PatientsLi-Fraumeni syndrome patients

The LIFSCREEN clinical trial (NCT01464086) was originally designed to measure the benefits of whole-body MRI screening to detect early-stage cancer cases in patients carrying germline TP53 mutations (gTP53m) [19, 20]. The primary outcome was to assess whether the inclusion of whole-body MRI in the evaluation of patients would influence their overall survival. The secondary aim of the study was to create a collection of serum and plasma samples to establish a library for future investigations into new tumor biomarkers. In total, 107 individuals, consisting of 78 women and 29 men from 75 distinct families, were recruited for the study. The present cohort includes the N = 38 patients bearing gTP53m who provided a signed agreement for the LIFSCREEN translational study part and for whom a sufficient volume of frozen plasma at inclusion was available. This sub-study was approved as part of a screening for inflammatory biomarkers by the steering committee of LIFSCREEN. For comparison, Li-Fraumeni syndrome (LFS) patients were matched by sex, BMI and age with cancer-free healthy patients from the previously published and publicly available DESIR cohort [9] to a 2:1 controls: cases ratio with the MatchIt R package. The resulting cohort has comparable values for sex, BMI and age between controls and LFS patients (Table S1).

Germline BRCA1/2 mutation carriers

Whole blood samples were collected at Gustave Roussy between 2013 and 2016, during a screening for BRCA1 and BRCA2 germline mutations among healthy individuals with a family history of breast or ovarian cancer. All samples were taken under similar material and psychological conditions: on the same premises, between around 10am and 12am, with the aim of screening, meaning that all patients were exposed to the same stress-inducing uncertainty. During the consultation, patients were asked to sign an informed consent form authorizing the use of the leftovers of the material collected for research purposes in addition to diagnostic. For each patient, 20 mL of peripheral blood was collected in EDTA tubes and kept at room temperature for less than 2 h until the plasma was separated. For this, the blood tubes were centrifuged for 15 min at 1700 g at 20 °C, plasma was homogenized, aliquoted and stored at -80 °C until use. Figure 1F represents the flowchart of the cohort. Table S2 summarizes the main patient characteristics.

Fig. 1

ACBP/DBI is elevated in the plasma from patients with genetic predisposition to cancer. Plasma from patients with Li-Fraumeni syndrome (LFS) bearing gTP53MUT were collected at inclusion in the LIFSCREEN trial for MRI-screening of new malignancies. Each patient with LFS was matched by sex, body mass index (BMI) and age with two healthy patients from the DESIR cohort (A), and plasma ACBP/DBI was measured by ELISA. LFS patients had increased ACBP/DBI levels compared to healthy volunteers (one-sided unpaired Student’s t-test, B). TP53 modulation in mice was achieved in vivo either in the long-term by genetic manipulations (tamoxifen-induced knockout or constitutive overexpression of the Trp53 gene) or at short-term by intraperitoneal injections of the TP inhibitor pifithrin-α (C), and plasma ACBP/DBI was measured by ELISA (D, E). Bacterial lipopolysaccharide (LPS) was used as positive control for short-term induction of ACBP/DBI in mice. Plasma specimens were collected from a cohort of women enrolled in a screening campaign for BRCA1/2 mutations. N = 45 mutated: control pairs were randomly created by age-matching patients bearing germline BRCA1/2 mutations with controls who were exempt of known previous or future cancer as well as of somatic BRCA1/2 mutations (F). Patients with germline mutations in either BRCA1 or BRCA2 had significantly higher plasma levels of ACBP/DBI than their age-matched controls (one-sided paired Student’s t-test, G). Breast cancer was induced by combination of a subcutaneous implant releasing medroxyprogesterone acetate (MPA) and weekly gavage with dimethylbenzanthracene (DMBA), after a 5-day tamoxifen induction period in Ubc: Cre+/−Acbpfl/fl (i.e. Acbp−/−, n = 9) or Ubc: Cre−/−Acbpfl/fl (i.e. Acbp+/+, n = 7) mice (H). Once diagnosed with breast cancer by palpation, Acbp−/− animals survived longer than their Acbp+/+ littermates (I). Female C57Bl/6J mice were regularly treated intraperitoneally with an ACBP/DBI monoclonal antibody (or the corresponding isotype control, mouse IgG2a, both 5 mg/kg). E0771 cells (5.0 × 105 per mouse) were injected into the mammary fat pad, and mice were treated with four cycles of anti-PD1 monoclonal antibody (or the corresponding isotype control, rat IgG2a, 200 µg per cycle) (J). The combination of anti-ACBP/DBI with immune checkpoint blockade slowed down tumor growth (K) and prolonged survival (L). Survival curves were compared by log-rank (Mantel-Cox) test, while tumor growth speeds were assessed by linear mixed effect modeling on the https://kroemerlab.shinyapps.io/TumGrowth/ website

Healthy volunteers

The healthy volunteers’ cohort was constituted of baseline samples from the randomized, double-blinded, placebo-controlled interventional study on the prevention of cancer and cardiovascular disease by supplementation of antioxidant vitamins and minerals (“SUpplémentation en Vitamines et Minéraux AntioXydants”, SU-VI-MAX, NCT00272428). A total of 13,017 patients were enrolled over a period of 8 years (1994–2002) and followed until 2007 [21, 22]. All patients signed informed consent forms to participate in the study, and this sub-study was approved by the steering committee of SUVIMAX. During the initial phase of the study, information regarding sociodemographic characteristics, smoking habits, medication usage, and overall health status was gathered through self-administered questionnaires. Additionally, trained nurses and physicians conducted a baseline clinical examination, which involved taking anthropometric measurements and obtaining blood samples from participants. The blood samples were collected in heparin tubes following a 12-hour fasting period. Among the 13,017 patients, we selected the N = 638 patients that had never had malignancies at baseline but developed cancer during follow-up. They were matched with a 2:1 controls: cases ratio by sex, age, BMI, nutritional intervention group (placebo vs. vitamins and minerals), smoking status, season of blood draw and, for women, menopausal status (at baseline and at the date of cancer occurrence). The clinical characteristics of the cancer patients and their matched controls are summarized in Table S3.

Statistics

Statistical analysis of patients’ datasets was performed on R (v. 4.2.0). The comparisons of ACBP/DBI concentrations between two groups were performed by unpaired, one-sided Student’s t-test, following the hypothesis that cancer occurrence was associated with increased circulating levels of ACBP/DBI. Correlation analyses were performed with the cor.test function: Pearson’s product moment correlation coefficients (r) were calculated, p-values were evaluated assuming that the r coefficients followed a t distribution and confidence intervals were computed based on Fisher’s Z transform. Classification power was evaluated by computing receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) with the pROC package [23]. Wilcoxon rank sum tests were used for computing p-values of the AUC (different from 0.5). AUCs with their 95% confidence intervals were represented as forest plots. Survival data were plotted as Kaplan-Meier curves and analyzed by log-rank (Mantel-Cox) test.

ACBP/DBI quantification

Plasma concentrations of ACBP/DBI were measured in the same laboratory and by the same experimenter for all presented cohorts. Enzyme-linked immunosorbent assay (ELISA) was performed as previously described [7, 24]. Briefly, heparin plasma samples were diluted (1/20 for mouse, 1/50 for human) and incubated on capture antibody (MyBioSource, Cat# MBS768488, RRID: AB_3083599) coated plates for 2 h at 18–22 °C (room temperature, RT). They were detected by means of a biotin-conjugated antibody (LS Bio, Cat# LS-C299614, RRID:3083603), incubated 2 h at RT, and avidin-coupled horseradish peroxidase (avidin-HRP, BioLegend, Cat# 405103). The HRP substrate (Thermo Fisher Scientific, Cat# 34028) was incubated until sufficient color appeared, at which point 2 M sulfuric acid was added to quench the reaction. Absorbance was read at 450 nm on an automated plate reader within 15 min, and out-of-standard curve measurements were excluded. For germline BRCA1/2 mutation carriers and their controls, plasma was collected in EDTA tubes only relative quantifications were available for subsequent analyses.

Cell culture

C57Bl/6-derived cancer cell lines (TC1 cells expressing the luciferase enzyme, TC1-Luc, RRID: CVCL_4699, and MCA205, RRID: CVCL_VR90) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 mM HEPES. MCA205 were transduced with lentiviral particles with the ZsGreen-OVA transgene that were produced in 293FT cells (Thermo Fisher, Cat# R70007) by co-transfecting the plasmid coding for the fluorescent protein ZsGreen coupled with ovalbumin (OVA) and puromycin resistance (pCDH_Zs-Green-OVA_puro, Fig. S5E), the lentiviral packaging plasmid psPAX2 (Addgene Cat# 12260), and the VSV-G envelope expressing plasmid (Addgene, Cat# 12259). After cytofluorometric sorting of ZsGreen+ cells, single clones were put to grow under puromycin selection (5 µg/mL) to obtain stably transduced cells.

Mouse experimentsAnimal housing and handling

C57Bl/6J mice were handled according to the Federation of European Laboratory Animal Science Associations (FELASA) guidelines, as approved by the local ethics committee (project numbers #24410, #31018, #49169, #50485 and #IACUC.015-2019). Animals were left untouched for at least one week of acclimatation, provided with food ad libitum and housed collectively in a temperature-controlled environment with 12-hour light-dark cycles.

Trp53 transgenic mice

Inducible knockout was induced in Trp53lox/lox mice [25], with either UBC-cre/ERT2+/T or UBC-cre/ERT2−/− [26] by feeding them with a tamoxifen diet (Envigo RMS, Cat. #TAM400/CreER) at 8 weeks of age for two weeks. Plasma from the resulting Trp53+/+ and Trp53−/− littermates was collected in EDTA tubes at 30 weeks of age. Overexpression of Trp53 was achieved by insertion of the Tg.Trp53[MS-2] (+/T) transgene [27] and plasmas were collected from p53-overexpressing mice and the corresponding controls between 20 and 25 weeks of age.

Induced breast cancer

Female Ubc: Cre, Acbpfl/fl mice under 8 weeks old were induced with five consecutive daily injections of tamoxifen (75 mg/kg i.p., Sigma, Cat# T5648). After a two-week washout period, breast cancer was induced as previously described [28]. Briefly, a small incision was made to insert a slow-release subcutaneous medroxyprogesterone acetate (MPA) pellet (50 mg, 90-day release, Innovative Research of America, Cat# NP-161) under the skin of the neck. One week after the implant, two cycles of three weekly gavages with dimethylbenzanthracene (DMBA, 1 mg, p.o., Sigma, Cat# D3254) were performed over the next 7 weeks. Starting from the second cycle, mice were palped manually three times per week to detect nodules in the mammary glands. When confirmed, an electronic caliper was used to measure the area of the tumors. Mice were sacrificed when humane endpoint was reached (total area > 1.8 cm2, ulceration, weight loss > 20% or distress).

Orthotopic breast cancer

0.5 × 106 E0771 cells were resuspended in 100 µL PBS and injected subcutaneously in the fourth left mammary fat pad from syngeneic C57Bl/6J mice (female, 8–10 weeks old) under 3% isoflurane anesthesia. Tumor sizes were measured with an electronic caliper and calculated in mm2 as A = π/4 × L × w. When the average tumor area became greater than 15 mm2, mice were randomized into groups of equal tumor sizes to be given either anti-PD1 (BioXCell, clone 29 F.1.A12, 200 µg per mouse i.p. at days 15, 19 and 23 and 27) or the corresponding isotype control (IgG2a, BioXCell, clone 2A3).

ACBP/DBI neutralization

For passive immunization, injections of anti-ACBP/DBI monoclonal antibody [6] or the corresponding isotype control (IgG2a) were administered i.p. at 5 mg/kg, following the schedules presented in the figures. For active immunization, animals were vaccinated against endogenous ACBP/DBI as described [24]. Briefly, recombinant murine ACBP/DBI was conjugated to keyhole limpet hemocyanin (KLH, Thermo Fisher, Cat# 77649) by glutaraldehyde cross-linking at a molar ratio of 20:1. The obtained aqueous solution was mixed 1:1 with the adjuvant Montanide ISA 51VG (Seppic, Cat# 36362/FL2R3) to form an injectable water-in-oil emulsion. The vaccine (KLH-ACBP/DBI or KLH only as a control) was injected i.p. once weekly for four consecutive weeks (30 µg, 30 µg, 30 µg and 10 µg, injected in a total volume of 100 µL). After immunization, plasma was collected to check reactivity against ACBP/DBI by immunoblotting.

Carcinogen-induced lung cancer

Six-week-old female C57Bl/6J mice were vaccinated with KLH or KLH coupled to ACBP/DBI. After a two-to-four-weeks washout period, lung cancer was induced by 10 weekly i.p. injections of 1 g/kg urethane (Sigma, Cat #U2500) dissolved in 200 µL warm saline (Sigma, Cat# S8776), as described [29]. Mice were euthanized 30 weeks after the first urethane injection, lungs were collected, and macroscopically visible tumors were counted.

Orthotopic NSCLC

0.5 × 106 non-small cell lung carcinoma TC1-Luc were resuspended in 100 µL PBS and injected intravenously into the lateral tail vein of each C57Bl/6J mouse (female, 8–10 weeks old). Tumor development in the lungs was monitored twice per week by bioluminescence imaging to quantify luciferase activity as previously described [30]. Mice were injected i.p. with 150 mg/kg D-luciferin (Promega, Cat# E1605) in 200 µL of PBS. Acquisition was performed on a Xenogen IVIS 50 (Caliper Life Sciences Inc., U.S.A.) 8 min after D-luciferin injection under light anesthesia (2% isoflurane), and the total photon flux was calculated on the region of interest (ROI) around the lungs. The total time of exposure was set at each measurement to avoid saturation: exposure time started with 4 min, and was gradually reduced to 3 min, 2 min, 1 min upon photon saturation. Chemotherapy was started (day 0) five days after tumors were detectable, and mice were randomized by tumor size. Tumor bearing mice showing photon saturation at 1 min of exposure were euthanized. After the end of the measurements, survival was monitored and the mice were sacrificed when reaching humane endpoints (distress, weight loss > 20%).

Skin fibrosarcoma

C57Bl/6J mice (female, 8–10 weeks old) were shaved and injected subcutaneously with 0.3 × 106 MCA205 cells under light isoflurane anesthesia (2% induction for ≤ 5 min). After 6–7 days, tumors were big enough to be measured by means of an electronic caliper (tumor volume was calculated with the following formula V = π/6 × L × w × h), and mice were randomized to create groups with equivalent tumor burden and body weight repartition [31]. Chemoimmunotherapy was started when tumors reached 50 mm3 (D0). From this point, tumor size was monitored thrice weekly until one of the following endpoints were reached: tumor larger than 1500 mm3, ulceration or weight loss > 20% or distress.

Chemoimmunotherapy

At day 0 (see each model for definition), chemoimmunotherapy was initiated with one cycle of oxaliplatin (Sigma, Cat# Y0000271, 10 mg/kg i.p.), followed by three cycles of PD-1 neutralizing antibody (BioXCell, clone 29 F.1.A12, 200 µg per mouse i.p. at days 8, 12 and 16). Vehicle and isotypes (IgG2a, BioXCell, clone 2A3) were injected following the same route and schedule into the control groups.

Vaccination experiment

On day 0, MCA205-OVA undergoing cell death induced by a 24-hour treatment with 500 µM oxaliplatin (Sigma, Cat# O9512) were collected, rinsed with PBS, and resuspended to a final concentration of 5 × 106 cells/mL. 100 µL of this solution was injected subcutaneously in the lower right back of each mouse two hours after anti-ACBP/DBI or isotype injection (5 mg/kg, i.p.) under light isoflurane anesthesia (Iso-vet isoflurane, 2% induction for ≤ 5 min). mAb injections were repeated according to the schedule described in Fig. 2. Half the mice were sacrificed at day 6 and the right-side inguinal lymph node was collected sterilely for ex-vivo stimulation. The other half of the mice was rechallenged at day 14 with 0.5 × 106 live MCA205-OVA cells per mouse, also injected subcutaneously. At endpoint (day 20), tumor sizes were measured with an electronic caliper.

Fig. 2

ACBP/DBI increases in healthy patients prior to (lung) cancer diagnosis. Plasma samples were selected from the inclusion specimens of the 12,749 healthy volunteers enrolled in the SU.VI.MAX study. Each of the 687 patients diagnosed with cancer at any time during follow-up (Cancer group) was matched with two individuals that stayeid disease-free (Control group), controlling for sex, age, body mass index (BMI), nutritional intervention (antioxidants/placebo), smoking status, menopausal status and season of blood sampling (A). In both these groups of still-healthy volunteers, population-scale positive Pearson correlations between ACBP/DBI and BMI (B) or age (C) were observed. The concentration of ACBP/DBI was slightly increased in future cancer patients (D). This increase was more pronounced in patients (N = 104) who developed cancer within 3 years after inclusion, but was lost in patients diagnosed at later time points (E). Among the most common cancers (N > 20), patients who developed lung cancer (n = 33) were the ones with the most prominent increase in ACBP/DBI (F). A cohort of mice was vaccinated with four weekly injections of keyhole limpet hemocyanin (KLH) coupled to ACBP/DBI (KLH-ACBP, n = 20) or with KLH alone (n = 28). After a wash-out period of 2–4 weeks, mice received weekly intraperitoneal injections of urethane (1 g/kg) for 10 weeks (G). Lungs were collected 30 weeks after the first urethane injection. Representative images are shown in (H) and the number of macroscopic lung tumors was compared between the two experimental groups (I). Statistical analyses were performed by one-sided, unpaired Student’s t-tests for normally distributed plasma ACBP/DBI measurements and by one-sided Mann Whitney tests for lung nodule counts

Statistics

Longitudinal comparison of the caliper-measured tumor sizes was performed on the online TumGrowth application (https://kroemerlab.shinyapps.io/TumGrowth/). In brief, tumor sizes were modeled by linear mixed effect modeling with treatment and time as fixed affects, and individual mice as random effect. Adequate fitting of the model was verified, and tumor sizes were log-transformed if needed to improve the linear fitting. Type II ANOVA was applied to compare the slopes of tumor size variations with time, and p-values were computed using the Wald test with one-sided hypothesis (reduction of slope by the treatment of interest). A factor based on Holm’s method was applied to correct for multiple comparisons. For bioluminescence imaging analyses, total flux over same-size ROIs were log-transformed prior to type-II ANOVA on the tumor sizes, and multiple comparisons between treatment groups were corrected with Sidak’s method. Survival curves were compared two-by-two by means of the Log-rank Mantel-Cox test.

Immunohistochemistry

For immunohistochemical detection of DBI staining, Bond Leica automated immunostainer instrument was used to perform immunohistochemistry. After fixation of tumors in 4% formaldehyde, 4 μm thick paraffin sections were processed for heat-induced antigen retrieval (Epitope Retrieval Solution 1, corresponding citrate buffer pH = 6, Leica, Cat# AR9961) for 20 min at 100 °C. Slides were incubated with a polyclonal rabbit anti-DBI antibody (Abcam, Cat# ab231910, 1:10000) and with Bond Polymer Refine Detection kit (Leica, Cat# DS9800). The signal was revealed with DAB and counterstained with haematoxylin.

Ex-vivo T cell stimulation

Lymph nodes were passed through a 70 μm strainer and thoroughly rinsed with PBS to obtain single-cell suspensions. Cells were resuspended in serum-free CTL-Test™ medium (Immunospot, Cat# CTLT-010) supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin (Gibco) and used for IFN-γ enzyme-linked immunospot (ELISPOT) following the manufacturer’s instructions (Mabtech, Cat# 3321-4APT-2). Briefly, 0.5 × 106 cells were seeded in each well of a 96-well PVDF ELISPOT plate pre-coated with anti-murine IFN-γ antibody (clone AN18). Each biological sample was tested in unstimulated condition (medium only) and either dominant MHC-I-restricted (SIINFEKL, 2 µg/mL) or MHC-II-restricted (ISQAVHAAHAEINEAGR, 2 µg/mL) OVA peptides for 16 h. Wells were rinsed thoroughly and IFN-γ signal was revealed with a biotinylated IFN-γ-reactive detection mAb (clone R4-6A2), streptavidin-ALP and BCIP-NBT plus enzymatic substrate. Spot counts were performed automatically on the Immunospot software (ISQAVHAAHAEINEAGR and unstimulated groups) or on ImageJ when the number of dots was high (SIINFEKL group). Visual inspection of the spots masks confirmed that the sensitivity of spots detection was comparable within each group (Fig. S5D). The specific spot count for each peptide was defined as max(0, NSPOTS, PEPTIDE – NSPOTS, UNSTIMULATED) and statistically compared by non-parametric, one-tailed Mann-Whitney test.

Flow cytometry analysis of the immune infiltrate

At day 10, MCA205-bearing mice were euthanized and tumors were collected and dissociated to a single cell suspension by mechanical and enzymatic disruption, following the manufacturer’s instruction (tumor dissociation kit, Miltenyi Biotec, Cat# 130-096-730). Cells were stained with a viability staining (Live-Dead Fixable Yellow Dye, Invitrogen, Cat# L34967) then Fc receptors were blocked by an uncoupled anti-mouse CD16/CD32 antibody (BD BioSciences, clone 2.4G2) and fluorophore-coupled antibodies were added for the detection of surface markers (CD45-BUV661, RRID: AB_2870247, CD3-APC, RRID: AB_10597589, CD4-APC-Vio770, RRID: AB_2751634, CD8a-PE, RRID: AB_394570, ICOS-BV421, RRID: AB_2738576, GITR-BV786, RRID: AB_2740641, LAG3-BV605, RRID: AB_2742805, PD1-BUV395, RRID: AB_2742320, TIGIT-BV711, RRID: AB_2742063 and VISTA-PerCP-Cy5.5, RRID: AB_2561400). Cells were fixed and permeabilized (eBioscience FoxP3/Transcription Factor staining buffer, Thermo Fisher, Cat# 00-5523-00) prior to intranuclear staining (FoxP3-FITC, RRID: AB_465243). Fluorescence data were acquired on a BD LSRFortessa X20 with the BD FACS Diva software. Compensation, scaling, gating and data analysis were performed on the omiq.ai online platform. In addition to the classical cell populations, specific subpopulations of interest were defined among CD4+ and CD8+ T cells by performing unsupervised clustering in one of the three independent experiments. More specifically, the opt-SNE algorithm was used to perform dimension reduction, and then the FlowSOM algorithm was applied to split the clusters. Gating strategies were inferred from the markers expressed by the clusters of interest (differentially present between the CT + anti-PD1 and anti-ACBP/DBI + CT + anti-PD1) and applied to the pooled data from three independent experiments for statistical analysis (Figure S6). Statistical comparison was performed on pooled data from all experiments, after elimination of the outliers (ROUT test, Q = 1%), by one-way ANOVA with Sidak’s correction for multiple comparisons.

Single-cell RNA sequencing of the intratumoral T cell populationsSample preparation

MCA205 tumors were collected and homogenized following the same protocol as the one used for flow cytometry. The single-cell suspension was incubated in anti-mouse CD16/CD32 antibody (BD Biosciences, Cat# 553142, RRID: AB_394656), then stained with a fluorescent mix of 4’,6-diamidino-2-phénylindole (DAPI, viability) and fluorescent-labelled antibodies. T cells were sorted on a BD Aria III as the cells that were negative for all lineage markers (CD11c, RRID: AB_647251, Ly6C, RRID: AB_1727557, Ly6G, RRID: AB_1877261, F4/80, RRID: AB_2733261, NK1.1, RRID: AB_394507, CD19, RRID: AB_394495, all in the PE-Cy7 channel) and positive for CD45(-AF488, RRID: AB_493531) and either CD4(-PerCp-Cy5.5, RRID: AB_1107001) or CD8(-PE, RRID: AB_394571). After sorting, cells were counted and resuspended at a ratio of 1 CD4+ to 1 CD8+ T cell before loading a total of 10,000 cells to the Chromium Next GEM Chip K for emulsion. All subsequent steps, including retro-transcription, cleanup, cDNA amplification and libraries construction, were performed according to the Single cell 5’ VDJ v2 manufacturer’s instruction (10× Genomics, USA). Libraries were sequenced on Illumina NovaSeq 6000, with paired-end 150 bp and 28/90 bp runs for gene expression and T cell receptor sequencing.

Data pre-processing

Single-cell 5’ and V(D)J data analyses was performed by GenoSplice technology (www.genosplice.com). Sequencing data quality was assessed using FastQC v0.11.5 on 6 mouse expression and VDJ samples. For read alignment, unique molecular identifiers (UMI) quantification and paired clonotype calling, the CellRanger software v7.0.0 was used on Mus Musculus 2020 A reference (genome mm10, gene annotation Ensembl 98 and VDJ reference) with default parameters. Cellranger multi and aggregate function were used. The 6 expression matrices containing the UMI counts were merged, and only the genes with UMI ≥ 1 in at least one cell were kept. The following filters were applied to generate a global matrix used in further analysis: cells with UMI ≥ 1300, number of detected genes ≥ 800, and cells with UMI in mitochondrial genes ≤ 10%. In order to estimate and suppress ambient RNA, DecontX (Yang et al., 2020) R package was applied on counts data from cellranger. DoubletFinder (McGinnis, Murrow, & Gartner, 2019) with 10x expected percentage of doublets was used to suppress doublets (8% for C1, C2, D1 and D3, 7.60% for C3 and 6.09% for D2). Single-cell 5′ and V(D)J were integrated in a Seurat object.

Clustering

For normalization and clustering, Seurat 4.0.3 was used [32] and SCTransform normalization was applied. To avoid bias due to Tcell receptor genes, all Trav/Traj/Trbc/Trbv genes were suppressed from variable genes. Based on elbow plot, 31 PC were used for UMAP calculation and clustering analysis. Clustering step was performed using default parameters from Seurat (FindNeighbors and FindClusters functions). To calculate markers for each cluster, a global-scaling normalization method was applied with a scale factor of 10,000 and log-transformation of data. Only genes expressed in at least 25% of cells with a log2FC minimum of 0.25 an adjusted p-value inferior at 0.05 were considered as markers using Seurat Wilcoxon test. Based on a treemap of clusters with different resolution parameters and clusters markers, we chose a resolution parameter of 0.2. Low-frequencies clusters without Cd3g/d/e expression, which resulted from sorting approximations, were excluded for downstream analysis (clusters 9 and 11). Cluster 5 was also excluded due to low UMI count, and the remaining 9 clusters were numbered from 0 to 8 (largest to smallest number of cells).

Statistics

Differentially expressed genes (DEG) analysis was performed in each cluster using a Wilcoxon test from Seurat, genes expressed in at least 25% of cells in one condition, with a log2FC minimum of 0.25 an adjusted p-value inferior at 0.05 were considered. Single cell pseudo-time trajectory analysis was performed with monocle3 [33] independently on CD4+ cells and CD8+ cells. Root was defined based on a clear expression pattern of the naïve T -cells markers Sell, Ccr7 and Tcf7. Differences between cluster compositions were compared by chi-square test.