Origin and differentiation of human memory CD8 T cells after vaccination

Abstract

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Measuring the turnover rate and half-life of human YFV-specific CD8 T cells using in vivo deuterium labelling.
Figure 2: Heavy water labelling during different stages of the immune response to investigate the in vivo kinetics of YFV-specific CD8 T cells.
Figure 3: Phenotypic, functional and transcriptional profiling of long-lived YFV-specific memory CD8 T cells.
Figure 4: Long-lived YFV-specific memory CD8 T cells retain the epigenetic marks of their effector history.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. 1

    Ahmed, R. & Gray, D. Immunological memory and protective immunity: understanding their relation. Science 272, 54–60 (1996)

    CAS  ADS  Article  Google Scholar 

  2. 2

    Lau, L. L., Jamieson, B. D., Somasundaram, T. & Ahmed, R. Cytotoxic T-cell memory without antigen. Nature 369, 648–652 (1994)

    CAS  ADS  Article  Google Scholar 

  3. 3

    Demkowicz, W. E., Jr & Ennis, F. A. Vaccinia virus-specific CD8+ cytotoxic T lymphocytes in humans. J. Virol. 67, 1538–1544 (1993)

    PubMed  PubMed Central  Google Scholar 

  4. 4

    Nanan, R., Rauch, A., Kämpgen, E., Niewiesk, S. & Kreth, H. W. A novel sensitive approach for frequency analysis of measles virus-specific memory T-lymphocytes in healthy adults with a childhood history of natural measles. J. Gen. Virol. 81, 1313–1319 (2000)

    CAS  Article  Google Scholar 

  5. 5

    De Boer, R. J. & Perelson, A. S. Quantifying T lymphocyte turnover. J. Theor. Biol. 327, 45–87 (2013)

    CAS  MathSciNet  Article  Google Scholar 

  6. 6

    Surh, C. D. & Sprent, J. Homeostasis of naive and memory T cells. Immunity 29, 848–862 (2008)

    CAS  Article  Google Scholar 

  7. 7

    Kaech, S. M. & Wherry, E. J. Heterogeneity and cell-fate decisions in effector and memory CD8+ T cell differentiation during viral infection. Immunity 27, 393–405 (2007)

    CAS  Article  Google Scholar 

  8. 8

    Akondy, R. S. et al. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response. J. Immunol. 183, 7919–7930 (2009)

    CAS  Article  Google Scholar 

  9. 9

    Busch, R., Neese, R. A., Awada, M., Hayes, G. M. & Hellerstein, M. K. Measurement of cell proliferation by heavy water labeling. Nat. Protocols 2, 3045–3057 (2007)

    CAS  Article  Google Scholar 

  10. 10

    Ladell, K. et al. Central memory CD8+ T cells appear to have a shorter lifespan and reduced abundance as a function of HIV disease progression. J. Immunol. 180, 7907–7918 (2008)

    CAS  Article  Google Scholar 

  11. 11

    Ahmed, R. et al. Reconciling estimates of cell proliferation from stable isotope labeling experiments. PLOS Comput. Biol. 11, e1004355 (2015)

    Article  Google Scholar 

  12. 12

    Hellerstein, M. K. et al. Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection. J. Clin. Invest. 112, 956–966 (2003)

    CAS  Article  Google Scholar 

  13. 13

    Choo, D. K., Murali-Krishna, K., Anita, R. & Ahmed, R. Homeostatic turnover of virus-specific memory CD8 T cells occurs stochastically and is independent of CD4 T cell help. J. Immunol. 185, 3436–3444 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Ahmed, R. et al. Human stem cell-like memory T cells are maintained in a state of dynamic flux. Cell Reports 17, 2811–2818 (2016)

    CAS  Article  Google Scholar 

  15. 15

    McCune, J. M. et al. Factors influencing T-cell turnover in HIV-1-seropositive patients. J. Clin. Invest. 105, R1–R8 (2000)

    CAS  Article  Google Scholar 

  16. 16

    Westera, L. et al. Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans. Blood 122, 2205–2212 (2013)

    CAS  Article  Google Scholar 

  17. 17

    Deaton, A. M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011)

    CAS  Article  Google Scholar 

  18. 18

    Sellars, M. et al. Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages. Nat. Immunol. 16, 746–754 (2015)

    CAS  Article  Google Scholar 

  19. 19

    Fuertes Marraco, S. A. et al. Long-lasting stem cell-like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination. Sci. Transl. Med. 7, 282ra48 (2015)

    Article  Google Scholar 

  20. 20

    Kaech, S. M., Hemby, S., Kersh, E. & Ahmed, R. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111, 837–851 (2002)

    CAS  Article  Google Scholar 

  21. 21

    Cui, G. et al. IL-7-induced glycerol transport and TAG synthesis promotes memory CD8+ T cell longevity. Cell 161, 750–761 (2015)

    CAS  Article  Google Scholar 

  22. 22

    Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011)

    CAS  Article  Google Scholar 

  24. 24

    Restifo, N. P. & Gattinoni, L. Lineage relationship of effector and memory T cells. Curr. Opin. Immunol. 25, 556–563 (2013)

    CAS  Article  Google Scholar 

  25. 25

    Youngblood, B. et al. Effector CD8 T cells dedifferentiate into long-lived memory cells. Nature https://doi.org/10.1038/nature25144 (2017)

  26. 26

    Miller, J. D. et al. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity 28, 710–722 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Doering, T. A. et al. Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory. Immunity 37, 1130–1144 (2012)

    CAS  Article  Google Scholar 

  28. 28

    Wherry, E. J. et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670–684 (2007)

    CAS  Article  Google Scholar 

  29. 29

    Wirth, T. C. et al. Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8+ T cell differentiation. Immunity 33, 128–140 (2010)

    CAS  Article  Google Scholar 

  30. 30

    Sarkar, S. et al. Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J. Exp. Med. 205, 625–640 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC–seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9 (2015)

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants U19AI057266 (R.A.), R01-AI43866-07 (M.H.), NIAID UM1 AI068618 (M.J.Mc.) and NIAID UM1 AI069481 (M.J.Mc.). The authors acknowledge technical support from R. Karaffa and S. Durham for cell sorting.

Author information

Affiliations

Authors

Contributions

R.A., M.H. and R.S.A. designed and analysed experiments. R.S.A., M.F., and S.Y. performed experiments with all other authors assisting with experiments and data analysis. H.T.K. and D.Z. analysed the genomics data. S.E., S.N. and M.J.Mu. directed the clinical part of human studies. M.J.Mc and K.W.C assisted with analysis of human CD8 T cells. R.S.A., K.W.L., H.T.K., R.A. and M.H. wrote the manuscript, with all authors providing feedback.

Corresponding authors

Correspondence to Marc Hellerstein or Rafi Ahmed.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 The dynamics of deuterium enrichment in body water.

Deuterium enrichment in body water for each vaccinee in study 1 (n = 10). Plasma or saliva was used to sample body water.

Extended Data Figure 2 Investigating the in vivo kinetics of YFV-specific CD8 T cells using heavy water labelling at a range of stages of the immune response to YFV-17D.

a, Kinetics of the CD8 T cell response tracked by MHC class-I tetramer staining for vaccinees in study 2. b, c, Graphs show dynamics of the deuterium enrichment in body water for each vaccinee in study 2 (b, n = 8) and study 3 (c, n = 8). The period of water intake is indicated by the shaded blue area. d, The cell division rate constant (mean and s.d.) of A2-NS4B214 tetramer+ CD8 T cells. This represents the rate per day of newly divided A2-NS4B214 tetramer+ CD8 T cells during the D2O labelling period. Deuterium incorporation at the end of the water intake period in each study was used for this calculation. e, The percentage of deuterium-labelled A2-NS4B214 tetramer+ CD8 T cells (mean and s.d.) at the end of the water intake period in each study. f, The cell division rate constants of YFV-specific memory CD8 T cells during the maintenance period (same data as in Fig. 2f). Each circle represents data from one donor; horizontal lines show mean and s.e.m. The division rate constants were calculated by dilution kinetics of deuterium enrichment (studies 1 and 2) or by incorporation kinetics of deuterium uptake (study 3).

Extended Data Figure 3 Transcriptional and epigenetic changes during effector and memory differentiation of YFV-specific CD8 T cells.

a, Schematic of time points used for transcriptional or epigenetic analysis of tetramer-sorted YFV-specific CD8 T cells. b, Gene expression (by microarray analysis) was normalized to fold change over naive samples, and unsupervised k-means clustering was performed. Four major expression patterns emerged: genes that were up- or downregulated during the effector phase and then reverted to the naive state, and genes that were up- or downregulated during the effector stage and persisted into the memory phase. Red lines show the mean expression and numbers on the graph indicate the number of transcripts within each cluster. Data represent mean of four donors for tetramer+ effector CD8 T cells and six donors for tetramer+ memory CD8 T cells. c, MeDIP–seq analysis of genome-wide DNA methylation in bulk naive and tetramer-sorted effector and memory CD8 T cell subsets. The figure shows the top 4,000 regions that were differentially methylated (2,000 methylated and 2,000 demethylated regions) in the YFV-specific effector CD8 T cells relative to the naive CD8 T cells. Each band represents a 1,000-bp region surrounding a differentially methylated region. Regions methylated in naive CD8 T cells (blue), YFV-specific effector CD8 T cells (red) and YFV-specific memory CD8 T cells (green) are shown. Each sample was obtained from a single donor.

Extended Data Figure 4 Pathways enriched in YFV-specific effector and memory CD8 T cells.

Genes from the transient or maintained clusters identified from the microarray analysis in Extended Data Fig. 3 were analysed using MSigDB to identify Reactome pathways and transcription factors associated with each expression pattern. Figures show the topmost ten significant pathways (a) and transcription factors (b) associated with each expression pattern and the corresponding negative logarithm of the P values.

Extended Data Figure 5 Naive and YFV memory CD8 T cell responses to homeostatic cytokines.

Proliferation of naive CD8 T cells (upper panels) or YFV-specific long-term (>8 years) memory CD8 T cells (lower panels) after stimulation in vitro with different doses of IL-7 or IL-15 (n = 3). Histogram plots are gated on naive CD8 T cells or tetramer+ CD8 T cells.

Extended Data Figure 6 RNA-seq analysis of YFV tetramer-sorted effector and long-term memory CD8 T cells.

Scatter graphs show the expression of selected genes plotted as normalized reads (mean ± s.d.) from RNA-seq analysis of naive, YFV-effector (day 14) and YFV-memory (4–13 years) CD8 T cells.

Extended Data Figure 7 Comparing human YFV-specific effector and memory CD8 T cell signatures with mouse effector and memory CD8 T cells.

The RNA-seq data from YFV-tetramer+ effector and long-term memory CD8 T cells was compared with published mouse transcriptional profiles. The following datasets were used: (1) from ref. 20; (2) from ref. 27; (3) from ref. 28; (4) from ref. 29; and (5) from ref. 30. a, Enrichment plots show the proportion of genes in the leading edge; the colour of the plots shows the normalized enrichment score. The size of the circles represents the number of genes in the mouse signatures that are present in the leading edge of the enrichment plots. b, The GSEA leading edge analysis tool was used to identify genes that were commonly correlated to the effector or memory phenotype and that overlapped with genes in the corresponding mouse signatures.

Extended Data Figure 8 Analysis of open chromatin sites in YFV-specific effector and memory CD8 T cells using ATAC–seq.

ATAC–seq analysis was performed using naive CD8 T cells (n = 15) and YFV-tetramer+ effector (n = 8), early memory (n = 5) and long-lived memory CD8 T cells (n = 3). a, Principal component analysis of ATAC–seq data showing principal component 1 (PC1) versus 2 (PC2). Each shape represents a sample from one donor. b, Comparison of the number of open chromatin peaks that are significantly different between CD8 T cell subsets. Bars to the left, number of open sites in the subset listed to the left; bars to the right, number of open sites in the CD8 T cell subset that was used for comparison. The day 90 YFV-memory (n = 5) and >4 year YFV-memory (n = 3) samples were all placed in the memory group for this comparison.

Extended Data Figure 9 Open chromatin sites near the TSSs of BCL2 and IL7R in YFV-specific effector and memory CD8 T cells.

Plots show traces of chromatin openness (y axis) at the IL7R and BCL2 loci (x axis). The dashed rectangles represent the ATAC–seq peaks identified as open that were significantly different between at least two subpopulations. The RefSeq gene tracks are represented in the bottom row. We identified open sites at the TSS as well as immediately upstream and downstream of the TSS for IL7R in naive cells that all transiently lost accessibility in effector CD8 T cells and then opened in the YFV-specific memory CD8 T cells. A similar temporary pattern was seen for open chromatin regions in the BCL2 gene. We found an accessible site downstream of the BCL2 TSS that partially closes in effector cells but then regains openness to an even greater extent in memory CD8 T cells than in naive cells, consistent with previous observations that memory cells are characterized by increased BCL-2 expression.

Extended Data Figure 10 Phenotypic comparison between YFV-specific long-term memory CD8 T cells and bulk stem-like memory (TSCM) CD8 T cell population.

Samples were obtained from individuals vaccinated with YFV-17D more than 8 years previously and CD8 T cells enriched by negative selection were used to compare bulk naive and bulk TSCM CD8 T cells with A2-NS4B214 tetramer+ CD8 T cells in the same stain. a, The gating schemes for naive, TSCM and A2-NS4B214 tetramer+ CD8 T cells. b, Histogram plots show the expression of CD58 (also known as LFA-3) and CD122 (also known as IL-2Rβ) on gated naive (filled grey histogram), TSCM (blue) and A2-NS4B214 tetramer+ CD8 T cells (red). Data are representative of three experiments.

Supplementary information

Life Sciences Reporting Summary (PDF 86 kb)

Supplementary Tables

This file contains supplementary tables 1-3. Supplementary Table1 - Raw data for deuterium labelling study 1. This table shows the cell division rate, cell growth raye and calculated death rate for each donor in the deuterium labelling study 1. Supplementary Table 2 - Comparing human YFV-specific effector and memory CD8 T cell signatures with mouse effector and memory CD8 T cells. The authors compared the RNA-seq data from YFV-tetramer+ CD8 T cells to published microarray data from mouse CD8 T cells. The authors performed gene set enrichment analysis (GSEA) to determine the degree of overlap of human YFV-specific CD8 T-cells with these mouse CD8 signatures. This table shows the p-value and FDR for each comparison. Supplementary Table 3- Donor Characteristics. The table shows the age, gender and race of the donors who participated in the heavy water labelling studies. (PDF 218 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Akondy, R., Fitch, M., Edupuganti, S. et al. Origin and differentiation of human memory CD8 T cells after vaccination. Nature 552, 362–367 (2017). https://doi.org/10.1038/nature24633

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing