1. Leukoreduction filter obtained from
RIBC 3-5 hours from blood draw
MACS isolation to obtain ‘untouched’ NK cells
NK cells prepared for flow
cytometry (within 36 hours)
and cell aliquots reserved
(-80⁰C) for nucleic acid isolation
[T=0]
NK cells cultured in the presence
of IL-2 and antibody-coated
microbeads (Miltenyi ) for
NK activation and expansion.
Culture cells
[T=16h and/or 9 days]
cytometry (within 24 hours)
and cell aliquots reserved
Functional assessment:
Cell killing assay
(T=9 days)
Bisulfite converted DNA;
run Illumina Infinium 450k
Methylation Bead chip
(T=0, 16 hrs, 9 days)
Hierarchical clustering of DNA
methylation at T=0 and 9 days
Prioritize candidate genes
(see insert B)
Repeat with a replication set
[A]
Suppl Figure 1. Experimental overview including target gene selection criteria [A] Experimental overview [B] To further narrow the list of significant loci for additional analyses, target gene selection criteria were implemented. The 1000 most significant loci (from the discovery phase) included 888 hypomethylated and 112 hypermethylated loci. Significant CpG sites were categorized by the average absolute difference in beta values between naïve and activated NK cells (called effect size), and evaluated for the number of significant probe within a genomic region. Loci with |>0.3| beta units and more than one significant probe were deemed “Class I”. Relatively few gene loci met the criteria for Class I. When the same criteria were applied to the replication set, 21 Class I loci (within 9 genes; all hypomethylated) were found in common between the two datasets. These loci/genes were given top priority for further investigation. Additionally, there were 44 loci (represented by 18 genes) that met criteria for Class II, 91 loci (represented by 52 known or hypothetical genes and 39 non-coding regions) that contain at least one probe with a large beta categorized as Class III, and the remaining loci were CpG sites fell into Class IV. [A complete list of loci can be found in Suppl Table 3.]
[B]
Class criteria:
I = both multiple probes and large effect size
II = multiple probes
III = large effect size not multiple probes
IV = neither multiple probes or large effect size
Design score criteria:
1 = Top hits in gene are very close (back to back), and
there are at least 4 CpGs within 150 bp
2 = Top hits somewhat close together, and there are
≥4 CpGs within 200 bp
3 = Top hits in gene are far away from one other, but at
least one of them has ≥4 CpGs with in 200 bp
4 = Top hits are far away from one another, and has only
2 or 3 CpGs within 200bp
5 = Top hits are far away from one another, and has
1 CpG within 200bp

2. [A]
[C]
[B]
[D]
[E]
Suppl Figure 2. Representative flow cytometry. [A] Representative plot of untouched MACS
isolated human NK cells (freshly isolated, T=0). The average purity of CD3- CD56+ (NK) cells at T=0
was 91.6% ± 5% (viability 98.4% ± 1.3%) and 96.1% ± 4.5% (viability 94% ± 2.6%) at T=9 days.
Populations of both CD3-CD56+bright and CD3-CD56+dim cells are present. [B] There is a clear shift
from virtually no activated NKs at T=0 (light gray) to 96% of cells expressing the cell surface marker
of activation, CD54, at day 9 (dark gray) following IL2/CD2/CD335 microbead stimulation. [C] Shift
in CD16+ at T=0 (light gray) to CD16- with cell activation (dark gray) was commonly observed. [D] A
shift from no detectable intracellular IFN-gamma at T=0 (light gray) to a small, but measurable
expression at T=9 days (dark gray). [E] Dot plot of IFN-gamma vs CD54 (activated) NK cells.

3. * IL5 IL13 Overall, p = 0.03; * p < 10-5 Overall, p < 10-10
Suppl Figure 3. Methylation status for NKs at the IL5 and IL13 loci. IL5 and IL13 are part of a cytokine gene cluster important in Th2 activation. The six probes from IL5 and the nine probes from IL13 were manually clustered by NK activation status and assessed for methylation status. A t-test was performed for each gene to compare naïve (T = 0) NKs with activated (T = 9) NKs, and p-values computed. The asterisk indicates a separate t-test for one of the IL5 probes, which is driving the overall association. It remains to be determined if this IL5 locus is important in NK activation. Similar to the observation by others in T-cells , the activated NK cells exhibit IL13 demethylation and expression is significantly increased (Suppl Fig 4).

4. * #
Suppl Figure 4. Expression among top ranked candidate genes in activated NK cells. Using criteria described in figure 1, several significantly hypomethylated candidate genes were tested for changes in gene expression. Expression was calculated using the comparative CT method (ΔΔCT) with three different reference genes. Results shown here are expressed relative to IPO8, the most stable of the three reference genes. Six genes exhibited significant increased mRNA (*) and one a small but significant decrease in expression (#), p<0.01.

5. Marker Stain Suppl Table 1. Flow cytometry panel.
Viability
eFluor450
CD3
PerCP-Cy5.5
CD56 PE
CD16
FITC
CD54
APC
IFN-γ
PE-Cy7
Suppl Table 1. Flow cytometry panel.
Freshly isolated untouched, 16 hour (subset of discovery phase only) and 9 day cultured human NK cells were routinely checked for viability, purity and activation using a 6-stain panel.

6. Life Technology assay #
Gene
Life Technology assay #
Reference Genes:
B2M
Hs _m1
GAPDH
Hs _g1
IPO8
Hs _m1
Target Genes:
BHLHE40
Hs _m1
CLEC16A
Hs _m1
DEXI
Hs _m1
IL-13
Hs _m1
IL-21R
Hs _m1
KSR1
Hs _m1
MAML-2
Hs _m1
MYO1G
Hs _m1
NCF4
Hs _m1
NFATC1
Hs _m1
TNFAIP3
Hs _m1
ZBTB32
Hs _g1
Suppl Table 2. Taqman gene expression assays. Expression analyses for several genes of interest were performed using Taqman assays (Life Technologies). Each assay was run against three reference genes previously characterized by Kaszubowska et al for stability in the NK-92 cell line: beta-2-microglobulin (B2M), importin 8 (IPO8) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Note that the NK-92 cell line used by Kaszubowska is not the same genetically modified line as used in this study, but rather, the parental NK-92 cell line to the CD16.176V.NK-92 used in this study.