Statistical evaluations were made by. Wilcoxon Matched Pairs test; **, P < 0.01. 0. 103. 104. 105. 0. 103. 104. 105. 0. 103. 104. 105. 0. 103. 104. 105. Ag. cNK.
Cell Reports, Volume 25
Supplemental Information
CMV Primes Functional Alternative Signaling in Adaptive Dg NK Cells but Is Subverted by Lentivirus Infection in Rhesus Macaques Spandan V. Shah, Cordelia Manickam, Daniel R. Ram, Kyle Kroll, Hannah Itell, Sallie R. Permar, Dan H. Barouch, Nichole R. Klatt, and R. Keith Reeves
Supplemental Figure 1
a
b
ns
1500
c
ns
1500
*
500
500
Siglec-7 MFI
1000
CD8α MFI
NKp46 MFI
400 1000
500
300 200 100
d
cNK
0
Δg
e
***
400
Eomes MFI
Helios MFI
200 100
Δg
f
***
250 200
300
0
0 cNK
150 100 50
cNK
0
Δg
cNK
Δg
*
100 90
% CD16+
0
80 70 60
cNK
Δg
50
cNK
Δg
Supplemental Figure 1. Disparate marker expression on Dg NK cells, Related to Figure 1. Density of surface NKp46 (a), CD8a (b), Siglec-7 (c), and intracellular Helios (d) and Eomes (e) in Dg and cNK cells in PBMC from rhCMV-infected macaques. (d) Percentage of CD16+ subpopulations among total Dg and cNK cells. Horizontal lines indicate medians and connecting lines are shown between individual animals. Statistical evaluations were made by Wilcoxon Matched Pairs test; *, P < 0.05; ***, P < 0.001. MFI, median fluorescence intensity.
1
Supplemental Figure 2
a
c
FAS
100
Granzyme B
80
% max
cNK
Δg
10 5
60 40 20
61.9
10 4
10
5
10
4
10 3
10 3
0
0
80.6
0 0
10 2
10 3
10 4
10 5
0
b 15000
10 3
% CD69+/GranB+
FAS MFI
10 5
CD69
0
10
3
10
4
10
5
d 100 **
10000
5000
0
10 4
cNK
80 60 40 20 0
Δg
cNK
Δg
Supplemental Figure 2. Increased FAS and cytotoxic arming of Dg NK cells, Related to Figure 1. (a) Representative histograms and (b) density measurements of cell surface FAS (MFI, median fluorescence intensity) on Dg and cNK cells in PBMC from CMV+ macaques. (c) Representative flow cytometry plots and (d) percentages of cytotoxic-armed Granzyme B+/CD69+ Dg and cNK cells in PBMC from rhCMV-infected macaques. Horizontal lines indicate medians and connecting lines are shown between individual animals. Statistical evaluations were made by Wilcoxon Matched Pairs test; **, P < 0.01.
2
Supplemental Figure 3
Syk
γ-chain 20
4.5
Helios
20
20
2.5
15
2
4
15
4
10
3.5
2.5
3
-5
2.5
bh-SNE2
bh-SNE2
3.5
5 0
10
3
10 5
2
0
1.5 1
-5
bh-SNE2
15
1.5
5
1
0
0.5
-5
0
0.5
-10
-10
2
-15
-10
0
10
0
-20 -20
20
bh-SNE1
-1
-10
0
10
-20 -20
20
-1
-10
FAS
NKG2A/C
15
2
15
15 5.5
1.5
10
10
4.5
-5
4
-10
3.5
-15
bh-SNE2
5
5
5
0
4.5
-5
1
bh-SNE2
10
0
20
20
6
5.5
5
10
Siglec-7
20
6
0
bh-SNE1
bh-SNE1
20
bh-SNE2
-0.5
-15
-0.5
-15
1.5
-20 -20
-10
5 0.5
0 0
-5
4
-10
-0.5
-10 -1
3.5
-15
-15 -1.5
3
-20 -20
-10
0
10
-20 -20
20
3
-10
0
10
20
-20 -20
-10
0
10
20
Supplemental Figure 3. Dg NK cell diversification, Related to Figure 1. Composite t-SNE of Dg NK cells from rhCMV-infected rhesus macaques as shown in Fig. 1d; individual markers are indicated. Data points represent individual cells and colors depict intensity of protein expression. . bh-SNE1
bh-SNE1
3
bh-SNE1
Supplemental Figure 4
a
inactive
pSyk
10
phosphorylated 105
5
104
104
0
3
103
102
102
10
25
0
0 0 102
103
104
105
b 80
0 10
2
10
3
% positive pSyk
4
10
5
Syk
* **
60
10
*
NK
****
ΔG
40
*
***
20
0
CMV- CMV+ SIV+
CMV- CMV+ SIV+
Supplemental Figure 4. Syk is inactive in Dg NK cells, Related to Figure 6. Representative plots from inactive and activated (a) phosphoSyk samples are shown. Dg and cNK cells were gated as shown in Figure 1. (b) Bars represent means ± SEM of 6-10 animal samples per group. Statistical comparisons between infection groups were conducted by Mann-Whitney U test, and statistical comparisons between conditions for individual animals were conducted using Wilcoxon-Matched pairs test; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.
4
Supplemental Figure 5 Representative 1 10
5
pSyk- pZAP70 40
NKG2a/c
cNK
10 3
10 2
10 2
0
0
0 10
10
10
3
10
4
10
5
pSyk- pZAP70 12.1
pSyk24.6
pSyk+ 69.9
4
10
3
pSyk- pZAP70 77.6
10 3
10 4
10 5
pSyk55.8
pSyk+ 36.5
10
4
10
3
pSyk14.1
0
0
10 2
10 3
10 4
10 5
0
10 4
10 4
pSyk- pZAP70 64.2
10 3
10 3
10 2
10 2
10 2
10 2
0
0
0
0
10
0 10 2
10 3
10 4
pSyk/pZAP70
10 5
0 10
2
10
3
10
4
10
5
0
pSyk
10 2
10 3
10 4
10 5
10 5
10 5
10 3
3
pSyk+ 81.9
10 2
0
5
10 4
10
10 5
10 2
0 10 2
10
5
10 4
Δg
10 4
10 3
2
10 5
5
10
10 4
Representative 2
NKG2a/c
a
10 2
10 3
10 4
pSyk/pZAP70
10 5
pSyk54.5
0
10 2
pSyk+ 37.6
10 3
10 4
10 5
pSyk
b **
% pSyk -ve (pZAP70+)
100 80 60 40 20
G Δ
cN
K
0
Supplemental Figure 5. Active ZAP70 is elevated in Dg NK cells, Related to Figure 6. (a) Representative gating strategy to identify pZAP70 in Dg and cNK cells. Since a fully specific pZAP70 antibody does not exist, frequencies of pSyk+ cells were subtracted from a dual pSyk/pZAP70-specific antibody to determine frequencies of pZAP70 expression. (b) Data are shown for percentages of pSyk- Dg and cNK cells between individual rhCMV-infected animals. Statistical comparisons were conducted using Wilcoxon-Matched pairs test; **, P < 0.01.
5
Supplemental Figure 6
40
% Δg
30 20 10
M B
LN O LN
0
Supplemental Figure 6. Dg NK cells in LN, OLN and bone marrow, Related to Figure 3. Frequencies among bulk NK cells in rhCMV-infected, but otherwise normal rhesus macaques. Bars indicate medians. LN, lymph nodes; OLN, oral lymph nodes.
6
