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Replicative history marks transcriptional and functional disparity in the CD8+ T cell memory pool

An Author Correction to this article was published on 23 May 2022

This article has been updated

Abstract

Clonal expansion is a core aspect of T cell immunity. However, little is known with respect to the relationship between replicative history and the formation of distinct CD8+ memory T cell subgroups. To address this issue, we developed a genetic-tracing approach, termed the DivisionRecorder, that reports the extent of past proliferation of cell pools in vivo. Using this system to genetically ‘record’ the replicative history of different CD8+ T cell populations throughout a pathogen-specific immune response, we demonstrate that the central memory T (TCM) cell pool is marked by a higher number of prior divisions than the effector memory T cell pool, owing to the combination of strong proliferative activity during the acute immune response and selective proliferative activity after pathogen clearance. Furthermore, by combining DivisionRecorder analysis with single-cell transcriptomics and functional experiments, we show that replicative history identifies distinct cell pools within the TCM compartment. Specifically, we demonstrate that lowly divided TCM cells display enriched expression of stem-cell-associated genes, exist in a relatively quiescent state, and are superior in eliciting a proliferative recall response upon activation. These data provide the first evidence that a stem-cell-like memory T cell pool that reconstitutes the CD8+ T cell effector pool upon reinfection is marked by prior quiescence.

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Fig. 1: DivisionRecorder activation is a proxy for replicative history.
Fig. 2: The DivisionRecorder can be applied to study T cell division kinetics in vivo.
Fig. 3: The multipotent TM cell pool is formed by replicative ‘mature’ cells.
Fig. 4: Replicative history identifies distinct transcriptional states within the TCM pool.
Fig. 5: Replicative history is linked to recall potential within the TCM pool.
Fig. 6: The secondary TEFF pool is predominantly generated by previously quiescent memory T cells.
Fig. 7: Modeled T cell responses are consistent with the presence of a replication-competent quiescent TCM population.

Data availability

Transcriptomic data presented in the manuscript have been deposited to the Gene Expression Omnibus (GEO), and can be accessed under the GEO accession numbers GSE169154 and GSE184947. The gp33-specific P14 T cell scRNAseq dataset was retrieved from GEO (accession GSE131847, sample GSM3822202). All statistical source data of the figures presented in the present study are provided with this paper. Indicated gene sets used in gene set enrichment analyses were retrieved from the Molecular Signatures Database (MSigDB) at http://www.gsea-msigdb.org/gsea/msigdb. Any additional data supporting the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

R scripts that were used to produce the main and extended data figures in the manuscript are available from GitHub (https://github.com/kasbress/DivisionRecorder_analysis).

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Acknowledgements

We would like to thank M. C. Wolkers (Sanquin, Amsterdam), C. Gerlach (Karolinska Institute, Stockholm), and K. van Gisbergen (Sanquin, Amsterdam) for helpful discussions regarding experimental procedures and sharing biological material, and D. Merkler (University of Geneva, Geneva) for kindly providing the artLCMV-OVA. In addition, we would like to thank the NKI Genomics Core Facility and Flow Cytometry Core Facility for providing experimental support. This work was supported by ERC AdG Life-his-T (Grant agreement ID: 268733) to T.N.S. and an NWO grant (ALWOP.265) to R.J.d.B.

Author information

Authors and Affiliations

Authors

Contributions

The study was designed by K.B., L.K., F.A.S. and T.N.S., and was supervised by T.N.S. and F.A.S.; K.B. and L.K. jointly performed, analyzed, and visualized all experimental work included in the manuscript; F.A.S. and K.B. designed and developed the retroviral DivsionRecorder construct. L.A.K. and L.J. performed optimization and validation experiments integral to the design of the DivisionRecorder; A.C.S. and R.J.d.B. performed mathematical modeling, together with T.S.W., L.P. and K.R.D.; K.B. and L.K. wrote the manuscript with the input of co-authors; T.N.S. and F.A.S. critically reviewed and revised the manuscript.

Corresponding authors

Correspondence to Ferenc A. Scheeren or Ton N. Schumacher.

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The authors declare no competing interests.

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Nature Immunology thanks Mohamed Abdel-Hakeem and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Simulation of different scenarios of memory T cell formation.

Simulated data depicting a responding antigen-specific T cell population (blue), comprised of TEFF undergoing clonal expansion and subsequent contraction (red), plus memory precursor T cells (MP, green) that develop into TM. Activated TEFF are modeled to divide rapidly for 6 days (expansion phase), die at a fixed rate throughout the response, and can differentiate into MP cells only during the expansion phase. Cell numbers (top row) and DRRFP percentages (bottom row) are shown for 3 scenarios: (left) TEFF can give rise to MP cells during the entire expansion phase, irrespective of the number of prior divisions, (middle) only TEFF that have gone through at most 24 divisions can give rise to MP cells, or (right) only TEFF that have gone through at most 10 divisions can give rise to MP cells. Note the strong decay in DRRFP percentage that is observed during memory formation in case T cell memory is founded by T cells that have undergone few divisions. See Supplementary Note 3 for detailed description and equations.

Extended Data Fig. 2 Evaluation of the division history of T cell subsets throughout a response to Lm-OVA.

a, Gating strategy used to identify indicated TM populations (d86) in spleen samples. b, DRRFP percentages within splenic TM populations (n = 6 mice) as identified in panel a. c, DRRFP percentages within the CD27HIKLRG1LO TCM subset in spleen and lymph nodes (LN) and within the CD27LOKLRG1HI TEM subset in spleen. d, Cell surface expression of CX3CR1, CD62L, and CD43 within splenic CD27LOKLRG1HI and CD27HIKLRG1LO populations at the peak of the TEFF phase (day 6 post infection) and in memory phase (day 86 post infection). e, Moving-average of surface marker expression of splenic DR+ OT-I T cells during effector phase (day 6), depicted as in Fig. 3g. f, Boxplots depicting DRRFP percentages within TEFF (day 6 post infection) subsets in spleen (n = 6 mice), relative to the total DRRFP percentage. g, Kinetics of DRRFP percentages within CD27LOKLRG1HI (left) and CD27HIKLRG1LO (right) DR+ OT-I T cell populations in blood. Values are relative to the percentage of DRRFP cells detected at the peak of the response (day 6). Grey lines represent individual mice (n = 22), red and blue lines indicate group mean. h, Simulation of the phenotype model (See Supplementary Note 5 for details) illustrating a scenario in which conversion of CD27HIKLRG1LO to CD27LOKLRG1HI cells occur only after the peak of the response at a low rate. Depicted are the overall cell numbers (left), and the percentage DRRFP cells of DR+ OT-I T cells (right) in CD27HIKLRG1LO cells (blue), CD27LOKLRG1HI cells (red) and the total T cell population (green). Note that in this scenario the fraction DRRFP within the terminally differentiated CD27LOKLRG1HI population would increase to almost twice the experimentally observed frequency. All depicted data are representative of at least two independent experiments. Boxplots (c, d, g) represent group median and 25th/75th percentiles, whiskers indicate the interquartile range multiplied by 1.5 (c, d) or min/max (g), dots indicate individual samples. P values were determined by one-way ANOVA followed by Tukey’s HSD post-hoc test (c and d), two-sided Student’s T test (c), two-sided repeated measurement correlation test (h), or two-sided Friedman test (g). All significant (< 0.05) P values are indicated in the plots.

Source data

Extended Data Fig. 3 Single cell mRNA sequencing of DivisionRecorder+ and unmodified memory T cells.

Single cell mRNA sequencing was performed on DivisionRecorder modified and unmodified OT-I memory T cells (Day 75 and 85 post Lm-OVA infection), isolated from spleens (n = 7 mice with DR + memory T cells; n = 4 with unmodified memory T cells). Obtained data were aggregated from two independent experiments (Experiment 1: M13; Experiment 2: M4-11). All cells were jointly analysed and clustered. a, Cell count per sample. b, Total cell count per MC. c, Sample composition of each MC. d, Relative contribution of DRGFP and DRRFP to the total DR+ pool within each MC.

Source data

Extended Data Fig. 4 TCM transcriptional states are preserved in DR+ OT-I T cells.

Comparison of transcriptional states of splenic memory T cells generated by either DivisionRecorder modified, or unmodified OT-I T cells (Day 75 and 85 post Lm-OVA infection). a-b, Memory OT-I T cells cluster into TCM (blue) and TEM (red). 2D projection colored by subset (a), and violin plots depicting normalized UMI counts of selected genes (b) are shown. c, 2D projection of either DR+ (left) or unmodified (right) memory OT-I T cells. d, Contribution of DR+ and unmodified memory T cells to the TCM and TEM subsets. e, Contribution of DR+ and unmodified OT-I T cells to the 19 MCs that jointly make up the TCM subset. Dots indicate individual mice (n = 3 per condition). Note that all TCM states are generated in near-equal proportions by DR+ and unmodified memory T cells. Depicted scRNAseq data was obtained from 6 individual mice, and was aggregated from 2 independent experiments. P values were determined by two-sided Student’s T test followed by Bonferroni correction for multiple testing (d and e). P values < 0.05 are indicated.

Source data

Extended Data Fig. 5 Replicative history identifies distinct transcriptional states within the TCM pool.

Single cell transcriptomic profiling of DR+ T cells obtained from spleen in memory phase (Day 75 and 85 post Lm-OVA infection). a, Log2 enrichment of selected genes in each MC cluster. Boxplots indicate group median and 25th/75th percentiles, whiskers indicate the interquartile range multiplied by 1.5, dots signify individual MCs. The phenotype clusters TEM, TCM(eff.) and TCM(mult.) contain 4, 9 and 10 MCs, respectively. For definition of TCM(eff.) and TCM(mult.), see Fig. 4b. b, Top and bottom marker genes of ldTCM (Top, MC2, 11, 14) and hdTCM (Bottom, MC6, 8, 18), see Fig. 4d for ldTCM and hdTCM definitions. c, Heatmaps depicting z-score transformed enrichment values of genes related to cell survival (left), cytotoxicity and effector function (middle), inhibitory markers (top-right), and transcription factors involved in T cell multipotency (bottom-right). Expression is depicted for the 3 ldTCM and 3 hdTCM MCs. d, Volcano plot depicting differentially expressed genes in ldTCM versus hdTCM. Significantly (adjusted P value < 0.05) differentially expressed genes are depicted in red. Selected genes are highlighted. e, Cytokine release of CD27HIKLRG1LO DR+ T cells (isolated from spleen at day >60 post infection) 4 hours post ex vivo stimulation. Percentage DRRFP cells within cytokine producers (+) and non-producers (-), relative to the average DRRFP percentage within each sample, is depicted. Lines connect individual ex vivo stimulated samples samples (n = 12), obtained from 3 mice. f, Ex vivo degranulation of CD27HIKLRG1LO DR+ T cells (isolated from spleen at day >60 post infection) 4 hours post ex vivo stimulation. Percentage DRRFP cells within the CD107a/b positive (+) or negative (-) cell populations is depicted. Lines connect individual samples ex vivo stimulated samples (n = 17), obtained from 5 mice. g, Enrichment of gene signatures from MSigDB (Hallmark) by gene set enrichment analysis comparing ldTCM and hdTCM. Data depicted was accumulated in two independent experiments (3-4 mice per experiment). P values were determined by Tukey’s HSD test (a), Wilcoxon Rank Sum test with Bonferroni correction (d), two-sided Wilcoxon signed-rank test (e, f), the FGSEA algorithm followed by the Benjamini-Hochberg procedure (g). P values < 0.05 are indicated.

Source data

Extended Data Fig. 6 gp33-specific P14 TCM with increased expression of genes associated with replicative quiescence resemble OT-I ldTCM.

Re-analysis of scRNAseq profiled splenic of P14 memory T cells, published in Kurd et al. (Kurd et al., Science Immunology, 2020). a-b, 2D projection of P14 memory T cells 90 days post LCMV infection, colors indicate individual MCs (a), or the relative expression of effector- and multipotency-associated genes (b). Gene list in Supplementary Table 1. c, P14 memory T cells cluster into TCM (blue) and TEM (red). 2D projection colored by subset (top), and violin plots depicting normalized UMI counts of selected genes (bottom) are shown. d, QstemScore of all TCM MCs in the Kurd et al. dataset. e, Pearson correlations between the Kurd et al. P14 TCM MCs that score high (MC1, 3) or low (MC6, 7) for QstemScore, and all OT-I TCM MCs described here. Data are depicted as waterfall plots, asterisks indicate significant correlations. TCM(eff.), TCM(mult.), ldTCM and hdTCM MCs are defined in Fig. 4. P values were determined by two-sided Pearson correlation test followed by Bonferroni correction (e). P values < 0.05 are indicated in the plots.

Source data

Extended Data Fig. 7 Single cell mRNA sequencing analysis of highly divided and less divided splenic TCM.

a, Volcano plot depicting differentially expressed genes in Div0-2 versus Div5+ TCM. Significantly differentially expressed genes (Adjusted P < 0.05) are depicted in red. Selected immune-related genes are highlighted. b, Cell count per MC. c, Number of sequenced cells per sample included in the analysis. d, Sample composition of each MC. e, 2D projection, colors indicate different MCs.Depicted scRNAseq data was collected from 4 individual mice. P values were determined by Wilcoxon Rank Sum test with Bonferroni correction (a).

Source data

Extended Data Fig. 8 Modelled T cell responses are consistent with the presence of a replication-competent quiescent TCM population.

a, Cartoon of the phenotype model depicting phenotypes, the considered interactions among them and the parameters associated with the interactions. Arrows indicate various events occurring during the response, such as cell division (denoted with λ), differentiation to a different phenotype (denoted with δ), cell death during contraction (denoted with μ), and recruitment toward the secondary response during recall infection (denoted with r). Subscripts indicate the phenotype of the cell that the parameter is affecting. Full list of parameters can be found in Supplementary Note 5. b-d, Best fit of the modelled T cell response to the experimental measurements depicting either cell numbers (top plot in each panel), or DRRFP percentages (bottom plot in each panel). The total number of quiescent T cells generated was either capped at 1% (b) or 0.1% (c, d) of the TEFF pool. Lines depict the modeled populations; Dots indicate the experimental measurements obtained from peripheral blood (b, d) or spleen (c). See Supplementary Note 5 for more details and calculations. Experimental data points are representative of at least two independent experiments, dots indicate individual mice (n = 6 mice per time point).

Extended Data Fig. 9 Model describing replicative behaviors in the CD8+ memory T cell pool.

Upon infection, antigen-specific CD8+ T cells activate and rapidly expand (phase 1, p1). Following pathogen clearance (p2), a subset of memory T cells continues to divide, resulting in a progressive increase in the replicative history of the overall T cell memory pool (dotted line). Within this population, three separate behaviors of transcriptionally disparate memory T cell pools can be distinguished. Top) Terminally differentiated TEM cells that cease division after the inflammation phase (p1) and that are marked by high transcription of effector- and minimal expression of multipotency-associated genes ([E], [M]). Upon reactivation, these cells exert rapid effector functions, but lack the potential to re-expand. Middle) A subgroup of TCM that continues to proliferate in the memory phase, exhibits diminished levels of multipotency-associated transcripts, and that abundantly expresses effector-associated genes. Although the functionality of these cells upon reinfection requires further study, their heightened expression of effector-associated genes suggests that these cells exert cytotoxic activity upon reinfection. The contribution of these cells to the secondary TEFF pool is limited. Bottom) A subgroup of TCM cells that shows low expression of effector-associated genes but increased expression of multipotency-associated genes, and that exists in a near-quiescent state after the inflammation phase. Upon renewed infection, this cell pool is primarily responsible for the generation of a new wave of secondary TEFF. Based on their transcriptional profile, these cells are expected to have limited immediate cytotoxic functions.

Extended Data Fig. 10 Gating strategy.

General gating applied to flow cytometry data presented in the study. Single lymphocytes were first selected using morphology gates, and were subsequently gated on CD8+ T cells and transferred OT-I T cells (Vβ5+CD45.2+). Next, DRRFP and DRGFP could be directly selected, or first separated by phenotype depending on the analysis. The data presented here was analyzed from blood of a recipient of DR+ cells, and was acquired 6 days post infection with Lm-OVA. Phenotype gates other than those shown here are defined in their respective figures.

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Bresser, K., Kok, L., Swain, A.C. et al. Replicative history marks transcriptional and functional disparity in the CD8+ T cell memory pool. Nat Immunol 23, 791–801 (2022). https://doi.org/10.1038/s41590-022-01171-9

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