P2RX7 signaling drives the differentiation of Th1 cells through metabolic reprogramming for aerobic glycolysis

Introduction This study provides evidence of how Th1 cell metabolism is modulated by the purinergic receptor P2X7 (P2RX7), a cation cannel activated by high extracellular concentrations of adenosine triphosphate (ATP). Methods In vivo analysis was performed in the Plasmodium chabaudi model of malaria in view of the great relevance of this infectious disease for human health, as well as the availability of data concerning Th1/Tfh differentiation. Results We show that P2RX7 induces T-bet expression and aerobic glycolysis in splenic CD4+ T cells that respond to malaria, at a time prior to Th1/Tfh polarization. Cell-intrinsic P2RX7 signaling sustains the glycolytic pathway and causes bioenergetic mitochondrial stress in activated CD4+ T cells. We also show in vitro the phenotypic similarities of Th1-conditioned CD4+ T cells that do not express P2RX7 and those in which the glycolytic pathway is pharmacologically inhibited. In addition, in vitro ATP synthase blockade and the consequent inhibition of oxidative phosphorylation, which drives cellular metabolism for aerobic glycolysis, is sufficient to promote rapid CD4+ T cell proliferation and polarization to the Th1 profile in the absence of P2RX7. Conclusion These data demonstrate that P2RX7-mediated metabolic reprograming for aerobic glycolysis is a key event for Th1 differentiation and suggest that ATP synthase inhibition is a downstream effect of P2RX7 signaling that potentiates the Th1 response.


Introduction
In the last decades, the relevance of purinergic signaling to T cell function has received increasing attention from the scientific community. Adenosine triphosphate (ATP) has been identified as a key molecule for communication between dendritic cells and T cells (1,2). ATP released by pannexin-1 hemichannels at immunological synapses activates ATP-gated P2RX cation channels on T cells and promotes the entry of Ca ++ . Environmental ATP released by stressed, damaged or dying cells from surrounding tissue as a danger signal can also increase T cell responses. Ca ++ influx leads to translocation of the nuclear factor of activated T cells (NFAT) to the cell nucleus and consequent production of IL-2. IL-2 signaling induces T cell proliferation and drives the differentiation of T helper 1 (Th1) cells together with some signals form dendritic cells, such as IL-12, and produce IFNg that activate macrophages. Th1 differentiation depends on the expression of transcriptional factors T-box expressed in T cells (T-bet) and B-lymphocyte-induced maturation protein 1 (Blimp-1) (3,4). As a regulatory mechanism, activated T cells express CD39 and CD73 ectonucleotidases that hydrolyze extracellular ATP (eATP) and generate adenosine, which exerts its regulatory activity through the engagement of G-coupled P1 receptors (5).
Among the seven members of the P2RX family, T cells express P2RX1, P2RX4 and P2RX7 (1). The latter receptor is particularly involved in pathological conditions as it is activated only at high concentrations of eATP and induces the formation of large pores in the cell membrane. The massive influx of Ca ++ through P2RX7induced membrane pores can potentiate cell activation or lead to cell death under prolonged stimulus (6,7). As an evidence of these effects, P2RX7 is upregulated in CD4 + T cells activated during acute murine malaria caused by Plasmodium chabaudi and promotes membrane pore formation, intense Ca ++ influx and IL-2 production, and thus induces a vigorous Th1 response that protects mice from death (8). Conversely, P2RX7 deficiency in CD4 + T cells during acute P. chabaudi infection leads to the predominance of early follicular helper T (Tfh) cells expressing the transcriptional factor B-cell lymphoma 6 (Bcl6), a cell population that induce antibody class switching and affinity maturation in B cells (9). Both Th1 and Tfh subsets contribute to protection against P. chabaudi infection, as in the human malaria, evidenced by the protective role of IFNg and parasite-specific antibodies (10). P2RX7 may also participate in the metabolic changes observed in activated T cells, particularly during intense immune responses and tissue damage. However, information on this subject is scarce, mainly addressing the contribution of P2RX7 to the metabolic fitness of long-lived memory CD8 + T cells developed after virus infection (11).
T cell subpopulations have metabolic differences that regulate cell fate and function (12,13). Resting and memory T cells oxidize pyruvate, lipids and amino acids to produce the basal level of ATP that is necessary for cell survival (14). Ca ++ input mechanisms, such as the stockade-calcium intake (SOCE), support T cell proliferation and differentiation to effector functions through metabolic reprogramming (15). After T cell activation, lipid oxidation is downregulated and aerobic glycolysis increases along with glutamine oxidation in order to supply the biosynthetic precursors required for rapid cell proliferation (16). Regarding CD4 + T cell subsets, Th1 cells are more proliferative and metabolically active than Tfh cells. In order to maintain an efficient Th1 response, activation of metabolic sensors Akt and the mammalian target of rapamycin (mTOR) drives CD4 + T cell fate through upregulation of aerobic glycolysis (17). As an indication that the ATP-P2RX7 axis also modulates T cell metabolism, overexpression of P2RX7 confers an intrinsic ability to upregulate aerobic glycolysis and mitochondrial reactive oxygen species (ROS) in fast-growing tumor cell lines (18). It has been shown that mitochondrial ROS is critical for T cell activation by inducing NFAT translocation to the cell nucleus and subsequent IL-2 production (19, 20).
Here, we investigated how P2RX7 modulates CD4 + T cell metabolism during strong Th1 responses. In vivo studies were performed in P. chabaudi-infected mice in view of the great relevance of malaria for human health, as well as the large amount of information available by flow cytometry and single cell RNAseq analysis concerning Th1/Tfh polarization in this experimental model (8, 21-23). The single cell RNAseq analysis highlights the metabolic profile as a determining factor for the fate of malaria-responsive CD4 + T cells during the Th1/Tfh subset bifurcation, as cells cycling fastest and exhibiting the greatest glycolytic activity are the first to acquire the ability to produce IFNg (22). Confirmation and further details on the effects of P2RX7 on CD4 + T cell metabolism were obtained by in vitro analysis under intense Th1 polarization. Our results demonstrate that P2RX7induced aerobic glycolysis is a key event for the development of efficient Th1 responses. P2RX7 signaling in CD4 + T cells upregulates Akt and mTOR, as well as glucose uptake, promoting proliferation and differentiation of Th1 cells. Inducing the production of ATP synthase inhibitors appears to be an important molecular mechanism by which P2RX7 signaling in CD4 + T cells upregulates aerobic glycolysis and, therefore, Th1 differentiation.

P2RX7 induces early upregulation of T-bet transcriptional factor in CD4 + T cells that respond to Plasmodium infection
Previous studies have shown that Th1/Tfh subsets emerge in parallel by day 7 post-infection (p.i.) with P. chabaudi in B6 mice (8), but subset bifurcation revealed by gene expression signatures occurs earlier around day 4 p.i. (22). Because P2RX7 has a fundamental role in the generation of Th1 cells during experimental malaria (8), we sought to determine the first cellular events through which this molecule supports the Th1 response. Initially, the expression of Blimp1, T-bet and Bcl6 transcriptional factors was evaluated in CD4 + T cells from B6 and P2rx7 -/mice at the acute phase of infection. These cells were stained for FoxP3 to exclude regulatory T cells in the phenotypic analysis. A single population of Foxp3 -CD4 + T cells co-expressed T-bet and Bcl6 at days 5 and 6 p.i., with comparable numbers of these cells per spleen being found in B6 and P2rx7 -/mice ( Figure 1A). Of note, T-bet expression was lower in Foxp3 -CD4 + T cells from P2rx7 -/mice at day 6 p.i., while Bcl6 and Blimp-1 were similarly upregulated in cells of both mouse groups ( Figure 1B). As previously reported (8), T-bet and Bcl6 expression distinguished Th1 (T-bet hi Bcl6 lo ) cells from early Tfh (T-bet lo Bcl6 hi ) cells in B6 mice at day 7 p.i., and a reduction in the Th1 subset was observed in P2rx7 -/mice ( Figure 1C). These findings indicate that P2RX7-induced T-bet upregulation is an early event that precedes Th1/Tfh polarization in murine malaria.
P2RX7 supports the Th1 response during experimental malaria by activating the glycolytic pathway In P. chabaudi malaria, CD4 + T cells proliferate intensely and are metabolically very active around day 4 p.i. and progressively stop dividing and differentiate into effector cells until day 7 p.i., after which most activated cells die in parallel with control of acute parasitemia (21,22,24). To investigate whether metabolic reprogramming of CD4 + T cells by P2RX7 is a key event for Th1 lineage differentiation, we performed a proteomic analysis on Foxp3 -CD11a + CD4 + T cells from B6 and P2rx7 -/mice at day 6 of infection. CD11a interacts with ICAM-1 and has been previously used to identify Plasmodium-specific T cells responding to infection (25). Either CD11a + cell populations, expressing or not P2RX7, were activated as evidenced by their large size (FSC-A) and positivity for T-bet and Bcl6, and were present in similar percentages among FoxP3 -CD4 + T cells ( Figure 2A). However, B6 CD4 + T cells were larger than P2rx7 -/-CD4 + T cells ( Figure 2B), which is indicative of increased proliferation and/or differentiation in effector cells in the presence of P2RX7; large cell size is a feature of both proliferating and IFNg-producing B6 CD4 + T cells (22).
According to heatmap analysis of label-free quantitative data, 121 of the upregulated proteins were found in P2rx7 -/-CD4 + T cells compared to B6 CD4 + T cells and 37 of the upregulated proteins were found in B6 CD4 + T cells compared to P2rx7 -/-CD4 + T cells ( Figure 2C and Table S1). Higher numbers of upregulated proteins were found in P2rx7 -/-CD4 + T cells for all assessed biological pathways as categorized using the Reactome program, including cell cycle and DNA replication pathways ( Figure 2D). Concerning the metabolic pathways, however, proteins involved in the glucose metabolism were selectively increased in B6 CD4 + T cells ( Figure 2E). Among these proteins, hexokinase 1 (Hk1), triosephosphate isomerase 1 (Tpi1) and fructose-biphosphatealdolase A (Aldoa) play a key role in the glycolytic pathway, as well as in the reverse gluconeogenesis pathway; alpha-enolase (Eno1) catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate required Early effects of P2RX7 on T-bet expression by splenic CD4 + T cells that respond to Plasmodium infection. B6 and P2rx7 -/mice were infected i.p. with 1 x 10 6 iRBCs. for pyruvate synthesis (26). Expression of glycolytic enzymes was associated with upregulation of isocitrate dehydrogenase subunit a (Idh3a) and ATP synthase subunit b (ATP5f1), which participate in the tricarboxylic acid (TCA) cycle and respiratory electron transport, respectively. In contrast, P2RX7 deficiency led to upregulation of enzymes involved in the synthesis of nucleotides and amino acids, such as phosphoserine aminotransferase (Psat1), D-3phosphoglycerate dehydrogenase (Phgdh), trifunctional purine biosynthetic protein adenosine-3 (Gart) and inosine-5' monophosphate dehydrogenase 2 (Impdh2). These proteins were associated with isocitrate dehydrogenase subunit g (Idh3g) and the F6 subunit of the ATP synthase peripheral stalk (ATP5j). In addition, P2RX7 deficiency promoted the upregulation of several proteins involved in lipid synthesis, such as acyl-CoA thioesterase (Acot7), fatty acid synthase (Fasn) and isopentenyl-diphosphate deltaisomerase 1 (Idi1).
To assess the impacts of P2RX7 expression in the glycolytic and oxidative capacity of CD4 + T cells at day 6 p.i., the extracellular consumption acidification rate (ECAR) and oxygen consumption rate (OCR) were evaluated. No difference was observed in basal rates, but maximal ECAR and OCR were reduced in P2rx7 -/-CD4 + T cells compared to B6 CD4 + T cells ( Figures 2F, G). It is Metabolic analysis of splenic CD4 + T cells from B6 and P2rx7 -/mice at early Plasmodium infection. Foxp3.GFP and Foxp3.GFP + P2rx7 -/mice were infected i.p. with 1 x 10 6 iRBCs and analyzed 6 days later. The first arrow indicates the injection of glucose to increase the glycolytic pathway, followed by oligomycin to enhance the glucose consumption (second arrow) and 2-deox-D-glucose (2DG) to inhibit glycolysis (third arrow). (G) The first arrow indicates the injection of oligomycin that promotes a metabolic stress, followed by a mitochondrial decoupler carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) to determine the maximal respiratory capacity (second arrow) and antimycin A/ rotenone to block the electron transport chain (third arrow). Data are shown as the mean ± SD (n = 4) of one representative experiment out of three. Significant differences were observed for the (-) indicated groups with p < 0.05, using the Mann Whitney U.
noteworthy that inhibition of oxidative phosphorylation by blocking ATP synthase with oligomycin upregulates ECAR in B6 and P2rx7 -/-CD4 + T cells, although the former population showed a greater increase. According to our proteomics and seahorse data it is possible to suggest that P2RX7 promotes a more energetic state of anabolic profile in the cell by inducing block synthesis pathways. Glycolysis appears to support the availability of substrates for this metabolic state and therefore allow rapid CD4 + T cell proliferation and differentiation to the Th1 profile. Otherwise, in the absence of P2RX7, most CD4 + T cells that respond to Plasmodium infection show an early Tfh profile and maintain a slow but sustained proliferative activity, giving rise to fully differentiated (PD1 hi CXCR5 + ) Tfh cells around day 14 p.i. (8, 23).
We concluded that cell-intrinsic P2RX7 expression is sufficient to sustain glucose metabolism, as evidenced by mTOR and Akt phosphorylation and glucose uptake upregulation, and induce Th1 differentiation. Increased ROS production and loss of MMP are signals of bioenergetic mitochondrial stress also induced by P2RX7 expression in CD4 + T cells that respond to Plasmodium infection.

ATP synthase inhibition restores Th1 differentiation in vitro in the absence of P2RX7
To assess whether P2RX7 is important for Th1 differentiation in vitro, B6 and P2rx7 -/-CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies and polarized to the Th1 profile in conditioning medium containing IL-12, IL-2 and anti-IL-4 antibody; stimulated but unpolarized Th0 cells were used as controls. Under Th1-polarizing condition, only B6 CD4 + T cells showed T-bet and Blimp-1 upregulation (Figure 4A), and these cells produced more IFNg than P2rx7 -/-CD4 + T cells ( Figure 4B). P2RX7 was also required for CD4 + T cell proliferation in this condition, as evidenced by both FSC-A ( Figure 4C) and CFSE staining ( Figure  S2A). Furthermore, the generation of CD44 + CD4 + T cells coexpressing CD25 (IL-2R) ( Figure 4D) and CD127 (IL-7R) ( Figure S2B) was impaired by the absence of P2RX7; IL-7R signaling controls T cell homeostasis (27). Notably, most P2rx7 -/-CD4 + T cells showed a Th0 phenotype when differentiated in the Th1 condition.
The role of metabolic reprogramming in the differentiation of Th1 cells that expressed or did not express P2RX7 was then investigated by inhibiting the glycolytic pathway or oxidative phosphorylation. Glycolysis inhibition with 2-deoxi-d-glucose (2DG) reduced the expression of Blimp-1 and T-bet, most evidently for Th1-polarized B6 CD4 + T cells that became phenotypically similar to P2rx7 -/-CD4 + T cells differentiated in the same condition ( Figure 4E). Remarkably, blocking ATP synthase with oligomycin that inhibits the oxidative phosphorylation and drives cellular metabolism for aerobic glycolysis was sufficient to promote Blimp-1 and T-bet expression in P2rx7 -/-CD4 + T cells. T-distributed stochastic neighbor embedding (t-SNE) analysis for T-bet, Bcl6, Blimp1, CD44, CD25, CD127 and FSC-A clearly demonstrated the major effect of P2RX7-mediated metabolic reprograming on Th1 differentiation ( Figure 4F). Treatment with 2DG impaired the proliferative response of Th1-polarized B6 CD4 + T cells, as evidenced by the reduction in cell size (FSC-A) and expression of CD44, CD25 and CD127; this phenotype was indistinguisheble from that of untreated P2rx7 -/-CD4 + T cells differentiated in the Th1 condition ( Figure 4G). Notably, ATP synthase blockade increased the proliferative response and expression of CD44, CD25 and CD127 in P2rx7 -/-CD4 + T cells differentiated in the Th1 condition to levels similar to those observed for Th1-polarized B6 CD4 + T cells.
We concluded that P2RX7-mediated metabolic reprogramming of CD4 + T cells for aerobic glycolysis is a general phenomenon, not restricted to malaria, which is a key event for Th1 differentiation ( Figure 5). Inducing the production of ATP synthase inhibitors is likely a molecular mechanism by which P2RX7 signaling in CD4 + T cells upregulates aerobic glycolysis and, therefore, Th1 differentiation. Column bar graphs show MTG hi TMRE + , MTG hi TMRE -, MTG lo TMRE + and MTG lo TMRE -CD11a + CD4 + T cell numbers per spleen. Data are shown as the mean ± SD (n = 4-5) of one representative experiment out of three. Significant differences were observed for the (-) indicated groups and (*) between infected and control mice with p < 0.05, using Mann Whitney U test.

Discussion
This study describes a sequence of events arising from P2RX7 signaling in CD4 + T cells that respond to Plasmodium infection, which, together with the data published in this malaria experimental model, provides a clear view on the role of this signaling pathway in Th1 differentiation. P2RX7 acts at the beginning of the malaria immune response, before Th1/Tfh Effects of glycolytic pathway and ATP synthase inhibition in vitro on Th1-conditionated CD4 + T cells that express or not P2RX7. Splenic naïve CD4 + T cells from B6 and P2rx7 -/mice were activated in vitro under Th1 conditions. Th0 condition was used as control. polarization, when activated CD4 + T cells constitute a single population that co-expresses T-bet and Bcl6, and contributes exclusively to the differentiation of Th1 cells, without affecting the generation of early Tfh cells. P2RX7 amplifies the Th1 response to malaria by inducing metabolic reprogramming for aerobic glycolysis, which supplies the anabolic intermediates needed to support rapid CD4 + T cell growth and proliferation (16). This was evidenced by its involvement in mTOR and Akt phosphorylation, glucose uptake and production of glycolytic enzymes such as hexokinase 1, triosephosphate isomerase 1, fructose-bisphosphatealdolase A and alpha-enolase. Supporting this, P2RX7 signaling is a key modulator of cancer cell metabolism, by increasing glucose uptake, glycolytic enzymes and glycogen stores (18). In contrast, Tfh cells are less proliferative and glycolytic than Th1 cells due to the lack of IL-2 signaling and mTOR and Akt activation (9) and, thus, do not require P2RX7 to differentiate. The induction of the glycolytic pathway by P2RX7 presumably occurs during the interaction of CD4 + T cells with activated phagocytes, which, as previously reported, is a determinant event for Th1 differentiation in Plasmodium infection (22). In contrast, the decision to differentiate in the Tfh phenotype relies on the interaction of activated CD4 + T cells with B cells in the primary lymphoid follicles (22), and occurs independently of P2RX7 signaling (8). Thus, the most likely scenario is that part of the activated CD4 + T cells interacts with red pulp phagocytes, which are strongly stimulated by infected red blood cells (iRBCs) and thus release high amounts of ATP through pannexin-1; the engagement of P2RX7 in CD4 + T cells induces the aerobic glycolysis and potentiates the Th1 response. Red pulp phagocytes include macrophages, monocytes and a large population of dendritic cells that actively uptake iRBCs and interact with CD4 + T cells during acute malaria (28,29); the optimization of phagocyte functional activities by IFNg helps to control the parasite (30). ATP released by parasites through ion channels (31,32) or upon erythrocyte lysis may also contribute to activating P2RX7 in CD4 + T cells that interact with red pulp phagocytes. This view is supported by a recent study showing that P2RX7 expression in CD4 + T cells is particularly important for cell migration, proliferation and production of IFNg in the pulmonary parenchyma during severe tuberculosis and influenza (33).
One of the main effects of P2RX7 signaling on activated CD4 + T cells is to enhance the efficiency of Ca ++ handling by the endoplasmic reticulum and, therefore, improve Ca ++ -dependent NFAT translocation to the cell nucleus, leading to IL-2 production and IL-2R expression (34,35). In agreement with this, P2RX7 signaling in CD4 + T cells increases intracellular Ca ++ concentration and IL-2 production during Plasmodium infection (8), as well as induces mTOR and Akt phosphorylation as shown in this study. These metabolic sensors upregulate the expression of several transcriptional factors that are critical for the expression of glycolytic enzymes such as hypoxia-inducible factor 1a (HIF1a), interferon regulatory factor-4 (IRF-4) and c-Myc (15). Increased ROS production and mitochondrial depolarization are also consequences of P2RX7 activation in CD4 + T cells during Plasmodium infection, being indicative of mitochondrial Ca ++ overload. Although mitochondrial ROS is critical for NFAT activation and IL-2 production (19, 20), uncontrolled Ca ++ influx from sustained P2RX7 signaling results in mitochondrial Ca ++ overload, and therefore in exacerbated ROS production and collapse of the MMP due to opening of the mitochondrial permeability transition pore (MPTP) (36). In this context, the selective expression of CD39 in Th1 cells (8), as well as the fact that Entpd1 (CD39 gene) is one of highest-ranking Th1 transcribed genes during Th1/Tfh subset bifurcation (22), indicates that tight regulation of eATP levels is crucial to minimize the undesirable effects of excessive P2RX7 signaling in Th1 responses.
More details on the critical role of P2RX7 signaling in the metabolic reprogramming of Th1 cells are given by in vitro experiments in which CD4 + T cells are stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, IL-12 and anti-IL-4 antibody. P2RX7 deficiency in CD4 + T cells prevents Th1 polarization, as well as pharmacological inhibition of the glycolytic pathway. Furthermore, the blockade of ATP synthase activity and consequent inhibition of oxidative phosphorylation, which drives cellular metabolism for aerobic glycolysis, is sufficient to induce IL- Schematic illustration showing the hypothetical role of P2RX7 in Th1 differentiation through metabolic reprogramming. High extracellular ATP concentrations activate P2RX7 in CD4 + T cells under stress conditions during inflammation. The intracellular ATP may be released to the extracellular space through pannexin-1 (Panx1) channels and maintain autocrine purinergic activation. P2RX7 signaling induces metabolic reprogramming required for the Th1 response by increasing the expression of phosphorylated mTOR and Akt, glucose uptake and glycolytic enzymes, which culminates in Tbet transcription. In addition, P2RX7-dependent pore formation promotes intense Ca ++ influx (8), leading to mitochondrial stress and membrane potential reduction (Dy). ATP synthase inhibition is likely a downstream effect of P2RX7 signaling that potentiates the Th1 response, as drug-induced blockade of this enzyme restores Th1 differentiation in the absence of P2RX7. Thus. a possible connection between P2RX7-mediated mitochondrial effects and Tbet upregulation is that P2RX7 signaling in CD4 + T cells induces ATP synthase inhibitors that promote the metabolic shift required to sustain the Th1 response.
2R and IL-7R expression, CD4 + T cell proliferation and polarization to the Th1 profile in the absence of P2RX7-mediated Ca ++ influx. These results show how glucose metabolism integrates with TCR, CD28, IL-2 and IL-12 signaling in order to ensure the differentiation of Th1 cells, in addition to highlighting the important role of P2RX7 as a catalyst for the metabolic shift to aerobic glycolysis. The fact that inhibition of ATP synthase has no important effect on polarized Th1 cells expressing P2RX7 is consistent with a previous study showing that proliferation of effector T cells does not depend on mitochondrial ATP production, although it is critical during the early activation of unpolarized T cells (37).
Our findings rise the possibility that inducing the production of ATP synthase inhibitors is a fundamental mechanism by which P2RX7 signaling promotes Th1 cell differentiation. The ATPase inhibitory factor 1 (IF1) is a master regulator of cell metabolism that once dephosphorylated inhibits oxidative phosphorylation and upregulates aerobic glycolysis (38). Dephosphorylated IF1 is highly expressed in colon, lung and breast carcinomas and in hypoxia (39); the overexpression or silencing of IF1 results in upregulation or downregulation of glycolysis in cancer cells, respectively (40-42). In addition, the TCA cycle metabolite aketoglutarate (43) and the oncometabolite (R)-2-hydroxyglutarate inhibit ATP synthase (44), as well as metabolites accumulated in some human pathologies such as neuronal ceroid lipofuscinosis and methylmalonic and glutaric acidurias (45). The induction of ATP synthase inhibitors by P2RX7 may provide the optimal metabolic conditions for CD4 + T cell proliferation and differentiation to the Th1 profile.
This study demonstrates that P2RX7-mediated metabolic reprogramming for aerobic glycolysis is a general phenomenon, which occurs both in vivo during acute P. chabaudi malaria and in Th1-polarized in vitro conditions, being a key event for the development of an efficient Th1 response in environments with high eATP levels. The molecular mechanism involved in this process should be better clarified from the evidence that points to the participation of ATP synthase inhibitors. Understanding the metabolic pathways involved in Th1 responses under intense stimulation can help to develop new therapeutic approaches for infectious, autoimmune and other inflammatory diseases. CD4 + T cell proliferation assay Spleen cells (3.0 × 10 7 ) were stained with 5 µM 5,6carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, USA) in phosphate-buffered saline with 0.1% bovine serum albumin (Sigma-Aldrich, USA). CD4 + T cells (5 × 10 5 ) were cultured with iRBCs (4 × 10 6 ) for 72 h at 37°C in a 5% CO 2 atmosphere, stained with PE-labeled mAb to CD4 and analyzed by flow cytometry.

Proteomic analysis
Splenic CD11a + CD4 + and CD11a -CD4 + T cells were sorted using a FACAria device, BD Biosciences) and frozen in a freezer -20°C ( Figure S3). For protein extraction, unfreezed cells were centrifuged (700g for 3 min, 4°C) and supernatants were discarded. After washing, the pellet was resuspended in 150 ml of 8M urea buffer containing 1x protease inhibitor cocktail (Merck, Germany). It was performed 6 cycles of thermal shock: the tubes were emerged in liquid nitrogen for 30s, transferred to a 30°C bath and mixed for 1 min. Protein was quantified using Qubit (Invitrogen) and ammonium bicarbonate was added to a final concentration of 50 mM. Proteins were reduced with dithiothreitol (Sigma-Aldrich) to a final concentration of 10 mM, incubated for 45 min at 30°C, subsequently alkylated with iodoacetamide (Sigma-Aldrich) to a final concentration of 5 mM and incubated for 15 min at 30°C. Next, the samples were diluted in urea 10x by adding ammonium bicarbonate 50 mM, pH 7, to perform trypsin (Sequencing Grade Modified Trypsin) (Promega Corporation, USA) digestion at a 1:50 enzyme:protein ratio (w/w), overnight at 30°C. The samples were acidified with trifluoroacetic acid to a final concentration of 1% and digested peptides were desalted using C18 microcolumns (10.1007/978-1-61779-148-2_21). The peptides were resuspended in 0.1% Trifluoroacetic acid (TFA; protein sequencer grade), injected, and loaded on ReproSil-Pur C18 AQ (Ammerbuch-Entringen, Germany) inhouse packed trap column (2 cm x 100 mm inner diameter; 5 mm). Peptides were separated at a flow of 300 nl/min on an analytical ReproSil-Pur C18 AQ packed in a house column (17 cm x 75 mm; 3 mm) by reversed-phase chromatography, which was operated on an EASY NanoLC II system (Thermo Scientific). The mobile phase was water/0.1% FA (A) and 95% ACN/0.1% FA (B). The gradient was 2-30% phase B (0.1% formic acid, 95% acetonitrile) for 80 minutes, 30-95% B for 5 minutes, and holding at 95%B for 20 minutes. The EASY NanoLC II was coupled into an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) operating in positive mode ion. The mass spectrometer acquired a full MS scan at 60,000 full width half maximum (FWHM) resolution with a 350-1500 Da mass range. The top 20 most intense peptide ions were selected from MS for Collision Ion Dissociation (CID) fragmentation (normalized collision energy: 35.0 V).

Proteomics data processing
Raw files were processed in the Maxquant software (version 1.5.3.8) using the Andromeda search engine in the Maxquant environment. Raw data were searched against Mus musculus reviewed Uniprot database (November 2018 release, 17,001 entries). The parameters used were trypsin full specificity; two missed cleavages allowed, protein common contaminants, and reverse sequences included. Carbamidomethylation of cysteine as a fixed modification; methionine oxidation, and protein N-terminal acetylation as variable modifications. Identification of protein was accepted with less than 1% false discovery rate (FDR).

Statistical analysis
Statistical analysis was performed by the Mann-Whitney U test to compare two groups. For more than two groups, data were analyzed by Kruskal-Wallis test. Survival curves were analyzed by the log-rank test using the Kaplan-Meier method. GraphPad Prism 6 software was used, in which differences between groups were considered significant when p< 0.05 (5%). Proteomic statistical analysis was performed in Perseus, using Student's T-test followed by Benjamini-Hochberg FDR correction (46). Proteins identified in at least 3 samples in total and regulated with FDR<0.05 were considered for further analyses. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (PubMed ID: 30395289) partner repository with the dataset identifier PXD037754.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PXD037754 (PRIDE).

Ethics statement
The animal study was reviewed and approved by Health Animal Committee of USP, with permit number 55/2017.