Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Abstract

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

Figure 1.. Tumor-infiltrating CD8⁺ T cells display a distinct phenotype in GIST.

(A-E) Bulk RNAseq was performed on CD8⁺ T cells sorted from tumors, tumor-draining lymph nodes (i.e., mesenteric nodes), and spleens of 4 untreated KitV558^Δ/+ mice. (A) Principal component analysis and (B) similarity of the transcriptomes of the sorted CD8⁺ T cells from tumor (T), spleen (S), and tumor-draining lymph node (N). (C) Hallmark inflammatory response, interferon gamma response, glycolysis, and IL2/STAT5 signaling pathway gene set enrichment (normalized enrichment score shown; all values p < 0.05), (D) heatmap depicting normalized expression of select antigen response genes, and (E) volcano plot comparing bulk RNAseq of the sorted CD8⁺ T cells from Kit^V558Δ/+ tumor or node. (F-H) Flow cytometry of tumors, tumor-draining lymph nodes (i.e., mesenteric nodes), and/or spleens of untreated KitV558^Δ/+ mice. (F) Frequency of T_EM, T_CM, and T_N among CD8⁺ T cells in tumor, spleen, or tumor-draining lymph node (n=4 mice/group). (G) Frequency of CCR7⁺, IL-7R⁺, Tbet⁺, Eomes⁺, and PD-1⁺ cells among CD8⁺ T cells from tumors of Kit^V558Δ/+ mice (n=4 mice/group). (H) Frequency of CD103⁺ and CD103⁻ effector CD8⁺ T cells from tumors of Kit^V558Δ/+ mice (n=3 mice/group). Each column in (D) represents an individual mouse. NS, non-significant; FC, fold-change. Data represent mean ± SEM; *, p < 0.05.

Figure 2.. ScRNAseq reveals intratumoral CD8⁺ T-cell heterogeneity.

Droplet-based scRNAseq was performed on immune cells (CD45⁺ cells) sorted from tumors from 3 untreated KitV558^Δ/+ mice. (A) Clusters of CD4⁺ T cells, CD8⁺ T cells, and Tregs within the CD3⁺ compartment based on unsupervised clustering by principal component analysis, defined by (B) cd4, cd8a, and foxp3 expression. (C) Numbered clusters within the intratumoral CD8⁺ T-cell compartment based on unsupervised clustering by principal component analysis. (D) Heatmap of gene expression modules, with stratified module enrichment by CD8⁺ T-cell clusters. (E) Violin plots of expression of select genes in CD8⁺ T-cell clusters. (A, C) UMAP plots display scRNAseq profiling of 11,641 cd3d/e/g and 8,065 cd8a/b T cells from 3 mice as detailed in the methods section.

Figure 3.. Imatinib expands intratumoral T_N and reduces T_EM.

(A-C) Kit^V558Δ/+ mice were treated with vehicle or imatinib for 1 week (n=4 mice/group). At that time, tumors, tumor-draining lymph nodes (i.e., mesenteric nodes), and spleens were harvested and bulk RNAseq was performed on CD8⁺ T cells sorted from the tissues. (A) Principal component analysis and (B) volcano plot of sorted CD8⁺ T cells from tumor. (C) Normalized expression of Il7r in node, spleen, or tumor. Each point in (A) represents a sample from an individual mouse. (D, E) Kit^V558Δ/+ mice were treated with vehicle or imatinib for 1 week (n=3 mice/group). At that time, scRNAseq was performed on CD45⁺ cells sorted from the tumors. (D) UMAP plot of CD8⁺ T-cell clusters and (E) cluster imatinib/vehicle fold change in tumors. (F) CD8⁺ T-cell clusters from (D), represented as grey triangles/isobars were projected onto a published reference atlas [29] and the difference in proportion of T-cell states including exhausted (T_EX), precursor exhausted (T_PEX), T_EM, early activation (T_EA), and T_N were compared between vehicle and imatinib groups. (G-J) Kit^V558Δ/+ mice were divided into 3 groups on Day 0 and all mice were sacrificed on Day 28. Mice were treated with vehicle or imatinib for 1 or 4 weeks and tumors were analyzed by flow cytometry for (G) Frequency of T_EM, T_CM, and T_N among CD8⁺ T cells; (H) CD44⁺ and CD62L⁺ expression in CD8⁺ T cells; (I) frequency of CCR7⁺, IL-7R⁺, Tbet⁺, Eomes⁺ cells among CD8⁺ T cells; (J) frequency of PD1⁺ cells among T_EM CD8⁺ T cells; and (K) frequency of CD103⁻ T_N cells among CD8⁺ T cells (n=3–4 mice/group). Data represent mean ± SEM; *, p < 0.05.

Figure 4.. Intratumoral CD8⁺ T cells have restricted PI3K signaling and lack clonal expansion in response to imatinib.

(A-B) Kit^V558Δ/+ mice were treated with vehicle or imatinib for 1 week (n=4 mice/group) and sorted CD8⁺ T cells from tumors were analyzed by bulk RNAseq for (A) hallmark TNF alpha response, inflammatory response, glycolysis, G2M checkpoint, interferon gamma response, and interferon alpha response gene set enrichment (normalized enrichment score shown; all values p < 0.05); and (B) Pdcd1 expression. (C) Following CD8⁺ T-cell depletion with one dose of anti-CD8 on day 0, Kit^V558Δ/+ mice were treated with vehicle or imatinib for 5 days. On day 4, mice received CSFE-labeled splenic CD8⁺ T cells from congenic CD45.1 mice by adoptive transfer. On day 5, frequency of CSFE⁺CD45.1⁺CD8⁺ T cells in the spleen and tumor was determined by flow cytometry, as well as the cell count in the tumor (n=4 mice/group, pooled from two independent experiments). (D) Normalized expression of Akt1, Akt2, Akt3, and Mtor in sorted CD8⁺ T cells in tumors from Kit^V558Δ/+ mice following bulk RNAseq, as in Fig. 4A,B (n=4 mice/group). (E) Median fluorescence intensity (MFI) and representative histograms of p-AKT⁺ and p-mTOR⁺ cells among CD45⁺CD3⁺NK1.1⁻CD4⁻CD8⁺ T cells in tumor and tumor-draining lymph node from untreated Kit^V558Δ/+ mice (5 mice/group, repeated twice). (F, G) Kit^V558Δ/+ mice were treated with vehicle (Veh) or imatinib (IM) for 1 week (n=4 mice/group). At that time, tumors, spleens and cecum were harvested and analyzed by high-throughput sequencing of the CDR3 TCRβ region. (F) Unique productive TCR sequences and (G) occupied repertoire space of small, medium, large, and hyperexpanded clonotype groups where units represent clonal frequency. Data represent mean ± SEM; *, p < 0.05.

Figure 5.. Human GIST display differential CD8⁺ T-cell subsets.

CD8⁺ T cells from human GIST specimens (20 tumor specimens from 20 patients) were analyzed by flow cytometry for (A) frequency of CD45RA and CD45RO expression among CD8⁺ T cells and (B) frequency of memory subsets among the CD8⁺ T-cell population. Line indicates median; *, p < 0.05

Figure 6.. IL15-mediated stimulation with immune checkpoint blockade improves the response to imatinib.

(A) Kit^V558Δ/+ mice were treated with IL15SA three times over the course of 9 days and given imatinib daily for 7 days. Controls received PBS for 9 days and vehicle or imatinib from days 0 to 7. (B) Tumor weights of Kit^V558Δ/+ GIST treated as described in (A) (n=5–6 mice/group, repeated twice). Flow cytometry of tumors was performed to determine: (C) Frequency of T_EM, T_CM, and T_N among intratumoral CD8⁺ T cells; and (D) Median fluorescence intensity (MFI) and representative histogram of p-AKT⁺ or p-mTOR⁺ CD8⁺ T cells in tumors from Kit^V558Δ/+ mice (n=5 mice/group, repeated twice). (E) Kit^V558Δ/+ mice were treated with IM (imatinib), IM + depleting anti-CD8 (−CD8), IM + IL15SA, or IM + IL15SA + −CD8 and assessed for tumor weight and frequency of CD8⁺ T cells among CD45⁺ cells, as assessed by flow cytometry (4–5 mice/group). (F) Kit^V558Δ/+ mice were treated with IM, IM + aPD1 (anti-PD1), IM + IL15SA, or IM + IL15SA + aPD1 for 4 weeks and assessed for tumor weight (5 mice/group, pooled from two independent experiments). Flow cytometry was used to assess these mice for (G) CD8⁺ T-cell subsets and (H) frequency of Ki67⁺ and Granzyme B⁺ cells among CD8⁺ T. Line indicates median; *, p < 0.05.