peptides under study were also observed after treatment. Benznidazole treatment had an effect on the proportion of both TEMRA and TEM effector memory cells, and a significant decrease was observed in the PFR2449-457specific CD8+ T cell population with a TEM phenotype. Moreover, a decrease in the percentage of TEM was also observed following treatment in the CD8+ T cells specific for PFR3428-436, TcCA2607-615 and K1. In PFR-specific T cells, treatment induced a decrease in the percentage of TEMRA antigen-specific CD8+ T cells and an increase in central memory CD8+ T cells. In contrast, the percentage of TcCA-2607-615-specific CD8+ T cells expressing a TEMRA phenotype was higher after treatment and the percentage of TEM and TCM subpopulations was lower. Likewise, the K1-specific CD8+ T cells showed a lower percentage of effector memory T cells (TEMRA and TEM) after treatment than before treatment.

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It has been reported that antigen-specific CD8+ T cells with an effector memory phenotype are maintained during persistent T. cruzi infection, and that CD8+ T cells with a central memory phenotype can be generated and maintained despite a persistent infection. In this regard, in murine models, complete pathogen clearance after benznidazole treatment induced the generation of a stable, parasite-specific CD8+ T cell population with phenotypic characteristics of central memory cells [38]. Additionally, it has been demonstrated that the differentiation and expansion of T. cruzi-specific CD8+ cytotoxic T cells (TEM) is dependent on parasite proliferation [39]. All these data, combined with the fact that treatment resulted in a decrease in the parasite antigens needed to induce the production of antigen-specific effector T cells [40], would explain the observed decrease in the percentage of antigen-specific CD8+ T cells with an effector memory phenotype (TEM) after benznidazole treatment. In addition, it has been reported that persistent antigen exposure during chronic infections (parasitic, viral or bacterial) leads to a progressive functional alteration in the CD8+ T lymphocyte response. This response is characterized by the loss of proliferative capacity and the inability to acquire the properties of antigen-independent memory T cells that are needed for survival in the absence of antigen [41, 42]. All of this implies a low expression of IL-7 (CD127), a marker for memory CD8+ T cell precursors capable of generating long-lived antigen-independent memory CD8+ T cells [43], and IL-15 (CD122) receptors as well as a decrease in the secretion of cytokines such as IL-2, TNF-

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α and IFN-γ [44, 45]. In this study, we observed that the treatment also influenced the proportion of antigen-specific CD8+ T cells expressing CD127, which was different depending on the peptide. In the CD8+ T cells specific for PFR2449-457, PFR3428-436, TcCA-2442-451 and TcCA-2607-615 (p≤0.05), the proportion of cells expressing CD127 was lower after treatment than before treatment. However, the percentage of K1specific T cells expressing this marker was higher following treatment. Our results also showed a higher percentage of antigen-specific CD8+ T cells with an advanced differentiation stage phenotype (TTD, CD45RA+CD127-) than those with an early differentiation stage phenotype (TED, CD45RA-CD127+), both before and after benznidazole treatment, with these differences being statistically significant. It has been reported that there is a higher prevalence of senescent T cells in highly differentiated memory T cell phenotypes (TEMRA). These senescent cells are characterized by the expression of the surface marker CD57 and have severely impaired proliferative capacity, suggesting that this molecule could be the best marker for replicative senescence [33]. In this regard, a significantly higher percentage of senescent CD8+ T cells specific for PFR2449-457 (p≤0.05), PFR3428-436 and K1 epitopes than non-senescent memory cells was detected, both before and after treatment, in contrast to the CD8+ T cells specific for TcCA-2442-451 and TcCA-2607-615 in which a predominant non-senescent phenotype memory cells was observed (p≤0.0001 and p≤0.01, respectively). In addition, after treatment, a considerable increase in the percentage of memory cells expressing CD57 was observed in CD8+ T cells specific for TcCA-2442-451 (p≤0.05), TcCA-2607-615 and K1 (p≤0.01).

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The cytokine secretion profiles of antigen-specific CD8+ T cells were analysed considering whether the patient did (+) or did not (-) meet the STEC, and differences in the functional profiles prior to benznidazole treatment were observed. Higher secretion of IFN-γ and TNF-α in response to each of the epitopes was observed in patients who met the STEC. The secretion of these cytokines has been previously correlated with improved protective CD8+-mediated immunity against T. cruzi infection [24, 46]. In addition, a higher cytotoxic capacity, determined by granzyme B secretion, was observed in the T cells stimulated with PFR2449-457, PFR3428-436, TcCA-2442-451, and K1 peptides. This finding is relevant given the crucial role played by CD8+ T cells to limit parasite expansion throughout the course of infection [47]. Furthermore, the patients who met the STEC showed a higher proportion of TcCA-2442-451- and TcCA-2607-615specific CD8+ T cells both before and after benznidazole treatment, which was statistically significant for the TcCA-2607-615-specific population. In these patients, there was a decrease in the percentage of PFR2449-457-, PFR3428-436-, TcCA-2442-451-, and TcCA-2607-615-specific CD8+ T cells with a terminal effector memory phenotype (TEMRA) after treatment. In patients who met the STEC, there was an increase in the frequency of CD8+ TEM cells both before (PFR3428-436-, TcCA-2442-451- and K1-specific CD8+ T cells) and after treatment (PFR2449-457-, PFR3428-436-, TcCA-2442-451- and K1specific CD8+ T cells) compared to those who did not meet the STEC. This phenotypic profile, observed even prior to the administration of the drug, could be conditioning the response to treatment given the importance of the effector response of CD8+ T cells. A

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Interestingly, it was also observed that patients who met the STEC presented: i) an increase in the percentage of cells expressing CD127 (antigen-independent memory cells) for PFR2449-457- (p≤0.05), PFR3428-436-, TcCA-2442-451- and K1-specific CD8+ T lymphocytes before treatment and PFR2449-457 (p≤0.05), TcCA-2442-451- and K1-specific CD8+ T cells after treatment. The maintenance of a CD8+ T cell population with capacity for antigen-independent survival, along with the recruitment of new effector cells from TCM and/or the naive T cell pool to maintain parasite control, suggests that low antigen levels preserve CD8+ T cell function while maintaining pathogen control during chronic infections [47]; ii) a decrease in the frequency of PFR2449-457- (p≤0.05), PFR3428-436-, and K1-specific CD8+ T cells with an advanced differentiation stage memory phenotype (TTD) before and after treatment; and iii) a considerably lower percentage of senescent CD8+ T cells (CD44+CD57+) specific for all peptides tested (PFR2449-457, PFR3428-436, TcCA-2442-451, TcCA-2607-615 (p≤0.05) and K1) before benznidazole treatment. After treatment, the population of senescent CD8+ T cells (CD44+CD57+) also showed an important decrease in the CD8+ T cells specific for PFR3428-436 (p≤0.05), TcCA-2442-451 (p≤0.05), TcCA-2607-615 (p≤0.05) and K1 (p≤0.05) in those patients who met the STEC compared to those who did not. Conversely, prior to treatment of patients who did not meet the STEC, the proportion of senescent cells (CD44+CD57+) specific for all the peptides tested (PFR2449-457, PFR3428-436, TcCA-2442451, TcCA-2607-615 and K1) was increased. This senescent cell profile may be associated with a lower functional response capacity, a higher percentage of late-differentiated cells, a lower percentage of effector cells and a lower percentage of antigen-independent cells. This, in turn, could be the cause of not generating an adequate response to treatment. The differential phenotype pattern observed in the patients who meet the STEC could be associated with a drastic reduction in parasite load, and the immunological status of the host might be a determining factor for successful treatment. Thus, the quality and profile of the T-cell response might be a key element not only for disease progression but also for the monitoring and early prediction of the response to anti-parasitic treatment. The observed functional and phenotypic modifications in CD8+ T cells are antigen-specific, which affirms the importance and usefulness of combining antigens that allow a broader spectrum of study. We conclude that the obtained results support the case for using and combining serological and cellular markers as tools that allow monitoring and predicting the treatment impact in Chagas disease patients in a short time. 5. Conclusions The obtained results allow us to elucidate that the memory, differentiation and senescence phenotype of epitope-specific cytotoxic CD8+ T cells from chronically T. cruzi-infected subjects is modulated by benznidazole treatment as well as

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associated with a significant decrease in the level of T. cruzi-specific antibodies. Interestingly, it has been observed that patients who met the previously reported criteria of therapeutic efficacy by the use of a set of serological BMKs showed a differential phenotypic profile with respect to patients who did not meet the criteria. The patients who met the criteria had a higher percentage of antigen-independent cells and a higher frequency of effector memory antigen-specific CD8+ T cells. This finding may be relevant because these cells have the capacity to reside for a long time in inflamed peripheral tissues, allowing rapid control of the infection. Furthermore, the patients who met the therapeutic success presented a lower percentage of terminally differentiated effector CD8+ T cells (TEMRA), which have been associated with severe disease. In addition, these patients also presented a lower percentage of antigen-specific CD8+ T cells with an advanced differentiation stage memory phenotype and a substantially lower percentage of epitope-specific CD8+ T cells expressing senescence markers even before treatment. In summary, we estimated that the host immunological status could be a determining factor for successful treatment and that the phenotypic and functional characterization of antigen-specific CD8+ T cells (before and after benznidazole treatment) might be a useful tool for monitoring and early predicting the response to anti-parasitic treatment.

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Acknowledgements We thank the patients for their participation in this study. We also thank Dr. Bartolomé Carrilero (Hospital Virgen de la Arrixaca, Murcia) for the recruitment, medical examination and characterization of Chagas disease patients.

References

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Funding This work was supported by grants SAF2016-81003-R, SAF2016-80998-R from the Programa Estatal I+D+i (MINECO); the Network of Tropical Diseases Research RICET (RD16/0027/0005 and RD16/0027/0016) and FEDER.

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Highlights: - Benznidazole treatment induces changes in the humoral and cellular responses - Modulation of the CD8 T cell phenotype could be associated with successful treatment

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- Higher proportion of cytotoxic T CD8 could be associated with successful treatment

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