Functional T cells are capable of supernumerary cell division and longevity

Abstract

Differentiated somatic mammalian cells putatively exhibit species-specific division limits that impede cancer but may constrain lifespans^1-3. To provide immunity, transiently stimulated CD8⁺ T cells undergo unusually rapid bursts of numerous cell divisions, and then form quiescent long-lived memory cells that remain poised to reproliferate following subsequent immunological challenges. Here we addressed whether T cells are intrinsically constrained by chronological or cell-division limits. We activated mouse T cells in vivo using acute heterologous prime-boost-boost vaccinations⁴, transferred expanded cells to new mice, and then repeated this process iteratively. Over 10 years (greatly exceeding the mouse lifespan)⁵ and 51 successive immunizations, T cells remained competent to respond to vaccination. Cells required sufficient rest between stimulation events. Despite demonstrating the potential to expand the starting population at least 10⁴⁰-fold, cells did not show loss of proliferation control and results were not due to contamination with young cells. Persistent stimulation by chronic infections or cancer can cause T cell proliferative senescence, functional exhaustion and death⁶. We found that although iterative acute stimulations also induced sustained expression and epigenetic remodelling of common exhaustion markers (including PD1, which is also known as PDCD1, and TOX) in the cells, they could still proliferate, execute antimicrobial functions and form quiescent memory cells. These observations provide a model to better understand memory cell differentiation, exhaustion, cancer and ageing, and show that functionally competent T cells can retain the potential for extraordinary population expansion and longevity well beyond their organismal lifespan.

Extended Data Fig. 1 |. Iteratively stimulated T cells populate non-lymphoid tissues.

a) CD45.1+ 48° ISTC memory CD8 T cells were transferred to naive mice, followed by three heterologous prime-boost-boost immunizations. Non-lymphoid tissues were analyzed 48 days after the 51° boost. Flow cytometry plots are gated on live CD8a+ lymphocytes that were not stained by intravascular in vivo antibody labeling. b) CD69 expression on endogenous 3° cells (CD45.1-) and 48° memory CD8 T cells (CD45.1+).Plots concatenated from four mice and experiment is representative of three similar experiments with similar results.

Extended Data Fig. 2 |. Gene set enrichment analysis of iteratively stimulated CD8 T cells.

Gene set enrichment analysis was performed on differentially expressed genes between 45° ISTCs and primary memory cells using the category “Biological process” of the Gene Ontology database. The top 20 upregulated (activated) and top 20 downregulated (suppressed) pathways in ISTCs are shown. Statistical significance was determined using Over-Representation analysis.

Extended Data Fig. 3 |. A single naive CD8 T cell shows the potential to produce >10⁴⁰ memory cell progeny.

a) Schematic showing estimate of cell expansion potential after 51 HPBB immunizations. HPBB: three successive Heterologous Prime, Boost, Boost immunizations. b) Schematic comparing the volume of earth with the theoretical volume of memory T cells implied by the calculated proliferation potential of a naive CD8 T cell.

Fig. 1 |. CD8⁺ T cells can undergo seemingly unlimited bursts of proliferation if rested between stimulations.

a, CD45.1⁺ VSV/N_52–59-specific memory CD8⁺ T cells were iteratively sorted, transferred to CD45.2⁺ mice, and then boosted three times per mouse with 60+ days between each of three heterologous immunizations. The graph shows the percentage of total lymphocytes in blood that are comprised of transferred CD45.1⁺ iteratively boosted cells (red) or newly generated CD45.2⁺ recipient H-2K^b/N_52–59-specific cells (blue). N-tet⁺ = H-2K^b/N_52–59-tetramer. b,c, As in a, except boosting was carried out at 28–34-day (b) or 7-day (c) intervals. Similar results were observed in trailing cohorts that have undergone 13, 21 or 31 (≥60-day interval), or 15 stimulations (28–34-day interval). c is representative of two experiments with similar results with n = 4 at 3° and n = 3 at 6° and 9°. Error bars show average and s.e.m. For flow cytometry gating strategies, see Supplementary Fig. 1. For exact number of mice at each time point in a,b, see Supplementary Table 1.

Fig. 2 |. Iteratively boosted T cells maintain telomere length, cell cycle control and durability.

a, Naive, 3° memory or 33° memory CD8⁺ T cells were sorted, and then tested for telomere length by quantitative PCR and compared to Mus spretus reference DNA. b, 3° and 48° cells were labelled with CellTrace Violet division-tracking dye, and then transferred to recipients without further boosting. Primary lymphocytic choriomeningitis virus (LCMV)-specific P14 memory CD8⁺ T cells were transferred for comparison. Cumulative cell divisions, indicated by dye dilution, were evaluated in spleen 34 days later. c, 45° and endogenous 3° memory CD8⁺ T cells were tracked in blood for 173 days following infection. a–c are representative of two experiments with similar results, n = 2 (a), n = 4 (b), n = 5 (c). Error bars show average and s.e.m. For flow cytometry gating strategies, see Supplementary Fig. 2.

Fig. 3 |. Iterative boosting induces progressive changes in gene and protein expression and acquisition of exhaustion markers.

a, RNA-seq was carried out on naive and N-specific memory CD8⁺ T cells that had experienced progressive boosts. Shown is a heatmap of the genes that were previously reported to be uniquely expressed by exhausted CD8⁺ T cells. b,c, The phenotype of naive and various generations of H-2K^b/N_52–59-specific memory cells isolated from blood was assessed by flow cytometry. All samples were run on the same day from staggered cohorts. The right column in c indicates days after last boost. a, n = 3 per group. b,c, Representative of n = 3 (7°), n = 4 (3°, 12°, 19° and 31°) or n = 9 (51° and naive), from at least two independent experiments with similar results. For flow cytometry gating strategies, see Supplementary Fig. 3.

Fig. 4 |. Transcriptional, epigenetic and functional profiling distinguishes ISTCs from exhausted T cells.

a, 18° CD45.1⁺ ISTCs were transferred to naive mice before a single immunization with VSVind. 317 days later, ISTCs and endogenous VSV/N_52–59-specific primary memory and naive CD8⁺ T cells were assessed for maintenance of PD1 expression in blood. b,c, Methylation (b) or chromatin accessibility (c) of the Pdcd1 locus (encoding PD1) was evaluated in naive, 3° or ISTC cells by bisulfite sequencing or assay for transposase-accessible chromatin using sequencing (ATAC-seq), respectively. Pdcd1 RNA as measured by RNA-seq is also shown. Mb, megabase. d, Gene expression modules that are shared or unshared with genes reported to be upregulated or downregulated by exhausted CD8⁺ T cells (Tex). e, Spleens with GP33-specific cells induced by chronic LCMV clone 13 (Cl13) or ISTCs were analysed by flow cytometry. f, 3° or 48° cells were labelled with CellTrace Violet division-tracking dye and transferred to recipients that were then infected with VSVind. The plots show cell division indicated by CellTrace Violet dilution 16 days after booster immunization. g, IFNγ and TNF staining 4 h after peptide stimulation. Representative flow cytometry (left panel) and response to peptide titration (right graphs). h, ISTCs or endogenous VSV/N_52–59-specific 3° memory cells were transferred to naive mice, which were then infected intravenously with Listeria monocytogenes expressing VSV-N (LM-N), and then assessed five days (5 d) later for bacterial burden in spleen. c.f.u., colony-forming units; LOD, limit of detection. n = 3 (a,c,d,g), n = 4 (b,e), n = 4 (4°) or 5 (49°) (f), n = 9 (3°) or 10 (no transfer and 33°) (h). a,e–g are representative of ≥2 similar experiments, b–d show data from a single experiment. g shows data combined from two experiments. Ordinary one-way analysis of variance with Dunnett’s multiple-comparison test was used on log₁₀-transformed data to test for significance between groups; **P = 0.0086 (for 3°) or P = 0.0028 (for 33°). Error bars show average and s.e.m. For flow cytometry gating strategies, see Supplementary Fig. 4. See Supplementary Information 3 for full statistical test details.