Functional T cells are capable of supernumerary cell division and longevity – Nature

Mice

Donor female B6.SJL-Ptprc^aPepc^b/BoyJ (CD45.1⁺ B6) and P14 CD8⁺ T cell transgenic mice were bred at the University of Minnesota animal facilities. Female C57BL/6J (CD45.2⁺ B6) mice were purchased from Jackson Laboratories and served as recipient mice, which were 8–10 weeks old at the time of first infection. Animals were housed with cycles of 14 h of light and 10 h of dark. Their environment was maintained at temperatures between 68 and 72 °F and humidity levels of 30–70%. Animals were treated according to the Institutional Animal Care and Use Committee guidelines and the protocols were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.

Viral infections

CD45.1⁺ tertiary memory cells were expanded through heterologous prime–boost–boost infection with 10⁶ plaque-forming units (p.f.u.) VSVnj, an experiment-specific period of rest, 2 × 10⁶ p.f.u. VVn, an experiment-specific period of rest, and 10⁷ p.f.u. of VSVind. Following transfer to recipient CD45.2⁺ mice, cells were expanded through heterologous prime–boost–boost infection with 10⁶ p.f.u. of VSVind, an experiment-specific period of rest, 2 × 10⁶ p.f.u. VVn, an experiment-specific period of rest, and 10⁷ p.f.u. VSVnj. All heterologous prime–boost–boost infections were delivered through the tail vein. For LCMV Armstrong infections, 2 × 10⁵ p.f.u. was delivered through an intraperitoneal injection. For LCMV clone 13, mice were given 200 μg of anti-mouse CD4 (GK1.5) from BioXCell through an intraperitoneal injection one day before and one day after an infection through the tail vein with 2 × 10⁶ p.f.u. of virus.

Tracking of ISTCs

At various time points post infections, blood was collected from the submandibular vein. Red blood cells were lysed using ACK lysis buffer, and blood cells were stained with a variety of antibodies that always included anti-mouse CD8a (53-6.7; 1:100) from BD Biosciences, anti-mouse CD44 (IM7; 1:200) from BioLegend, anti-mouse CD45.1 (A20; 1:400), Ghost Dye Red 780 (1:1,000) from Tonbo Biosciences, and major histocompatibility complex I tetramer with the H2Kb-binding RGYVYQGL peptide from VSVind nucleoprotein (N-tetramer; 1:200). Major histocompatibility complex tetramers were prepared as previously described⁴³. Flow cytometry data were collected on a BD LSR II, BD Fortessa or Cytek Aurora and analysed using BD FlowJo. After a cell transfer, values for ISTCs were calculated on the basis of the number of cells transferred and 10% survival whereas values for endogenous cells were based on the reported number of naive H2K^b/RGYVYQGL-binding cells in B6 mice⁴⁴.

Fluorescence-activated cell sorting and cell transfers

A single-cell suspension was prepared from the spleen and macroscopic lymph nodes of donor mice. In some cases, red blood cells were lysed with ACK lysis buffer before the samples were stained with surface antibodies. In other cases, CD8⁺ T cells were enriched through negative selection before they were stained with surface antibodies. CD8⁺ T cell enrichment was carried out using the Stem Cell EasySep Mouse CD8⁺ T Cell Isolation Kit following the manufacturer’s instructions or using a prepared cocktail of biotinylated antibodies. Briefly, single-cell suspensions were resuspended at 10⁸ cells per millilitre in phosphate-buffered saline (Gibco) supplemented with 2% heat-inactivated fetal bovine serum (Peak Serum) and 1 mM EDTA (Promega) and then incubated with 5% rat serum (Stem Cell) and 0.0275 mg ml⁻¹ anti-mouse CD4 (GK1.5) from Invitrogen and anti-mouse CD19 (1D3), anti-mouse CD11b (M1/70), anti-mouse NK1.1 (PK136), anti-mouse F4/80 (BM8.1), anti-mouse TER119, anti-mouse CD45R (RA3-6B2), anti-mouse LY6G (GR1) and anti-mouse CD16/32 (2.4G2) from Tonbo Biosciences. All antibodies were conjugated to biotin. After antibody incubation, antibody-bound cells were removed using the Stem Cell EasySep Mouse Streptavidin RaphidSpheres Isolation kit following the manufacturer’s instructions. Cells were then stained with anti-mouse CD8a (53-6.7; 1:200) from BD Biosciences, anti-mouse CD45.1 (A20; 1:200), anti-mouse CD45.2 (104; 1:200), N-tetramer (1:200) and Ghost Dye Red 780 (1:1,000) from Tonbo Biosciences. Live CD8a⁺VSV-N-tetramer⁺CD45.1⁺CD45.2⁻ cells were sorted on a BD FACS Aria II, and 10⁵ sorted cells were transferred through the tail vein into recipient mice. Infections resumed the following day.

Quantitative-PCR-based measurement of mouse telomere length

Telomere length was measured using quantitative PCR, including the primers (synthesized by Integrated DNA Technologies) and control gene as described previously³⁷. Data were collected using the QuantStudio 5 system (Applied Biosystems). Isolated M. spretus DNA was purchased from Jackson Laboratories to serve as a source of mouse DNA with relatively short telomeres.

CellTrace Violet labelling of cells

The cells of a single-cell suspension of cells isolated from the spleen and macroscopic lymph nodes were labelled with CellTrace Violet (Invitrogen) following the manufacturer’s instructions. CellTrace Violet-labelled cells were transferred into mice through the tail vein.

RNA-seq

Bulk RNA was isolated from 10⁵ sorted cells using the Qiagen RNeasy Plus Micro kit following the manufacturer’s instructions. Libraries were prepared using the Takara/Clontech Stranded Total RNA-seq pico input mammalian kit. Naive, 1°, 3° and 27° cells were prepared using kit version 1 and all other samples were prepared used kit version 2. Samples prepared with kit version 1 were sequenced on an Illumina HiSeq; samples prepared with kit version 2 were sequenced on the Illumina NovaSeq 6000. Quality of fastq files was assessed with FastQC. Adapters and low-quality segments were trimmed with Trimmomatic. Filtered reads were aligned to the mouse genome GRCm38 using Hisat2 and the count matrix was generated using featureCounts. All subsequent gene expression data analyses were carried out in the R software. Genes expressed at low levels were filtered using the filterByExpr function and TMM-normalized in edgeR. Differentially expressed genes were determined using limma. The RNA-seq samples were sequenced in two batches. Genes were considered significantly different if log[fold change] > 1 and false discovery rate < 0.05. Heatmaps were generated with the ComplexHeatmap package. We noticed a batch effect between the first and the second run of RNA-seq samples. However, differential gene expression analysis between the two naive groups revealed only few differentially expressed genes, most being undefined genes or ribosomal genes, indicating that the observed batch effect is not confounding our analysis.

Cell phenotyping through flow cytometry

Phenotyping was carried out on either blood cells or splenocytes that were treated with ACK lysis buffer. Cells were stained extracellularly with various combinations of: anti-mouse CD8a (53-6.7; 1:100), anti-mouse CD4 (GK1.5; 1:1,000), anti-mouse CD45.1 (A20; 1:400), anti-mouse CD122 (TM-β1; 1:100), anti-mouse CD62L (MEL-14; 1:800) and anti-mouse KLRG1 (2F1; 1:200) from BD Biosciences, anti-mouse CD44 (IM7; 1:200), anti-mouse CD38 (90; 1:100), anti-mouse CD45.1 (A20; 1:400), anti-mouse CD28 (E18; 1:100), anti-mouse PD1 (RMP1-30; 1:100), anti-mouse CD200R (OX-110; 1:50) and anti-mouse TIM3 (RMT3-23; 1:100) from BioLegend, anti-mouse CD45.1 (A20; 1:400), anti-mouse CD127 (A7R34) (1:100), anti-mouse CD45.2 (104; 1:200) and Ghost Dye Red 780 (1:1,000) from Tonbo Biosciences and either N-tetramer or the H2Db-binding KAVYNFATM peptide from lymphocytic choriomeningitis virus (GP33-tetramer), fixed and permeabilized using Tonbo Foxp3/Transcription Factor Staining Kit, and then stained intracellularly in Tonbo Permeabilization buffer with anti-mouse TOX (TXRX10; 1:50), and anti-mouse EOMES (Dan11mag; 1:50) from Invitrogen, anti-mouse BCL-2 (10C4; 1:50) from eBioscience, and anti-mouse TCF1/TCF7 (C63D9; 1:50) from Cell Signaling Technology. Flow cytometry data were collected on either a BD Fortessa or Cytek Aurora and analysed with BD FlowJo.

ATAC-seq

ATAC-seq was carried out following a previously described protocol⁴⁵. Library preparation on transposed DNA was carried out with a Nextera DNA library preparation kit following the manufacturer’s instructions. Samples were sequenced on an Illumina HiSeq. FastQC was used to assess the quality of fastq files. Reads were aligned to the mouse Genome (UCSC version mm10) using bowtie2 with valid alignment per read and allowed numbers of mismatches set to 1. Samtools was used to generate sorted bam files that contain mapped reads alone, and Picard was used to mark duplicates. All subsequent data analyses were carried out in the R software. Blacklist regions and mitochondrial reads were removed before peak regions were called with csaw using a window width of 200 base pairs. Windows were considered enriched over background if the log₂[fold change] was >3. As biological sample groups strongly differed in the enrichment of reads within accessible chromatin over background, log[c.p.m.] TMM-scaled peak counts were quantile-normalized as previously described⁴⁶. Differential accessibility analysis was carried out with limma, and peak regions were subsequently merged with a maximum distance between adjacent windows of 200 base pairs. Genomic regions were plotted using Gviz.

PD1 methylation analysis

Genomic DNA was isolated from the purified cells and subjected to bisulfite treatment using the Zymo EZ DNA methylation kit as per the manufacturer’s instructions. Bisulfite-modified DNA was PCR-amplified using PD1 promoter-specific primers⁴⁷. The amplicon was cloned into TA vector and transformed into bacteria. Vector from individual colonies was sequenced and analysed using the BISMA software (Bremen, Germany), as previously described⁴⁷.

In vitro cytokine stimulation

Blood cells were collected from the submandibular vein, red blood cells were lysed using ACK lysis buffer, and cells were resuspended in RPMI supplemented with 5% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U ml⁻¹ penicillin–streptomycin and 0.05 mM β-mercaptoethanol. Cells were added to the wells of a 96-well plate and medium containing brefeldin A from Tonbo Biosciences and various concentrations of RGYVYQGL VSV-N peptide synthesized by New England Peptide Inc (now named Vivitide) to yield a final concentration of 3 μM brefeldin A and labelled concentrations of peptide. After 4–5 h, cells were stained extracellularly with anti-mouse CD8a (53-6.7; 1:100) from BD Biosciences, anti-mouse CD45.1 (A20; 1:400), anti-mouse CD45.2 (104; 1:400) and Ghost Dye Red 780 (1:1,000) from Tonbo Biosciences and N-tetramer (1:200), fixed and permeabilized using Tonbo Foxp3/Transcription Factor Staining Kit, and then stained intracellularly in Tonbo Permeabilization buffer with TNF (MP6-XT22; 1:100) from BD Biosciences and IFNγ (XMG1.2; 1:100) from BioLegend. Flow cytometry data were collected on a BD Fortessa and analysed with BD FlowJo software.

Listeria protection

One day after transfer of 10⁵ specified sorted N-specific cells, mice were injected through the tail vein with 7 × 10³ colony-forming units of L. monocytogenes expressing VSV-N (LM-N). Five days after LM-N infection, spleens were removed and cells were lysed through homogenization in sterile 0.5% Igepal CA-630 (Sigma Aldrich). Various dilutions of cell homogenate were plated onto Petri dishes with BBL Brain Heart Infusion Agar (BD Biosciences) prepared according to the manufacturer’s instructions, incubated at 37 °C overnight and colonies were counted the next day.

Non-lymphoid tissue analysis

Non-lymphoid tissues were treated as previously described⁴⁸, including the use of 3 μg of an intravascular anti-CD8a antibody (53-6.7) from BD Biosciences as previously described⁴⁹ to prepare a single-cell suspension. Single-cell suspensions from each tissue were stained extracellularly with anti-mouse CD8a (53-6; 1:00) from BD Biosciences, anti-mouse CD45.1 (A20; 1:200) and Ghost Dye Red 780 (1:1,000) from Tonbo Biosciences, anti-mouse CD69 (H1.2F3; 1:50) from Invitrogen and N-tetramer (1:200). Flow cytometry data were collected on a BD Fortessa and analysed with BD FlowJo software.

Data measurements

Cells that were tracked over time were measured repeatedly after different infections with discreet measurements made on populations of cells within different mice.

Statistical methods

No statistical test was used to determine sample sizes. Mice were randomly assigned to different experimental groups. Researchers were not blinded to experimental groups. Specific statistical tests used to determine significance, group sizes (n) and P values are provided in the figure legends. P value < 0.05, significant. All statistical analysis was carried out using Prism (GraphPad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.