Download DNA methylation signature of activated human natural killer cells

[A]
Leukoreduction filter obtained from
RIBC 3-5 hours from blood draw
MACS isolation to obtain ‘untouched’ NK cells
[T=0]
NK cells prepared for flow
cytometry (within 36 hours)
and cell aliquots reserved
(-80⁰C) for nucleic acid isolation
NK cells cultured in the presence
of IL-2 and antibody-coated
microbeads (Miltenyi ) for
NK activation and expansion.
[T=16h and/or 9 days]
Culture cells
NK cells prepared for flow
cytometry (within 24 hours)
and cell aliquots reserved
(-80⁰C) for nucleic acid isolation
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
[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
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.]
[A]
[B]
[C]
[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.
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).
Log2 Fold Change
20
18
16
14
12
10
8
6
4
2
0
-2
*
*
*
*
*
*
#
Genes
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.
Marker
Stain
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.
Gene
Life Technology
assay #
Reference Genes:
B2M
Hs00984230_m1
GAPDH
Hs039290975_g1
IPO8
Hs00183533_m1
Target Genes:
BHLHE40
Hs01041212_m1
CLEC16A
Hs00322376_m1
DEXI
Hs003060234_m1
IL-13
Hs00174379_m1
IL-21R
Hs00222310_m1
KSR1
Hs01075790_m1
MAML-2
Hs00418423_m1
MYO1G
Hs01070818_m1
NCF4
Hs00241129_m1
NFATC1
Hs00542678_m1
TNFAIP3
Hs00234713_m1
ZBTB32
Hs00998475_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.