Full sequences of the DNA oligos used are supplied in Supplementary table 4. In order to prevent expression of Cre recombinase during bacterial cloning, a synthetic intron—containing a splice donor, a branch site, a pyridine rich region, and a splice acceptor— was inserted into the Cre gene through three-fragment isothermal assembly. The synthetic intron was produced as two overlapping DNA fragments (1st: CTGGTTATGCGGCGGAAGGTAAGTCAGTAGGCCATGTTAGAGGGTGGCCCAGGTATTGA, 2nd: TGGCCCAGGTATTGACCTATTTCCACCTTTCTTCTTCATCCTTAGATCCGAAAAGAAAAC, IDT). Subsequently, a pCDH plasmid containing Cre recombinase was digested with BamHI to split the Cre gene (at basepair position 353), and the resultant linear plasmid (100 mM) was assembled together with the synthetic fragments. To prevent low level Cre translation occurring from alternative start sites, two ATG codons (position 78 and 84) were replaced by TGT codons. Finally, the Cre start codon was replaced by an EcoRI-spacer-XhoI site, to facilitate subsequent introduction of synthetic STRs. To generate the DivisionRecorder vector, two lox511 sites were introduced into the multiple cloning site of the pMX retroviral vector. Subsequently, an eGFP gene and the modified Cre recombinase gene were introduced directly upstream and downstream of the 5’ lox511 site, respectively. Finally, a P2A element was inserted directly in between the eGFP gene and the 5’ Lox511 site. Together, this resulted in a cassette comprising from 5’ to 3’: Kozak, an eGFP gene, a P2A site, a lox511 site, an EcoRI restriction site, spacer, an XhoI restriction site, a Cre recombinase gene, and a lox511 site. In its base configuration, Cre recombinase is out of frame. Synthetic STR domains were ordered as oligonucleotides (Invitrogen) and subsequently dimerized. STR dimers were inserted via the EcoRI and XhoI sites, sequences of STR oligonucleotides are supplied in Supplementary Table 4.
Cre-activity reporter vector generation
LoxP sites were introduced into the multiple cloning site of the pCDH-CMVp-MCS-PGK-BlastR vector through two sequential rounds of digestion/ligation. First, the 3’ loxP site was introduced using EcoRI and BamHI restriction sites. The introduced region included a short spacer sequence and a XhoI site directly upstream of the LoxP. Second, the 5’ loxP site was introduced using the XbaI and EcoRI restriction sites. This resulted in a modified MCS, comprising XbaI-loxP-EcoRI-spacer-XhoI-loxP-BamHI. Next, a scrambled non-sense open reading frame, containing multiple stop codons, was introduced into the EcoRI and XhoI sites. Finally, a katushka open reading frame was introduced via the BamHI site. This resulted in a vector containing from 5’ to 3’; The CMV promoter, a floxed scrambled open reading frame, a Katushka open reading frame, the PGK promoter, and a blasticidin resistance gene.
Establishment of cell lines
The Cre-activity reporter cell line (Fig. S1) was generated by retroviral transduction of HEK 293T cells with the Cre-activity reporter plasmid. Successfully transduced cells were selected using 2 µg/ml Blasticidin (InvivoGen). Transduced cells were subsequently seeded at 1% confluency, and resulting single cell-derived colonies were transferred to individual wells. Clones were then examined for efficiency of induction of Katushka expression upon transfection with Cre recombinase, and the best-performing clone was selected. Cre-activity reporter cells were cultured in IMDM (Gibco) supplemented with 8% fetal calf serum (FCS, Sigma), 100 U/ml penicillin (Gibco), 100 µg/ml streptomycin (Gibco) and 2 mM Glutamax (Gibco).
A mouse embryonic fibroblast (MEF) cell line from the Ai9 mouse strain was generated by modification of E14.5 embryonic fibroblasts with a retroviral vector encoding short-hairpin RNA directed against the p53 mRNA. Resultant cells were cultured in IMDM supplemented with 8% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM Glutamax.
C57BL/6J-Ly5.1, OT-I and Ai9 mice were obtained from Jackson Laboratories, and strains were maintained in the animal department of The Netherlands Cancer Institute (NKI). Ai9 and OT-I mice were crossed to obtain the Ai9;OT-I strain. All animal experiments were approved by the Animal Welfare Committee of the NKI, in accordance with national guidelines.
Generation of DivisionRecorder+ OT-I T cells
Platinum-E cells cultured in IMDM supplemented with 8% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM Glutamax were transfected with the DivisionRecorder vector using FuGeneTM6 (Roche). Retroviral supernatant was harvested 48h after transfection and stored at -80°C. Spleens from Ai9;OT-I mice were harvested and mashed through a 70 µm strainer (Falcon) into a single cell suspension and resulting splenocytes were subsequently treated with NH4Cl to remove erythrocytes. Subsequently, splenocytes were cultured in T cell medium (RPMI (Gibco Life Technologies) with 8% FCS, 100 U/ml penicillin, 100 ug/ml streptomycin, Glutamax, 10mM HEPES, MEM Non-Essential Amino Acids (Gibco), 1mM Sodium pyruvate (Gibco), 50µM 2-mercaptoethanol), supplemented with 1ng/ml recombinant murine IL-7 (PeproTech) and 2 µg/mL ConcanavalinA (CalBiochem). After 48h, splenocytes were re-seeded on RetroNectin (Takara) coated plates in T cell medium supplemented with 60IU/mL human IL-2 and DivisionRecorder virus, and were centrifuged for 90min at 400g to allow spinfection. virus concentration was chosen such that a transduction efficiency of approximately 10-15% was achieved, in order to minimize the occurrence of multiple retroviral integrations. Cells were harvested 24h later and a small aliquot was stained with anti-CD8-PercpCy5.5, anti-Vb5-PeCy7, anti-CD45.2-AF700 and DAPI to determine the fraction viable OT-I T cells (DAPI-CD8+Vb5+CD45.2+) by flow cytometry (Fortessa, BD Bioscience), which generally was around ~80%. CD8+Vb5+CD45.2+ that expressed GFP were considered as DivisionRecorder+ OT-I cells. Within the initial population of DivisionRecorder+ OT-I cells the fraction of cells that already showed reporter activation (as inferred by tdTomato expression) 24h after transduction was consistently between 0.4 and 0.8%. Activated splenocytes were prepared for adoptive transfer (see below).
Infection, adoptive transfer and cell recovery
C57BL/6J-Ly5.1 mice were infected with 5,000-10,000 CFU of a recombinant Listeria monocytogenes strain that expresses ovalbumin or with 5,000 PFU artLCMV-OVA25, kindly provided by Doron Merkler, University of Geneva. Approximately 24h later, infected mice received 5,000-40,000 DivisionRecorder+ OT-I T cells through intravenous tail vein injection. To analyze OT-I T cell responses in peripheral blood over time, 25-50 mL blood samples were obtained from the tail vein at the indicated time points, and were treated with NH4Cl supplemented with 0.2 mg/ml grade-II DNaseI (Roche) to remove erythrocytes (see Methods, Flow Cytometry). To obtain spleen and liver samples, mice were sacrificed, organs were harvested, and single cell suspensions were prepared by means of mashing through a 100µM or 70µm strainer (Falcon), respectively. Subsequently, erythrocytes were removed by treatment with NH4Cl. To purify leukocytes from single cell suspensions of liver tissue, cell suspensions were separated over a 37.5% Percoll (Sigma) density gradient. Following Percoll density separation, the pelleted cells consisted predominantly of leukocytes, while hepatocytes precipitated at the interphase. Obtained blood, spleen and liver samples were further processed for flow cytometric analysis, scRNA-sequencing or functional in vitro assays, as indicated.
Validation of DivisionRecorder functionality
To assess the ability of the DivisionRecorder as represented in Fig. 1D to faithfully report on the replicative history of T cell populations, an alternative experimental set-up was employed. First, splenic CD8+ T cells were isolated using the Mouse CD8 T Lymphocyte Enrichment Set (BD Biosciences) and were subsequently stained with CellTraceä Violet (Thermofisher). Next, cells were activated for 16h in T cell medium supplemented with 0.05 µg/mL SIINFEKL peptide and 60 IU/mL IL-2. Following this activation step, cells were seeded onto RetroNectin® (Takara Bio) coated plates and were transduced with DivisionRecorder virus by spinfection for 4h in the presence of IL-2 and SIINFEKL peptide. Analysis of CellTraceä Violet signal by flow cytometry indicated that the cells had not undergone a full cell division post labeling. Subsequently, 6x106 OT-I T cells were transferred into Lm-OVA infected recipients. Spleens were harvested 48h after adoptive transfer, processed into single cell suspensions and prepared for flow cytometric analysis. In order to reliably determine the fraction of DRRFP cells per division during the initial stages of the proliferative burst, analysis of a large number of DivisionRecorder+ OT-I T cells events is required. For this reason, a transduction efficiency of ~60% was chosen in these experiments, instead of the 10-15% transduction efficiency used in other experiments. Note that a high transduction efficiency will result in the more frequent occurrence of cells that carry multiple retroviral integrations. The presence of cells with multiple integrations will result in a higher, yet stable, DRRFP acquisition rate, as compared to the experimental set-up used in the remainder of the study.
Ex vivo analysis of degranulation and cytokine secretion potential of memory T cells
Spleens were harvested from recipient mice at >60 days post-infection, and CD8 T cells were isolated using the Mouse CD8 T Lymphocyte Enrichment Set (BD Biosciences). Following isolation, T cells were plated at 106 cells per well in 96-well U bottom plates in T cell medium supplemented with 0.05 µg/mL SIINFEKL peptide to selectively activate OVA-specific T cells. Following a 4hr incubation, capacity of indicated T cell populations to either produce the indicated cytokines or to degranulate was assessed. To allow analysis of cytokine production, Brefeldin A (GolgiPlugä, BD Biosciences) was added 30 minutes after initiation of T cell stimulation. To allow analysis of degranulation, T cell medium was supplemented with anti-CD107a and anti-CD107b antibodies at the initiation of T cell stimulation, and Brefeldin A (GolgiPlugä, BD Biosciences) and Monensin (GolgiStopä, BD Biosciences) were added 30 minutes after initiation of T cell stimulation. At the end of the T cell stimulation period, cells were stained for KLRG1 and CD27 and prepared for flow cytometric analysis (see below).
Flow cytometric analysis
Cells were taken up in PBS (Gibco) supplemented with 0.5% bovine serum albumin (BSA, Fisher Scientific), and stained with antibodies directed against the indicated cell surface proteins (1:200 dilution), for 30min on ice. To allow detection of intracellular cytokine production, cells were fixed and permeabilized with CytoFix/CytPermä (BD Biosciences) according to the manufacturer’s protocol and subsequently stained using antibodies against IL-2, TNFa and IFNg. To detect intranuclear Ki-67 expression, the Foxp3/Transcription factor Staining buffer set (eBioscience) was used. See Supplementary table 6 for specifics on all antibodies used. All samples were acquired on a BD LSR Fortessaä (BD Bioscience); DRGFP and DRRFP cells were identified as CD8+Vb5+CD45.2+GFP+tdTomato- and CD8+Vb5+CD45.2+GFP+tdTomato+, respectively. Flow cytometry data analysis was performed using FlowJo V10 (See Supplementary table 5).
For the moving average analysis depicted in Fig3A and Fig S2, CD8+Vb5+CD45.2+GFP+ events were exported and further processed using the R package FlowCore47. In brief, outlier events (i.e. antibody aggregates/cell doublets) were removed, fluorescence intensities of each of the cell surface proteins were normalized using an inverse hyperbolic sine transformation and subsequently scaled between 0 and 1. To obtain the depicted moving averages, the fraction of DRRFP cells was calculated within windows that each contained 10% of total cells, starting with the 10% of cells with the lowest expression levels for the indicated marker, and with subsequent windows moving up by steps of 2.5%.
Single cell RNA sequencing and data analysis
Spleens of DivisionRecorder+ OT-I T cell recipient mice (n=3) were harvested >85 days post-infection. Splenocytes were stained with fluorochrome-conjugated antibodies directed against CD8, CD45.2 and Vβ5 (See Supplementary table 5), to allow purification of DivisionRecorder+ cells by FACS. In addition, to infer surface protein abundance, splenocytes were stained with barcode-labeled TotalSeqä antibodies directed against CD27 (TotalSeq-A0191, Biolegend) and KLRG1 (TotalSeq-A0250, Biolegend). Following the isolation of DRGFP and DRRFP memory T cells by FACS (FACSAria Fusion, BD Biosciences), obtained cell populations were barcode-labeled with distinct anti-mouse TotalSeqä Hashtag antibodies (TotalSeq-A0301, TotalSeq-A0302, TotalSeq-A0303, TotalSeq-A0304, TotalSeq-A0305, TotalSeq-A0306, Biolegend), and mixed 1:1, with an equal number of cells from each mouse to form the total pool of cells for scRNA-sequencing. Single-cell RNA isolation and library preparation was performed according to the manufacturer’s protocol of the 10X Genomics Chromiumä Single Cell 3’ kit, and the cDNA library was sequenced on two separate NextSeqTMruns. Accumulating data of the two individual runs, tallied to a total of ~7x108 reads, and resulted in the detection of ~15,000 cells with a median of 2,143 detected genes per cell. Feature-barcode matrices were generated using the Cell Ranger software of the 10X Genomics Chromiumä pipeline. Cells that could be ascribed to multiple mice or to no mouse (inferred from the detection of multiple or no Hastags), cells with a transcript (UMI) count lower than 2,000 and cells with a gene count higher than 3,000 genes, or a mitochondrial gene fraction higher than 0.1 were excluded from downstream analysis. Subsequent transcriptional profiling of the remaining 11,767 cells was performed using the Seurat48 and MetaCell29 algorithms.
UMI counts derived from the anti-CD27 and anti-KLRG1 TotalSeq antibodies were used to classify cells as either TCM or TEM. Thresholds used to assign cells were set manually, guided by the expression profile measured by flow cytometry. TCM were defined as KLRG1LOCD27HI, TEM were defined as KLRG1HICD27LO. Remaining cells were unclassified.
To examine enrichment or depletion of DRRFP cells within the different MetaCells, cell counts were first normalized across hashtags. Data obtained from the different mice were subsequently aggregated and used to calculate the ratio of DRRFP versus DRGFP cells in each MetaCell.
The immune signature gene list (Fig 4D and F) was composed of gene clusters involved, or proposed to be involved in T cell function. The full gene list is described in Supplementary table 6.
The core TEFF signature was generated by selecting genes that were present in at least 2 out of 7 TEFF gene signatures downloaded from the GSEA database (M9475, M5836, M5820, M3041, M3039, M3027 and M3013; resulting list in Supplementary table 3). To determine the shared upregulated genes of both highly and lowly divided cells, the top 1,000 enriched genes from the 3 highest and 3 lowest DRTOM/DRGFP MetaCells were selected. Shared upregulated genes were defined as genes that appeared in the top 1,000 of at least 2 out of 3 MetaCells. Shared downregulated genes were defined similarly, using the top 1,000 depleted genes of each MetaCell.
All statistical analyses were performed either with R (V3.6.1, ‘Action of the Toes’) or Graphpad (V8.4.1, Prism software). All data shown is representative of at least 2 independent experiments.