Human Bone Marrow-Resident Natural Killer Cells Have a Unique Transcriptional Profile and Resemble Resident Memory CD8+ T Cells

Abstract

Human lymphoid tissues harbor, in addition to CD56^bright and CD56^dim natural killer (NK) cells, a third NK cell population: CD69⁺CXCR6⁺ lymphoid tissue (lt)NK cells. The function and development of ltNK cells remain poorly understood. In this study, we performed RNA sequencing on the three NK cell populations derived from bone marrow (BM) and blood. In ltNK cells, 1,353 genes were differentially expressed compared to circulating NK cells. Several molecules involved in migration were downregulated in ltNK cells: S1PR1, SELPLG and CD62L. By flow cytometry we confirmed that the expression profile of adhesion molecules (CD49e^-, CD29^low, CD81^high, CD62L^-, CD11c^-) and transcription factors (Eomes^high, Tbet^low) of ltNK cells differed from their circulating counterparts. LtNK cells were characterized by enhanced expression of inhibitory receptors TIGIT and CD96 and low expression of DNAM1 and cytolytic molecules (GZMB, GZMH, GNLY). Their proliferative capacity was reduced compared to the circulating NK cells. By performing gene set enrichment analysis, we identified DUSP6 and EGR2 as potential regulators of the ltNK cell transcriptome. Remarkably, comparison of the ltNK cell transcriptome to the published human spleen-resident memory CD8⁺ T (Trm) cell transcriptome revealed an overlapping gene signature. Moreover, the phenotypic profile of ltNK cells resembled that of CD8⁺ Trm cells in BM. Together, we provide transcriptional and phenotypic data that clearly distinguish ltNK cells from both the CD56^bright and CD56^dim NK cells and substantiate the view that ltNK cells are tissue-resident cells, which are functionally restrained in killing and have low proliferative activity.

Keywords: CD8+ T cells; RNA sequence; lymphoid tissue; natural killer cells; tissue-resident.

Figure 1

RNA sequence analysis confirms distinctiveness of CD56^bright, CD56^dim, and ltNK cells. (A) The applied gating strategy to isolate the different natural killer (NK) cell subsets from bone marrow (BM) and blood is depicted. A representative sample of BM NK cell enriched mononuclear cells is shown. Lymphocytes were gated based on forward and sideward scatter and the doublets were excluded. Residual B and T cells were excluded by selecting CD3⁻CD19⁻ lymphocytes. LtNK cells were defined as CD56⁺CXCR6⁺CD69⁺CD54⁺. CD56⁺CXCR6⁻ NK cells were divided into CD56^brightCD16^+/− and CD56^dimCD16⁺ NK cells. The blood-derived CD56^dim and BM derived ltNK cells were further divided into a NKG2A⁺ and NKG2A⁻ fraction. (B,C) Venn-diagrams illustrating the number of differentially expressed genes (up- and downregulated) between the NK cell subsets in blood and BM. (D) Unsupervised hierarchical clustering of the individual donor samples of BM and blood (PB) derived CD56^bright, CD56^dim, and ltNK cells. The clustering is based on the expression pattern of all the 9,382 genes, which were included in the differential expression analysis.

Figure 2

LtNK cells are Eomes^highTbet^low. (A) Heatmap illustrates normalized mRNA expression values of transcription factors, which have the highest or lowest mRNA expression [false discovery rate (FDR) <0.05] in 1 of the 3 bone marrow (BM)-derived natural killer (NK) cell subsets. The column side bars represent the log2-fold change (FC) of gene expression levels in one NK cell subset versus another. The color indicates in which NK cell population the gene is expressed at the highest level (green = ltNK, red = CD56^bright, blue = CD56^dim). The color intensity represents the magnitude of the FC. (B) Eomes and Tbet expression of CD56^bright (red), CD56^dim (blue), and ltNK cells (green), as determined by flow cytometry. Shown are representative dot plots of BM-derived NK cells. MFI, mean fluorescence intensity. *P < 0.05, **P < 0.01, by one-way ANOVA. (C) Normalized mRNA expression levels of selected transcription factors. *FDR < 0.05, ***FDR < 0.001. CPM, counts per million of uniquely mapped reads. FDR, false discovery rate. Error bars represent mean ± SD in (B,C).

Figure 3

Surface molecule profile of ltNK cells reveals altered adhesion molecule repertoire. (A) Heatmap shows scaled mRNA expression values of genes, which encode surface molecules and have the highest or lowest mRNA expression [false discovery rate (FDR) < 0.05] in 1 of the 3 bone marrow (BM)-derived natural killer (NK) cell subsets. The column side bars represent the log2-fold change (FC) of gene expression levels in one NK cell subset versus another. The color indicates in which NK cell population the gene is expressed at the highest level (green = ltNK, red = CD56^bright, blue = CD56^dim). The color intensity represents the magnitude of the FC. (B) Normalized mRNA expression levels of differentially expressed surface molecules involved in migration/adhesion. *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001. CPM, counts per million of uniquely mapped reads; FDR, false discovery rate. (C,D) Validation of mRNA expression of surface molecules by flow cytometry. Shown are representative dot plots of BM-derived NK cells. MFI, mean fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA. Mean ± SD are shown in (B–D).

Figure 4

Effector molecule profile of ltNK cells highlights low killing capacity. (A) Heatmap depicts normalized mRNA expression levels of genes, which encode effector molecules and have the highest or lowest expression in 1 of the 3 bone marrow (BM) derived natural killer (NK) cell subsets [false discovery rate (FDR) < 0.05]. The column side bars represent the log2-fold change (FC) of gene expression levels in one NK cell subset versus another. The color indicates in which NK cell population the gene is expressed at the highest level (green = ltNK, red = CD56^bright, blue = CD56^dim). (B,C) Normalized mRNA expression values of differentially expressed (B) cytolytic molecules (granzyme B, granzyme H, and granulysin) and (C) chemokines (XCL1, XCL2, CCL3) and the growth factor TGFα. CPM, counts per million of uniquely mapped reads. *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001. FDR, false discovery rate. (D) Intracellular IFN-γ expression of CD56^bright, CD56^dim, and ltNK cells. BM mononuclear cells (n = 7) were stimulated for 4 h with PMA/ionomycin or the combination of IL12, IL15, and IL18. After 1 h, Golgistop was added to the culture. *P < 0.05, **P < 0.01, by one-way ANOVA. Mean ± SD are shown in (B–D).

Figure 5

Gene set enrichment analysis (GSEA) reveals potential inducers of ltNK cell transcriptome. (A) The gene sets epidermal growth factor (EGF) signaling (M16311) and bone marrow (BM) stimulation (M5929) were enriched in ltNK cells, as identified by GSEA using CAMERA and the Broad institute gene set collections. The log2 fold change of ltNK versus CD56^bright or ltNK versus CD56^dim is plotted against the false discovery rate (FDR) in volcanoplots. Genes that were induced by either EGF, BM stromal cells, or both are depicted. SPRY1 and SPRY2 are two inhibitors of growth factor signaling. FDR, false discovery rate. (B) GSEA revealed that targets that are repressed by EGR2 (M12804) or DUSP6 (M7339) are downregulated in ltNK cells. Heatmaps show the normalized expression values of the corresponding genes, which are repressed by EGR2 (n = 73) and DUSP6 (n = 39). The column order is based on the combined Z score of each donor natural killer cell population (high → low). The combined Z score is a quantification of the overall expression level of the target genes. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, by one-way ANOVA.

Figure 6

Proliferative capacity of ltNK cells is lower compared with CD56^bright and CD56^dim natural killer (NK) cells. (A) Gene set enrichment analysis using CAMERA and the Broad institute gene collections revealed that many cell-cycle related genes are downregulated by ltNK cells. Heatmaps show the normalized expression values of the hallmark gene sets G2M checkpoint (M5901) and E2F targets (M5925). The column order is based on the combined Z score of each donor NK cell population (high → low). The combined Z score is a quantification of the overall expression level of the genes per donor derived NK cell population. *P < 0.05, **P < 0.01, by one-way ANOVA. Shown is mean ± SD. (B) Proliferative capacity was assessed by stimulating sorted bone marrow-derived NK cell populations for 6 days with IL2, IL15, or IL21. Intracellular Ki67 expression was measured by flow cytometry. The number of CD56⁺ NK cells is based on flow cytometer counts. Shown are dot plots of medium control and IL15 stimulated NK cell populations.

Figure 7

LtNK cells and tissue-resident CD8⁺ memory T cells share a core transcriptional and phenotypic profile. (A) The datasest GSE94964 contains RNAseq data of spleen-derived CD8⁺ effector memory T cells (CD3⁺CD8⁺CD45RA⁻CCR7⁻), which were subdivided into CD69⁺ tissue-resident memory (Trm) and CD69⁻ effector memory T (Tem) cells. We compared this gene set to our RNAseq data to determine a core gene signature among Trm and ltNK cells in lymphoid tissues. The log2-fold change (FC) of 8,700 genes [which were both expressed in T and natural killer (NK) cells] was calculated. The log2 FC of ltNK versus CD56^dim was plotted against the log2 FC of CD8⁺ Trm against Tem cells. Figure S5 in Supplementary Material depicts the plot, which contains the log2 FC of ltNK versus CD56^bright NK cells. Core resident genes, which are depicted in turquoise were identified as genes with a log2 FC ≥ 1 or ≤−1 in all three comparisons. (B) Frequency of CD69⁻ non-resident (Tem) and CD69⁺ resident CD8⁺ memory T (Trm) cells in bone marrow (BM). CD8⁺ memory T cells were defined as CD3⁺CD8⁺CCR7⁺ and included both effector memory and end-stage effector memory T cells. (C,D) Protein expression of surface molecules and transcription factors, which were differentially expressed by ltNK versus circulating NK cells were determined on CD8⁺ memory T cells (CD69⁺ versus CD69⁻). For comparison, plots visualizing the ltNK, CD56^bright, and CD56^dim NK cells are shown. The Eomes MFI on the CD8⁺ memory T cells was determined only on the Eomes⁺ CD8⁺ memory T cells, rather than on the whole population (as was done for the NK cells). Statistics of NK cell protein expression comparisons are shown in Figure 3. Dot plots depict a representative BM donor. MFI, mean fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed paired t-test. Median and interquartile range are shown in (B). Mean ± SD are shown in (C,D).