NKG2C/ KLRC2 tumor cell expression enhances immunotherapeutic efficacy against glioblastoma

Abstract

Background: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment.

Methods: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response.

Results: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2C^high-expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association.

Conclusions: This study explored the role of neoplastic NKG2C/KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials.

Keywords: Central Nervous System Cancer; Immune Checkpoint Inhibitor; Macrophage; NK Cell Lectin-Like Receptor Subfamily K; Tumor microenvironment - TME.

Figure 1. NKG2C is expressed on neoplastic cells in gliomas. (A) UMAP plot annotated according to each cell type of the integrated object (control and glioma scRNA-seq databases) (online supplemental table 1C). (B) List of the main genes and their corresponding proteins that are involved in the activation or inhibition of immune cell function; particularly regarding natural killer (NK) cells. (C) Dot plot showing the percentage and average expression of activation and inhibition markers in the different cell types of the integrated object (control and glioma scRNA-seq databases). The dot.min value was set to 0.01, meaning only genes with expression levels above 1% are represented by a dot. (D) UMAP plot with the cells expressing KLRC2 detailing the clusters annotated according to each cell type of the integrated object. (E) UMAP plot of cells with positive KLRC2 expression in non-tumor brain, newly diagnosed glioblastoma (GBM) and recurrent GBM. (E) UMAP plot of cells with positive KLRC2 expression in non-tumor brain, newly diagnosed GBM and recurrent GBM. (F) Human KLRC2 relative expression in human GBM patient-derived xenografts (PDXs) (n=3). GAPDH expression was used for normalization. (G) Representative image of the immunofluorescence analysis of SOX2 (magenta) and NKG2C (green) in patients with GBM. The white arrow points to a double-positive cell. Nuclei were counterstained with DAPI. Scale: 10 µm. DAPI, 4′,6-diamidino-2-phenylindole; IDHwt, isocitrate dehydrogenase-wild type; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Figure 2. Intertumoral and intratumoral heterogeneity of NKG2C expression in IDHwt GBM. (A) KLRC2 relative expression (qRT-PCR) in the periphery (n=30) versus tumor core (n=18) of IDHwt GBM. P values using unpaired t-test: **p≤0.01, ***p≤0.001. (B) UMAP plot annotated according to each cell type of the integrated GBM scRNA-seq database (GSE117891). A detailed description of the samples and counts used for the study can be found in online supplemental table 1C). (C) UMAP plot of cells with positive KLRC2, KLRC1 and KLRD1 expression in the periphery and tumor core of the GBM scRNA-seq database (GSE117891). (D) Bar chart of the percentage of cells expressing KLRC2 in each cell type in both the periphery and tumor core of the GBM scRNA-seq database (GSE117891). Significant differences were observed in the proportions of KLRC2-positive cells, especially in neoplastic cells (p=5.2e-22), oligodendrocytes (p=7.1e-7), and T cells (p=7.0e-3). The statistical analysis was conducted using the χ² test. The statistical analysis was conducted using the χ² test and can be found in more detail in online supplemental table 3B. GBM, glioblastoma; IDHwt, isocitrate dehydrogenase-wild type; qRT-PCR, quantitative real time-polymerase chain reaction; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Figure 3. Intertumoral and intratumoral heterogeneity of NKG2C expression in three patients with GBM. (A) Representative high-magnification image showing the co-expression of SOX2 (magenta), SOX4 (cyan), and NKG2C (green) in tumor cells. The upper panels display individual DAPI/NKG2C, DAPI/SOX2, and DAPI/SOX4 staining. (B–D) Representative images of the immunofluorescence analysis of the expression of SOX2 (magenta), SOX4 (cyan), and NKG2C (green) in the periphery (top images) and tumor core (bottom images) of three different patients with GBM. Nuclei were counterstained with DAPI (blue). The left images show the overall tissue structure with boxes indicating regions of interest magnified on the right. Scale: 10 µm (A) 100 µm for the main images and 50 µm for the insets (B–D). DAPI, 4',6-diamidino-2-phenylindole; GBM, glioblastoma; qRT-PCR, quantitative real time-polymerase chain reaction; UMAP, uniform manifold approximation and projection.

Figure 4. Effect of KLRC2 overexpression in murine and human glioma. GFP cDNA-expressing GL261 or KLRC2 cDNA-expressing GL261 cells were injected into the striatum of C57BL/6 mice. (A) Western blot analysis quantified NKG2C expression in GL261 brain tumors with Rho as the normalization control (GFP, n=6; NKG2C, n=5). (B) Representative H&E staining of tumor sections. KLRC2 cDNA-expressing GL261 brain tumors show microcystic formations (arrow). (C) Representative immunohistochemical staining of tumor sections using a CD68 or a CD206-specific antibody. Quantification of each staining is shown above (n=5). (D–F) qRT-PCR analysis of the expression for several macrophage-related (D) immune-related (E) or KLRC2- associated (F) gene markers in GFP versus KLRC2 cDNA-expressing GL261 brain tumors (n=7). (G–H) Flow cytometry assessment of immune cell population percentages in human glioma categorized by KLRC2 expression levels (n=8). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Scale: 2.5 mm and 100 µm (B) 50 µm (C). cDNA, complementary DNA; GFP, green fluorescent protein; qRT-PCR, quantitative real time-polymerase chain reaction.

Figure 5. KLRC2 cDNA expression enhances the antitumor immune response against a brain tumor after treatment with PD-1 mAb. (A) qRT-PCR-based KLRC2 expression in tumor tissues comparing GFP control with KLRC2 in the PD-1 mAb-treated group (n=6). (B) Mouse survival rates post-injection of GFP (n=7) or KLRC2 (n=9) GL261 cells and treatment that is depicted by Kaplan-Meier curves (p value=0.0145). (C–D) Representative immunohistochemical staining of tumor sections using CD68 and CD206 (n=5) (C) or CD3 and CD8 (GFP, n=4; NKG2C, n=7) (D) antibodies. Quantification of each staining is shown on the top. (E) Heatmap of normalized expression levels of genes associated with the immune environment. The heatmap’s color gradation corresponds to the relative expression levels of targeted markers. *p<0.05, **p<0.01. Scale: 50 µm (C–D). GFP, Green Fluorescent Protein; mAb, monoclonal antibodies; PD-1, programmed cell death protein-1; qRT-PCR, quantitative real time-polymerase chain reaction.

Figure 6. Relationship between NKG2C Expression and PD-1 mAb treatment efficacy in IDHwt patients with GBM. (A) Representative image of the immunohistochemical analysis (red) of NKG2C in patient tumor sections. (B) Correlation analysis between NKG2C+staining and overall survival (OS), stratified by the extent of tumor resection (complete (n=7) or partial (n=6)). The correlation coefficient (R) and p values are provided for each resection group. (C) Kaplan-Meier survival curves of IDHwt GBM divided according to the KLRC2 expression in the TCGA and CGGA studies. Scale: 100 µm. CGGA, Chinese Glioma Genome Atlas; GBM, glioblastoma; IDHwt, isocitrate dehydrogenase-wild type; mAb, monoclonal antibodies; PD-1, programmed cell death protein-1; TCGA, the Cancer Genome Atlas.

Figure 7. Association of the neoplastic expression of KLRC2 and other NK receptors with the response to anti-PD-1 in patients. (A) UMAP plot annotated by cell type within the isocitrate dehydrogenase-wild type glioblastoma single cell RNA sequencing database (CRA011176) (online supplemental table 1C). (B) Dot plot showing the percentage and average expression of KLRC2, KLRB1, KLRC1, and KLRD1 markers among responders and non-responders to anti-PD-1. The dot.min value was set to 0.01, meaning only genes with expression levels above 1% are represented by a dot. (C) UMAP plot of cells with positive KLRC2, KLRC1, and KLRD1 expression among responder and non-responder patients. cDCs, conventional dendritic cells; NK, natural killer; OPCs, oligodendrocyte progenitor cells; PD-1, programmed cell death protein-1; UMAP, uniform manifold approximation and projection.