Human single cell RNA-sequencing reveals a targetable CD8+ exhausted T cell population that maintains mouse low-grade glioma growth

Abstract

In solid cancers, T cells typically function as cytotoxic effectors to limit tumor growth, prompting therapies that capitalize upon this antineoplastic property (immune checkpoint inhibition; ICI). Unfortunately, ICI treatments have been largely ineffective for high-grade brain tumors (gliomas; HGGs). Leveraging several single-cell RNA sequencing datasets, we report greater CD8⁺ exhausted T cells in human pediatric low-grade gliomas (LGGs) relative to adult and pediatric HGGs. Using several preclinical mouse LGG models (Nf1-OPG mice), we show that these PD1⁺/TIGIT⁺ CD8⁺ exhausted T cells are restricted to the tumor tissue, where they express paracrine factors necessary for OPG growth. Importantly, ICI treatments with α-PD1 and α-TIGIT antibodies attenuate Nf1-OPG tumor proliferation through suppression of two cytokine (Ccl4 and TGFβ)-mediated mechanisms, rather than by T cell-mediated cytotoxicity, as well as suppress monocyte-controlled T cell chemotaxis. Collectively, these findings establish a previously unrecognized function for CD8⁺ exhausted T cells as specialized regulators of LGG maintenance.

Fig. 1. CD8⁺ exhausted T cells are present in human pilocytic astrocytomas.

A UMAP visualization of T cells from human pLGG ([pilocytic astrocytoma, PA] PA set 1 and PA set 2 datasets; WHO grade 1 PA tumors) (n = 2 datasets, 11 tumors, GSE222850 and dbGAP phs001854.v1.p1), human pediatric HGG (pHGG [diffuse midline glioma, DMG] datasets; WHO grade 4 tumors) (n = 1 dataset, 5 tumors, GSE102130), and human adult HGG ([glioblastoma, GBM], aHGG1 and aHGG2 datasets; WHO grade 4 tumors) (n = 2 datasets, 7 tumors, Annotare [ArrayExpress] accession # E-MTAB-9435 and GSE138794) scRNAseq datasets. B Charts showing the percentage of T cells from human PA (PA set 1 and PA set 2 datasets; WHO grade 1 PA tumors) (n = 2 datasets, 11 tumors), human pediatric HGG (pHGG [diffuse midline glioma, DMG] datasets; WHO grade 4 tumors) (n = 1 dataset, 5 tumors), and human adult HGG ([glioblastoma, GBM], aHGG1 and aHGG2 datasets; WHO grade 4 tumors) (n = 2 datasets, 7 tumors) scRNAseq datasets. C CD8A expression in human PA (n = 2 datasets), pediatric HGG (n = 1 dataset) and adult HGG samples (n = 2 datasets). Each dot is an individual cell. D Table showing the P value, false discovery rate (FDR), and fold change of CD8A expression in human PA, pediatric and adult HGGs. Differential analyses were performed using gene specific analysis (GSA). E Dot plot showing the genes used to identify CD8⁺ exhausted T cells in human PA (n = 2) and HGG datasets (pediatric, n = 1; adult, n = 2). F UMAP visualization of scRNAseq data from five human PA specimens (PA1 [4463 cells], PA2 [4829 cells], PA3 [10404 cells], PA4 [7210 cells], PA5 [4636 cells]) based on tumor sample. G UMAP visualization of scRNAseq data from five human PA specimens (PA1, PA2, PA3, PA4, PA5) based on cell type. H UMAP visualization of scRNAseq data from five human PA specimens (PA1, PA2, PA3, PA4, PA5) color-coded by gene expression (CD8A, PDCD1, TIGIT, LAG3, TOX, CTLA4). I Dot plot showing the genes used to identify CD8⁺ exhausted T cells in human PA specimens. J Top 20 transcripts in CD8A⁺ PDCD1⁺ T cells relative to other T cells calculated with DESeq2 and filtered P values ≤ 0.05, false discovery rates ≤0.1, and log fold changes greater or equal to ±2. Differential analyses were performed using gene specific analysis (GSA).

Fig. 2. CD8⁺ exhausted T cells are present in the optic nerve of Nf1-OPG mice.

A t-SNE visualization of scRNAseq data of optic nerves collected from 12-week-old Nf1-OPG mice, based on cell type; n = 10 pooled mice. Microglia and monocytes constitute tumor associated monocytes (TAM). B t-SNE visualization of the T cell populations from 12-week-old Nf1-OPG mouse optic nerves, color-coded by T cell type. NOS, not otherwise specified. C Dot plot showing the genes used to identify CD8⁺ exhausted T cells in 12-week-old Nf1-OPG mouse optic nerves. D Stacked bar plot representing the barcode frequency of the top clones in the different T cell populations in 12-week-old Nf1-OPG mouse optic nerves. The graph is color coded to show the 1–10 clones and 11–10,000 clones.

Fig. 3. PD1⁺ TIGIT⁺ CD8⁺ exhausted T cells are enriched in the tumors from Nf1-OPG mice.

A Schematic representation of T cell circulation from the blood through the dural meninges (dura), optic nerve (ON), and draining deep cervical lymph nodes (dCLN). Created in BioRender. Chatterjee, J. (2023) BioRender.com/f03c341. B t-SNE visualization of scRNAseq data from 12-week-old Nf1-OPG mice peripheral blood mononuclear cells (PBMCs), based on cell type; n = 10 pooled mice. C t-SNE visualization of scRNAseq data from 12-week-old Nf1-OPG PBMCs, color-coded by gene expression (Cd8a, Pdcd1, Tigit, Lag3). D t-SNE visualization of the T cell populations from 12-week-old Nf1-OPG mouse meningeal dura, color-coded by T cell type, n = 10 pooled mice. E Dot plot showing the genes used to identify CD8⁺ exhausted T cells in the 12-week-old Nf1-OPG mouse meningeal dura scRNAseq dataset. Representative gating strategy for flow cytometry of 12-week-old PMBC T cells (n = 9 mice) (F), meningeal dura (n = 9 mice) (G), optic nerve (n = 4, 3 pooled optic nerves per sample) (H), and deep cervical lymph nodes dura (n = 9 mice) (I). J Dot plot showing the genes used to identify CD8⁺ exhausted T cells in 12-week-old Nf1-OPG mouse optic nerves (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice), meningeal dura (corresponds to the dataset shown in (D); n = 10 pooled mice), and PBMC (corresponds to the dataset shown in (B); n = 10 pooled mice) scRNAseq datasets. K Graph representing the percentages of CD8⁺/PD1⁺ and CD8⁺/TIGIT⁺ cells in the optic nerves (ON) and dura of 12-week-old Nf1-OPG mice (ON, n = 4, 3 pooled optic nerves per sample; dura, n = 9 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. L Graph comparing the percentages of CD8⁺/PD1⁺/TIGIT⁺ cells in the PBMCs, dura, optic nerve (ON), and dCLN, of 12-week-old Nf1-OPG mice (PBMCs, n = 8 mice; dura, n = 9 mice; ON, n = 4, 3 pooled optic nerves per sample; dCLN, n = 8 mice). Data are shown as mean ± SD. A one-way ANOVA test was performed followed by a Holm-Šídák post-test correction. Exact P values are included. M Representative gating strategy for flow cytometry of one representative human PA1 (n = 1; corresponds to PA1 sample in Fig. 1F).

Fig. 4. CD8⁺ exhausted T cells express Ccl4.

A Schematic representation of the optic glioma immune circuit. Created in BioRender. Chatterjee, J. (2024) BioRender.com/e28x983. B Ccl4 expression in the dura, optic nerve (ON), cerebellum (CBL), brainstem (BSTM), cortex (CTX), hippocampus (HC), deep cervical lymph nodes (dCLN), and superficial cervical lymph nodes (CLN) of 12-week-old Nf1-OPG mice, following normalization to the optic nerve. Data are represented as mean ± SD. (dura, n = 6 mice; ON, n = 3, 2 pooled optic nerves per sample; CBL, n = 6 mice; BSTM, n = 5 mice; CTX, n = 5 mice; HC, n = 6 mice; dCLN, n = 6 mice; CLN, n = 6 mice). A one-way ANOVA test was performed followed by a Holm-Šídák post-test correction. Exact P values are included. C Dot plot showing Ccl4 expression from 12-week-old Nf1-OPG mouse optic nerves (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice), meningeal dura (corresponds to the dataset shown in Fig. 3D; n = 10 pooled mice), and PBMC (corresponds to the dataset shown in Fig. 3B; n = 10 pooled mice) scRNAseq datasets. D Volcano plot showing fold change and P value comparing CD8⁺ exhausted T cells and the other T cell populations in the optic nerves of 12-week-old Nf1-OPG mice (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice). Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). E Volcano plot showing fold change and P value comparing CD8⁺ exhausted T cells in the optic nerves (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice) and PMBC T cells of 12-week-old Nf1-OPG mice (corresponds to the dataset shown in Fig. 3B; n = 10 pooled mice). Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). F Volcano plot showing fold change and P value comparing CD8⁺ exhausted T cells in the optic nerves (corresponds to the dataset shown in Fig. 2, Supplementary Fig. 2; n = 10 pooled mice in each dataset) and T cells from the dura of 12-week-old Nf1-OPG mice (corresponds to the dataset shown in Fig. 3D; n = 10 pooled mice). Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). G Volcano plot showing fold change and P value comparing CD8⁺ TEX in the optic nerves (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice, and to the dataset shown in Supplementary Fig. 2; n = 10 pooled mice) and CD8⁺ non-TEX T cells of 12-week-old Nf1-OPG mice (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice, and to the dataset shown in Supplementary Fig. 2; n = 10 pooled mice). Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). H Graph showing levels of secreted Ccl4 in the TCM by ELISA of unstimulated and CD8⁺ exhausted T cells (α-CD3+α-CD28 antibody stimulated) with and without FK506 treatment (none, n = 4; α-CD3+α-CD28, n = 4; α-CD3+α-CD28 + FK506, n = 4). A one-way ANOVA test was performed followed by a Holm-Šídák post-test correction. Data are represented as mean ± SD. Exact P values are indicated.

Fig. 5. Presence of CD8⁺ exhausted T cells mirrors optic glioma growth and Ccl4/Ccl5 expression.

A t-SNE visualization of scRNAseq data from optic nerves of 6-week-old Nf1-OPG mice, based on cell type, n = 10 pooled mice. B t-SNE visualization of the T cell populations from 6-week-old Nf1-OPG mice, color-coded by T cell type. Created in BioRender. Chatterjee, J. (2023) BioRender.com/p15a720. C t-SNE visualization of scRNAseq data from optic nerves of 24-week-old Nf1-OPG mice, based on cell type, n = 10 pooled mice. Microglia and monocytes constitute tumor associated monocytes (TAM). D t-SNE visualization of the T cell populations from 24-week-old Nf1-OPG mice, color-coded by T cell type. Created in BioRender. Chatterjee, J. (2023) BioRender.com/p15a720. E Dot plot showing genes used to identify CD8⁺ exhausted T cells in 6- (corresponds to the dataset shown in Fig. 5A; n = 10 pooled mice), 12- (corresponds to the datasets shown in Fig. 2 and Supplementary Fig. 2; n = 10 pooled mice in each dataset), and 24-week-old Nf1-OPG mouse optic nerves (corresponds to the dataset shown in (C); n = 10 pooled mice). F Graph showing changes in the percentage of CD8⁺ exhausted T cells (blue) in 6-week-old (corresponds to the dataset shown in (A); n = 10 pooled mice), 12-week-old (corresponds to the datasets shown in Fig. 2 and Supplementary Fig. 2; n = 10 pooled mice in each dataset), and 24-week-old (corresponds to the dataset shown in (C); n = 10 pooled mice) Nf1-OPG mice and proliferating (Ki67⁺ cells; red) tumor cells over time (6 weeks, n = 5 mice; 12 weeks, n = 4 mice; 24 weeks, n = 4 mice). Data are represented as mean ± SD. G Quantification CD8⁺ T cells in the optic nerves of Nf1-OPG mice at 12 and 24 weeks of age (12 weeks, n = 5 mice; 24 weeks, n = 5 mice). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. ns, not significant. H Ccl4 RNA expression in the optic nerves of 6-week-old, 12-week-old, and 24-week-old Nf1-OPG mice. Data are represented relative to the 12-week-old group (6-week-old mice, n = 6, 2 pooled optic nerves per sample; 12-week-old mice, n = 4, 2 pooled optic nerves per sample; 24-week-old mice, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. ns, not significant. I Dot plot showing Ccl4 expression in T cells from optic nerves of 6-week-old (6 W; corresponds to the dataset shown in (A); n = 10 pooled mice), 12-week-old (12 W) (corresponds to the datasets shown in Fig. 2 and Supplementary Fig. 2; n = 10 pooled mice in each dataset), and 24-week-old (24 W) (corresponds to the dataset shown in (C); n = 10 pooled mice) Nf1-OPG mice. J Ccl5 RNA expression in the optic nerves of 6-week-old, 12-week-old, and 24-week-old Nf1-OPG mice. Data are represented relative to the 12-week-old group (6-week-old mice, n = 3, 2 pooled optic nerves per sample; 12-week-old mice, n = 5, 2 pooled optic nerves per sample; 24-week-old mice, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. ns, not significant. K Dot plot showing Ccl5 expression in TAM from the optic nerves of 6-week-old (6 W; corresponds to the dataset shown in (A); n = 10 pooled mice), 12-week-old (12 W) (corresponds to the datasets shown in Fig. 2 and Supplementary Fig. 2; n = 10 pooled mice in each dataset), and 24-week-old (24 W) (corresponds to the dataset shown in (C); n = 10 pooled mice) Nf1-OPG mice.

Fig. 6. Anti-PD1⁺/TIGIT⁺ ICI treatment reduces CD8⁺ T cell infiltration by decreasing TAM-mediated Ccl12 and Cxcl13 chemoattraction.

A Schematic representation of the anti-PD1 (α-PD1), anti-TIGIT (α-TIGIT), and combined (α-PD1 + TIGIT) antibody treatments. Nf1-OPG mice were treated (200 µg/dose/ i.p., twice per week) from 12 to 16 weeks of age, and tissues were analyzed at 16 weeks. The control group was injected with anti-IgG isotype control antibodies. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. B Immunohistochemistry and quantification CD8⁺ T cells in the entire optic nerve (IgG, n = 10 mice; α-TIGIT, n = 10 mice; α-PD1, n = 6 mice; α-PD1 + TIGIT, n = 5 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0054; α-PD1, P = 0.0003; α-PD1 + TIGIT, P = 0.0032). Scale bar, 200 µm. C Ccl2 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. D Volcano plot showing fold change and P value comparing Nf1-OPG TAM to Nf1^+/ and WT monocytes in the optic nerves of 12-week-old mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). E Ccr2, Cxcr3, and Cxcr5 expression in T cell populations from 12-week-old Nf1-OPG mouse optic nerves, color-coded by T cell type. F Cxcl9, Ccl12, and Cxcl13 RNA expression in the optic nerves of 12-week-old WT and Nf1-OPG mice. Data are represented relative to the WT group (Cxcl9; WT n = 3, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample; Ccl12; WT n = 4, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample; Cxcl13; WT n = 4, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. ns, not significant. G Graph showing the percentage of migrated Nf1^+/- CD8⁺ T cells treated with medium (Control) (n = 5), Ccl12 (n = 6), or Cxcl13 (n = 5). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. H Ccl12 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 3, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0012; α-PD1, P = 0.0187; α-PD1 + TIGIT, P = 0.0013). I Cxcl13 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 4, 2 pooled optic nerves per sample; α-TIGIT n = 4, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post test correction. Exact P values are indicated (α-TIGIT, P = 0.0003; α-PD1, P = 0.0007; α-PD1 + TIGIT, P = 0.0001).

Fig. 7. Anti-PD1⁺/TIGIT⁺ ICI treatment reduces tumor cell proliferation through two distinct mechanisms.

A Immunohistochemistry and quantification of proliferating (%Ki67⁺) cells in the optic nerves of Nf1-OPG mice (IgG, n = 9 mice; α-TIGIT, n = 10 mice; α-PD1, n = 9 mice; α-PD1 + TIGIT, n = 6 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated (α-PD1, P < 0.0001; α-PD1 + TIGIT, P = 0.001). Scale bar, 200 µm. B, C Ccl4 and Ccl5 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post test correction. Exact P values are indicated. D Immunocytochemistry and quantification of proliferating (%Ki67⁺) optic glioma tumor cells (o-GTCs) treated with TCM from unstimulated (none) and exhausted (α-CD3+α-CD28 antibody stimulated) CD8⁺ T cells (Control, n = 8; TCM none, n = 4; TCM α-CD3+α-CD28, n = 4). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. Scale bar, 200 µm. E Volcano plot showing fold change and P value comparing CD8⁺ PD1⁺ T cells (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice) and in CD8⁺ PD1^- T cells (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice) the optic nerve of 12-week-old Nf1-OPG mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). F Graph showing levels of secreted TGFβ in the TCM of unstimulated (none) and exhausted (α-CD3+α-CD28 antibody stimulated) CD8⁺ T cells by ELISA (none, n = 4; α-CD3+α-CD28, n = 4). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. G Tgfb1 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Holm-Šídák post-test correction. Exact P values are indicated. H Schematic representation of the anti-TGFβ treatment (α-TGFβ). Nf1-OPG mice were treated (200 µg/dose, i.p., twice per week) from 12 to 16 weeks of age, tissues were analyzed at 16 weeks. The control group was injected with anti-IgG isotype control antibodies. Created in BioRender. Chatterjee, J. (2024) BioRender.com/z17k582. I Immunohistochemistry and quantification of proliferating (%Ki67⁺) cells in the optic nerves of Nf1-OPG mice (IgG, n = 5 mice; α-TGFβ, n = 4 mice). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. Scale bar, 200 µm. J Immunohistochemistry and quantification of CD8⁺ T cells in the entire optic nerve of Nf1-OPG mice (IgG, n = 5 mice; α-TGFβ, n = 4 mice). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. Scale bar, 200 µm. K Schematic representation of the direct and indirect mechanisms by which CD8⁺ exhausted T cells regulate Nf1-OPG growth. Created in BioRender. Chatterjee, J. (2024) BioRender.com/z17k582.