Mice
C57BL/6 J mice, aged 6 to 8 weeks, were purchased from ENVIGO and housed in the animal care facility at Institut Pasteur under specific pathogen-free conditions. Vav-iCre x Percevalfl/fl (SPICY), GFP-OT-I-Rag1-/-, CD45.1 mice were bred on-site at our facility. Experimental/control animals were co-housed. Both male and female mice were used, except for circadian experiments, which were conducted on males. Mice have been euthanized using a CO2 chamber in agreement with ethical requirements. All animal experiments were conducted with approval from the Institut Pasteur Safety Committee, in compliance with French and European guidelines (CETEA 230038) and were validated by the French Ministry of Education and Research.
Sample preparation
Blood was collected by 0.5 G insulin syringes containing 50 µL of 2 mM EDTA by cardiac puncture. 1 mL of blood sample was processed using RBC lysis buffer (eBioscience), following manufacturer’s instructions. Lymph nodes were crushed on 70 µm cell strainers (Corning) with Injekt Luer Solo 2 mL syringes plug, lymphocytes have been collected in R-10 complete media (RPMI 1640 Glutamax without phenol-red; 10% heat-inactivated FCS,; 50 µ/ml Penicillin-streptomycin; 10 mM HEPES, 50 µM b-mercaptoethanol, Gibco) and centrifuged. After the last centrifugation, blood and lymph node cells were resuspended in R-10 complete media for further staining.
SPICE-Met Flow cytometry
Cells expressing PercevalHR were analyzed by flow cytometry. ATP:ADP ratios (R) in individual cells were calculated from two fluorescence signals. In brief, excitation with a violet laser (405 nm) and signal detection with a 525 band-pass filter were used to measure ADP contribution, while ATP levels were estimated by excitation with a blue laser (488 nm) and signal detection with a 525 band-pass filter. To calculate ATP:ADP ratio, we derived a new parameter (FlowJo) corresponding to fluorescence signals collected in the ATP channel divided by the fluorescent signals from the ADP channel. To reveal OXPHOS/glycolysis contribution, cells have been treated for 10 min at 37 °C with 1 µM oligomycin (oligo, Sigma); to reveal glucose/fatty and amino acids contribution, cells have been treated with 1 mM iodoacetic acid (IA, Sigma) or 100 mM 2-deoxy-D-glucose (2DG, Sigma). A combination of oligomycin with iodoacetic acid treatment was used to reveal the minimal level of energetic resources. Control samples were treated with H2O and DMSO. Relative OXPHOS/glycolysis dependencies were calculated as ((RDMSO+H2O – ROligomycin)/(RDMSO+H2O – ROligo+IA)) × 100 and ((ROligomycin – ROligo+IA)/(RDMSO+H2O – ROligo+IA)), respectively. Absolute OXPHOS/glycolysis dependencies were calculated as (RDMSO+H2O – ROligomycin) and (ROligomycin – Roligo+IA), respectively. Relative glucose/fatty and amino acids dependencies were calculated as ((RDMSO+H2O – RIA)/(RDMSO+H2O – ROligo+IA)) × 100 and ((RIA – ROligo+IA)/(RDMSO+H2O – ROligo+IA)), respectively. Absolute glucose/fatty and amino acids dependencies were calculated as (RDMSO+H2O – RIA) and (RIA – Roligo+IA), respectively.
MVA vaccination
The recombinant MVA-HIV-B, which encodes full-length HIV Gag along with three Pol and two Nef fragments12 was supplied by the Agence Nationale de Recherche sur le Sida (ANRS). Mice were injected with 2 × 10⁶ p.f.u. of MVA-HIV-B via footpad injection.
Adoptive transfer
Lymphocytes were isolated from lymph nodes of Vav-iCre x Percevalfl/fl (SPICY) mice (expressing CD45.2 congenic marker), and the resulting cell suspension was injected intravenously (2 × 107 cells per mouse) in CD45.1 animals of the same sex. Recipients were killed 3 h later, and lymphocytes isolated from blood and lymph nodes were processed for SPICE-Met profiling.
Generation of T cells expressing the glucose reporter iGluco
The construct of encoding the iGlucoSnFR-mRuby2 (iGluco) glucose probe17 was obtained from Addgene and subcloned into the retroviral vector MSCV. T cells were activated in plates coated with anti-CD3 monoclonal antibody (mAb) (2.5 μg ml−1; clone 17.A2; BioLegend) in the presence of soluble anti-CD28 mAb (2.5 μg ml−1; clone 37.51; BioLegend) and murine IL-12 (10 ng ml−1; I8523; Sigma-Aldrich)36. Two rounds of spin infection were performed at 24 and 48 hours after T cell activation using retroviral particles supplemented with polybrene (8 μg ml−1; Merck). T cells were cultured for four additional days in the presence of hIL-2 (10 ng ml−1; 202-IL; R&D Systems). Calibration of the iGluco probe was performed in R10 glucose-free media, supplemented with different glucose concentrations (D-Glucose, Sigma).
Impact of nutrient availability
Lymphocytes, isolated from Vav-iCre/Percevalfl/fl (SPICY) mice, were isolated and stained as described above. Later, they were subjected to different media for at least 20 min at 37 °C 5% CO2 incubator, and their ATP: ADP ratio was analyzed. Media were prepared based on the SILAC RPMI 1640 Flex Media without glucose (Gibco), with the following additives, according to experimental condition: D-glucose (Gibco), fatty acids supplement (1:100, Sigma), GlutaMAX (1:100, Gibco), Glutor (4 µM, Sigma), 2DG (100 mM, Sigma). HEPES, penicillin, streptomycin and b-mercaptoethanol were added in all conditions, as described above for complete R-10 media composition. The addition of metabolic inhibitors and the reading of ATP:ADP ratio were performed directly in the experimental media.
Circadian experiments
In all cases, Vav-iCre/Percevalfl/fl (SPICY) and C57BL/6J male mice were entrained to a 12-h light:12-h dark cycle for at least 3 weeks before experimentation, using Leddy programmable IVC lighting control system (Techniplast). Zeitgeber time (ZT) 0 was defined as the time of lights on, and ZT12 is the time of lights off.
Generation of effector OT-I CD8+ T cells
In vitro OT-I T cell stimulation was performed during 4 h in complete R10 media, supplemented with 0.1% brefeldin (BD GolgiPlug, BD Biosciences) and, according to experimental conditions, supplemented with phormol 12-myristate 13-acetate (0.02 µg/mL PMA, Sigma) and ionomycin calcium salt from Streptomyces conglobatus (0.5 µg/mL, Sigma); or plated on anti-CD3 antibody coated plates (2.5 µg/mL, clone 17A2, Biolegend) and, in this case, supplemented with anti-CD28 (2.5 µg/mL, clone 37.51, Biolegend). In case of N4-peptide re-stimulation, SIINFEKL peptide (10 µM, AS-60193-5, Anaspec) was used.
Flow cytometry
For standard cell surface staining, the cells were incubated in R-10 complete media. All antibodies were purchased from Biolegend, if not noted otherwise. For extracellular staining cells have been incubated for 20 min at 4 °C with constant agitation with CD8-BUV395 (1:200, clone 53-6.7, BD Biosciences), CD11b-BUV736 (1:200, clone M1/70, BD Biosciences), CD44-BV605 (1:200, clone IM7), CD4-BV786 (1:200, clone RM4-5), NK1.1-BV650 (1:200, clone PK136), TCRβ-APCCy7 (1:200, clone H57-597), Ly6G-Alexa647 (1:200, clone 1A8), CD19-PECy7 (1:200, clone 6D5), CD62L-BV421 (1:200, clone MEL-14), CD45.1-PECy7 (1:50, clone A20), CD45.2-BUV736 (1:50, clone 104, BD Biosciences), GLUT1 (1:50, Sigma 07-1401-AF647 or Almone labs AGT-041 or Biotechne clone 202915), H2-Kb-TSYKFESV MHC:peptide tetramers were generously provided by the National Institutes of Health Tetramer Core Facility. Tetramer staining was conducted first, followed by staining with the antibody cocktail. For intracellular staining, cells have been processed with BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit, according to manufacturer instructions. Incubations with anti-IFN-γ (1:50, clone XMG1.2) and anti-Perforin (1:50, clone S16009B) antibodies were done overnight at +4 °C with constant agitation. Isotype controls were PE-Rat IgG1, κ; clone RTK2071 (for IFN-γ staining) and APC-Rat IgG2a, κ; clone RTK2758 (for perforin staining). Dead cells were stained and excluded by fixable viability dye eF780 (1:1000, Invitrogen), added to extracellular antibodies cocktail mix. Data were collected using a Cytoflex LX flow cytometer (Beckman Culture) and analyzed using FlowJo software v10.8.1 (BD Biosciences).
Statistical analysis
All statistical analyses were conducted using Prism v.9.5.0 (GraphPad). Data are presented as mean ± SEM. The Wilcoxon signed-rank test was used to compare paired groups. The Mann-Whitney U test was used for comparing two independent groups or a paired t-test if normality was validated by a Shapiro-Wilk test. For comparison of multiple (>2) independent groups, we used Kruskal-Wallis-Dunn’s test or one-way ANOVA if normality was validated by a Shapiro-Wilk test. All statistical tests were two-tailed with a significance level of 0.05. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, non-significant.
Graphical design
Figures 1 A, 2A, D, 3A, 4A, D, G, J, 5A, 6A, F, H, supplementary Fig. 6A, were created in-house using Adobe Illustrator software. Clock images in Figs. 5A, 6F, supplementary Fig. 6A, were created in BioRender, and are under license CHIKINA, A. (2025) https://BioRender.com/zx9rjpl.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.