Functional heterogeneity of human tissue-resident memory T cells based on dye efflux capacities

Abstract

Tissue-resident memory T cells (TRMs) accelerate pathogen clearance through rapid and enhanced functional responses in situ. TRMs are prevalent in diverse anatomic sites throughout the human lifespan, yet their phenotypic and functional diversity has not been fully described. Here, we identify subpopulations of human TRMs based on the ability to efflux fluorescent dyes [efflux(+) TRMs] located within mucosal and lymphoid sites with distinct transcriptional profiles, turnover, and functional capacities. Compared with efflux(-) TRMs, efflux(+) TRMs showed transcriptional and phenotypic features of quiescence including reduced turnover, decreased expression of exhaustion markers, and increased proliferative capacity and signaling in response to homeostatic cytokines. Moreover, upon activation, efflux(+) TRMs secreted lower levels of inflammatory cytokines such as IFN-γ and IL-2 and underwent reduced degranulation. Interestingly, analysis of TRM subsets following activation revealed that both efflux(+) and efflux(-) TRMs undergo extensive transcriptional changes following TCR ligation but retain core TRM transcriptional properties including retention markers, suggesting that TRMs carry out effector function in situ. Overall, our results suggest a model for tissue-resident immunity wherein heterogeneous subsets have differential capacities for longevity and effector function.

Keywords: Adaptive immunity; Immunology; T cells.

Figure 1. A subset of memory CD8⁺ T cells across human tissues efflux fluorescent dyes.

Human T cells from the indicated tissue sites were loaded with mitochondrial dyes and analyzed by flow cytometry. (A) MitoTracker fluorescence within memory (CD45RA^–CCR7^–) CD8⁺ T cells in tissues of a representative donor. Numbers within plots indicate percentage of cells that are Mito^hi (right) or Mito^lo (left). (B) Comparison of CMXRos (upper) and MitoTracker Green (lower) staining of memory CD8⁺ T cells from the spleen and lung of a representative donor. (C) Inhibition of dye efflux by cyclosporine A (CSA) and verapamil. CD8⁺ T cells from spleen were labeled with MitoTracker green as in panel A in the presence or absence of CSA (top row) or verapamil (bottom row) at the indicated concentrations. Results are representative of 3 different donors. (D) Fraction of memory CD8⁺ T cells that are efflux(+) from the indicated tissue sites. LN, lymph node. Bar graph shows mean + SEM with individual samples shown from 4–20 donors for each tissue. (E) A subset of CMV-specific CD8⁺ T cells across tissues efflux dyes. Upper: Detection of CMV-specific CD8⁺ T cells within memory CD8⁺ T cells from indicated tissue sites. Lower: Frequency of efflux(+) and efflux(–) cells within the CMV tetramer⁺ population. Results are representative of 2 donors.

Figure 2. Efflux(+) memory CD8⁺ T cells are enriched in the TRM fraction across human tissues.

(A) Upper: CD69 expression by efflux(+) (Mito^lo) and efflux(–) (Mito^hi) subsets of memory CD8⁺ T cells in the indicated tissues. Lower: Compiled frequency of efflux(+) cells within the CD69⁺ (TRM-enriched) and CD69^– subsets of memory CD8⁺ T cells in each tissue site, with each line connecting subsets from 1 individual donor (spleen, 17 donors; bone marrow, 7 donors; lung, 6 donors). (B) Frequency of TRMs that are efflux(+) in each tissue site, with bars showing mean ± SEM and individual donors represented by single dots. (C) Upper: CD103 expression by efflux(+) and efflux(–) TRMs in spleen and lung. Lower: Compiled frequency of CD103 expression among efflux(+) and efflux(–) TRMs in the spleen (n = 10) and lung (n = 6), with each line connecting subsets within individual donors. (D) Upper: Expression of CD49a and CD101 among efflux(+) and efflux(–) TRMs (CD69⁺) from the spleen of a representative donor. Lower: Compiled frequency of CD49a- and CD101-expressing cells within efflux(+) and efflux(–) splenic TRMs (n = 6). For all panels, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by paired t test.

Figure 3. Efflux(+) TRMs have a unique phenotype.

(A) Left: CD127 expression by efflux(+) and efflux(–) TRMs and TRMs from the spleen of 1 representative donor. Right: Compiled frequencies of CD127⁺ efflux(+) and efflux(–) TRMs and TEM subsets from spleen (n = 5) and bone marrow (BM, n = 7). (B and C) Left: CD27 (B) and CD28 (C) expression by efflux(+) and efflux(–) TRMs and TRMs from the spleen of 1 representative donor. Right: Compiled frequencies of CD27⁺ (B) and CD28⁺ (C) efflux(+) and efflux(–) TRMs and TEM subsets from spleen (n = 7). (D) Left: Expression of PD-1 by efflux(+) and efflux(–) TRM and TEM subsets from the spleen of a representative donor. Right: Compiled expression of PD-1 by efflux(+) and efflux(–) TRMs from spleen (n = 8), lung (n = 7), and BM (n = 6). (E) Left: Histograms of CD57 expression by efflux(+) and efflux(–) TRMs and TEM subsets from the spleen of 1 representative donor. Right: Compiled frequencies of CD57⁺ TRMs and TEMs from spleen (n = 7) and BM (n = 3). (F) Left: Plots show CD39 expression by efflux(+) and efflux(–) TRM and TEM subsets from the spleen of 1 representative donor. Right: Compiled frequencies of CD39⁺ TRMs and TEMs from spleen (n = 6). For all panels, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by paired t test. ns and n.s., not significant.

Figure 4. Efflux(+) cells are a transcriptionally distinct subset of TRMs.

Whole transcriptome profiling by RNA sequencing was performed on efflux(+) and efflux(–) CD69⁺ (TRM) memory CD8⁺ T cells from the spleen of 3 donors. (A) PCA of efflux(+) and efflux(–) TRM samples, based on the global transcriptome. (B) Functional annotation analysis by DAVID software. Select gene ontology (GO) terms with significant adjusted P values (Adj. p) are displayed, along with fold enrichment. (C) Heatmap of normalized expression levels of all genes with significant differential expression between the 2 groups, defined as FDR ≤ 0.05 and absolute value of log2 fold change ≥ 1. (D) Select significantly differentially expressed genes that are upregulated [“up in efflux(+)”]or downregulated [“down in efflux(+)”] grouped by category. Shown are the log2 fold changes (log2FC) of select genes between efflux(+) and efflux(–) cells for each donor, designated by a unique shape (see legend in bottom left panel). Genes marked with a “*” did not meet FDR criteria (0.05), but had log2FC ≥ 1 and significant P values and were included for potential biological relevance.

Figure 5. Efflux(+) TRM subset stability and function following stimulation.

(A) Sorted efflux(+) and efflux(–) TRMs were stimulated with anti–CD3/CD28/CD2 beads, and efflux capacity was reassessed after 48 hours along with CD25 expression. Plots show MitoTracker and CD25 expression from the spleen of 1 donor for the indicated subsets. (B) Cytokine production following TCR stimulation. Efflux(+) and efflux(–) TRMs were sorted and stimulated with anti–CD3/CD28/CD2 beads for 72 hours, and cytokines in supernatant were quantified using cytometric bead array (see Methods). Graphs show levels of indicated cytokines in supernatants compiled from 8 donors. (C) Degranulation of efflux(+) and efflux(–) TRM subsets. Sorted cells were pulse labeled with CD107a antibody followed by PMA/ionomycin stimulation. Representative plots and quantification of CD107a⁺ from splenic efflux(+) and efflux(–) TRM subsets from 4 donors. For all panels, *P ≤ 0.05. **P ≤ 0.01 ****P ≤ 0.0001 by paired t test. ns, not significant.

Figure 6. Efflux(+) TRMs have enhanced proliferative capacity and responses to homeostatic cytokines.

(A and B) Proliferative capacity of TRM subsets. Sorted efflux(+) and efflux(–) TRMs were labeled with cell proliferation dye and stimulated with anti–CD3/CD28/CD2 beads. Proliferation was assessed on day 4 of culture. (A) Left: Histogram showing cell proliferation dye dilution at day 4. Results are representative of 4 independent experiments. Middle: Quantification of the percentage of proliferating cells. Right: Quantification of the percentage of proliferating cells at each division number. Significance assessed by 2-way ANOVA with Sidak’s multiple comparisons test. (B) Differential IRF4 induction in efflux(+) and efflux(–) TRMs following TCR stimulation. Left: IRF4 expression as a function of cell division at day 4 after stimulation; plots are representative of 3 independent experiments. Rectangular gate identifies proliferating cells that have upregulated IRF4. Middle: IRF4 expression within proliferating cells from efflux(+) and efflux(–) subsets. Right: Quantification of IRF4 mean fluorescence intensity within activated proliferating cells from 3 donors. (C) Left: Expression of Ki67 within TRM subsets from the spleen of 1 representative donor. Right: Frequency of effluxing cells in Ki67⁺ and Ki67^– TRMs. (D) Efflux(+) TRMs exhibit increased responses to IL-7. Left: STAT5 phosphorylation following IL-7 stimulation ex vivo. Right: Quantification of pSTAT5⁺ cell percentage within efflux(+) and efflux(–) TRMs. For all panels, *P ≤ 0.05. **P ≤ 0.01 ****P ≤ 0.0001 by paired t test. n.s., not significant.

Figure 7. The transcriptional response of efflux(+) and efflux(–) TRMs to stimulation.

RNA-Seq analysis was performed on splenic efflux(+) and efflux(–) TRM subsets isolated as in Figure 3 following stimulation with anti–CD3/CD28 for 12 hours. (A) Differential expression was assessed using DESEQ2, and plots display the number of significantly upregulated and downregulated genes following stimulation in both efflux(+) and efflux(–) subsets. (B) Both efflux(+) and efflux(–) TRMs preserve TRM-like characteristics after stimulation. Venn diagrams show overlap between genes that are differentially expressed when comparing stimulated versus unstimulated TRMs (current data set) and genes that are differentially expressed when comparing human spleen CD8⁺ TRMs and TEMs (“TRM core genes”; from ref. 12). (C) TRMs downregulate egress receptors after stimulation. Plot shows log2 fold changes (log2FC) of CCR7, S1PR1, and KLF2 when comparing stimulated versus unstimulated efflux(+) and efflux(–) TRM samples. (D) Scatterplot displays all genes found to have significant differential expression in either efflux(+) or efflux(–) stimulated versus unstimulated samples as in A. Value on the x axis represents the log2FC of the gene between stimulated versus unstimulated efflux(+) samples, and the y axis represents the log2FC of the same gene between stimulated versus unstimulated efflux(–) samples. Samples are color coded by whether the differential expression was significant in only efflux(+) TRMs, only efflux(–) TRMs, or both groups. (E) Ingenuity Pathway analysis (IPA) analysis. Select pathways that had significant P values (≤0.01) when comparing stimulated versus unstimulated samples in both efflux(+) and efflux(–) TRMs are displayed. Direction of enrichment in stimulated samples is proportional to the color intensity of each bar. (F) Plot shows log2FC of select genes related to T cell function when comparing stimulated versus unstimulated TRM samples for both efflux(+) and efflux(–) subsets. (G) Expression of genes (log2FC) from C that have opposite-direction changes for efflux(+) and efflux(–) samples after stimulation across all 3 donors. Plot shows log2FC of the selected genes, comparing expression by stimulated versus unstimulated samples for efflux(+) (orange triangles) and efflux(–) (blue circles) TRMs.