Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors

Abstract

Background: Glioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (T_ex) in many malignancies, has not shown efficacy in glioblastoma. Loss of CD4 T cells can lead to an exhausted CD8 T-cell phenotype, and terminally exhausted CD8 T cells (T_ex ^term) do not respond to PD-1 blockade. GL261 and CT2A are complementary orthotopic models of glioblastoma. GL261 has a functional CD4 T-cell compartment and is responsive to PD-1 blockade; notably, CD4 depletion abrogates this survival benefit. CT2A is composed of dysfunctional CD4 T cells and is PD-1 blockade unresponsive. We leverage these models to understand the impact of CD4 T cells on CD8 T-cell exhaustion and PD-1 blockade sensitivity in glioblastoma.

Methods: Single-cell RNA sequencing was performed on flow sorted tumor-infiltrating lymphocytes from female C57/BL6 mice implanted with each model, with and without PD-1 blockade therapy. CD8⁺ and CD4⁺ T cells were identified and separately analyzed. Survival analyses were performed comparing PD-1 blockade therapy, CD40 agonist or combinatorial therapy.

Results: The CD8 T-cell compartment of the models is composed of heterogenous CD8 T_ex subsets, including progenitor exhausted CD8 T cells (T_ex ^prog), intermediate T_ex, proliferating T_ex, and T_ex ^term. GL261 is enriched with the PD-1 responsive T_ex ^prog subset relative to the CT2A and CD4-depleted GL261 models, which are composed predominantly of the PD-1 blockade refractory T_ex ^term subset. Analysis of the CD4 T-cell compartments revealed that the CT2A microenvironment is enriched with a suppressive T_reg subset and an effector CD4 T-cell subset that expresses an inhibitory interferon-stimulated (Isc) signature. Finally, we demonstrate that addition of CD40 agonist to PD-1 blockade therapy improves survival in CT2A tumor-bearing mice.

Conclusions: Here, we describe that dysfunctional CD4 T cells are associated with terminal CD8 T-cell exhaustion, suggesting CD4 T cells impact PD-1 blockade efficacy by controlling the severity of exhaustion. Given that CD4 lymphopenia is frequently observed in patients with glioblastoma, this may represent a basis for resistance to PD-1 blockade. We demonstrate that CD40 agonism may circumvent a dysfunctional CD4 compartment to improve PD-1 blockade responsiveness, supporting a novel synergistic immunotherapeutic approach.

Keywords: CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; brain neoplasms; immunotherapy; lymphocytes, tumor-infiltrating.

Figure 1

Single-cell RNA-sequencing characterization of infiltrating CD8⁺ T cells in six glioma models. (A) UMAP with cells colored according to graph-based cluster on the left (T_ex subsets defined in subsequent analyses) and by glioma model on the right. (B) Gene expression dot plot of key genes associated with terminal and progenitor exhausted T-cell (T_ex) populations, which defines cluster 1 as T_ex^prog. (C) UMAP and violin plot of left exhaustion module score and right expression of Tcf7. (D) UMAP feature plot of Ki67 expression supports clusters 3 and 7 as a T_ex^prolif subset. T_ex, exhausted CD8 T cell; T_ex^prog, progenitor exhausted CD8 T cell; T_ex^prolif, proliferating T_ex cell.

Figure 2

Effect of murine glioma model and PD-1 blockade therapy on CD8⁺ T-cell compartment. For each model: left UMAP of CD8⁺ T cells, right histogram of cell composition in each cluster. While GL261 shifts from a T_ex^prog to T_ex^inter predominant model following PD-1 blockade therapy, the CD4-deficient models (CT2A and CD4-depleted GL261) are composed of T_ex^term cells that are not affected by PD-1-blockade therapy. T_ex, exhausted CD8 T cell; T_ex^inter, intermediate exhausted CD8 T cell; T_ex^prog, progenitor exhausted CD8 T cell; T_ex^prolif, proliferating T_ex cell; T_ex^term, terminally exhausted CD8 T cell.

Figure 3

Differences in TCF1 and TOX expression among CD8⁺ TIL isolated from GL261 and CT2A with and without PD-1 blockade therapy. (A) Bar graph depicting percentage TCF1⁺PD-1⁺CD8⁺ TIL among total PD-1⁺CD8⁺ TIL in GL261 and CT2A with and without anti-PDL1 antibody treatment (GL261 TIL vs CT2A TIL, p=0.0025; GL261 TIL vs GL261+PDL1 TIL, p=0.021). (B) Bar graph depicting percentage TOX⁺PD-1⁺CD8⁺ TIL among total PD-1⁺CD8⁺ TIL in GL261 and CT2A with and without anti-PDL1 antibody treatment (GL261 TIL vs CT2A TIL, p=0.0003; GL261 TIL vs GL261+PDL1 TIL, p=0.0057). (C) Bar graph representing mean fluorescent intensity of TOX expression in TOX⁺PD-1⁺CD8⁺ TIL (GL261 TIL vs CT2A TIL, p=0.0069). Data pooled from at least two independent experiments with n=3 mice per group per experiment. TIL, tumor-infiltrating lymphocyte.

Figure 4

Tumor-infiltrating T-cell TCR clonality and diversity (A) TCR heterogeneity of each glioma model, wherein ‘clonal index’ represents each clonotype’s rank-based frequency. (B) Clonal expansion categories superimposed onto UMAP of all conditions concatenated. (C) Visualization of the top five expanded clonotypes of each tumor model superimposed onto individual UMAP.

Figure 5

Single-cell RNA sequencing characterization of infiltrating CD4 T cells. (A) UMAP with cells colored according to graph-based cluster. (B) Graph-based clustering and cluster composition of the CD4 T-cell compartment separated by glioma model. (C) Characterization of the regulatory T-cell (T_reg) compartment: scaled Foxp3 expression defines clusters 3 and 4 as T_reg (left/top); expression dot plot of key T_reg genes defines cluster 3 as activated and cluster 4 as resting T_reg (right); and histogram of T_reg composition shows CT2A is predominantly activated T_reg, while GL261 is composed of equivalent proportions of activated and resting T_reg (left/bottom). (D) Expression dot plot of effector CD4 T-cell genes in the non-T_reg clusters. (E) Expression dot plot of genes comprising the Isc signature, which is more highly expressed in cluster 2. (F) Feature plot demonstrating scaled gene expression of IL-21 (left) and bar graph depicting protein expression represented as mean fluorescent intensity of IL-21 within Foxp3⁻ CD4⁺ TIL (right; data pooled from at least two independent experiments with n=3 mice per group per experiment.; GL261 TIL vs CT2A TIL, p=0.030). IL, interleukin; TIL, tumor-infiltrating lymphocyte; T_reg, regulatory T cell.

Figure 6

Circumventing a dysfunctional CD4 T-cell response through CD40 agonism. Kaplan-Meier survival curves evaluating CD40 agonist and PD-1 blockade monotherapy and combinatorial therapy in (A) CT2A (p=0.0003) and (B) GL261 (p=0.1129) demonstrate CD40 agonism delivers a synergistic benefit with PD-1-blockade therapy, exclusively in a CD4-deficient environment. For each experiment, n=5 per group and pooled from two independent experiments.