Abstract
Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called ‘memory inflation’ is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family’s capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family’s long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.
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Data availability
RNA sequencing data generated for this study have been deposited in the Gene Expression Omnibus under accession number GSE157644. All other data generated or analyzed during this study are included in the published article (and its supplementary information files) or are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Code availability
No custom code or algorithms were used in this study.
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Acknowledgements
We thank thank J. Sun and N. Adams as well as all members of the Buchholz laboratory for discussion and support. This work was supported by the Else Kröner-Fresenius-Stiftung – EKFS 2019_A91 (‘Dissecting the induction of inflationary CD8+ T cell memory on the single-cell level’) to V.R.B., the DFG – SFB 1054 (project no. 210592381) to V.R.B. and D.H.B., the TUM Seed Funds (SCIMAP) to V.R.B., the BMBF project Quan-T-cell (e:Med initiative on Systems Medicine, FKZ 01ZX1505) to M.F., the BMBF project TIDY (Computational Life Sciences, FKZ 031L0170A) to T.H., the Helmholtz association project PIE-008 to L.C.S. and the DFG – TRR 179 (project number 031L0170A) to M.S.
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S.G. and V.R.B. designed the study. S.G., L.M., R.R., S.F., I.H., J.L., L.K. and L.O.P. performed experiments. S.G., L.M., J.M., A.K., A.J. and M.F. performed data analysis. Q.Z., A.J. and T.H. provided sample processing and RNA sequencing. M.Z.C. and L.C.-S. generated virus stocks. M.S. provided cell sorting. K.S. and D.H.B. contributed to study design and critically read the manuscript. S.G., L.M. and V.R.B. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Virus schematic, gating strategy and tracking of T cell families until day 412 post infection.
a, Schematic of MCMV-ie2-SIINFEKL virus. The SIINFEKL peptide sequence was inserted at the 3’ end of the ie2 gene. b, Gating strategy for single-cell fate mapping. Following doublet and dead-cell exclusion, transferred cells are identified by expression of congenic markers. Further gating depends on the method used for single-cell transfer (exemplified in Figs. 2b and 4b, respectively). T cell families are characterized phenotypically using antibodies directed against CD27 and CD62L. c, Longitudinal quantification of T cell family sizes in blood, measured as percent of living leukocytes, for a cohort of T cell families from Fig. 1e traced until day 412 p.i. n = 15 T cell families.
Extended Data Fig. 2 Gene ontology term enrichment analysis of early gene signature in long-term dominators (sc-derived RNA-seq, day 8 p.i.).
a–c, Gene ontology (GO) term enrichment analysis of early gene signature in long-term dominating T cell families acquired via sc-derived RNA-seq at day 8 p.i. a, Top 10 GO terms that were underrepresented in long-term dominators (n = 2) compared to all remaining T cell families (n = 14). b,c, Top 10 GO terms that were significantly underrepresented (b) or overrepresented (c) in long-term dominators (n = 2) compared to acutely dominating T cell families (n = 2). d, GSEA analysis of genes differentially expressed in long-term dominating vs. acutely dominating T cell families associated with cellular senescence (GO:0090398). Genes up- (red) and down-regulated (blue) in long-term dominators vs. acutely dominating T cell families. P values were determined based on the default method in the goseq (a–c) and fgsea packages (d). See methods for details.
Extended Data Fig. 3 Expression of genes enriched in TCM, TEM and TE clusters (scRNA-seq, day 60 p.i.).
a,b, Analysis of scRNA-seq data derived from an established inflationary T cell population in spleen at day 60 p.i. a, Dimensional reduction using Uniform Manifold Approximation and Projection (UMAP) showing Leiden clusters and expression of genes enriched in the corresponding TCM, TEM and TE clusters. b, Cell cycle scores for S and G2M-phase projected onto UMAP embedding. These scores indicate both a weak proliferative activity and a uniform distribution of scores across different Leiden clusters. Scores were computed based on lists for S- and G2M-phase associated genes using the software package SCANPY.
Extended Data Fig. 4 Retrogenic color-barcoding and single-cell transfer.
a, Schematic of the generation of retrogenic color-barcoded OT-1 T cells: Bone marrow of CD45.1+ OT-1 Rag1–/– mice was harvested and enriched for hematopoietic stem cells (HSCs) by flow cytometric sorting for Sca1+ cells. These were transduced retrovirally with different combinations of fluorescent proteins (GFP, YFP, BFP, CFP and T-sapphire) and transplanted into sublethally irradiated C57BL/6 mice. More than 6 weeks after HSC engraftment, naive color-barcoded OT-1 T cells were harvested for experiments from peripheral blood of retrogenic chimeras. b, Exemplary gating strategy for identification of color-barcoded naive OT-1 T-cells. c, Sort and transfer of single naive color-barcoded OT-1 T cells (see methods section for details).
Extended Data Fig. 5 Temporal dynamics of single-cell-derived memory inflation correlated to early abundance of TEs, EMPs and CMPs.
a, Scatter plots comparing the amount of TEs (top row), EMPs (middle row) and CMPs (bottom row) in each T cell family on day 8 p.i. to the total size of the respective T cell families at various time points between day 8 and day 140 p.i. The amount of CMPs, EMPs and TEs and the total size of T cell families is measured as % of living leukocytes. Top-10 long-term dominating T cell families are marked (blue circles). P values from left to right: TE: p < 0.0001, p < 0.0001, p = 0.009, p = 0.95, p = 0.91, p = 0.97. EMP: p < 0.0001, p < 0.0001, p = 0.003, p = 0.88, p = 0.30, p = 0.48. CMP: p = 0.001, p = 0.001, p = 0.007, p = 0.0018, p = 0.0008, p = 0.0007. b, Scatter plots comparing the relative amount of TEs (top row), EMPs (middle row) and CMPs (bottom row) in each T cell family on day 8 p.i. to the total size of the respective T cell families at various time points between day 8 and day 140 p.i. The relative amount of CMPs, EMPs and TEs is measured as % of the corresponding T cell family’s overall size. Top-10 long-term dominating T cell families are marked (blue circles). P values from left to right: TE: p = 0.99, p = 0.07, p = 0.15, p = 0.13, p = 0.006, p = 0.002. EMP: p = 0.59, p = 0.73, p = 0.90, p = 0.82, p = 0.39, p = 0.10. CMP: p < 0.0001, p = 0.58, p = 0.57, p = 0.42, p = 0.0008, p = 0.0004. ‘r’ indicates Spearman’s correlation coefficient. Significances were measured using a two-sided Mann-Whitney test. * p < 0.05, ** p <0.01, *** p <0.001, **** p < 0.0001. n = 104 T cell families (zero values are not shown and not used for determining Spearman’s correlation coefficients and respective p values).
Extended Data Fig. 6 CMPs constitute only a minute fraction of MPECs and are enriched for non-cytolytic T cells.
a–d, Adoptive transfer of 100 congenically labelled OT-1 T cells followed by immunization with MCMV-ie2-SIINFEKL. a,b, Analysis of CD27, CD127, KLRG-1 and CD62L expression in expanded T cell populations (n = 6) at day 8 p.i. in spleen. a, Percent of CD62L+CD27+ CMPs among all responding T cells or among KLRG-1–CD27+ MPECs b, Percent of CD62L+CD27+ CMPs among all responding T cells or KLRG-1–CD127hi MPECs or KLRG-1+CD127lo SLECs. c,d, Analysis of Gzmb expression in T cell subsets defined as in (a) measured in expanded T cell populations (n = 5) at day 8 p.i. c, Percent of Gzmb– cells among MPECs or SLECs (d) Percent of Gzmb– cells among CMPs, EMPs or TEs. e,f, Adoptive transfer of 100 congenically labelled OT-1 T cells followed by immunization with 5000 cfu L.m.-OVA. Analysis of Gzmb expression in T cell subsets defined as in (a) in expanded T cell populations (n = 4) at day 8 p.i. e, Percent of Gzmb– cells among MPECs or SLECs (f) Percent of Gzmb– cells among CMPs, EMPs or TEs. Stacked bars in 6a–f show mean, error bars indicate SD. g,h, Further analysis of experiments described in Fig. 4: Scatter plots comparing the amount of CD27+ Memory precursors (MPs) (g) or the percentage of MPs in each T cell family (h) on day 8 p.i. to the total size of the respective T cell families at various time points between day 8 and day 140 p.i. The amount of MPs and the total size of T cell families is measured as % of living leukocytes. The relative amount of MPs is measured as % of the corresponding T cell family’s overall size. Top-10 long-term dominating T cell families are marked (blue circles). P values from left to right: (g): TE: p < 0.0001, p < 0.0001, p = 0.008, p = 0.69, p = 0.16, p = 0.30. (h): p = 0.14, p = 0.21, p = 0.27, p = 0.19, p = 0.01, p = 0.002. ‘r’ indicates Spearman’s correlation coefficient. Significances were measured using a two-sided Mann-Whitney test. * p < 0.05, ** p <0.01, *** p <0.001, **** p < 0.0001. n = 104 T cell families (zero values are not shown and not used for determining Spearman’s correlation coefficients and respective p values).
Supplementary information
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Statistical source data and lists of differentially expressed genes.
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Source Data Extended Data Fig. 1
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Lists of differentially expressed genes.
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Grassmann, S., Mihatsch, L., Mir, J. et al. Early emergence of T central memory precursors programs clonal dominance during chronic viral infection. Nat Immunol 21, 1563–1573 (2020). https://doi.org/10.1038/s41590-020-00807-y
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DOI: https://doi.org/10.1038/s41590-020-00807-y
