CD8+ T cell response quality is related to parasite control in an animal model of single and mixed chronic Trypanosoma cruzi infections.

CD8 + T Cell Response Quality Is Related to Parasite Control in an Animal Model of Single and Mixed Chronic Trypanosoma cruzi Infections Jose Mateus 1 *, Paola Nocua 1 , Paola Lasso 2 , Manuel Carlos Lo ´pez 3 , M. Carmen Thomas 3 , Adriana Egui 3 , Claudia Cuervo 1 , John Mario Gonza ´lez 4 , Concepcio ´n J. Puerta 1 and Adriana Cue ´llar 5 * 1 Grupo de Enfermedades Infecciosas, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota ´, Colombia, 2 Grupo de Inmunobiologı ´a y Biologı ´a Celular, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota ´, Colombia, 3 Instituto de Parasitologı ´a y Biomedicina Lo ´pez Neyra, Consejo Superior de Investigaciones Cientı ´ficas, Granada, Spain, 4 Grupo de Ciencias Ba ´sicas Me ´dicas, Facultad de Medicina, Universidad de los Andes, Bogota ´, Colombia, 5 Grupo de Ciencias de Laboratorio Clı ´nico, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota ´, Colombia Chagas disease (ChD) is a chronic infection caused by Trypanosoma cruzi. This highly diverse intracellular parasite is classified into seven genotypes or discrete typing units (DTUs) and they overlap in geographic ranges, vectors, and clinical characteristics. Although studies have suggested that ChD progression is due to a decline in the immune response quality, a direct relationship between T cell responses and disease outcome is still unclear. To investigate the relationship between parasite control and immune T cell responses, we used two distinct infection approaches in an animal model to explore the histological and parasitological outcomes and dissect the T cell responses in T. cruzi-infected mice. First, we performed single infection experiments with DA (TcI) or Y (TcII) T. cruzi strains to compare the infection outcomes and evaluate its relationship with the T cell response. Second, because infections with diverse T. cruzi genotypes can occur in naturally infected individuals, mice were infected with the Y or DA strain and subsequently reinfected with the Y strain. We found different infection outcomes in the two infection approaches used. The single chronic infection showed differences in the inflammatory infiltrate level, while mixed chronic infection by different T. cruzi DTUs showed dissimilarities in the parasite loads. Chronically infected mice with a low inflammatory infiltrate (DA-infected mice) or low parasitemia and parasitism (Y/Yinfected mice) showed increases in early-differentiated CD8 + T cells, a multifunctional T cell response and lower expression of inhibitory receptors on CD8 + T cells. In contrast, infected mice with a high inflammatory infiltrate (Y-infected mice) or high parasitemia and parasitism (DA/Y-infected mice) showed a CD8 + T cell response distinguished by an increase in late-differentiated cells, a monofunctional response, and enhanced expression of inhibitory receptors. Overall, our results demonstrated that the infection outcomes Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231211 Edited by: Christophe Chevillard, TAGC Theories and Approaches of Genomic Complexity, France Reviewed by: Galadriel Hovel-Miner, George Washington University, United States Andre Talvani, Universidade Federal de Ouro Preto, Brazil *Correspondence: Jose Mateus [email protected] Adriana Cue ´llar [email protected] Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 10 June 2021 Accepted: 10 September 2021 Published: 12 October 2021 Citation: Mateus J, Nocua P, Lasso P, Lo ´pez MC, Thomas MC, Egui A, Cuervo C, Gonza ´lez JM, Puerta CJ and Cue ´llar A (2021) CD8 + T Cell Response Quality Is Related to Parasite Control in an Animal Model of Single and Mixed Chronic Trypanosoma cruzi Infections. Front. Cell. Infect. Microbiol. 11:723121. doi: 10.3389/fcimb.2021.723121 ORIGINAL RESEARCH published: 12 October 2021 doi: 10.3389/fcimb.2021.723121 caused by single or mixed T. cruzi infection with different genotypes induce a differential immune CD8 + T cell response quality. These findings suggest that the CD8 + T cell response might dictate differences in the infection outcomes at the chronic T. cruzi stage. This study shows that the T cell response quality is related to parasite control during chronic T. cruzi infection. Keywords: T cells, Trypanosoma cruzi, Chagas disease, reinfections, immune quality INTRODUCTION The T cell responses induced after infection or vaccination might be used as a possible predictor of protection against pathogens. Although T cells are heterogeneous, their immune quality is defined as the efficient maintenance of memory T phenotypes and a competent antigen-specific T cell response (Appay et al., 2002;Darrah et al., 2007;Seder and Darrah, 2008;Riou et al., 2012;Ferreira et al., 2019). Efficient maintenance of early memory T cell subsets has been associated with the protection induced by successful vaccination and infection models (Ribeiro et al., 2014;Vigano et al., 2015;Akondy et al., 2017;Mpande et al., 2018). Memory T cell subsets encompass populations with distinct levels of differentiation, including central memory (T CM ), effector memory (T EM ), and effector cells (Brummelman et al., 2018). Early differentiated stem memory cells ( TSCM ), an antigen-experienced T cell subset endowed with the ability to self-renew and reconstitute memory and effector T phenotypes, were recently described (Gattinoni et al., 2009; Gattinoni et al., 2011;Brummelman et al., 2018). Additionally, a competent cellular response is defined as the capacity of individual T cells to simultaneously produce several cytokines or molecules participating in the cytotoxic response (Darrah et al., 2007;Westerhof et al., 2019), and this feature is affected by the expression of inhibitory molecules, such as programmed cell death-1 (PD-1 or CD279) and cytotoxic T-lymphocyte– associated antigen 4 (CTLA-4 or CD152) (Wherry, 2011; Wherry and Kurachi, 2015). Studies in diseases caused by viruses, bacteria, fungi, or protozoa have shown that the lack of infection control is associated with an increase in the percentages of late-differentiated T cells, the loss of functional T cell capacities, and an increase in immune inhibition due to augmented expression of inhibitory molecules on T cells (Darrah et al., 2007;Gigley et al., 2012;Attanasio and Wherry, 2016; McLane et al., 2019). Indeed, similar findings from studies on T cells in cancer have led to the development of immunotherapy with antibodies against inhibitory molecules such as anti-PD-1 or anti-CTLA-4. A treatment that reverses the observed impairments in the immune response quality and boosts the antigen-specific T cell response (Moreira et al., 2020). Protozoan infections are responsible for many fatal human diseases in undeveloped countries, including malaria, sleeping sickness, visceral leishmaniasis, and Chagas disease (ChD) (Barrett et al., 2019). Trypanosoma cruzi, the causal agent of ChD, is an intracellular microorganism with a predilection for cardiac muscle or gastrointestinal tract tissues that can cause serious human pathologies such as cardiomyopathy or megasyndromes in chronically infected patients (Perez-Molina and Molina, 2018;WHO, 2020). T. cruzi exhibits high genetic diversity, which has led to classification into seven genotypes or discrete typing units (DTUs, TcI-TcVI and TcBat) based on genetic markers. The parasite genotypes show overlaps in their geographic ranges, vectors, and clinical characteristics (Messenger et al., 2015;Zingales, 2018). A comprehensive study that included several genetic T. cruzi groups found strains from different genotypes induced a high degree of heterogeneity in inflammation degree or immune response in acutely and chronically infected mice (Santi-Rocca et al., 2017). For example, TcI and TcII strains, which are the most predominant T. cruzi genotypes in Latin America, have shown high diversity in inflammation outcomes ranging from mild to severe both chagasic patients and animal models (Barrera et al., 2008;Cruz et al., 2015;Sales-Campos et al., 2015;Hernandez et al., 2016;Zingales, 2018;Ledezma et al., 2020). Indeed, epidemiological studies of individuals with positive Chagas serological tests have shown that continuous exposure to infections with distinct T. cruzi genotypes increases the risk of progression to chronic Chagas cardiomyopathy (Zicker et al., 1990;Basquiera et al., 2003;Sabino et al., 2013;Perez et al., 2014). Similar results have been observed in animal models of ChD, which revealed that reinfections with T. cruzi strains might determine the severity of cardiac damage (Bustamante et al., 2002;Bustamante et al., 2007). However, although various studies have investigated chronic ChD, it is unknown why approximately 30-40% of T. cruzi-infected patients develop cardiac or gastrointestinal illnesses decades after the initial infection (Acevedo et al., 2018;Bonney et al., 2019). A strong effector T cell response is induced by T. cruzi infection, and this response results in the secretion of cytokines and the release of cytotoxic granules upon antigen recognition. Several lines of evidence indicate that T lymphocyte type I responses (T helper (Th) or T cytotoxic (Tc) type I responses) are the central mediator of protection against T. cruzi infection; however, Th17 and regulatory T cell (Treg) responses might influence the infection outcome (Cai et al., 2016;Ersching et al., 2016;Araujo Furlan et al., 2018). Additionally, studies in humans and mice have shown that chronic T. cruzi infection leads to a decay in the T cell response quality accompanied by a marked increase in late-differentiated T cells, a decreased multifunctional T cell capacity, and higher expression of inhibitory receptors (Laucella et al., 2004;Albareda et al., 2013;Lasso et al., 2015; Mateus et al., 2015), suggesting that T cells might be associated with the progression of disease severity. Indeed, the administration of antiparasitic therapy to individuals with Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231212 chronic T. cruzi infection and in mouse models of chronic T. cruzi infection has shown that treatment reverses the deterioration of the T cell response (Bustamante et al., 2008; Vallejo et al., 2016;Mateus et al., 2017;Perez-Anton et al., 2018; Egui et al., 2020). Overall, although the mechanisms involved in the pathogenesis of ChD are unknown, the available evidence suggests that parasite persistence drives the impairment of the T cell response and leads to uncontrolled T. cruzi infection and progression of the disease. Several studies in Chagasic patients or animal models with chronic T. cruzi infection have suggested a relationship between TcellresponsesandChDseverity(Pinazo et al., 2015; Cortes-Serra et al., 2020). However, validating this hypothesis has been difficult because of the natural ChD history, which makes the long-term follow-up of patients for decades after the initial infection complex. Thus, to investigate whether the infection outcome is related to the T cell response quality during chronic T. cruzi infection, we used two distinct infection approaches in an animal model to explore the histological and parasitological outcomes and dissect the T cell responses in T. cruzi-infected mice. We examined the T cell response focused on the following parameters: memory/effector subsets, antigen-specific response, and expression of inhibitory receptors on T cells. First, we performed a set of single infection experiments with T. cruzi DA (TcI) or Y (TcII) strains to compare the infection outcomes and evaluate the relationship with the T cell responses in acute and chronic parasite infection. We selected these two T. cruzi strains since they represent the most predominant genetic groups in Latin-American countries and have shown different inflammatory profiles and infection outcomes in animal models (Santi-Rocca et al., 2017;Zingales, 2018). Next, because infections with diverse T. cruzi genotypes can feasibly occur in naturally infected individuals, mice were infected with either the Y or DA strains and subsequently reinfected with the Y strain, and the mice with homologous or heterologous infection with T. cruzi strains (Y/Y or DA/Y) were assessed to determine the relationship of the infection outcome with the T cell response. MATERIALS AND METHODS Mice and Parasites Female inbred BALB/cAnNCr mice (aged 6 to 8 weeks) were purchased from Charles River Laboratories International, Inc. (Wilmington, MA, USA) and housed in specific pathogen-free (SPF) animal facilities from the Unidad de Biologı  a Comparativa at the Pontificia Universidad Javeriana. The BALB/c mouse strain was selected to minimize variability compared with previous studies (Hoft and Eickhoff, 2002;Mariano et al., 2008; Sanoja et al., 2013;Egui et al., 2017;Mateus et al., 2019). The animals were housed in polycarbonate cages (4 or 5 animals/ cage) with sterile soft wood shaving bedding, which was changed weekly, and these cages were maintained in ventilated racks in an animal biosafety level 2 (ABSL-2) room under constant noisefree environmental conditions. The mice received filtered water (changed weekly) and a standard mouse maintenance diet ad libitum. Stress and microbiological monitoring (including behavioral and animal welfare analyses and microbiological and serological testing) were performed according to IACUC guidelines. T. cruzi trypomastigotes from the DA (MHOM/CO/ 01/DA; discrete typing unit (DTU) TcI) or Y strain (MHOM/BR/ 00/Y; TcII) were used in the present study. Trypomastigotes of both strains were maintained by tissue culturing involving serial passage through a monolayer of renal fibroblast-like cells or VERO cells (ATCC CCL-81, Manassas, VA, USA). Trypomastigotes of the Y strain were passaged in female inbred BALB/cAnNCr mice at least three times to maintain virulence. Both T. cruzi strains represent the most predominant genetic groups in Latin America and have shown different inflammatory profiles and infection outcomes in animal models (Barrera et al., 2008;Cruz et al., 2015;Sales-Campos et al., 2015;Santi-Rocca et al., 2017;Zingales, 2018;Ledezma et al., 2020). For instance, an infection with the T. cruzi DA strain has shown a prolonged infection with preferential migration to cardiac tissue and reappearance of parasitemia in chronic stages of infection (Barrera et al., 2008;Cruz et al., 2015). In contrast, strain Y shows an aggressive illness with high parasitemia levels in the acute phase and tropisms towards the liver and colon (Mateus et al., 2019). Infection and Challenges Experiments in Mice Forty BALB/c mice were randomly divided into two experimental groups and infected intraperitoneally (i.p.) with 10 5 trypomastigotes of the Y or DA strain in 100 ml of PBS under aseptic conditions. Five mice per group were euthanized by CO 2 inhalation at 10, 30, 100, or 260 days postinfection (dpi). We selected the time periods for the acute (10 and 30 dpi) or chronic phases (100 and 260 dpi) based on a previous study carried out by our research group that demonstrated differential infection outcomes and T cell immune responses in T. cruzi-infected mice (Mateus et al., 2019). This is in the sense that we and others evaluate in similar days post-infection the acute and chronic infection stages evaluating immunological parameters of T. cruzi-infected mice (Bustamante et al., 2002;Hoft and Eickhoff, 2002;Bustamante et al., 2007;Bustamante et al., 2008;Sanoja et al., 2013;Mateus et al., 2019). For the assessment of homologous and heterologous infection with T. cruzi strains, 20 BALB/c mice were randomly divided into two experimental groups and infected i.p. with trypomastigotes of either the DA or Y strain under the abovementioned conditions, and five mice per group were then challenged at 10 or 100 dpi with 10 5 trypomastigotes of the Y strain. Both mouse groups were euthanized by CO 2 inhalation at 260 dpi as described above. We obtained spleen samples from all the mice for cell purification and samples of the cardiac blood, skeletal muscle of the posterior leg, heart, colon and liver tissue for DNA extraction or histopathology analyses. The sample size was determined based on the average number of mice used in previous studies of T. cruzi infection in mice (Maranon et al., 2001;Planelles et al., 2001;Egui et al., 2012;Sanoja et al., 2013). Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231213 Various animal welfare indicators (score sheet) were recorded weekly during the first 30 days and then every month thereafter. One experiment was conducted to evaluate either single or mixed T. cruzi infections in mice. Each mouse group included five biological replicates. If the mouse groups were pooled, each group included nine or ten biological replicates. Each figure legend describes the number of mice included in each group. T. cruzi Soluble Antigens The T. cruzi soluble antigens (TcSA) were obtained from the Y strain using previously described methods (Martinez-Calvillo et al., 2007;Fernandez-Villegas et al., 2011;Mateus et al., 2013). Briefly, amastigotes and trypomastigotes (1:1 ratio) were collected from the VERO cell culture supernatants at 96-120 hours postinfection (Fernandez-Villegas et al., 2011). The parasites were then washed twice with cold 1× PBS (Eurobio) and resuspended at a density of 1 x 10 6 parasites/ml in lysis buffer as previously reported (Martinez-Calvillo et al., 2007). The parasites were incubated on ice for 30 minutes, and supernatants containing TcSA were collected by centrifugation at 12,000 x gand 4°C for 15 minutes and stored at -80°C until use. The protein concentrations were determined using the Bradford assay, and the protein profiles were analyzed by SDSPAGE followed by Coomassie blue staining (Gibco BRL; Grand Island, NY, USA). Noninfected VERO cell supernatants were subjected to all above-described procedures and used as a mock control in the flow cytometry assays. Flow Cytometry The antibodies used in the present study are listed in Supplemental Table 1.TheFixableAquaDeadCellStain viability marker (LIVE/DEAD) (Invitrogen; Eugene, OR, USA) was used to exclude dead cells. All conjugated antibodies were titrated and evaluated using FMO controls as previously described (Mateus et al., 2013). Spleen cells were plated in 96well round-bottom tissue culture plates and stained with multicolor immunofluorescence panels for assessment of CD4 + and CD8 + T cell responses. First, cells were treated with Fc block antibodies (BD Biosciences) for 5 minutes at 4°C and subsequently stained with the LIVE/DEAD marker for 20 minutes at room temperature. To evaluate the memory/effector phenotypes, the cells were stained with antibodies against CD3, CD4, CD8, CD44, CD62L, CD122, CD127, and KLRG1 antigens for 30 minutes at 4°C. To evaluate antigen-specific T cell-producing cytokines, the cells were cultured with mock (as a negative control) and TcSA (1 µg/ml) in the presence of anti-CD28 (1 mg/ml, clone 37.51, BD Pharmingen) for 1 hour at 37°C in a humidified atmosphere containing 5% CO 2 and then incubated in the presence of brefeldin A (1 mg/ml) and monensin (0.7 mg/ ml) (BD Biosciences) for 5 hours. After incubation, the cells were stained with the viability marker and then with antibodies against CD3, CD4, and CD8 molecules for 30 minutes at 4°C and washed with staining buffer. The cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) according to the manufacturer’s instructions and incubated with anti-IFNg, anti-TNFa, and anti-IL-2 antibodies for 30 minutes at 4°C. To evaluate the functional subsets of CD4 + T cells, the cells were cultured under the above-described conditions, stained with viability markers and then with antibodies against CD3, CD4, CD25, and LAP markers for 30 minutes at 4°C and washed with staining buffer. The cells were fixed and permeabilized with Foxp3 Transcription Factor Fixation/Permeabilization (Invitrogen) according to the manufacturer’s instructions and incubated with anti-Foxp3, anti-RORgt, anti-IL-10, anti-IL17A, and anti-IL-21 antibodies for 30 minutes at 4°C. To evaluate the expression of inhibitory receptors on T cells, the cells were stained with antibodies against CD3, CD4, CD8, PD-1 (CD279), 2B4 (CD244), and CD160. The cells were then fixed and permeated using Cytofix/Cytoperm buffer according to the manufacturer’s instructions and incubated with anti-CTLA-4 (CD152) antibody for 30 minutes at 4°C. At least 50,000 events gated on live CD3 + cells were acquired withaFACSAriaIIflow cytometer (BD Biosciences). Supplemental Table 2 provides the cell counts collected for the CD3 + CD4 + and CD3 + CD8 + cells. The data were analyzed using FlowJo 9.3 (Tree Star; Ashland, OR, USA), Pestle 1.7 (National Institutes of Health (NIH), Bethesda, MD, USA), and SPICE 5.3 (NIH) software. A Boolean analysis was performed to define the multifunctional profiles and the coexpression of inhibitory receptors on FlowJo. The Boolean analysis for the multifunctional profiles included IFNg, TNFa, and IL-2 and for the coexpression of inhibitory receptors included PD-1, 2B4, CD160, and CTLA-4 gated on CD4 + and CD8 + T cells. Dead and doublet cells were excluded from the analysis (Supplemental Figure 1). A positive cytokine response was defined for each measured profile, and this response was determined as the median frequency of the T cell response obtained from uninfected mice after stimulation with TcSA plus 1 SD after background subtraction (cells from each mouse cultured with Mock). Parasite Quantification by qPCR Sample tissues from each mouse were collected and processed as described previously (Mateus et al., 2019). Briefly, DNA from tissue samples was extracted using a High Pure PCR template preparation kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). For the assessment of DNA integrity and to exclude the presence of inhibitors in the sample, PCR was then performed using the CytB Uni fw 5’- TCATCMTGATGAAAYTTYGG-3’and CytB Uni rev 5’- ACTGGYTGDCCBCCRATTCA-3’primers, which amplify the cytochrome B gene of small mammalian species, as described previously (Schlegel et al., 2012). Subsequently, qPCR was performed with the Cruzi 1 5’-ASTCGGCTGATCGT TTTCGA-3’and Cruzi 2 5’-AATTCCTCCAAGCAGCG GATA-3’primers and the Cruzi 3 5’-6FAM-CACACACTG GACACCAA-BBQ-3’probe, which amplify a 166-bp segment of T. cruzi satellite DNA (Piron et al., 2007). Each sample was analyzed in duplicate. The parasite load was estimated based on a standard curve and constructed with different DNA concentrations of the Y strain mixed with 50 ng of DNA from tissue sampled from an uninfected mouse, which ranged from 10 -1 –10 4 parasite equivalents per 50 ng of DNA as described Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231214 previously (Cummings and Tarleton, 2003;Mateus et al., 2019). Parasite loads below the limit of quantification (LOQ) were set to LOQ/2 (0.05 parasite equivalents per 50 ng of DNA) as previously described (Hecht et al., 2018). Amplification was performed using the Applied Biosystems™QuantStudio™3 Real-Time PCR System (Applied Biosystems, USA) and previously described qPCR conditions (Duffy et al., 2013). Each PCR performed in this analysis included the following controls: reaction (water added in the room containing the reaction mixture), gray (water added in the room where the sample was added to the reaction), negative (genomic DNA from one uninfected mouse), and positive (DNA from the DA and Y strains). Histopathology Sample tissues from each mouse were stained with hematoxylin and eosin (H&E) for blinded analysis according to the following features: presence of inflammation, type of cellular infiltration, and pathological changes. Histopathological scores were assigned as follows using previously described methods (Guarner et al., 2001): absent, mild, moderate, or severe. Representative pictures of different inflammatory infiltrate scores observed in tissue samples from DA and Y-infected mice are shown in Supplemental Figure 2. Statistical Analysis Statistical analyses between two groups were performed using the Mann–Whitney U test. The correlations between the T cell response and the parasite loads in tissue were analyzed using Spearman’s rank correlation coefficient. The tests were twotailed, and p<0.05 indicated statistical significance. GraphPad Prism 8.0 for Mac OS X software (GraphPad, San Diego, CA, USA) was used for the statistical analyses. RESULTS Distinctive Outcomes in Mice With Acute and Chronic Infections With T. cruzi Y or DA Strains Because several studies have documented that the genetics and phenotypic heterogeneity of T. cruzi strains may be associated with distinct infection outcomes (Santi-Rocca et al., 2017; Zingales, 2018), we first analyzed the parasitological and histological outcomes during T. cruzi infection in experimentally infected mice with T. cruzi trypomastigotes belonging to two strains of different genotypes (Y or DA) as described in the Materials and Methods section. The Y and DA strains were isolated from infected patients and belong to the TcII and TcI genotypes of T. cruzi, respectively (Pinto et al., 1999;Barrera et al., 2008;Amato Neto, 2010;Cruz et al., 2015). We pooled acutely and chronically infected mice from days 10 and 30 and days 100 and 260, respectively, as described previously (Mateus et al., 2019). The parasite load and the inflammatory infiltrate scores in tissue samples corresponded to the infection outcomes evaluated in the acutely and chronically DAor Y-infected mice. The colon (p= 0.0005), heart (p= 0.0007), liver (p= 0.0325), skeletal muscle (p= 0.0007), and blood (p= 0.0037) samples from the acutely Y-infected mice showed significantly higher parasite loads than those from the acutely DA-infected mice (Figure 1A). We found a greater inflammatory infiltrate score in liver samples (p= 0.0027) from Y-infected mice than in DA-infected mice (Figure 1B). In chronically infected mice, the parasite loads in tissues were similar in samples from Yor DA-infected mice (Figure 1C). However, the cellular infiltrate scores obtained from the colon (p= 0.0008) and liver (p= 0.0036) were higher in Y-infected mice than in DA-infected mice. Similar infiltrate scores were found in the heart and skeletal muscle samples from chronically infected mice with both T. cruzi strains (Figure 1D). Overall, increased parasitism and inflammation were observed in tissue samples from acutely Y-infected mice, and reduced but identifiable inflammatory scores were observed in the chronically DAinfected mice. Moreover, the acutely and chronically DAinfected mice had lower parasitic and inflammatory outcomes. Thus, our findings indicate that acutely and chronically Yor DA-infected mice exhibit different outcomes. Differential CD8 + T Cell Responses Induced by the DA or Y Strain During Acute and Chronic T. cruzi Infection Since distinctive outcomes in mice infected with T. cruzi YorDA strains were observed, we evaluated the relationship between the T cell response in Yor DA-infected mice and infection outcomes. We examined the CD8 + T cell response focused on the following parameters: memory/effector subsets, antigenspecific response, and expression of inhibitory receptors on T cells. Each parameter was defined as described in the Materials and Methods and as shown in Supplemental Figure 1. The memory CD8 + T cell subsets analyzed in the Yor DAinfected mice included stem cell memory cells (T SCM cells, CD44 - CD62L + CD122 + CD127 + ), central memory cells (T CM , CD44 + CD62L + ), and effector memory cells (T EM cells, CD44 + CD62L - ). As shown in Figure 2A, the Y-infected mice presented reduced percentages of CD8 + T SCM (at 10 dpi [p= 0.0079], 100 dpi [p= 0.0079], and 260 dpi [p= 0.0079]) and T CM cells (at 10 dpi [p= 0.0079], 30 dpi [p= 0.0159], and 260 dpi [p= 0.0159]) and increased percentages of CD8 + T EM cells (at 10 dpi [p= 0.0079], 30 dpi [p= 0.0079], 100 dpi [p= 0.0317], and 260 dpi [p= 0.0079]) compared with the DA-infected mice (Figure 2A). These results showed that the Y-infected mice exhibited a memory profile of T cell subsets characterized by a predominant proportion of effector cells consisting of terminally differentiated memory phenotypes (T EM cells), whereas the DAinfected mice exhibited early-differentiated memory phenotypes (T SCM and T CM cells). Because the effector CD8 + T cell response in mice can be examined based on KLRG1 and CD127 expression (Joshi et al., 2007;Obar and Sheridan, 2015), we analyzed these effector subsets in Yor DA-infected mice. The effector CD8 + T cell subsets scrutinized in this study included early effector cells (EECs, KLRG1 - CD127 - ), short-lived effector cells (SLECs, KLRG1 + CD127 - ), memory precursor effector cells Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231215 (MPECs, KLRG1 - CD127 + ), and double-positive effector cells (DPECs, KLRG1 + CD127 + )(Figure 2B). The results shown in Figure 2B indicated that Yor DA-infected mice showed between the acute and chronic infection stages similar proportions of effector CD8 + T cell subsets. Then, we analyzed the effector subsets at 10 or 260 dpi. The obtained data show that Y-infected mice presented high percentages of EECs at 10 dpi and SLEC CD8 + T cells at 10 and 260 dpi compared with DAinfected mice. Additionally, the Y-infected mice showed low percentages of MPEC CD8 + T cells at 10 and 260 dpi compared with the DA-infected mice (Figure 2B). Thus, the Y-infected mice presented short-lived effector cells (EECs and SLECs), whereas the DA-infected mice exhibited memory precursor effector cells (MPECs) in both acutely and chronically infected mice. To determine whether the functional properties exhibited by antigen-specific T cells are influenced by the T. cruzi strain, we subsequently compared the functional antigen-specific CD8 + T cells by measuring the production of IFNg, TNFa, or IL-2 in response to stimulation with soluble antigens from T. cruzi. The Y-infected mice exhibited high percentages of IFNg-producing CD8 + T cells at 10 (p= 0.0079) and 100 dpi (p= 0.0317) and TNFa-producing cells at 10 dpi (p= 0.0079) in the Y-infected mice, whereas the DA-infected mice presented a greater percentage of TNFa-producing cells at 30 dpi (p= 0.0476) (Figure 2C). IL-2 was the cytokine produced at the lowest level in the mice infected with either the Y or DA strain in acute or chronic phases, with no differences in the percentages of IL-2-producing CD8 + T cells in either group (Supplemental Figure 3). The analysis of the monofunctional and multifunctional responses in T. cruzi-specificCD8 + T cells showed that Y-infected mice exhibited CD8 + T cells with three or two functions at 10 or 100 dpi. In contrast, the DA-infected mice displayed a stronger multifunctional response with three or two functions at 30, 100, or 260 dpi (Figure 2D). Overall, Yinfected mice showed monofunctional T. cruzi-specific CD8 + T cell responses at 30 and 260 dpi, whereas DA-infected mice exhibited monofunctional T. cruzi-specific CD8 + T cell responses at 10 dpi. Additionally, the multifunctional capacities of Agspecific CD8 + T cells in DA-infected mice were maintained for up to 260 dpi (Figure 2D). To assess the expression of inhibitory receptors on T cells in mice infected with the T. cruzi strains, cells from the Yor DAinfected mice were stained with antibodies against 2B4, CD160, CTLA-4, and PD-1. The frequency of 2B4-expressing CD8 + T cells was higher (p= 0.0079) at 10 dpi in Y-infected mice (median 4.01% [3.52-4.35]) than in DA-infected mice (median 1.12% [0.98-1.28]). Similar values of 2B4-expressing CD8 + T B A D C FIGURE 1 | Parasite loads and inflammatory infiltrate scores of tissues from Yor DA-infected mice. (A, C) Parasite loads in colon, heart, liver, skeletal muscle, and blood samples from acutely and chronically Yor DA-infected mice, respectively. The bar graphs show the medians and ranges of the parasite equivalent per 50 ng of DNA (LOG 10 ) in tissues from each group of infected mice. The dotted line represents the cutoff for the limit of detectable quantification (LOQ) based on serially diluted T. cruzi-spiked tissue DNA as described in the Materials and Methods (0.1 parasite equivalents per 50 ng of DNA). (B, D) Inflammatory infiltrate scores obtained for colon, heart, liver, and skeletal muscle samples from the acutely and chronically Yor DA-infected mice, respectively. The bar graphs show the average inflammatory infiltrate scores found for the tissues from each group of infected mice. The fold change (FC) shown in B and C was determined as the average inflammatory score detected in the Y-infected mice divided by the average infiltrate inflammatory score found in the DA-infected mice. Each point represents the value of the parasite load or the infiltrate inflammatory score detected in each mouse infected with the Y (white) or DA (gray) strain of T. cruzi. Results are pooled from one experiment with ten mice per group. The pvalues were calculated using the Mann–Whitney U test. Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231216 cells were detected in Yand DA-infected mice at 30, 100, and 260 dpi (Y-infected mice, 1.01%, 1.3%, 1.25%, respectively; DAinfected mice, 1.13%, 1.37%, 0.83%, respectively). CD8 + T cells from the Y-infected mice showed increased expression of CD160 (at 10 dpi [p= 0.0079] and 30 dpi [p= 0.0159]), CTLA-4 (at 10 dpi [p= 0.0079] and 260 dpi [p= 0.0317]), or PD-1 (at 10 dpi [p= 0.0079] and 260 dpi [p= 0.0317]) compared with the DAinfected mice (Figure 2E). Additionally, CD8 + T cells expressing several inhibitory receptors exhibited a greater coexpression profile in the Y-infected mice than in the DA-infected mice at 10 dpi. The predominant profile of coexpression in the chronically infected mice corresponded to CD8 + Tcells coexpressing CD160, CTLA-4, and PD-1. CD8 + T cells from the Yor DA-infected mice coexpressed similar proportions of inhibitory receptors at 100 and 260 dpi (Supplemental Figure 4). Altogether, our results suggest that CD8 + T cell responses were strain-specific and differentially modulated during acute and chronic T. cruzi infection. CD4 + T Cell Responses in Yand DA-Infected Mice in the Acute and Chronic Phases Previous studies have revealed that T. cruzi infection promotes the activation of different CD4 + Tcellprofiles involved in CD A E B FIGURE 2 | CD8 + T cell responses in the acutely and chronically Yor DA-infected mice. (A) Frequency of CD8 + T SCM ,T CM , and T EM cells in acutely and chronically Yor DA-infected mice. (B) Proportion of EEC, SLEC, MPEC, and DPEC CD8 + T cells in the acutely and chronically Yor DA-infected mice at 10 and 260 dpi. Significant differences of effector CD8 + T cell subsets between Yand DA-infected mice are shown with an asterisk (*). (C) Antigen-specific CD8 + T cells producing IFNgor TNFain acutely and chronically Yor DA-infected mice. Background-subtracted data analyzed in all cases. (D) Proportion of antigen-specific CD8 + T cells with one, two, or three functions, as defined by the production of IFNg, TNFa, and IL-2 based on a Boolean strategy, in the acutely and chronically Yor DA-infected mice. (E) Frequency of CD8 + T cells expressing CD160, CTLA-4, or PD-1 from acutely and chronically Yor DA-infected mice. The bar graphs in (A, C, E) show the geometric mean and geometric SD. Log data analyzed in (A, C, E) Each point represents a mouse. Results are pooled from one experiment with five mice per group. The pvalues were calculated using the Mann–Whitney U test. T SCM , stem cell memory; T CM , central memory; T EM , effector memory; EECs, early effector cells; SLECs, short-lived effector cells, MPECs, memory precursor effector cells; DPECs, double-positive effector cells. Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231217 activating and regulating immune response processes that may determine the infection outcome (Acevedo et al., 2018). We analyzed the CD4 + T cell response in Yand DA-infected mice, and slight differences were found between the T. cruzi-specific CD4 + T cell immune responses in Yand DA-infected mice. Similar to the observed for the CD8 + T cell response in Yand DA-infected mice, the results obtained indicated that chronically DA-infected mice had higher CD4 + T EM cell frequencies and increased multifunctional CD4 + T cells than Y-infected mice (Figures 3A–C), as reported previously in mouse models and humans (Padilla et al., 2007;Albareda et al., 2009;Padilla et al., 2009). The Y-infected mice exhibited high percentages of IFNgproducing CD4 + T cells (at 10 dpi, p= 0.0079) and TNFaproducing CD4 + T cells (at 10 dpi [p= 0.0159] and 100 dpi [p= 0.0079]) compared with the DA-infected mice (Figure 3B). CD4 + T cells from the Y-infected mice showed increased expression of CD160 (at 10 dpi [p= 0.0079]), CTLA-4 (at 10 dpi [p= 0.0079] and 30 dpi [p= 0.0159]), or PD-1 (at 10 dpi [p= 0.0079], 30 dpi [p= 0.0079] and 260 dpi [p= 0.0079]) than those from DA-infected mice (Figure 3D). Overall, our results suggest that the CD4 + and CD8 + T cell responses were strainspecific and differentially modulated during acute and chronic T. cruzi infection. The heterogeneous response of CD4 + T cells can be analyzed through the expression of master transcription factors (TBX21, Foxp3, or RORgt) or the profiles of cytokine-producing cells (IFNg, IL-10, or IL-17A) (Oestreich and Weinmann, 2012). Since an early CD4 + T cell response could define T. cruzi infection (Guo and Cobb, 2009;Miyazaki et al., 2010;Sanoja et al., 2013; Cai et al., 2016), we next analyzed the CD4 + T cell profiles expressing RORgt and Foxp3 transcription factors in Yand DAinfected mice for up to 100 dpi. As observed in Figure 4,at10 dpi, the Y-infected mice showed a higher percentage (p= 0.0079) of double-positive CD4 + T cells expressing Foxp3 and RORgt than the DA-infected mice; however, at 30 or 100 dpi, the Yor DA-infected mice presented similar values of T cells that were positive for both of these transcription factors (Figure 4A). Moreover, increased percentages of RORgt + Foxp3 - cells were detected in the Y-infected mice at 10 dpi (p= 0.0079), whereas in the DA-infected mice, the percentages of these cells were C A D B FIGURE 3 | CD4+ T cell responses in acutely and chronically Yor DA-infected mice. (A) Frequency of CD4 + T SCM ,T CM , and T EM cells in acutely and chronically Yor DA-infected mice. (B) Antigen-specific CD4 + T cells producing IFNgor TNFain acutely and chronically Yor DA-infected mice. Background-subtracted data analyzed in all cases. (C) Proportion of antigen-specific CD4 + T cells with one, two, or three functions, as defined by the production of IFNg, TNFa, and IL-2 based on a Boolean strategy, in the acutely and chronically Yor DA-infected mice. (D) Frequency of CD4 + T cells expressing CD160, CTLA-4, or PD-1 from acutely and chronically Yor DA-infected mice. The bar graphs in (A, B, D) show the geometric mean and geometric SD. Log data analyzed in (A, B, D). Each point represents a mouse. Results are pooled from one experiment with five mice per group. The pvalues were calculated using the Mann–Whitney U test. Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231218 increased at 100 dpi (p= 0.0079). In addition, the proportions of RORgt - Foxp3 + cells at 10 dpi were lower in the Y-infected mice than in the DA-infected mice (p= 0.0079). Notably, the percentages of CD4 + T cells expressing Foxp3 in the mice infected with Y or DA remained similar throughout the other days evaluated in this study (Figure 4A). Subsequently, we examined two T regulatory phenotypes, including cells expressing Foxp3 at high levels and CD25, which are known as thymic Tregs (tTregs), and cells expressing latency-associated peptide (LAP), which identifies a population of cells producing TGFb. Notably, both CD4 + T regulatory phenotypes presented similar patterns during acute infection and were dependent on the T. cruzi strain because Foxp3 Hi CD25 + and LAP + cells were significantly increased in the DA-infected mice at 10 dpi (p= 0.0079) and were found at higher proportions in the Y-infected mice at 30 dpi (p= 0.0317 and p= 0.0159) (Figure 4B). In C A D B FE FIGURE 4 | CD4 + T helper profiles dissected during early T. cruzi infection with two distinct strains. (A) Percentages of CD4 + T cells showing different expression of RORgt and Foxp3 in the Yor DA-infected mice. The left panel shows the representative dot plot of the gating strategy for RORgt + Foxp3 + , RORgt + Foxp3 - , or RORgt - Foxp3 + CD4 + T cells. (B) Percentages of CD4 + T cells coexpressing Foxp3 and CD25 in the Yor DA-infected mice. The left panel shows the representative dot plot of the gating strategy for expressing Foxp3 High CD25 + CD4 + T cells. (C) CD4 + T cells expressing LAP in the Yor DA-infected mice. The left panel shows the representative dot plot of the gating strategy for CD4 + T cells expressing LAP. (D) Representative dot plot of CD4 + T cells producing IL-10, IL-17A, or IL-21. The gates applied for the identification of cytokine production among the total population of CD4 + T cells were defined according to the cells from each mouse that were cultured with mock. (E) Percentages of CD4 + T cells producing IL-10, IL-17A, or IL-21 in the Yor DA-infected mice. (F) Proportion of antigen-specific CD4 + T cells with one, two, or three functions, as defined by the production of IL-10, IL-17A, and IL-21 based on a Boolean strategy, in the Yor DA-infected mice. The bar graphs show the geometric meam and geometric SD. Each point represents a mouse. The pie charts show the medians of the CD4 + T cells with one, two, or three functions detected in the acutely and chronically Yor DA-infected mice (E). Results are pooled from one experiment with five mice per group. The pvalues were calculated using the Mann–Whitney U test. Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 7231219 AUTHOR CONTRIBUTIONS Conceptualization: JM, PL, ML, CP, and AC. Formal analysis: JM, JG, CP, and AC. Funding acquisition: JM, PL, CP, and AC. Methodology: JM, PN, and CC. Writing –first draft: JM. Writing –review & editing: JM, PN, PL, ML, MT, AE, CC, JG, CP, and AC. All authors contributed to the article and approved the submitted version. FUNDING JM was supported by Ph.D. scholarships from the Departamento Administrativo de Ciencia, Tecnologi  a e Innovacio n (COLCIENCIAS) and PUJ (Convocatoria 727 Doctorados Nacionales). ML and MT were supported by grant SAF201680998-R from Programa Estatal I+D+I (MINECO, Spain). This work was supported by grants from COLCIENCIAS (code: 120365842534, contract no. 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Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Mateus, Nocua, Lasso, Lo pez, Thomas, Egui, Cuervo, Gonza lez, Puerta and Cue llar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Mateus et al. T Cells in T. cruzi-Infected Mice Frontiers in Cellular and Infection Microbiology | www.frontiersin.org October 2021 | Volume 11 | Article 72312119