Changes in expression landscape during NK cell development

3. RESULTS

3.3.1 Changes in expression landscape during NK cell development

To check how expression profiles change during NK cell development, the analysis was done on ex vivo NK cell samples (n=53) including two samples from NK stage 1, four samples from stage 2, eleven samples from stage 3, nine samples from stage 4, and 29 samples from stage 5. Among all analyzed genes, 20459 exceeded the threshold of LoD=1;

PCA and HC were performed based on the top 100 differentially expressed genes.

On PCA score plot (Figure 5) samples were grouped according to developmental stages and partly according to the origin of the tissue. Groups including stage 4 and stage 5 samples were clearly separated; only one out of 30 stage 5 samples (toNKst5) was localized together with samples from stage 4. Also eight samples from tonsil-originated stage 3 were grouped together, while stage 3 samples from bone marrow were localized closer to earlier developmental stages. The stage 3 sample from cord blood and stage 2 sample from tonsils had an intermediate position between the group consisting of other stage 3 samples and the group including stages 1 and 2 samples. For such an outlying position of these two samples two explanations are possible: either their expression phenotypes are intermediate between early progenitors (stages 1 and 2) and stage 3 NK precursor, or cells included in them represent a mixture of several cell types.

According to the HC heatmap (Figure 6), a gradual change in the gene expression profiles was observed from stage 1 to stage 5. The following gene expression patterns were observed:

•Genes with the highest expression among stage 1 and 2 samples included myeloid-lineage related genes (MMP8, LTF, DEFA3, MPO, AZU1, CTSG, CLC, PRTN3, TAL1, AHSP, RHAG, SPTA1, HBD), as well as other genes known being expressed by CD34⁺ NK progenitor cells (MS4A3, CPA3, ELANE) (Lakschevitz et al., 2015; Sánches et al., 1992; Thomas et al., 2014).

•Among the genes, expression of which continues till stage 3, were typical markers of early NK cell development (MAP7, KIT, MYB, SPINK2, GNAI1, GRB10, CDH1), other NK-lineage specific genes (e.g. ADGRG6, CXCL8), T-lineage associated gene TGM2, and myeloid-specific genes CA2, CDC42BPA, PRSS57 (Eissens et al., 2012; Surmiak et al., 2012). Besides this, VWDE, CASC15, MRC2, STAC, EREG were also expressed at the highest levels within stages 1 to 3.

• Genes with the highest expression in stage 3 and lower expression at stage 4

Heatmap of Expression (Global Z-Score)

1:annotation_width toNKst2 cbNKst3 cbNKst2 bmNKst2_3 bmNKst2_1 bmNKst1_1 bmNKst1_2 bmNKst3_1 bmNKst3_2 toNKst3_6 toNKst3b toNKst3_4 toNKst3_5 toNKst3_7 toNKst3_2 toNKst3_1 toNKst3a bmNKst4_1 bmNKst4_2 cbNKst4_2 cbNKst4 pbNKst4_2 pbNKst4_1 toNKst4_2 toNKst5 toNKst4_1 1:ncpbNKst4_3 pbNKst5nonl_3 pbNKst5mem_4 pbNKst5lic_12 pbNKst5mem_6 pbNKst5mem_5 cbNKst5_2 bmNKst5_1 bmNKst5_2 pbNKst5mem_1 pbNKst5mem_3 pbNKst5lic_8 pbNKst5mem_2 pbNKst5_1 cbNKst5 pbNKst5lic_2 pbNKst5lic_1 pbNKst5_3 pbNKst5_2 pbNKst5nonl_2 pbNKst5lic_9 pbNKst5nonl_1 pbNKst5lic_10 pbNKst5lic_11 pbNKst5lic_3 pbNKst5lic_7 pbNKst5lic_6 pbNKst5lic_5 pbNKst5lic_4 cbNKst5pbNKst5lic_3pbNKst5lic_4pbNKst5lic_5 pbNKst5lic_6pbNKst5lic_7

Figure 5 PCA score plot of ex vivo NK samples based on expression levels of the top 100 differentially expressed genes

Genes were selected from the total number of 20459, LoD=1.

myeloid lineage (LIF and SLC4A10) and a set of genes known to be expressed in lymphocytes, for which, however, no specific function for NK stage 3 is yet known (IL4I1, B3GALT5, CSPG4, ENPP1, GPR55, KRT81) (Gurnett et al., 2008; Eissens et al., 2012;

Takatori et al., 2009). In addition, a gene group was identified for which no specific function in lymphocytes was yet described (KIAA1211L, KIAA1324, LINGO4, LPAR1, PKDCC, RRAD, THEM5, SMIM10L2A), as well as non-protein-coding genes LINC00892, RP11-330A16.1, RP5-1028K7.2, and AL450992.2.

• A group of genes with equally high expression among stage 3 and 4 samples included mainly NK-lineage specific ones (DLL1, XCL1, TNFSF11, TOX2), but also IL7R (ILC3-specific), IGFBP4 and KRT86 (Cupedo et al., 2009; Eissens et al., 2012; Spits et al., 2013).

•Genes expressed among stages 3 to 5 were all typical for NK cells: CD2, GNLY, GPR68, TNFRSF18, XCL2 (Seillet et al., 2014).

• Genes with the highest expression among stage 4 and 5 samples included mainly markers of mature NK cells: TBX21, GZMB, PDZD4, IFNG, GZMM, GZMH, GZMA, EOMES, FCRL6, KLRD1, TGFBR3, KLRF1, KIR3DX1, CD8A, FGFBP2, S1PR5, FCGR3A, FCRL3, PDZD4, but also several genes for which expression in NK lymphocytes was not yet described (DTHD1, PYHIN1, RP11-222K16.2, TENM1) (Eissens et al., 2012; Seillet et al., 2014).

•Finally, genes expressed only among stage 5 samples included FEZ1, B3GAT1, AKR1C3 known to be expressed in CD56⁺NK cells, as well as SLC1A7, BNC2, LGR6 (Jakobs et al., 2001).

Consistent to the picture observed on the PCA score plot (Figure 5), on the HC heatmap, the stage 3 samples of bone marrow origin were clustered together with developmental stages 1 and 2, and shared gene expression pattern with them, suggesting that they might have a more immature phenotype in comparison to stage 3 samples of tonsil origin.

To check, how expression profiles change during development, pairwise comparisons between developmental stages were performed (Figure 7A). If gene expression changes sequentially from one stage to the next one, total expression profiles should correlate between neighboring stages, but not with those that are far from each other along the NK cell developmental flow. The correlation was calculated using the top 100 differentially expressed genes selected upon LoD=1; correlation coefficients of >0.5 were accepted as a true positive correlation. Indeed, the correlation coefficient between

stages 1 and 2, stages 2 and 3, as well as between stage 4 and 5 were highly positive, namely 0.74, 0.679, and 0.664 respectively, while there was no significant correlation between stages 3 and 4. Correlation coefficients between non-neighboring stages were in the range from -0.507 to 0.331.

To prove this tendency, an analogical analysis was performed based on the top 400 mostly differentially expressed genes (Figure 7B). In that case the highest correlations were observed between stages 1 and 2 (correlation coefficient 0.787) and between stages 4 and 5 (0.735). The correlation coefficient between stages 2 and 3 was lower (0.568), but

AKR1C3

Heatmap of Expression (Gene Z-Score)

pbBcbBcbMonopbMonocbNKst3bmNKst1_1cbCD34pos_2cbCD34pos_1daNKst2_2daNKst2_1cbCD34fe_negcdD34fe_posbmNKst3_1bmNKst3_2bmNKst2_1bmNKst2_3cbNKst2bmNKst1_2pbTKIRneg_3pbTKIRpos_7pbTKIRpos_2pbTKIRpos_1cbTCD8pos_2cbTCD8pos_1cbTCD4pos_1cbTCD4pos_2pbTKIRpos_6pbTKIRpos_5pbTKIRneg_4pbTKIRneg_5pbTCKIRneg_2pbTKIRpos_4pbTKIRneg_1toNKst2toNKst3_7toNKst3btoNKst3_4toNKst3_5toNKst3_6toNKst3_1toNKst3_2toNKst3adaNKst3_3daNKst3_2daNKst3_1daNKst4_3daNKst5_2daNKst4_1daNKst5_1daNKst4_2bmNKst4_1toNKst4_2bmNKst4_2pbNKst5nonl_3pbNKst5mem_6pbNKst5mem_5pbNKst5lic_12pbNKst5mem_4liNKCXCR6ptoNKst5toNKst4_1cbNKst4_2pbNKst4_3cbNKst4pbNKst4_2pbNKst4_1bmNKst5_1bmNKst5_2cbNKst5_2pbNKst5_1cbNKst5liNKCXCR6n_2liNKCXCR6n_1pbNKst5_3pbNKst5lic_2pbNKst5lic_1pbNKst5_2pbNKst5lic_3pbNKst5lic_11pbNKst5lic_9pbNKst5lic_4pbNKst5lic_5pbNKst5lic_7pbNKst5lic_6pbNKst5nonl_2pbNKst5nonl_1pbNKst5lic_10pbNKst5mem_3pbNKst5mem_1pbNKst5mem_2pbNKst5lic_8

Heatmap of Expression (Gene Z-Score)

-3 -2 -1 0 1 2 3

Heatmap of Expression (Global Z-Score)

Figure 6 HC heatmap of ex vivo NK samples based on expression levels of the top 100 differentially expressed genes

Genes were selected from the total number of 20459, LoD=1.

still higher than between any of non-neighboring stages, while correlation between stages 3 and 4 remained under the threshold (coefficient 0.265).

st1

0.787 0.366 0.149 -0.039

0510

st2

0.568 0.051 -0.229

0510

st3

0.265 -0.138

0510

st4

0.735

0510

0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

st5 Pairwise Comparsion of Sample Groups

Figure 7 Pairwise comparison of gene expression levels within ex vivo NK developmental stages A – based on top 100 differentially expressed genes; B – based on top 400 genes.

st1

0.740 0.331 0.159 -0.071

051015 st2

0.679 0.0058 -0.366

051015 st3

0.005 -0.507

051015 st4

0.664

051015

0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

st5 Pairwise Comparsion of Sample Groups

A plausible reason for the exceptional position of stage 3 samples is the known heterogeneity of stage 3. First, it is known that stage 3 cells sorted in FACS according to their CD34^-CD117⁺CD94^-surface phenotype are not a homogeneous population of cells, but also can include precursors of T lymphocytes, DCs and ILC3 cells. Another theoretical possibility would be heterogeneity between the analyzed stage 3 samples. To test this hypothesis, a correlation analysis between gene expression levels of individual stage 3 samples was performed (Table 4). According to Spearman’s correlation analysis, correlation between all samples was between 0.84 and 0.93. Considering that this ratio was not lower than correlation rates between individual samples within other stages (not shown), it is unlikely that heterogeneity between stage 3 samples is the cause of the unique position of stage 3.

Table 4 Correlation between gene expression profiles of individual stage 3 ex vivo NK samples

bmNKst3_1 cbNKst3 bmNKst3_2 toNKst3_1 toNKst3_2 toNKst3a toNKst3b toNKst3_4 toNKst3_5 toNKst3_6 toNKst3_7

bmNKst3_1 1

cbNKst3 0.85 1

bmNKst3_2 0.92 0.85 1

toNKst3_1 0.86 0.86 0.88 1

toNKst3_2 0.86 0.86 0.88 0.92 1

toNKst3a 0.86 0.86 0.88 0.91 0.93 1

toNKst3b 0.82 0.83 0.83 0.87 0.89 0.88 1

toNKst3_4 0.83 0.83 0.85 0.88 0.90 0.89 0.87 1

toNKst3_5 0.83 0.83 0.84 0.87 0.90 0.89 0.87 0.91 1

toNKst3_6 0.78 0.79 0.80 0.82 0.84 0.83 0.82 0.85 0.86 1 toNKst3_7 0.81 0.82 0.83 0.85 0.87 0.87 0.83 0.87 0.87 0.87 1

ANOVA analysis was then performed to specify the change in gene expression during transition from one developmental stage to the next one. Among a total of 100 of the most differentially expressed genes, 56 genes were differentially expressed between stages 3 and 4, 54 genes between stages 4 and 5, 45 genes between stages 2 and 3, and only 4 were differentially expressed between stages 1 and 2 (Appendix 3). In order to account for genes specifically expressed in small size groups, the whole analysis and ANOVA comparison was performed for only stage 1 and stage 2 groups separately.

However, even in that case no additional genes were identified (data not shown).

Im Dokument Analysis of the transcriptome of human NK lymphocytes (Seite 47-53)