Fig 1.
Ki67+ TSCM cells exhibit increased CD95 expression.
T cells from healthy volunteers were isolated and stained with Ki67 and canonical T cell differentiation markers. (A) Median Fluorescence Intensity (MFI) of CD95 for Ki67− and Ki67+ TSCM cells. Each dot represents one donor (N = 25, Cohort 1); P = 3 × 10−7 paired Wilcoxon (B) TSCM cells are classified as CD95hi and CD95int and the percentage of Ki67+ cells is compared among TN, CD95int, CD95hi and TCM subsets. Each dot represents one donor (N = 15, from Cohort 1). TN v CD95int P = 0.0005, CD95int v CD95hi P = 0.003, CD95hi v TCM P = 0.002, TN v TCM P = 0.002, paired Wilcoxon, number of independent tests = 3. **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represents the median, the box the interquartile range (IQR) and whiskers show lower quartile − 1.5 * IQR and upper quartile + 1.5 * IQR; all data points (including outliers) are shown by filled circles. Abbreviations: TN: T naïve, TSCM: T stem-cell memory, TCM: T central memory, MFI: median fluorescence intensity. The data underlying this figure can be found in S1 Data.
Fig 2.
CD95hi TSCM cells exhibit increased self-renewal and functionality compared to both CD95int TSCM cells and TCM cells.
(A) Self-renewal in response to homeostatic cytokines was investigated by culturing sorted CD95hi, CD95int and TCM for 7 days with 25 ng/ml IL-7/IL-15, 25 ng/mL IL-15 or 6.1 ng of IL-2 (N = 13, 10, 5, respectively). The self-renewal (defined as percentage of divided cells that have maintained their input phenotype) of each subset on day 7 is shown. The self-renewal of CD95hi TSCM was consistently higher than that of CD95int TSCM or TCM in response to all 3 conditions (CD95hi v CD95int: IL-7/IL-15 P = 0.0001, N = 13; IL-15 P = 0.0002, N = 10; IL-2 P = 0.1, N = 5. CD95hi v TCM: IL-7/IL-15 P = 0.0008, N = 13; IL-15 P = 0.1, N = 10; IL-2 P = 0.5, N = 5. Unpaired Wilcoxon, number of independent tests per dataset = 2). (B) Self-renewal in response to TCR stimulation was investigated by culturing sorted T cell subpopulations for 7 days with 25 ng/ml IL-7/IL-15, 2 μg/ml aCD3 and 1 μg/ml aCD28. (C) Multipotency in response to TCR stimulation was investigated by culturing sorted CD95hi, CD95int and TCM for 7 days with 25 ng/ml IL-7/IL-15, 2 μg/ml αCD3 and 1 μg/ml αCD28. The phenotype of the starting (sorted) population is shown in the grey bars above each panel and the subpopulations they differentiated into are plotted. (D) The data from panel C is used to calculate a multipotency index, this captures the diversity in subpopulations that a subpopulation generates and is defined for a subpopulation j as where pi is the proportion of differentiated cells (i.e., those that have changed phenotype) that are represented by subpopulation i. N = 9 but due to low frequencies of some cell populations N = 4 for CD95hi TSCM and N = 7 for TCM. (E) FACS-isolated T cell subsets were stimulated for 12 h with 2 μg/ml αCD3 and 1 μg/ml αCD28. Boxplots on left summarize the percentage of TNFα, IFNγ and IL-2 expressing cells among the different subsets. Boxplot on right shows markers associated with cytotoxicity. N = 5. An extension of panel E to show TN and TEM is shown in Fig E in S1 Text. In all cases bars represents the median, the box the interquartile range (IQR) and whiskers show lower quartile − 1.5 * IQR and upper quartile + 1.5 * IQR; all data points (including outliers) are shown by filled circles. All subjects from Cohort 1. A replicate of this figure but with individuals identified by different symbols is provided in Fig S in S1 Text. The data underlying this figure can be found in S1 Data.
Fig 3.
CD8+ TN, CD95int TSCM, CD95hi TSCM, TCM, TEM and TEMRA populations were sorted. Cells were either unstimulated or stimulated with αCD3αCD28. RNA was extracted and sequenced. (A) PCA representation of the most variable genes showed that, for unstimulated cells, CD95int TSCM clustered near TN, whereas CD95hi TSCM clustered with TCM. Upon stimulation these four populations separated with both TSCM subpopulations distinct from each other and from TN and TCM cells. (B) Gene set enrichment analysis of genes differentially expressed between unstimulated CD95int TSCM and unstimulated CD95hi TSCM found, transcriptionally CD95int TSCM were closer to naïve CD4 and CD8+ T cells while CD95hi TSCM were correspondingly closer to various memory populations (i.e., genes that were down in naïve v memory CD4, naïve v effector memory CD4 and naïve v PD1 hi CD8 were also down in CD95int TSCM). (C) The same analysis for of genes differentially expressed between stimulated CD95int TSCM and stimulated CD95hi TSCM was harder to interpret. The only T cell pathway (GSE21360, PRIMARY VS QUATERNARY MEMORY CD8 TCELL UP) implies that genes upregulated in CD95hi TSCM are enriched in genes upregulated in memory CD8 T cells following multiple antigen challenge whereas genes upregulated in CD95int TSCM are associated with memory CD8+ T cells following primary antigen challenge.
Fig 4.
CD95hi and CD95int frequencies differ between the blood and the lymph nodes.
PBMCs and lymph node cells isolated from the blood and axillary lymph nodes, respectively, of 7 healthy vaccinated volunteers from Cohort 3 were isolated and stained. Cells were gated on the population of (A) CD95int cells and (B) CD95hi cells. PBMC shown in red and lymph node cells in blue. Each dot represents one sample. CD95int TSCM (as a proportion of total TSCM) are significantly enriched in the LN, P = 0.029 unpaired two-tailed Wilcoxon test. Conversely CD95hi TSCM cells show a non-significant trend to be enriched in the blood P = 0.079. *P < 0.05. Bars represents the median, the box the interquartile range (IQR) and whiskers show lower quartile − 1.5 * IQR and upper quartile + 1.5 * IQR; all data points (including outliers) are shown by circles. The data underlying this figure can be found in S1 Data.
Fig 5.
CD95hi cells have a lower TREC content and their frequency increases with age.
(A) TREC (T-cell receptor excision circle) content was quantified by ddPCR for FACS-sorted T cell subpopulations from N = 7 healthy donors, from Cohort 4. Boxplots indicate the number of TREC copies per cell for each subset (bars represents the median, the box the interquartile range (IQR) and whiskers show lower quartile − 1.5 * IQR and upper quartile + 1.5 * IQR; all data points, including outliers, are shown by circles). Graph on right is a zoom in (truncated y axis) to show TREC content of memory populations more clearly. Statistics: Wilcoxon paired test (B) PBMCs isolated from the blood of 21 healthy individuals (23−65 yrs, Cohort 4) were stained to identify CD95hi and CD95int TSCM cells. The percentage of TSCM that were CD95hi (orange circles) and CD95int (blue circles) was quantified. The results are plotted against the age of the donor. We found that the proportion of TSCM that were CD95hi increased significantly with age (Rs = 0.82, P = 6.78 × 10−6 Spearman). The data underlying this figure can be found in S1 Data.
Fig 6.
The CD95hi subset incorporated more label in vivo compared to CD95int.
(A) Workflow of heavy water labelling technique and timepoints of sample collection. Participants from Cohort 5 drank 70% deuterated water for 7 weeks (100 ml twice daily for one day, then 50 ml twice daily thereafter). Saliva and PBMC were collected at multiple time-points during and after labelling. Cells of interested were sorted and deuterium enrichment was measured by gas chromatography/mass spectrometry (B) M + 1 enrichment of DNA from selected cell populations is shown per individual for all timepoints for the three different subsets. Error bars represent analytic variance and show the standard deviation of 4 replicate measurements. Note, DW19 attained higher levels of label in body water hence the higher enrichment in DNA (the mathematical modelling allows for this variation when calculating cell kinetics). Here lines connect neighboring timepoints for ease of viewing, they do not represent model fits. The data underlying this figure can be found in S1 Data.
Table 1.
Best fit estimates of parameters obtained by fitting the winning model (Model D, linear model with CD95int TSCM first) to the labelling, telomere and YFV data. Interquartile range (estimated by bootstrapping the data) shown in square brackets below the corresponding estimates. Note, the clonal half-life corresponds to the half-life of the clone (ln2/d-p) not the half-life of the cell as is typically reported (ln2/d). Best fit estimates for the other 4 models are provided in Tables G–J in S2 Text. We found that CD95int TSCM cells proliferated much more slowly than CD95hi TSCM cells with an average proliferation rate of 0.0008 per day (equivalent to one division every 3.4 yrs) whereas CD95hi TSCM cells had an average proliferation rate of 0.002 per day (equivalent to one division every 1.4 yrs). The data in this table can be found in S1 Data.
Fig 7.
Models capturing different kinetics and relationships between the cell populations of interest.
(A) Independent homogeneous model. Each population (TN, CD95int, CD95hi) is independent and homogeneous. (B) Independent heterogeneous model. Each population (TN, CD95int, CD95hi) is independent and allowed (but not constrained) to be heterogeneous. (C) Forked differentiation model. Upon meeting cognate antigen, TN cells undergo a clonal burst (of size 2k); a fraction (f) differentiate into the CD95int pool with the remainder differentiating into the CD95hi pool. (D) Linear differentiation model (CD95int first). Upon meeting cognate antigen, TN cells undergo a clonal burst (of size 2k) and differentiate into the CD95int pool which subsequently differentiate into the CD95hi pool. (E) Linear differentiation model (CD95hi first). Upon meeting cognate antigen, TN cells undergo a clonal burst (of size 2k) and differentiate into the CD95hi pool which subsequently differentiate into the CD95int pool. Note for compactness, in the cartoons, CD95int TSCM are designated Tint and CD95hi TSCM are designated Thi. In every model each population was assumed to be of constant size. The models were fitted to the data to identify which model was most consistent with the experimental observations and to quantify the population parameters.
Fig 8.
Fit of the three viable models – Model C (fork), Model D (linear, CD95int first) and Model E (linear, CD95hi first) – to all data (labelling, telomere, YFV) simultaneously.
(A) Fit of models to the labelling data (each individual in a different row, each cell population in a different column); black dots represent data, colored lines represent fitted model predictions (solid: Model C, dashed: Model D, dotted: Model E). (B) Fit of models to the telomere data. Observed differences in mean telomere length between TN and TSCM for 5 individuals shown on the left in black, predictions for each individual and each model shown by colored points on the right (DW19 in blue, DW20 in pink, DW25 in orange. squares: Model C, diamonds: Model E, triangles: model D). (C) Fit of the model to the YFV data. Observed data (YFV-specific TSCM as a % of CD8+ T cells) shown by black points, fitted model predictions for each individual shown by colored lines (DW19 in blue, DW20 in pink, DW25 in orange) and for each model shown by line type (solid: Model C, dashed: Model D, dotted: Model E). The data underlying this figure can be found in S1 Data.
Fig 9.
Predictions of winning model – Model D (linear, CD95int first) overlaid (NOT fitted) onto a novel data set.
(A) The winning model and best fit parameters were used to predict the long-term dynamics of YFV-specific CD95int TSCM (left) and CD95hi TSCM (right) for each individual (shown by colored lines: DW19 in blue, DW20 in pink, DW25 in orange). For CD95int the predictions overlay and only one line can be seen. The predictions are plotted against but not fitted to a proxy for CD95int and CD95hi TSCM frequencies measured in the cross-sectional cohort post-vaccination (the same cohort for which total TSCM were fitted). (B) As above but also including 100 predictions obtained by fitting bootstrapped data (sampled with replacement) shown as grey lines. A comparison of all models to this unseen data set is provided in Table L in S2 Text. The data underlying this figure can be found in S1 Data.
Fig 10.
A population of YFV-specific cells enriched for CD95int TSCM declines much more slowly over time than a population enriched for CD95hi TSCM.
Here we reproduce the analysis from Fuertes Marraco and colleagues [2] who investigated the decline of YFV-specific memory subpopulations with time post-vaccination in a cross-sectional cohort. Fuertes Marraco and colleagues note that there is large between-individual variation in the sizes of CD8+ memory subpopulations and that this noisy interindividual variation can be reduced by quantifying the frequency of YFV-specific cells within each subpopulation rather than within the total CD8+ population (e.g., quantify YFV-specific TCM CD8+ cells as a percent of total TCM rather than as a percent of total CD8+ T cells) (A). Analysis of the raw data shows that a proxy for YFV-specific CD95int TSCM cells (YFV-specific “naïve hi” cells) – left panel – decline significantly more slowly than a proxy for YFV-specific CD95hi TSCM cells (YFV-specific “naïve int” cells) – right panel (P = 0.039, linear regression). The blue and orange lines are the best fit straight lines to the CD95int and CD95hi TSCM data, respectively. (B). Analysis in Fuertes Marraco and colleagues [2] differs slightly in that they chose to exclude three individuals with very low frequencies of YFV-specific CD8+ T cells and to include 4 individuals who had received multiple YF vaccinations. Our conclusions are robust to these two steps (if anything they appear to strengthen the conclusion that YFV-specific CD95int TSCM cells decline more slowly than YFV-specific CD95hi TSCM cells). Recipients of multiple vaccinations shown by open symbols.
Fig 11.
Schematic of the best performing model and parameters.
An overview of the model and parameters that provides the best description of the data sets. Average residency time is average time a cell spends in that population (“Materials and methods”).
Fig 12.
Summary of TSCM CD95 int and TSCM CD95hi properties.
As well as being distinct from one another in many respects, CD95int TSCM cells and CD95hi TSCM cells are distinct from both TN and TCM cells. Furthermore, cellular properties do not vary in a monotonic way from TN to CD95int to CD95hi to TCM. A blank cell indicates that combination of property and cell population was not investigated in this study.