IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors

Abstract

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8⁺CD62L⁺TCF7⁺IRF4^- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7⁺ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Keywords: CAR T cells; IL-15; T(CM); TCF1; TCF7; anti-PD-1; cancer; checkpoint blockade; solid tumor.

Graphical abstract

Figure 1

Preconditioning Anti-Her2 CAR T Cells with IL-7/IL-15 Leads to Increased Expression of Memory-Associated Genes Anti-Her2 CAR T cells were generated in IL-2 (100 U/mL) and IL-7 (200 pg/mL) or IL-7 and IL-15 (10 ng/mL). (A) Schematic of CAR T cell generation. (B) Expression of CD62L and CD44 on CD8⁺NGFR⁺ T cells at day 6 post-activation. Representative FACS plot (left) or pooled data (mean ± SEM; right) from n = 5 experiments. ∗∗∗p < 0.001, ∗p < 0.05 paired t test. (C) Proportion of CD8⁺NGFR⁺ cells with a T_SCM phenotype (defined by gating as in (B) following culture in indicated cytokine combinations). Data represent mean ± SD of triplicate cultures from a representative experiment. (D–F) At day 6 post-activation, CD8⁺NGFR⁺ CAR T cells were FACS sorted into T_EM, T_CM, and T_SCM populations following culture with IL-2/IL-7 or IL-7/IL-15 and their transcriptome analyzed by RNA-seq. (D) Principal-component analysis (PCA) plot based upon the top 100 most variable genes. (E) Heatmap of indicated genes. (F) GSEA plots showing enrichment for a gene-set isolated from TCF7⁺ cells and previously indicated to be responsive to immune-checkpoint blockade. (G-I) At day 6 post-activation, anti-Her2 CAR T cells were cocultured with E0771-Her2 or E0771 parental tumor cells for 3 consecutive days. Supernatants were analyzed for their expression of IFN-γ at 1 day and 3 days postinitiation of coculture (G), and the CD8⁺NGFR⁺ CAR T cell phenotype was determined at the end of the experiment. (H and I) Data represent the mean ± SD of triplicate conditions from a representative experiment (I) or concatenated FACS plots from these 3 cocultures (H). ∗∗p < 0.01 paired t test.

Figure 2

Preconditioning CAR T Cells with IL-7/IL-15 Leads to Enhanced Anti-Tumor Efficacy When Combined with Anti-PD-1 (A–F and H) C57BL/6 Her2 transgenic (Tg) or (G) RAG^−/− mice were injected subcutaneously with 2 × 10⁵ MC38-Her2 CAR T cells or with 2 × 10⁵ E0771-Her2 tumor cells into the fourth mammary fat pad and treated with anti-Her2 CAR T cells, generated as per Figure 1. 1 × 10⁷ CAR T cells were injected intravenously on days 7 and 8 (E0771-Her2) or 5 and 6 (MC38-Her2) following total body irradiation (4 Gy or 0.5 Gy, respectively). Mice were treated with 50,000 U of IL-2 on days 0–4 post-CAR T cell treatment and with either anti-PD-1 (200 μg/mouse) or 2A3 isotype control on days 0, 4, 8, and 12 post-treatment. (A, B, F, and G) Tumor growth, shown as the mean ± SEM of n = 6–8 mice per group. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 two-way ANOVA. (B) 9 days post-treatment, spleens and tumors were excised, and the number of CD8⁺NGFR⁺ CAR T cells in the indicated organs was determined by flow cytometry. Data represent the mean ± SEM of 19–20 mice per group from 3 replicate experiments. ∗∗∗∗p < 0.0001, ∗p < 0.05 unpaired t test. (C) Tumor growth, shown as the mean ± SEM of 12–14 mice per group from 2 pooled experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 two-way ANOVA. (D) Survival of mice with end point defined as when tumors exceeded 100 mm². ∗p < 0.05 log rank test. (E and H) Expression of CD62L, PD-1, and TCF7 within CD8⁺NGFR⁺ CAR T cells. FACS plots represent concatenated data from n = 8 mice. Pooled data represent the mean ± SEM of 10–17 mice per group from 2 to 3 replicate experiments. ∗p < 0.05 one-way ANOVA.

Figure 3

The CD8⁺CD62L⁺ CAR T Cell Population Elicits the Majority of Transcriptional Changes Following PD-1 Blockade C57BL/6 Her2 Tg mice were injected with 2 × 10⁵ E0771-Her2 tumor cells into the fourth mammary fat pad and treated with IL-7/IL-15-generated anti-Her2 CAR T cells as per Figure 2. Mice were treated on day 7 with anti-PD-1 (200 μg/mouse) or 2A3 isotype control, and on day 9, tumors were excised, and CD8⁺NGFR⁺CD62L⁺ and CD8⁺NGFR⁺CD62L⁻ were isolated by FACS sorting. Transcriptional changes were analyzed by RNA-seq. Data represent a pool of cells obtained from 3 mice for each biological replicate. (A) Differential gene-expression analysis; genes in red indicate significance based upon an FDR of <0.05. (B) Gene expression by heatmap for indicated cell populations in mice treated with 2A3 isotype control. (C) GSEA plot showing enrichment for genes upregulated in CD62L⁺ (relative to CD62L⁻) compared to a gene set identified in TCF7⁺ cells responsive to immune-checkpoint blockade. (D) Differential gene-expression analysis following anti-PD-1 treatment in CD62L⁻ and CD62L⁺ cells. Volcano plots (top) and Venn diagram showing overlapping genes (bottom) with significance cutoff set at FDR < 0.05. (E) Principal component plot based upon the top 1,000 most variable genes. (F and G) Enriched pathways within the gene ontology (GO) biological processes database based upon differentially expressed genes within CD62L⁺ cells following anti-PD-1 treatment. (F) Most significantly enriched Gene Ontology (GO) pathways (G) Heatmaps indicate gene expression within selected GO pathways.

Figure 4

IL-7/IL-15 Preconditioned CAR T Cells Elicit a More Potent Effector Response Following Anti-PD-1 Treatment C57BL/6 Her2 Tg mice were injected with 2 × 10⁵ E0771-Her2 tumor cells into the fourth mammary fat pad and treated with anti-Her2 CAR T cells as per Figure 2, having been generated with IL-2/IL-7 or IL-7/IL-15. At day 9 post-treatment, tumors were excised, and tumor-infiltrating CD8⁺NGFR⁺ CAR T cells were analyzed by flow cytometry. (A) The expression of IFN-γ and TNF following 3 h of PMA (5 ng/mL) and ionomycin (1 μg/mL) in the presence of GolgiPlug and GolgiStop. Top: data shown as mean ± SEM of 14–18 mice per group from 3 replicate experiments. ∗p < 0.05, ∗∗∗p < 0.001, one-way ANOVA. Bottom: concatenated data from 8 mice per group. (B and C) The expression of (B) Ki-67, granzyme B, and TIM-3 and (C) IRF4 in CD8⁺NGFR⁺ CAR T cells. Data shown as mean ± SEM of 14–18 mice per group from 3 replicate experiments. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA. (D) The expression of IRF4 in CD62L⁻ and CD62L⁺ subsets. Data are from concatenated mice from a representative experiment of n = 2. Bottom: pooled data from 11–13 mice per group from 2 individual experiments. ∗p < 0.05, ∗∗∗p < 0.001, one-way ANOVA.

Figure 5

IL-15 Preconditioning Results in a Favorable Epigenetic Landscape for Enhanced Effector Functions Following PD-1 Blockade Anti-Her2 CAR T cells were generated in IL-2 (100 U/mL) and IL-7 (200 pg/mL) or IL-7 and IL-15 (10 ng/mL). (A–D) At day 6 post-activation, CD8⁺NGFR⁺ CAR T cells were FACS sorted into T_EM, T_CM, and T_SCM populations and their genome accessibility analyzed by ATAC-seq. (A) PCA plot based on the 5,000 most variable peaks. (B and C) Analysis was restricted to sites within −5 kb or +10 kb of TSS. (B) MA plot of log2 fold change (FC) differences in accessibility peaks between T_CM CAR T cells generated with IL-2/IL-7 or IL-7/IL-15 versus the mean of normalized log CPM. Colored peaks are statistically significant. (C) Heatmap of normalized log CPM for peaks associated with individual genes. Green and blue indicate sites significantly more/less accessible, respectively, after IL-15 treatment. Numbers indicate position of peak relative to TSS. (D) Accessibility tracks for selected genes and indicated cell types. (E and F) C57BL/6 Her2 Tg mice were injected with 2 × 10⁵ E0771-Her2 tumor cells into the fourth mammary fat pad and treated with IL-7/IL-15-generated anti-Her2 CAR T cells and CAR T cells FACS sorted at day 9 post-treatment as per Figure 3. (E) PCA plot based on the 5,000 most variable peaks. (F) MA plot of log 2FC differences in accessibility peaks between CD62L⁺ or CD62L⁻ CAR T cells treated with either 2A3 or anti-PD-1 on day 5 post-treatment versus the mean of normalized log CPM.