A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool

Abstract

An increasing body of evidence emphasizes the role of tissue-resident memory T cells (TRM) in the defense against recurring pathogens and malignant neoplasms. However, little is known with regard to the origin of these cells and their kinship to other CD8+ T cell compartments. To address this issue, we followed the antigen-specific progeny of individual naive CD8+ T cells to the T effector (TEFF), T circulating memory (TCIRCM), and TRM pools by lineage-tracing and single-cell transcriptome analysis. We demonstrate that a subset of T cell clones possesses a heightened capacity to form TRM, and that enriched expression of TRM-fate-associated genes is already apparent in the circulating TEFF offspring of such clones. In addition, we demonstrate that the capacity to generate TRM is permanently imprinted at the clonal level, before skin entry. Collectively, these data provide compelling evidence for early stage TRM fate decisions and the existence of committed TRM precursor cells in the circulatory TEFF compartment.

Graphical abstract

Figure 1.

Proportional contribution of individual T cell clones to the systemic and skin effector response. OT-I thymocytes were transduced with the barcode library and intrathymically transferred into recipient mice. After maturation, barcode-labeled GFP⁺ OT-I T cells were transferred into secondary recipients that were subsequently exposed to skin vaccination. (A) Schematic overview of experimental setup. (B) Barcode-labeled GFP⁺ OT-I T cell response to DNA vaccination, measured in blood (n = 11 mice, gray lines). Black line represents group average. (C) Representative flow cytometry plots showing the presence of GFP⁺ memory T cells within CD8⁺ cells in blood and skin >60 d after vaccination. (D and E) Spleen, skin, draining LNs, and whole blood were collected from vaccinated recipient mice 12 d after start of vaccination. (D) Analysis of the contribution of individual T cell clones to the spleen, blood, and draining LN effector stage T cell compartment, relative to the skin effector–stage T cell compartment. Spearman correlation r was calculated over clones detected in both samples. Left: Spearman correlations for individual mice (n = 4), mean with whiskers representing SD. Right: Dots represent individual clones; P values were <0.0005. (E) Clonal output in all examined tissues of the 5% of largest clones detected in skin tissue. Heat map depicts log10-transformed clone sizes (read counts). D and E are representative data of two independent experiments.

Figure S1.

Quality of barcode quantification and analysis of blood-borne T cell contamination in effector-phase skin samples. (A and B) Recipients of barcode-labeled T cells were vaccinated, and whole blood and organs were harvested 12 d after first vaccination. (A) Measured clone sizes detected in representative technical replicates of blood (left) and skin (right) samples. (B) Measured clone sizes detected in blood (left) and skin (right) of independent mice. Dots represent individual clones. (C) Analysis of the presence of blood-borne T cells in skin preparations. Recipients of GFP⁺ OT-I T cells were DNA vaccinated. 10 d after vaccination, mice received 1.5 × 10⁶ Tomato⁺ OT-I T cells and were sacrificed 5 min later. Top: Pie charts depicting the relative percentage of GFP⁺ and Tomato⁺ cells in blood (left) and skin (right) preparations. Bottom: Representative flow cytometry plots. Cells are gated on live lymphocytes. Data are representative of four mice.

Figure 2.

Clonal bias in T_RM generation. (A) Representation of experimental timeline. Barcode-labeled T_RM and T_CIRCM were isolated from the skin and circulatory compartment (spleen, LN, and blood) of DNA-vaccinated mice (or HSV-OVA_257–264–infected mice; C), and clonal output was quantified. (B) Comparison of clonal contribution to the skin T_RM and T_CIRCM compartment after DNA vaccination. (C) Comparison of clonal contribution to the skin T_RM and T_CIRCM compartment after HSV-OVA_257–264 infection. (D) Clones responding to DNA vaccination were defined as T_RM biased, T_CIRCM biased, or nonbiased, based on their relative contribution to either memory compartment. Scatterplot similar to B highlighting T_RM-biased (blue), T_CIRCM-biased (red), and nonbiased (gray) T cell clones. Small clones for which clone size measurements were less reliable were excluded from analysis and are not depicted. (E and F) Comparison of effector stage burst size of nonbiased (gray), T_RM-biased (blue), and T_CIRCM-biased (red) T cell clones. In E, values on y axis depict (clone size T_RM − clone size T_CIRCM)/(clone size T_RM + clone size T_CIRCM) and represent the degree of preferential contribution to T_RM or T_CIRCM. Dashed lines indicate bias threshold of 4.8-fold. In F, median with whiskers representing minimum/maximum, Kruskal–Wallis test with Dunn’s multiple comparisons test; N.S., not significant. In B and C, Spearman correlation r was calculated over all clones that contributed to both samples, P < 0.0005 (B) and P = 0.01 (C). Dots represent individual clones. Data from four mice, representative of two individual experiments.

Figure S2.

Quality control and analyses of the barcode-labeled T_RM and T_CIRCM compartment. (A) Measured clone sizes detected in technical replicates of T_RM (left) and T_CIRCM (right) samples derived from the mice described in Fig. 2 B. Spearman correlation r was calculated over clones that were detected in both technical replicates: P < 0.0005 (left); P < 0.0005 (right). (B) Measured clone sizes detected in T_RM (left) and T_CIRCM (right) of independent mice described in Fig. 2 B. (C) Step-by-step description of the strategy used to filter biological data and define biased clones, as depicted in Fig. 2 D. First, unreliably detected clones (indicated in red) are removed. Second, a bias threshold (dashed lines) is set, such that 98% of the clones in technical replicates fall below this threshold. This threshold is subsequently applied to the biological data to identify clones with a bias in output that goes beyond the variation that occurs because of technical noise. For the data presented in Fig. 2 B, the threshold identified only clones contributing >4.8 times to one sample than to the other to be considered biased. Dots represent individual clones.

Figure 3.

Nonstochastic formation of tissue-resident and systemic T cell memory. (A) Contribution of T cell clones to the T_RM (left) or T_CIRCM (right) pool, relative to the effector stage blood compartment. Spearman correlation r was calculated over T cell clones that were detected in both samples; n = 4 mice. (B) Spearman correlations of clone sizes in skin (left) and spleen (right) samples collected during effector (n = 4 mice) and memory (n = 4 mice) phase to day-12 effector blood. (C) Clone size disparity of skin (left) and spleen (right) T cell pools in the effector and memory phase from the day-12 effector blood T cell pool. See Fig. S3 A for the definition of disparity. (D) Illustration depicting the strategy used to assess whether stochasticity can explain the observed clonal skewing during memory formation. Based on observed clone distribution in the T_EFF pool, a virtual pool of T_EFF cells is generated in silico, from which cells are sampled to form a randomly selected T_RM or T_CIRCM memory pool. The number of randomly sampled cells is equal to the number of observed cells in the biological memory (T_M) pool (α), to 10% of the observed T_M pool (α/10), or to the number of observed clones in the biological T_M pool (β), which represents the smallest theoretically possible T_M founder pool. The Spearman correlation coefficient between the randomly sampled cell pool and the experimentally observed T_EFF pool is calculated (Y) and compared with the Spearman correlation coefficient between the experimentally observed T_M pools and the experimentally observed T_EFF pool (X). Only if Y approaches X, stochastic engraftment can explain the observed skewing in clonal output in the T_M pool. (E) Stochastically formed T_RM (left) and T_CIRCM (right) pools were modeled 10,000 times in silico, as described in D, and the Spearman correlation between the modeled memory pools and the observed T_EFF pool was calculated (Y). Graphs indicate individual mice (n = 4); histograms represent the distribution of Spearman r. Red vertical line indicates the correlation between the clonal distribution of the T_EFF pool and the experimentally observed memory pool (X). Spearman correlations r were calculated over all clones detected in either the effector pool or the (modeled or experimental) memory pool. In A, dots represent output of individual clones. In B and C, dots represent individual mice. Spearman correlation r was calculated over clones that were detected in both samples: P < 0.0005 (A, left) and P < 0.0005 (A, right); Mann–Whitney U test, *, P < 0.05 (C). Representative data of two independent experiments.

Figure S3.

Remodeling of the skin-resident and circulating memory compartment. (A) Example plots depicting the strategy to determine the disparity between two cellular compartments, as applied in Figs. 3 C and 7 F. Disparity of compartments B and C to compartment A can be assessed by plotting the fraction of cumulative reads of clones in compartments B and C, which are ordered based on their size (largest to smallest) in compartment A (y axis), to the cumulative reads of the ordered clones in compartment A (x axis). Area between the compartment A reference curve and compartment B (left) and C (right) curves is calculated to generate a measure of disparity. (B) Left: Illustration of the subdivision of ordered effector-stage T cell clones (large to small) into four bins, with each bin containing 25% of all observed clones. Middle and right: Quantitative contribution of binned clones detected in effector blood to the T_RM and T_CIRCM compartment. Median with whiskers representing minimum/maximum; ***, P < 0.0005, Mann–Whitney U test. (C) Relative contribution of T_EFF clones in bins 1–4 (highlighted in blue) to the T_RM and T_CIRCM compartments. In B and C, data are representative of two independent experiments; dots represent individual clones.

Figure 4.

scRNA-seq reveals a transcriptional T_RM-like MP state in the circulating T_EFF pool. Barcode-labeled T_EFF were isolated from blood of recipient mice at day 12 after skin vaccination, and scRNA-seq was performed to map transcriptional profiles of circulating effector cells. In addition, barcode sequences were specifically amplified from single cell–derived cDNA and subsequently sequenced (single-cell barcode sequencing). Matching of cellcode sequences (sequences marking all transcripts derived from a single cell) in scRNA-seq and single-cell barcode sequencing datasets allows for the coupling of transcriptional profile and clone of origin of individual T_EFF cells. (A) Schematic overview of the experimental procedure. (B) Left: 2D projection of 5,383 T_EFF cells that, based on transcriptional profile, are grouped into 14 distinct MCs. MC colors indicate assigned T_EFF state, as defined in C (red/brown, T_CIRCM-like; blue, T_RM-like MP; purple, TE; gray, Int). Right: 2D projections with superimposed expression of Il7r and Klrg1. Legend indicates gene expression Z-score. (C) Left: Hierarchical clustering of 14 transcriptionally distinct MCs based on the expression of TE- and MP-associated genes. Legend indicates log2 enrichment Z-score. Right: Hierarchical clustering, based on the expression of core T_RM and T_CIRCM genes, of the seven MCs that are classified as MP. (D) Gene expression comparison of genes associated with skin T_RM biology between the four defined T_EFF transcriptional states (i.e., T_CIRCM-like, T_RM-like MP, TE, and Int). Values on axis represent log2 enrichment Z-score. Spearman correlation r, P < 0.005. In B–D, data were obtained from three mice.

Figure S4.

Transcriptional T_EFF profile of T cell clones is linked to memory generation capacity. scRNA-seq and single-cell barcode sequencing were performed on barcode-labeled T_EFF cells responding to skin vaccination. The T_EFF cells were grouped into 14 transcriptionally distinct MCs. (A) Relative contribution of each mouse to the 14 MCs. (B) Number of cells assigned to each MC. Colors indicate the transcriptional T_EFF subset individual MCs were assigned to; see Fig. 4 C for the definition of the identified T_EFF subsets. (C) Pie chart depicting the relative frequency of the four identified T_EFF states. (D) Number of T_EFF cells observed for each reliably detected clone. (E) Comparison of T_CIRCM clone size to the relative contribution of clones to the indicated T_EFF states (i.e., T_CIRCM-like, T_RM-like MP, TE, or Int). Spearman correlation r (when significant) and Spearman correlation P value are depicted. Dots represent individual clones. Clones that were not detected in the T_CIRCM compartment were excluded. Red line represents the linear regression line. (F) Analysis as depicted in Fig. 6 B, but here depicting mean effector phase TE and Int output of large and small T_CIRCM and T_RM clones. (G) Comparison of the absolute production of T_RM-like MP (top) and T_CIRCM-like MP (bottom) of clones to their production of mature T_RM. Black line indicates linear regression line with 95% confidence interval in gray.

Figure 5.

Differential contribution of antigen-specific T cell clones to distinct T_EFF states, independent of clone size. The relative contribution of individual antigen-specific T cell clones to the four T_EFF states was assessed. (A) Heat map depicting the contribution of 91 clones to the 4 identified T_EFF states. The sum of each row equals 100%. (B) Comparison of the relative contribution to a transcriptional state (i.e., T_CIRCM-like, T_RM-like MP, TE, or Int) and clone size in day-12 effector blood. Spearman correlation P values are depicted. Dots represent individual clones. Red line represents the linear regression line. Note that biased output toward the four T_EFF states is observed for both small and large clones and is not explained by stochasticity. Data obtained from three mice.

Figure 6.

Skewed production of circulating T_RM-like MP cells by T cell clones in T_EFF phase is associated with enhanced T_RM generation. Clonal output in the T_RM and T_CIRCM compartments was assessed and compared with the transcriptional profiles of matched clones in the circulating T_EFF compartment. (A) Comparison of T_RM clone size in memory with the relative output of individual clones to the distinct transcriptional T_EFF states (i.e., T_CIRCM-like, T_RM-like MP, TE, or Int) during the effector phase. Spearman correlation r (when significant) and Spearman correlation P value are depicted. Dots represent individual clones. Clones that were not detected in the T_RM compartment were excluded. Red line represents the linear regression line. (B) Top: Analysis of relative production of T_CIRCM-like and T_RM-like MPs during the effector phase by either large or small T_RM clones in memory phase. 15 clones (of a total of 49) were randomly selected 10,000 times (gray histogram, middle), selected with a bias toward large T_RM clones (red histogram, front), or selected with a bias toward small T_RM clones (blue histogram, back). The distribution of mean T_CIRCM-like and T_RM-like MP production of the sampled clones is plotted. Dotted line represents the most frequently observed mean production of randomly selected clones. Bottom: Similar analysis as in top, but performed for large and small T_CIRCM clones, using random sampling of 69 clones. (C) Comparison of bias in memory generation of individual clones to their production of T_CIRCM-like and T_RM-like MP cells in the effector phase. Clones that contributed to both the T_RM and T_CIRCM pool (n = 40) were selected according to their bias in memory production: 10 clones per selection window, moving five clones with each step in the direction of T_CIRCM-biased clones, depicted as red dots in scatterplots. The first selection window represents clones with the most prominent bias to T_RM generation; the last selection window represents clones with the most prominent bias toward T_CIRCM generation. Mean production of T_RM-like (left) and T_CIRCM-like MP (right) is plotted per window. Red lines represent smoothing spline curves. (D) Difference in expression of core T_RM (top) and T_CIRCM (bottom) genes between the most T_RM-biased T_EFF-stage clones (n = 10) and most T_CIRCM-biased T_EFF-stage clones (n = 10). X axis represents log2 fold difference of mean expression of T_RM-biased clones over T_CIRCM-biased clones (fold difference calculated as mean expression T_RM-biased clones/mean expression T_CIRCM-biased clones). Blue and red numbers indicate the sum of the log2 fold differences of genes enriched in T_CIRCM- (red) or T_RM- (blue) biased clones. Data obtained from three mice.

Figure 7.

T_RM generation capacity is a clone-imprinted attribute that is preserved upon secondary antigen encounter. (A) Schematic timeline used in B–D. (B) Contribution of T cell clones to the T_RM pool present at two separate sites of primary vaccination (T_RM-LEFT, T_RM-RIGHT) relative to the T_CIRCM pool (left) and relative to each other (right). Dots represent individual clones. (C) Spearman correlations (left) and ratios (right) of nine individual mice, comparing the clonal composition of the T_RM-LEFT compartment to the T_CIRCM and to the T_RM-RIGHT compartment. Left: Mean with whiskers representing SD. Right: **, P < 0.005, Wilcoxon signed-ranked test. Data from nine mice from two independent experiments. (D) Output of individual OT-I T cells to different T_RM and T_CIRCM pools, as indicated in the columns. Heat map depicts log10-transformed clone sizes (read counts), clustered using Euclidian distance. Data from six mice from two independent experiments. (E and F) Recipient mice were vaccinated on the right hind leg (primary site) and >60 d later on the left hind leg (secondary site), and clonal composition at both sites was assessed >60 d after secondary vaccination. Top: Schematic time line used in E and F. Bottom left: Contribution of T cell clones to the T_RM-SEC pool relative to the T_RM-PRIM pool. Dots represent individual clones. Bottom right: Spearman correlations of six individual mice, mean with whiskers representing SD. (F) Disparity between T_RM-LEFT and T_CIRCM pool (red) and between the T_RM-LEFT and T_RM-right pool (cyan) in case of simultaneous or staggered vaccination. prim/prim, simultaneous vaccination; prim/sec, primary and secondary vaccination separated by >60 d. N.S., not significant; *, P < 0.05; ***, P < 0.0005; Mann–Whitney U test. Mean with whiskers representing SD. See Fig. S3 A for the definition of disparity. Dots represent individual mice. prim/prim and prim/sec groups each consisted of nine mice. Data from three independent experiments. (G) Illustration of proposed T_RM differentiation model. After priming in the skin-draining lymph node, naive T cells undergo clonal expansion and a selection of activated T cells commit to the T_RM fate. During the effector phase, these transcriptionally distinct T_RM precursor cells migrate, along with non-T_RM precursor cells, to the inflamed skin tissue. At the inflamed site, T_RM precursors display a heightened capacity to mature into long-term persisting T_RM in response to tissue-derived external signals, such as TGFβ, IL-15, and antigen. Note that formation of T_RM precursor cells may occur early during clonal expansion, as depicted here, or may reflect heterogeneity in T cell potential that already exists before priming.

Figure S5.

De novo T_RM generation upon secondary vaccination in previously unperturbed sites. (A and B) Mice received GFP⁺ OT-I T cells (A) or barcode-labeled OT-I T cells (B) and were subjected to DNA vaccination on the right hind leg, whereas the other hind leg remained unperturbed. (A) Analysis of T_RM frequencies in vaccinated (right leg) and nonvaccinated (left leg) skin sites >60 d after vaccination. (B) More than 60 d after primary vaccination, the nonvaccinated (left leg) skin site was subjected to DNA vaccination. More than 60 d after secondary vaccination, the primary and secondary vaccinated skin sites were harvested, and GFP⁺ T_RM at the two sites were quantified. Number of barcode-labeled T_RM detected at the primary and secondary skin vaccination site of nine mice. *, P < 0.05, Wilcoxon signed-rank test. Dots represent individual mice.