The aged tumor microenvironment limits T cell control of cancer

Abstract

The etiology and effect of age-related immune dysfunction in cancer is not completely understood. Here we show that limited priming of CD8⁺ T cells in the aged tumor microenvironment (TME) outweighs cell-intrinsic defects in limiting tumor control. Increased tumor growth in aging is associated with reduced CD8⁺ T cell infiltration and function. Transfer of T cells from young mice does not restore tumor control in aged mice owing to rapid induction of T cell dysfunction. Cell-extrinsic signals in the aged TME drive a tumor-infiltrating age-associated dysfunctional (T_TAD) cell state that is functionally, transcriptionally and epigenetically distinct from canonical T cell exhaustion. Altered natural killer cell-dendritic cell-CD8⁺ T cell cross-talk in aged tumors impairs T cell priming by conventional type 1 dendritic cells and promotes T_TAD cell formation. Aged mice are thereby unable to benefit from therapeutic tumor vaccination. Critically, myeloid-targeted therapy to reinvigorate conventional type 1 dendritic cells can improve tumor control and restore CD8⁺ T cell immunity in aging.

Extended Data Fig. 1 ∣. Phenotyping of CD8⁺ T cells in young vs. aged tumors.

Related to Fig. 1. (a) Representative flow cytometry plots (left) and frequencies of splenic CD8⁺ T cells that were either naive (PD-1⁻CD44⁻; right-top), activated (PD-1⁺CD44⁺; right-center), or antigen experienced (PD-1⁺Tim-3⁺; right-bottom) cells in mice that ranged in 5-85 weeks of age. (b) Representative flow cytometry gating strategy utilized to quantify tumor-infiltrating CD8⁺ T cells as well as tetramer (Tet⁺) specific, and BrdU⁺ CD8⁺ T cells in young and aged tumor-bearing mice. (c) Representative flow cytometry gating strategy used to measure lymphatic CD8⁺ T cells as well as tetramer (Tet⁺) specific CD8⁺ T cells in young and aged tumor-bearing mice. (d) ScRNA-seq UMAP projection of 5862 aged tumor-infiltrating CD8⁺ T cells defined in Fig. 1d. (e) Corresponding violin plots of scRNA-seq that denote RNA read counts (nFeature_RNA; left), and the percentage of mitochondrial genes per cell (% mitochondrial reads; right) for each cluster. (f) Hashtag of scRNA-seq profiles reveals distinct biological replicates, denoted by color, in young (left) and aged (right) tumor-bearing mice. (g) Expression of indicated genes (grey: low, purple: high). (h) Heatmap shows top cluster-defining genes in each CD8⁺ T cell cluster/subset identified in Fig. 1d. (i) Quantification of subsets within live tetramer (Tet⁺) antigen-experienced (CD44⁺ PD-1⁺) CD8⁺ T cells, classified based on the Tim-3 and Slamf6 expression in young (blue, n = 8) and aged (red, n = 4) tumor-bearing mice. (j) Geometric mean fluorescence intensity (MFI) of IL-2, IFN-γ, TNF, Gzmb, and BrdU in live antigen-experienced (CD44⁺ PD-1⁺) tumor-infiltrating CD8⁺ T cells from young (blue; n = 7) and aged (red; n = 4) tumor-bearing mice, separated by subsets. For panels a, i and j: Summary of at least two independent experiments. Mean ± s.d. For i and j, significance was calculated using a two-sided Student’s t-test. Asterisks used to indicate significance corresponds to the following: N.S. (not significant, P > 0.05), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Extended Data Fig. 2 ∣. Transcriptional profiling of T cell-intrinsic defects in aging.

Related to Fig. 2. (a) Schematic of single and co-transfers OT-1 CD8⁺ T cells into young tumor bearing recipient mice and the corresponding representative flow cytometry gating strategy utilized to quantify endogenous and transferred OT-1 CD8⁺ T cells in young tumor bearing recipient mice. (b) Schematic of young and aged splenic OT-1 CD8⁺ T cell co-transfer into young recipient mice experiments. scRNA-seq UMAP projection of 7731 tumor-infiltrating OT-1 CD8⁺ T cells from co-transfer of young and aged splenic OT-1 CD8⁺ T cell into young recipient mice, colored by cluster. Expression of indicated genes (grey: low expression, purple: high expression). (c) Violin plots that denote RNA read counts (nFeature_RNA; top), total number of genes in each cell (nCount_RNA; top second), and the percentage of mitochondrial genes (% mitochondrial reads; top third) and ribosomal genes (% ribosomal reads; bottom) per cell. (d) Heatmap showing top cluster-defining genes in each CD8⁺ T cell cluster/subset identified in Fig. 2b. (e) Relative proportion of young (OT-1_Y>Y, n = 5) and aged (OT-1_A>Y, n = 5) CD8⁺ T cells within progenitor-like exhausted (T_Prog), terminally exhausted (T_Term), and dividing (T_Divi) clusters. (f) Experimental design for in vivo transfer assay to study T_Prog formation and maintenance at early time points, such as days 7 and 9, between OT-1_Y>Y (blue) and OT-1_A>Y (red) in young B16-OVA tumor-bearing mice on days 7, 9, and 15. Proportion of T_Prog, T_Tran, and T_Term between OT-1_Y>Y and OT-1_A>Y on days 7, 9, and 15 (n = 9-10). (g) UMAP projection of scRNA-seq profiles from 6231 live splenic OT-1 CD8⁺ T cells from young and aged mice, colored by cluster (C). C1, young naive OT-1 CD8⁺ T cells (Young Spl T_Naiv); C2, aged naive OT-1 CD8⁺ T cells (Aged Spl T_Naiv); C3, aged memory-like OT-1 CD8⁺ T cells (Aged Spl T_Mem-like); C4, aged effector-like OT-1 CD8⁺ T cells (Aged Spl T_Eff-like); C5, young effector-like OT-1 CD8⁺ T cells (young Spl T_Eff-like); C6, aged exhausted-like OT-1 CD8⁺ T cells (Aged Spl T_Exh). Expression of indicated genes (grey: low expression, purple: high expression). i: Tcf7, ii: Pdcd1, iii: Ccr7, iv: Sell, v: Tox. (h) Heatmap showing top cluster-defining genes in each of splenic OT-1 CD8⁺ T cell cluster/subset identified in Extended Data Fig. 2e, with a specific focus of the top 7 genes in cluster 2 (C2). (i) UMAP projection of scRNA-seq profiles of young splenic OT-1 (Spl OT-1_Y), aged splenic OT-1 PD-1⁻ (Spl OT-1_A), aged splenic OT-1 PD-1⁺ (Spl OT-1_{A PD-1+}), and wild-type young splenic (WT_Y) CD8⁺ T cells. Frequencies of each sample within different cell clusters. Mean ± s.d. For e and f, significance was calculated using a two-sided Student’s t-test. Asterisks used to indicate significance corresponds to the following: N.S. (not significant, P > 0.05), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Extended Data Fig. 3 ∣. Epigenetic profiling of aged T cell-intrinsic defects at baseline.

Related to Fig. 3. (a) Transcriptional start site (TSS) enrichment of chromatin accessibility profiles from aged and young pre-transfer splenic as well as tumor-infiltrating OT-1 CD8⁺ T cell subsets. (b) Representative ATAC-seq tracks of aged (yellow) and young (green) splenic naive OT-1 CD8⁺ T cells at the indicated gene loci. (c) Pathway enrichment in splenic young OT-1 CD8⁺ T cells (Spl OT-1_Y, green) and splenic aged OT-1 CD8⁺ T cells (Spl OT-1_A, yellow) chromatin accessible regions (ChARs). The FDR values (hypergeometric test) are presented as −log10(q-value).

Extended Data Fig. 4 ∣. Cell-extrinsic effects of aging on differentiation and function of CD8⁺ T cells.

Related to Fig. 4, part 1. (a) Experimental design and tumor growth curves for B16-OVA tumors in young (10w; blue; n = 17) and aged (68w; red; n = 16) recipient mice treated with young spleen OT-1 CD8⁺ T cells (Spl OT-1_Y). (b) Frequency of transferred OT-1 CD8⁺ T cells isolated from young (10w, blue; n = 17) and aged (68w; red; n = 16) recipient B16-OVA tumor-bearing mice. (c) B16-OVA tumor volumes at day 15 from the cell-intrinsic model (left, young mice receiving either young or aged splenic OT-1), and cell-extrinsic model (right, young or aged mice received young spleen OT-1). (d) Experimental design for in vitro killing assay and percentage of cytotoxicity of sorted young (blue) and aged (red) OVA tetramer⁺ (Tet⁺) intratumoral CD8⁺ T cell subsets at 1:1 effector to target (E:T; OT-1: B16-OVA) ratio (n = 4-6). (e) Experimental design for in vivo transfer and T cell differentiation. (f) Schematic of young and aged intratumoral Tet⁺ CD8⁺ T cell subsets transfer into young recipient mice experiments. The corresponding representative flow cytometry gating strategy is utilized to quantify transferred Tet⁺ CD8⁺ T cell subsets in young tumor-bearing recipient mice. (g) Frequency of transferred OT-1 CD8⁺ T cells differentiated into different CD8⁺ T cell subsets (n = 4-5). Mean ± s.d. For b, d, and g, significance was calculated using a two-sided Student’s t-test. Asterisks used to indicate significance corresponds to the following: N.S. (not significant, P > 0.05), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Extended Data Fig. 5 ∣. Epigenetic and transcriptional profiling of T cell-extrinsic defects in aged tumors.

Related to Fig. 4, part 2. (a) ScRNA-seq UMAP projection of 4263 tumor-infiltrating OT-1 CD8⁺ T cells from transfer of young splenic OT-1 CD8⁺ T cell into young or aged recipient tumor-bearing mice, colored by cluster. Expression of indicated genes (grey: low, purple: high). (b) Heatmap showing top cluster-defining genes in each CD8⁺ T cell cluster/subset identified in Fig. 4e. (c) Violin plots that denote RNA read counts (nFeature_RNA; top), total number of genes in each cell (nCount_RNA; top second), and the percentage of mitochondrial genes (% mitochondrial reads; bottom) per cell, related to Fig. 4e, f. (d) Volcano plot of differentially expressed genes between transferred young OT-1 (OT-1_Y) subsets: progenitor-like exhausted (T_Prog; brown) and terminally exhausted (T_Term; blue) as well as the differentially expressed genes between T_Term (blue) and tumor-infiltrating age-associated dysfunctional (T_TAD; red) OT-1 CD8⁺ T cells. (e) Heatmap showing Z-scored TF regulon activity within cell-type clusters identified by Seurat. (f) Principal component analysis (PCA) projection and transcriptional start site (TSS) enrichment of chromatin accessibility profiles from tumor-infiltrating CD8⁺ T cell subsets in young and aged tumor-bearing mice. (g) Violin plots that denote RNA read counts (nFeature_RNA; top), total number of genes in each cell (nCount_RNA; top second), related to Fig. 4j-l. (h) Heatmap showing top cluster-defining genes in each CD8⁺ T cell cluster/subset identified in Fig. 4j. (i) Volcano plot of differentially expressed genes between human tumor-infiltrating T cell subsets: T_Term vs. T_TAD CD8⁺ T cells.

Extended Data Fig. 6 ∣. Phenotyping of immune cells in young vs. aged tumors.

Related to Fig. 5. (a) Representative flow cytometry gating strategy (top) utilized to quantify intratumoral CD45⁺ cells in young and aged tumor-bearing mice, including conventional CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, and subsets of dendritic cells (DCs), macrophages (Mac), Ly6C⁺ monocytes (Mono), and conventional dendritic cells type1 (cDC1s) and 2 (cDC2s) within DCs. Representative flow cytometry gating strategy (bottom) utilized to lymphatic CD8⁺ T cells, tetramer (Tet⁺) specific CD8⁺ T cells, subsets of dendritic cells (DCs), and cDC1/cDC2 within DCs in young and aged tumor-bearing mice. (b) Heatmap shows top cluster-defining genes in each cell cluster/subset identified in Fig. 5c. (c) Volcano plot of differentially expressed genes between intratumoral T, NK, and myeloid cells from young (blue) and aged (red) tumor-bearing mice. (d) All the significant ligand-receptor interactions between NK, cDC1, and CD8⁺ T cells in young (blue) and aged (red) tumor-bearing mice. The dot color and size represent the calculated communication probability and p-values. (e) Percentage of intratumoral CD8⁺ T cells and OVA tetramer⁺ (Tet⁺) intratumoral CD8⁺ T cells from B16-OVA tumor-bearing mice with different ages, from 10w, 24w, 44w, up to 68w old (n = 6-8). (f) Percentage of intratumoral CD8⁺ T cell subsets (such as T_Term and T_TAD) from B16-OVA tumor-bearing mice with different ages, from 10w, 24w, 44w, up to 68w old (n = 6-8). Mean ± s.d. For b, d, e, f, and g, significance was calculated using a two-sided Student’s t-test. Asterisks used to indicate significance corresponds to the following: N.S. (not significant, P > 0.05), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Extended Data Fig. 7 ∣. Transcriptional profiling of CD8⁺ T cells after myeloid-targeted immunotherapy.

Related to Fig. 6. (a) ScRNA-seq UMAP projection of 3454 tumor-infiltrating CD8⁺ T cells defined in Fig. 6h. (b) Expression of indicated genes (grey: low, purple: high). (c) Corresponding violin plots of scRNA-seq that denote RNA read counts (nFeature_RNA; up), and the percentage of mitochondrial genes per cell (% mitochondrial reads; down) for each cluster. (d) Heatmap shows top cluster-defining genes in each CD8⁺ T cell cluster/subset identified in Fig. 6h. (e) ScRNA-seq UMAP projection of different tumor-infiltrating CD8⁺ T cell cluster proportion from untreated young/aged, and young/aged treated with CD40 agonist antibody mice. (f) Quantification of naive (T_Naiv), transitory exhausted (T_Tran), IFN-reponse (T_IFN), dividing (T_Divi), and Lars2⁺ (T_Lars2+) subsets based on scRNA with individual mouse hashtags (n = 5). (g) Volcano plot of differentially expressed genes between transferred terminally exhausted (T_Term; brown) and different subsets, including tumor-infiltrating age-associated dysfunctional (T_TAD; red), progenitor-like exhausted (T_Prog; golden yellow), memory-like (T_Mem-like), and effector-like (T_Eff-like) subsets. (h) Dot plot of different expression levels of critical surface markers and transcriptional regulators among each CD8⁺ T cell cluster. Mean ± s.d. For f, significance was calculated using a two-sided Student’s t-test. Asterisks used to indicate significance corresponds to the following: N.S. (not significant, P > 0.05), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Fig. 1 ∣. Aging promotes tumor growth and alters CD8⁺ T cell fate and effector function.

a, Schema of the experimental design for the B16-OVA melanoma model used to assess tumor growth in mice of the indicated ages; D0, day 0; D15, day 15. b, Tumor growth curves for B16-OVA and LLC-OVA tumors in young (10 weeks (w); blue; n = 17 and n = 8) and aged (68 weeks; red; n = 12 and n = 4) mice. c, Percentage of day 15 total tumor-infiltrating CD8⁺ T cells as well as tetramer-specific (Tet⁺) CD8⁺ T cells isolated from B16-OVA and LLC-OVA tumors, tdLNs and non-draining LNs (ndLN) from young (10 weeks; blue; n = 20) and aged (68 weeks; red; n = 12) mice. d, Uniform manifold approximation and projection (UMAP) of scRNA-seq profiles from 5,862 live tumor-infiltrating CD8⁺ T cells from day 15 B16-OVA tumor-bearing young and aged mice colored by cluster. Expression profiles of the indicated genes are shown on the right (gray, low expression; purple, high expression). e, Galaxy plots depicting the cell density in UMAP space for tumor-infiltrating CD8⁺ T cells from young (left) and aged (right) tumor-bearing mice. Cooler colors indicate low density, and warmer colors indicate high density. The relative proportion of CD8⁺ T cells from young (blue, n = 5) and aged (red, n = 5) tumors within T_Prog, T_Term, dividing T (T_Divi) and T_TAD cell clusters is shown. f, Representative flow plots of exhausted subsets in mice that were 10 weeks (young; blue; n = 19), 68 weeks (aged; red; n = 8) and over 90 weeks old (aged; purple; n = 2 groups; each biological replicate group contains three individual mice) bearing either B16-OVA or LLC-OVA tumors for 15 days. g, Quantification of T_Prog, T_Term, T_Divi and T_TAD cell subsets within live antigen-experienced (CD44⁺PD-1⁺) CD8⁺ T cells based on TIM-3 and SLAMF6 expression. h, Geometric mean fluorescence intensity (MFI) of IL-2, IFNγ, TNF, GZMB and BrdU in live antigen-experienced (CD44⁺PD-1⁺) tumor-infiltrating CD8⁺ T cells from young (blue; n = 16) and aged (red; n = 10) tumor-bearing mice. For b, c and f–h, the data represent a summary of at least two independent experiments and are shown as mean ± s.d. For b, c and e–h, significance was calculated using a two-sided Student’s t-test; NS, not significant (P > 0.05); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 2 ∣. CD8⁺ T cell-intrinsic defects in aging lead to premature terminal differentiation and impaired proliferation.

a,b, Schema for in vivo co-transfer experiment (a) and scRNA-seq UMAP (b, left) of 7,731 live tumor-infiltrating OT-1 CD8⁺ T cells from day 15 B16-OVA tumor-bearing young mice colored by cluster. Expression of the indicated genes is shown (gray, low; purple, high). Density plots (b, right) in UMAP space for young T cells transferred into young mice (OT-1_Y>Y, top) and aged T cells transferred into young (OT-1_A>Y, bottom) mice are shown. Cooler colors indicate low density, and warmer colors indicate high density. c, Ratio of T_Term cells to T_Prog cells between OT-1_Y>Y and OT-1_A>Y T cells in scRNA-seq data. d, Volcano plot of differentially expressed genes between T_Term OT-1_Y>Y and OT-1_A>Y cells. e, Schema for the in vivo transfer assay to study T_Prog formation and maintenance at the indicated time points; i.v., intravenous. f, Representative flow plots of subset proportions within OT-1_Y>Y (blue) and OT-1_A>Y (red) T cells on the indicated days. g, Ratio of T_Term cells to T_Prog cells between OT-1_Y>Y and OT-1_A>Y on the indicated days (n = 9). h, scRNA-seq UMAP projection of 6,231 live splenic (Spl) OT-1 CD8⁺ T cells from young (10 weeks; blue; n = 5) and aged (68 weeks; red; n = 5) T_Naiv cells colored by cluster. i, Volcano plot of differentially expressed genes between young and aged splenic OT-1 CD8⁺ T_Naiv cells. j, Correlation of genes between aged and young splenic OT-1 CD8⁺ T_Naiv cells (before transfer to young tumor-bearing mice) and between aged and young tumor-infiltrating T_Term OT-1 cells (after transfer). k, Preranked GSEA of the indicated gene signatures between aged and young splenic OT-1 T_Naiv cells. l, Schema for in vitro killing assay and percent cytotoxicity of splenic aged (n = 4) and young (n = 4) OT-1 CD8⁺ T cells at different effector-to-target (E:T) ratios. m, Schema for the in vivo transfer assay to study CD8⁺ T cell-intrinsic responses. Young mice (n = 32) received either young (n = 63; blue) or aged (n = 43; red) OT-1 CD8⁺ T cells before B16-OVA tumor challenge. The data in l and m are derived from at least two independent experiments and are shown as mean ± s.d. For c, g, l and m, the significance was calculated using a two-sided Student’s t-test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3 ∣. Cell-intrinsic alternations in aged CD8⁺ T cells underlies low proliferation but not the formation of the T_TAD subset.

a, Principal component analysis (PCA) projection and transcription start site enrichment of chromatin accessibility profiles from aged and young pretransfer splenic and tumor-infiltrating OT-1 CD8⁺ T cell subsets. b, Representative assay for transposase-accessible chromatin with sequencing (ATAC-seq) tracks of aged (yellow) and young (green) splenic OT-1 CD8⁺ T_Naiv cells (Spl T_Naiv) at the indicated gene loci. c, Pathway enrichment in T_Term OT-1_Y>Y (purple) and T_Term OT-1_A>Y (pink) chromatin-accessible regions (ChARs). The false discovery rate (FDR) values (hypergeometric test) are presented in −log₁₀ (q value); eNOS, endothelial nitric oxide synthase. d, Representative flow cytometry plots and geometric MFI of γH2AX (left) and p21 (center) after 1–3 h of UV exposure in young and aged splenic OT-1 CD8⁺ T cells; FMO, fluorescence minus one. e, Frequency of BrdU⁺CD8⁺ T cells derived from the endogenous model (Endo.; top; young (n = 8) and aged (n = 4) mice without OT-1 T cell transfer), cell-intrinsic model (center; young mice that received either young (n = 5) or aged splenic OT-1 T cells (n = 4)) and cell-extrinsic model (bottom; young (n = 8) or aged mice (n = 7) that received young splenic OT-1 T cells). f, Representative flow cytometry plots of SLAMF6/TIM-3 gating and frequency of endogenous or transferred T_Term CD8⁺ T cells in young (10 weeks; blue; n = 14) or aged (68 weeks; red; n = 14) recipient mice 15 days after tumor inoculation. g, Representative flow cytometry plots of SLAMF6/TIM-3 gating and frequency of endogenous or transferred age-associated T_TAD cells in young (10 weeks; blue; n = 14) or aged (68 weeks; red; n = 14) recipient mice 15 days after tumor inoculation. For d–g, data are representative of at least two independent experiments and are shown as mean ± s.d. For e–g, significance was calculated using a two-sided Student’s t-test. For c, enriched ChARs between each group were identified using GREAT analysis; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 4 ∣. Cell-extrinsic signals from the aged TME drive the T_TAD subset state, which is distinct from CD8⁺ T cell exhaustion.

a, Schema and growth curves for B16-OVA tumors in young (10 weeks; blue; n = 17) and aged (68 weeks; red; n = 16) mice that received young splenic OT-1 CD8⁺ T cells (Spl OT-1_Y). b, Geometric MFI of IL-2, IFNγ, TNF, GZMB and γH2AX within transferred young OT-1 T_Term (red) and T_TAD (red dot) cell subsets in aged recipient mice (n = 7). c, In vitro killing assay and percent cytotoxicity of sorted young (blue) and aged (red) OT-1 intratumoral CD8⁺ T cell subsets at an effector:target ratio of 0.5:1 (n = 5). d, Schema for in vivo tumor control by transferring different OVA Tet⁺CD8⁺ T cell subsets from young (blue) and aged tumors (red) to young B16-OVA tumor-bearing mice (n = 4–6); w/o, without. e, UMAP projection of scRNA-seq profiles from 4,263 young OT-1 CD8⁺ T cells transferred into young and aged mice colored by cluster. Expression of the indicated genes is shown on the right (gray, low; purple, high). f, Density plots in UMAP space of young (left, OT-1_Y>Y) and aged (right, OT-1_Y>A) groups. Cooler colors represent low density, and warmer colors represent high density. Relative proportions of young (OT-1_Y>Y, n = 5) and aged (OT-1_Y>A, n = 5) CD8⁺ T cells within T_Prog, T_Term, T_Divi and T_TAD cell clusters are shown. g, Preranked GSEA enrichment of the indicated gene signatures within transferred OT-1 CD8⁺ T_Term and T_TAD cells. h, Hypergeometric enrichment of effector, exhausted and memory core programs within ChARs differentially open in CD8⁺ T_Term and T_TAD cells. i, Superenhancer plots based on ChARs differentially open in CD8⁺ T_Term and T_TAD cells. The red dot indicates the superenhancer cutoff. j, scRNA-seq UMAP projection of 7,242 tumor-infiltrating CD8⁺ T cells from human pan-cancer aggregated tumor biopsies (n = 57) colored by cluster. Expression of the indicated genes (gray, low; red, high) is shown on the right; Hu., human; Mel, melanoma; LC, lung cancer; BC, breast cancer; CRC, colorectal cancer; OC, ovarian cancer. k, Density plots in UMAP space of CD8⁺ T cells from individuals with cancer at various age ranges. Cooler colors indicate low density, and warmer colors indicate high density. l, GSEA enrichment of mouse T_TAD and T_Term cell signatures within a human intratumoral CD8⁺ T cell UMAP projection. The dark blue color indicates low enrichment, and the yellow-green color indicates high enrichment. The data in a–d are representative of at least two independent experiments and are shown as mean ± s.d. For a–d and f, the significance was calculated using a two-sided Student’s t-test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 5 ∣. The aged TME erodes NK cell–cDC1–CD8⁺ T cell cross-talk and impairs CD8⁺ T cell priming and expansion.

a, Schema to assess intratumoral CD45⁺ cells from young (blue) and aged (red) tumor-bearing mice. b, Frequency of live intratumoral CD45⁺ cells in young and aged mice challenged with B16-OVA (young (blue), n = 15; aged (red), n = 8) and LLC-OVA (young (blue), n = 8; aged (red), n = 4) tumors. c, scRNA-seq UMAP projection of 2,660 intratumoral T cells, NK cells and myeloid cells subclustered from CD45⁺ cells in young and aged tumor-bearing mice colored by cluster. Expression of the indicated genes is shown on the bottom (gray, low; purple, high). Broad cell types were designated as follows: macrophages (Mac; clusters (C) 2, 3 and 5), NK cells (cluster 4), CD8⁺ T cells (clusters 1, 7 and 9), DCs (clusters 6, 8 and 10), CD4⁺ T cells (cluster 11) and monocytes (Mono; cluster 12). d, Volcano plot of differentially expressed genes between young (blue) and aged (red) intratumoral DCs (clusters 6, 8 and 10). e, scRNA-seq CellChat differential number of interactions between macrophages, CD4⁺ T cells (CD4), monocytes, type 2 cDCs (cDC2), migratory DCs (migDC), NK cells, cDC1s and CD8⁺ T cells (CD8) and relative value within each cell–cell interaction between young (blue) and aged (red) cells. f, Percentage of cDC subsets from B16-OVA tumor-bearing mice of the indicated ages (n = 6–10). g, Geometric MFI of CD40, CD86, FLT3, MHC class I and MHC class II in intratumoral cDC1s from day 15 tumors of tumor-bearing mice of the indicated ages (n = 6–10). h, Representative flow cytometry plots of cDC subsets from tumors (left), tdLNs (center) and non-draining LNs (right) in young (blue; top) and aged (red; bottom) mice. i, Percentage of cDC subsets from B16-OVA (top; n = 14) and LLC-OVA (bottom; n = 14) tumor-bearing young (blue; n = 10) and aged (red; n = 8) mice. j, Schema of potential CD8⁺ T cell-intrinsic and CD8⁺ T cell-extrinsic mechanisms of age-related immune dysfunction with progressive aging or in the aged TME, respectively. The data in b and f–i are representative of at least two independent experiments and are shown as mean ± s.d. For b, f, g and i, the significance was calculated using a two-sided Student’s t-test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 6 ∣. Limited CD8⁺ T cell priming and expansion by cDC1s in the aged TME can be rescued by myeloid-targeted immunotherapy.

a, Schema and growth curves after OVA-mRNA LNP vaccination in young (10 weeks; n = 10) and aged (68 weeks; n = 8) B16-OVA tumor-bearing mice; tx, treatment. b, Day 15 tumor volumes from a. c, Frequencies of cDC1s (first and second columns) and CD8⁺ T cells (third and fourth columns) within live CD45⁺ cells in tumor and tdLNs. The frequencies of indicated cells from OVA-mRNA LNP-treated (young tumor (top), n = 16; young tdLN (top), n = 8; aged tumor (bottom), n = 12; aged tdLN (bottom), n = 7) or nontreated (young tumor (top), n = 16; young tdLN (top), n = 8; aged tumor (bottom), n = 8; aged tdLN (bottom), n = 4) tumor-bearing mice are shown. d, Schema and growth curves after treatment with CD40 agonist antibody in young (10 weeks; blue; n = 16) and aged (68 weeks; red; n = 7) B16-OVA tumor-bearing mice. e, Day 15 tumor volumes from d. f, Frequencies of cDC1s (first and second columns) and CD8⁺ T cells (third and fourth columns) within live CD45⁺ cells in tumor and tdLNs. The frequencies of indicated cells from CD40 agonist antibody-treated (young tumor (top), n = 8; young tdLN (top), n = 8; aged tumor (bottom), n = 7; aged tdLN (bottom), n = 7) or nontreated (young tumor (top), n = 8; young tdLN (top), n = 8; aged tumor (bottom), n = 7; aged tdLN (bottom), n = 4) tumor-bearing mice are shown. g, Geometric MFI of CD40, CD86, FLT3, MHC class I and MHC class II in intratumoral cDC1 cells from day 15 tumors of young (10 weeks (Y)) and aged (68 weeks (A)) tumor-bearing mice with or without CD40 agonist antibody treatment. h, scRNA-seq UMAP projection of 3,454 live tumor-infiltrating CD8⁺ T cells from day 15 B16-OVA tumor-bearing young and aged mice with or without CD40 agonist antibody treatment colored by cluster. Expression of the indicated genes is shown along the bottom (gray, low; purple, high). i, Density plots in UMAP space for tumor-infiltrating CD8⁺ T cells from young (left top), young treated with CD40 agonist antibody (left bottom), aged (right top) and aged treated with CD40 agonist antibody (right bottom) tumor-bearing mice. Cooler colors indicate low density, and warmer colors indicate high density. j, Quantification of T_Prog, T_Term, T_TAD, T_Mem-like and T_Eff-like cell subsets. Data in a–g are representative of at least two independent experiments and are shown as mean ± s.d. For the data in a–g and j, the significance was calculated using a two-sided Student’s t-test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.