NKG7 is a Stable Marker of Cytotoxicity Across Immune Contexts and Within the Tumor Microenvironment

Abstract

Cytotoxicity is a cornerstone of immune defense, critical for combating tumors and infections. This process relies on the coordinated action of granzymes and pore-forming proteins, with granzyme B (GZMB) and perforin (PRF1) being key markers and the most widely studied molecules pertaining to cytotoxicity. However, other human granzymes and cytotoxic components remain underexplored, despite growing evidence of their distinct, context-dependent roles. Natural killer cell granule protein 7 (NKG7) has recently emerged as a crucial cytotoxicity regulator, yet its expression patterns and function are poorly understood. Using large publicly available single-cell RNA sequencing atlases, we performed a comprehensive profiling of cytotoxicity across immune subsets and tissues. Our analysis highlights NKG7 expression as a strong marker of cytotoxicity, exhibiting a strong correlation with overall cytotoxic activity (r = 0.97) and surpassing traditional markers such as granzyme B and perforin in reliability. Furthermore, NKG7 expression is notably consistent across diverse immune subsets and tissues, reinforcing its versatility and robustness as a cytotoxicity marker. These findings position NKG7 as an invaluable tool for evaluating immune responses and a reliable indicator of cytotoxic functionality across biological and clinical contexts.

Keywords: CD8+ T‐cells; NKG7; cytotoxicity; granzyme B; natural killer cells; single‐cell RNA sequencing; tumor microenvironment.

FIGURE 1

Pattern of cytotoxic molecule expression in human PBMC subsets. (A) The cellular subsets of human PBMC as identified using integrated scRNAseq and scCITEseq data visualized on weighted‐nearest neighbor (wnn) UMAP coordinates. (B) The density of expression for cytotoxicity score overlayed on wnnUMAP. (C) Violin plots showing the “cytotoxicity score” across immune populations. (D) Violin plots showing the imputed expression of cytotoxicity markers that contribute to the cytotoxicity score. Cellular subsets are abbreviated as follows: CD14⁺ monocyte (CD14‐Mono), CD16⁺ monocyte (CD16‐Mono), CD56BrightCD16⁻ (NK‐Bright), CD56DimCD16⁺ (NK‐Dim), central memory (CM), conventional dendritic cell type 1 (cDC1), conventional dendritic cell type 2 (cDC2), cytotoxic T‐lymphocyte (CTL), double‐negative T‐cell (dnT; CD4⁻CD8⁻ T‐cells), effector memory (EM), gamma delta T‐cell (gdT), hematopoietic stem and progenitor cell (HPSC), innate lymphoid cell (ILC), mucosal‐associated invariant T‐cell (MAIT), plasmacytoid dendritic cell (pDC), proliferating (Prolif), regulatory T‐cell (Treg), Vγ9Vδ2 gamma delta T‐cells (gdT‐V9D2).

FIGURE 2

NKG7 correlates with cytotoxicity across human PBMC subsets. (A) Scatter plots demonstrating the correlation between y‐axis gene expression and cytotoxicity signature across PBMC subsets. Pearson correlation is displayed and the shaded area represents the 95% CI. (B) Histograms showing the expression of NKG7 in NK cell populations and the corresponding geometric mean fluorescence intensity in CD56Bright and CD56Dim populations. p‐values from Welch's t‐test. (C–D) Histograms showing the expression of NKG7 in EM, CM, and Naïve CD8+ T‐cells or EM, CM, and Naïve CD4+ T‐cells, respectively; and dot plots showing the frequency of NKG7+ populations. P values from ordinary one‐way ANOVA. (E) Histograms showing the expression of GZMA, GZMB, PRF1, and in NKG7 positive and negative CD4+ T‐cells, and corresponding dot plots indicating the frequency of positive cells. Dashed lines in the histograms do not represent gating thresholds but are included for visual comparison. Gating was determined based on unstained controls, isotype controls, or fluorescence minus one (FMO) controls, depending on the most appropriate approach for each marker. p‐values from unpaired t‐test. *p < 0.05; ****p value < 0.0001.

FIGURE 3

NKG7 correlates with cytotoxicity across organs. (A) Violin plots showing the gene expression of cytotoxic molecules in NK (left) and CD8+ T‐cells (right) across major immunological organs. (B) Scatter plots of the correlation between NKG7 gene expression and cytotoxicity score within NK or CD8+ T‐cells across all organs of the Tabula Sapiens dataset. Pearson correlation is displayed and the shaded area represents the 95% CI. (C) Violin plots showing the expression of NKG7 across immune populations within the Tabula Sapiens dataset.

FIGURE 4

NKG7 is consistently co‐expressed with other cytotoxic molecules. Co‐expression pattern of cytotoxic molecules across NK and T‐cell subsets. UpSet plots (top row) demonstrate the co‐expression pattern and frequency observed. Meanwhile, heatmaps (bottom row) demonstrate the expression profile of individual cells. n value represents the number of cells of a particular subset included in the analysis.

FIGURE 5

NKG7 correlates with cytotoxicity in tumor‐infiltrating immune subsets. (A) Density of cytotoxicity score expression overlaid on UMAP coordinates of the tumor immune cell atlas. (B) UMAP plot of the different cell subsets identified within the tumor immune cell atlas. (C) Violin plots showing the cytotoxicity score of various cell subsets within tumor immune cell atlas. (D) Scatterplot of the correlation between y‐axis gene expression and cytotoxicity signature. Pearson correlation is displayed and the shaded area represents the 95% CI. (E) UMAP plot highlighting the NK subsets identified within the pancancer NK atlas (left). The violin plot depicts the expression of NKG7 across various NK subsets within the pancancer NK atlas. (F) Scatterplot of the correlation between NKG7 or Granzyme B (GZMB) expression and cytotoxicity score across NK subsets. Pearson correlation is displayed and the shaded area represents the 95% CI.