CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory

Abstract

Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8⁺ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8^- CCR8⁺ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8⁺ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Keywords: CCR8; TCR repertoire; autoimmunity; cancer immunotherapy; regulatory T cells.

Fig. 1.

CCR8 is specifically expressed by clonally expanding Tregs in tumor tissues. (A) Search for the genes specifically expressed by clonally expanding tumor Tregs. Tumor-infiltrating CD3⁺ T cells in CT26-bearing mice on day 18 after tumor cell inoculation were analyzed for gene expression by single-cell RNA-seq. The Treg population defined by the expression of Treg function-associated genes was divided into the clonally expanded population (i.e., more than two Tregs commonly expressing a particular TCR clonotype) (red dots) and the clonally nonexpanded one (i.e., every Treg expressing a nonreplicated unique TCR) (blue dots) by TCR clonotyping. Volcano plot shows gene expression profiles of the two cell populations. (B) Clonotyping of TCRα and TCRβ genes expressed by tumor Tregs. Frequency of each TCR composed of indicated TRAV-TRAJ genes for TCRα chain or TRBV-TRBJ genes for TCRβ chain in CCR8^– and CCR8⁺ tumor Tregs (Left). The proportion of each TCR clonotype size in CCR8⁺ and CCR8^– tumor Tregs (Right). Representative data of two biological replicates. (C) CCR8 protein expression by CD3⁺CD4⁺ T cells in LNs, DLNs, and tumors. CT26-, EMT6-, or Renca-bearing mice 18, 10, or 13 d, respectively, after tumor inoculation were analyzed by flow cytometry (n = 5 to 8 each) in two independent experiments. ****P < 0.0001 (one-way ANOVA followed by Dunnett's multiple comparisons test). Vertical bars: means ± SD.

Fig. 2.

Specificity of CCR8 expression in human cancers. (A) UMAP plots showing expression of indicated genes by each CD3⁺ T cell in a kidney cancer and peripheral blood (PB) from a healthy donor. Red squares indicate Tregs. CCR8⁺ rates were calculated as the percentage of CCR8⁺ cells among Tregs or CD4⁺ Tconvs. (B) CCR8⁺ rate in Treg L-group and CD4⁺ Tconv L-group, as defined in A, in various cancers. (C) CCR8 and Treg-associated gene expression by CD3⁺CD4⁺ T cells in PB and the tumor tissue from a kidney cancer patient. (Left) Cell fractions separated by CD45RA and CD25 expression for bulk RNA-seq analysis of each fraction. (Right) The expression of designated genes in each cell fraction. (D) CpG methylation status around the human FOXP3 CNS2 region in naïve or activated Tregs and CD4⁺ Tconvs in PB of a healthy donor (Left). Data from Ohkura et al. was reanalyzed (28). (Right) Cell fractions of CD3⁺CD4⁺CD45RA^– T cells in a bladder cancer tissue and the FOXP3 CNS2 CpG methylation rate in each cell fraction. (E) FOXP3 and CCR8 protein expression by CD4⁺ and CD8⁺ T cells in a kidney cancer tissue. Representative staining (Left) and the percentages of CCR8⁺ cells among CD8⁺, FOXP3^–CD4⁺, and FOXP3⁺CD4⁺ cells in 15 renal cancer tissue samples (Right). Vertical bars: means ± SD ****P < 0.0001 (one-way ANOVA followed by Dunnett's multiple comparisons test). TPM, transcripts per million.

Fig. 3.

Antitumor effects of cell-depleting anti-CCR8 mAb. (A) Tumor growth in tumor-bearing mice treated with anti-CCR8 or control mAb. Antibodies were administered on day 5 for CT26 and EMT6, and on days 5 and 12 for Renca tumors (n = 8 each). Average and individual tumor volumes are shown by thick and thin lines, respectively. The complete remission (CR) rates are also shown. P values were calculated with tumor volume on day 19 (CT26 and EMT6) or day 25 (Renca) using Mann–Whitney U test. (B) In vitro ADCC activity (Upper) and neutralizing activity against CCL1-CCR8 interaction (Lower) assessed for anti-CCR8 mAb and its derivative (αCCR8ΔADCC). (C) Ratios of Foxp3⁺ cells among CD4⁺ T cells in CT26 tumors after antibody treatment. CT26-bearing mice were treated with PBS (control), anti-CCR8, or anti-CCR8ΔADCC mAb on day 3 and analyzed on day 10 after tumor inoculation (n = 8 each). (D) Tumor growth of CT26-bearing mice and the CR rates (n = 8 each). The mice were treated twice with PBS (control), anti-CCR8, or anti-CCR8ΔADCC mAb on days 3 and 10. (E) PD-1 and CCR8 expression by CD8⁺, Foxp3^–CD4⁺, and Foxp3⁺CD4⁺ T cells in tumor tissues and DLNs in CT26-bearing CB6F1 mice. Representative staining of tumor-infiltrating T cells 10 d after tumor inoculation (Upper) and percentages of PD-1 single-positive, CCR8 single-positive, and the double-positive cells in indicated cell populations from DLNs and tumors on day 10 (n = 6 each) (Lower). (F) Tumor growth of CT26-bearing CB6F1 mice treated with control, anti–PD-1, anti-CCR8 mAb or the combination of anti–PD-1 and anti–CCR8 mAbs on days 5 and 12 (n = 8 each). Data are presented as means with SD for C–F. ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001 (one-way ANOVA followed by Tukey’s multiple comparisons test for C, Kruskal–Wallis test followed by Dunn's multiple comparisons test for tumor volume on day 24 for D or day 20 for F). Data are representative (A–D and F) or summary (E) of two independent experiments.

Fig. 4.

Minimal autoimmune inflammatory responses in anti-CCR8–treated mice. (A) Ratios of Foxp3⁺ cells to CD4⁺ T cells from peripheral blood (PB), spleen, LNs, DLNs, and tumors of CT26-bearing FDG mice on day 8 (Left) or anti-CCR8–treated BALB/c mice on day 10 (Right). PBS or DT was administered to CT26-bearing FDG mice on days 5 and 7. Control or anti-CCR8 mAb was administered to BALB/c mice on day 5 (n = 3 or 5 each). (B) Tumor growth in CT26-bearing mice after Foxp3- (Left) or CCR8-targeted Treg depletion (Right) (n = 5 or 8 each). (C) Changes in spleen and LN sizes and in lymphocyte numbers (in an inguinal LN) in CT26-bearing mice treated as in B and assessed on day 13 after tumor inoculation (n = 3 or 5 each). (D) Serum concentrations of antiparietal cell antibody and anti-dsDNA antibody (n = 5 or 10, respectively) in CT26-bearing mice treated and assessed as in C. (E) Histologies and histopathological scores of H&E-stained stomach sections from CT26-bearing mice treated and assessed on day 13 as in C (n = 5 or 6 each). Arrowheads indicate infiltration of mononuclear cells. (Scale bars, 100 μm.) Data are presented as means with SD (A–D) or median (E). ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (multiple unpaired t tests with Welch correction followed by Holm-Šídák method for A, Mann–Whitney U test for B and E, Welch's t test for D). Data of anti-CCR8–treated mice in C–E are from one experiment. Other data are representative (A from FDG mice, C from FDG mice, B and D from FDG mice for anti-dsDNA Ab) or summary (A from anti-CCR8–treated mice, D for anti-parietal cell Ab, and E from FDG mice) of two independent experiments.

Fig. 5.

Changes in cell composition and function of tumor-infiltrating T cells after anti-CCR8 mAb treatment. (A) Experimental design for single-cell RNA-seq of tumor-infiltrating T cells isolated on day 12 or 16 from CT26-bearing mice treated with anti-CCR8, anti–PD-1, or control mAb on day 9. Data from each sample at each time point were merged for analysis. (B) A representative tumor growth in CT26-bearing mice treated with the mAbs shown in A in two independent experiments (n = 6 each). Data are presented as means with SD. ns, P ≥ 0.05; *P < 0.05 (Kruskal–Wallis test followed by Dunn's multiple comparisons test). (C) Single-cell analysis of tumor-infiltrating T cells on day 12. Three treatment groups were combined for analysis, plotted by UMAP, and grouped by Leiden (Upper Left). Cell density of each group (Upper Right Top), marker gene expression (Upper Right Bottom and Lower Left Right), and cell phase (Lower Left Left) are shown. Shown Lower Right is GSEA of up-regulated genes in L-group 2 (reduced in the anti-CCR8 mAb-treated group) compared with L-group 8 (retained in other groups) regarding the Treg vs. Tconv UP gene set (34). Blue square in Upper Left figure indicates Tregs. (D) Single-cell analysis of tumor-infiltrating T cells on day 16. Panels are represented as in C. Shown Lower Right is GSEA of up-regulated genes in L-group 0 (high in the anti-CCR8 mAb-treated group) compared with L-group 5 (high in other groups) regarding the secondary vs. quaternary memory CD8 T cell UP gene set (35). NES, normalized enrichment score.

Fig. 6.

Changes in TCR clonotype composition of Tregs after anti-CCR8 mAb treatment. (A) Experimental design for assessing the changes in TCR clonotypes in tumors before and after anti-CCR8, anti–PD-1, or control mAb treatment. Two tumors were inoculated on one mouse in each group, and each tumor was removed before (Pre, day 9) or after (Post, day 14) each treatment. All data were merged for analysis. (B) UMAP plots of single-cell RNA-seq data with TCR clonotypes of tumor-infiltrating TCRβ⁺ T cells before and after anti-CCR8, anti–PD-1, or control mAb treatment. Cells were annotated to Tregs (green), CD4⁺ Tconvs (orange), CD8⁺ Tconvs (blue), and ambiguous cells (red) by Foxp3, Cd4, and Cd8a expression. Expression of Ccr8 and clonotype size are shown for tumor Treg population. (C) Percentages of Tregs among TCRβ⁺ T cells and their clonotype frequencies before and after antibody treatments. (D) Maintained T cell clones before (Pre) and after (Post) antibody treatment. Expanded clones at Pre, singletons at Pre, and clones not detected at Pre are colored in red, blue, and gray, respectively. Treg populations indicated by green squares (Left) are reanalyzed and shown in Middle panels. Right panel shows the percentages of Tregs among TCRβ⁺ T cells and the proportion of their clonotypes before (Pre) and after (Post) antibody treatment in each group. (E) TCR clonotype similarity of tumor Tregs and Tconvs before (Pre) and after (Post) antibody treatment in each group.

Fig. 7.

Establishment of antitumor immune memory by depleting CCR8⁺ Tregs. (A) Experimental design for assessing the establishment of antitumor immune memory. CT26-bearing mice were treated with anti-CCR8 or control mAb and the tumors on the control-treated mice were surgically removed on day 20. On day 85, mice eradicated of tumors by anti-CCR8 mAb treatment or surgery were reinoculated with CT26 or EMT6. Intact mice inoculated with these tumors were used as no treatment control. (B) Changes in serum concentration of anti-CCR8 mAb after administration (n = 5 each). Dotted line indicates the detection limit. ND, not detected. (C) Tumor growth after secondary inoculation of the same (CT26 → CT26, Left) or different (CT26 → EMT6, Right) tumors in the mice eradicated of the primary CT26 tumor by anti-CCR8 antibody or surgical removal (n = 6 each), with tumor volumes of the individual secondary tumors (CT26 → CT26) on day 15 (Center). (D) Tumor-specific CD8⁺ T cells revealed by gp70 tetramer staining in the PB collected from mice before (Pre) and 6 d after (Post) the secondary challenge with CT26 (n = 11 each). (E) Correlation between the frequencies of gp70 tetramer⁺CD8⁺ T cells in the peripheral blood 6 d after the secondary CT26 challenge and tumor volume 14 or 15 d after the challenge (n = 11 each). Data are presented as means with SD. ns, P ≥ 0.05; *P < 0.05 (Mann–Whitney U test for C, multiple unpaired t tests with Welch correction followed by Holm-Šídák method for D). Data in B are from one experiment. Other data are representative (C) or summary (D and E) of two independent experiments.