Functional-­‐specific  differences

At the next step of an analysis it was investigated, how the licensing status may influence the NK cell expression profile. For this purpose only those stage 5 samples were selected, where the licensing status was known. Besides this, two KIR^-NKG2A^- samples were also excluded, as they might have represented less mature cell population in comparison to KIR⁺ and/or NKG2A⁺ NK cells. In this case, the analysis was based on 22 samples from peripheral blood: 3 of nonlicensed, 11 licensed, and 7 memory-like NK cells.

Of note, when PCA and HC for these groups were performed, no group-dependent distribution was observed at the plot (Figure 14A, B).

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When these three groups were compared pairwise by ANOVA, the analysis revealed that there is indeed significant difference in gene expression between them (listed in Appendix 8). Among the top 100 genes, significantly (p<0.05) differently expressed between licensed and nonlicensed were 22 of them (all with higher expression in

Figure 14 PCA score plot (A) and HCA (B) of licensed, nonlicensed and memory stage 5 ex vivo NK samples based on expression levels of the top 100 differentially expressed genes

SLC16A10

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 (Gene Z-Score)

1:annotation_width pbNKst5lic_1 pbNKst5lic_2 pbNKst5lic_9 pbNKst5nonl_1 pbNKst5lic_6 pbNKst5lic_7 pbNKst5lic_5 pbNKst5lic_4 pbNKst5lic_10 1:ncpbNKst5nonl_2 pbNKst5lic_8 pbNKst5mem_2 pbNKst5mem_1 pbNKst5mem_3 pbNKst5mem_5 pbNKst5nonl_3 pbNKst5mem_6 pbNKst5mem_4 pbNKst5lic_12

Heatmap of Expression (Gene Z-Score)

1:annotation_width pbNKst5lic_1 pbNKst5lic_2 pbNKst5lic_9 pbNKst5nonl_1 pbNKst5lic_6 pbNKst5lic_7 pbNKst5lic_5 pbNKst5lic_4 pbNKst5lic_10 1:ncpbNKst5nonl_2 pbNKst5lic_8 pbNKst5mem_2 pbNKst5mem_1 pbNKst5mem_3 pbNKst5mem_5 pbNKst5nonl_3 pbNKst5mem_6 pbNKst5mem_4 pbNKst5lic_12

Heatmap of Expression (Gene Z-Score)

1:annotation_width cbNKst3 bmNKst3_1 bmNKst3_2 daNKst3_3 daNKst3_1 daNKst3_2 toNKst3b 1:nc toNKst3_4 toNKst3_5 toNKst3_6 toNKst3_7 toNKst3_1 toNKst3a toNKst3_2

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nonlicensed group, than in licensed), and 66 genes between licensed and memory (only ADGRA3 was expressed higher in licensed group, all other genes had higher expression in memory group), and 2 genes between nonlicensed and memory group (LTF and ADGRA3).

This, however, contradicted current knowledge about memory-like cells. As memory NK cells further differentiated licensed NK cells, they were supposed to share at least the same differences in expression landscape, as between licensed and nonlicensed sample groups, so the observed result was unlikely due to functional differences.

To search for another explanation, the data were revised again. As it was shown before, among stage 5 was a group of five sample, the expression profile of which differed obviously from all other stage 5 samples, and that group included both nonlicensed (n=1), licensed (n=1) and memory-like (n=3) NK samples, and such prominent difference could have affected the analysis. Looking back at the sorting data, it was found out, that all these five samples came from the same donor (donor 6, Table 5). Taking this into account, it was suggested, that specific characteristics of the donor determined an outstanding expression profile of these samples; this dominating expression profile, in turn, influenced an analysis, so that no other differences could be identified.

Table 5 Donor origin of selected stage 5 NK samples

Sample Donor Sample Donor

pbNKst5lic_1 1 pbNKst5lic_9 5

pbNKst5lic_2 2 pbNKst5nonl_1 5

pbNKst5lic_3 3 pbNKst5lic_10 5

pbNKst5lic_4 1 pbNKst5nonl_2 5

pbNKst5lic_5 1 pbNKst5mem_4 6

pbNKst5lic_6 4 pbNKst5mem_5 6

pbNKst5lic_7 4 pbNKst5lic_12 6

pbNKst5mem_1 2 pbNKst5mem_6 6

pbNKst5mem_2 2 pbNKst5nonl_3 6

pbNKst5mem_3 2 pbNKst5lic_11 7

pbNKst5lic_8 2

To check this hypothesis, ANOVA analysis was done again excluding the five abovementioned samples. Indeed, the results in this case were rather different. Among the top 100 genes, five were expressed higher in the nonlicensed group, than in the licensed group (TGFBI, MS4A6A, PLD4, NAPSB, KCTD12, and CD4), nine genes were differentially expressed between nonlicensed and memory cells (DEFA3, LTF, MGAM2, SELENBP1, MPO, ADGRA3, LAG3, RP11-706O15.3, and DEFA4), and 25 genes were differentially expressed between licensed and memory cells (LTF, SELENBP1, G0S2,

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LAG3, ADGRA3, SERPINA1, S100A9, MPO, CDA, HLA-DQA2, CXCL8, FCN1, HBA1, RXRG, RP11-873E20.1, IL1B, HBA2, CCDC144A, CD4, RP11-706O15.3, LYZ, TNFAIP2, HCK, DEFA4, and DEFA3).

However, the higher difference between licensed and memory group, than between nonlicenced and memory, as well as between nonlicensed and licensed groups was still unexpected. Furthermore, when ANOVA pairwise comparison was performed based on top 400 genes, the ratio between gene numbers remained comparable: 14 genes were differentially expressed between licensed and nonlicensed cells, 27 genes were differentially expressed between nonlicensed and memory cells, and 74 genes were differentially expressed between licensed and memory cells (Appendix 9). Also when HC was performed using the same sample set, no clear gene expression patterns specific for a certain functional group was observed, even though in a PCA plot memory-like samples were grouped together and rather separately from other samples (Figure 15).

When the expression levels of the abovementioned genes were examined, it became clear that most of the top 100 genes were expressed relatively low in all stage 5 samples, e.g. DEFA3 with 1.5 fpkm in nonlicensed samples and 287 fpkm in memory samples or ADGRA3 with average levels 205 fpkm licensed cells, 47.9 fpkm in nonlicensed and 7.8 in memory cell group. Mathematically, the fold change between them is big enough, however, the biological significance of such difference is questionable. Such low expressed genes might play role, when rather homogenic group of samples is examined, or when differences within compared groups are relatively big. As an outcome, no biologically significant difference can be detected.

Considering all observations mentioned above, it was suggested, that besides tissue origin and functional state, also individual differences between stage 5 NK cells derived from different donors influence their expression profiles; and these influence is big enough to blur other differences.