Download Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV

Supplemental Digital Content
Supplementary Methods
Study population
The cohort was formed in 1990 as an open prospective cohort for whom regular yearly
serological and clinical examinations were offered. The cohort included 4878 individuals at
its closure in 2011. In 2013 participants of the current study were recalled for another followup visit. Antiretroviral treatment was available in Guinea-Bissau at the time of sampling, but
blood samples from the individuals that, at any time, had received treatment were not
analysed in this study.
FACS staining
Immunophenotyping of NK and NKT cells was performed on Cytochex (Streck) stabilized
whole blood by multicolor flow cytometry. Before antibody labelling, red blood cells were
lysed using lysis buffer (BD FACS™) and washed with PBS containing 2mM EDTA. All
antibodies used were anti-human (see Table S1). The surface staining was carried out in two
staining rounds. Cells were washed between staining procedures with PBS containing 2mM
EDTA. Prior to intracellular staining, cells were permeabilized using Fixation/
Permeabilization solution and washed with permeabilization buffer (eBiosciences).
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Measurement of CD38, PLZF and T-bet
As CD38 is commonly expressed on T and NK cells the MFI of the CD38 expression was
used as a proxy for activation rather than the percentage of cells expressing CD38. In our
evaluation of flow cytometry data generated from blood samples collected in Cyto-Chex
BCT tubes we noted that CD38 expression levels were not affected by the preservatives (data
not shown). However, we were uncertain as to how the preservative in the tubes would affect
the expression and staining of the transcription factors (TFs), PLZF and T-bet. We therefore
decided to measure the MFI of the TFs in cell types determined to express these factors and
relate these to cells from the same sample that did not express the TFs (internal negative
control) (Fig. S1), allowing us to record a patient specific increase for the TFs and minimise
any potential effect of the Cyto-Chex tubes.
2
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Supplementary Tables
Table S1: Fluorochrome conjugated antibodies used in the study.
Antibody
Conjugate
Clone
Company
Comment
CD14
V500
M5E2
BD Horizon™
1. surface staining
Identification of invariant natural killer T cells (iNKT)
CD3
Alexa Fluor® 700
UCHT1
BD Pharmingen™ 1. Surface staining
CD4
R-PE-Cy®5.5
S3.5
Lifetechnologies
TCR Vα24
biotin
C15
Beckman Coulter™
TCR Vβ11
PE
C21
Beckman Coulter™
Streptavidin
Qdot® 655
Q10123MP Lifetechnologies
2. Surface staining
Identification of natural killer cells (NK)
CD56 (NCAM) Brilliant Violet 711™
HCD56
BioLegend
1. Surface staining
HIT2
BD Horizon™
1. Surface staining
Intracell. staining
Markers of immune activation
CD38
PE-CF594
Transcription factors
PLZF
APC
6318100
R&D Systems™
T-bet
Brilliant Violet 605™
4B10
BioLegend
3
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Table S2: Results from the univariate general linear models
Tests of Between-Subjects Effects
Dependent Variable: CD38 MFI on iNKT
Type III Sum of
Source
Squares
df
Mean Square
F
Sig.
12281120.639a
7
1754445.806
7.362
.000
Intercept
1408390.404
1
1408390.404
5.910
.019
Age
190194.644
1
190194.644
.798
.376
Log_VL
627054.329
1
627054.329
2.631
.111
Log_CD4%
6038228.503
1
6038228.503
25.338
.000
HIV
149532.340
2
74766.170
.314
.732
Gender
67962.295
1
67962.295
.285
.596
HIV * Gender
309275.479
1
309275.479
1.298
.260
Error
12153513.346
51
238304.183
Total
30021450.600
59
Corrected Total
24434633.986
58
Corrected Model
a. R Squared = .503 (Adjusted R Squared = .434)
4
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Tests of Between-Subjects Effects
Dependent Variable: CD38 MFI on CD4 iNKT
Type III Sum of
Source
df
Mean Square
F
Sig.
1498617.757a
7
214088.251
2.810
.020
14856.163
1
14856.163
.195
.662
149.676
1
149.676
.002
.965
Log_VL
359869.177
1
359869.177
4.723
.037
Log_CD4%
130460.106
1
130460.106
1.712
.199
HIV
355244.876
2
177622.438
2.331
.113
.714
1
.714
.000
.998
396.071
1
396.071
.005
.943
Error
2590655.184
34
76195.741
Total
7264858.575
42
Corrected Total
4089272.941
41
Corrected Model
Intercept
Age
Gender
HIV * Gender
Squares
a. R Squared = .366 (Adjusted R Squared = .236)
5
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Tests of Between-Subjects Effects
Dependent Variable: CD38 MFI on CD56 dim NK
Type III Sum of
Source
df
Mean Square
F
Sig.
132081629.060a
8
16510203.632
5.253
.000
Intercept
12558699.228
1
12558699.228
3.996
.050
Age
1773616.578
1
1773616.578
.564
.455
48474.267
1
48474.267
.015
.902
Log_CD4%
39229823.555
1
39229823.555
12.483
.001
HIV
9492717.945
2
4746358.972
1.510
.229
Gender
14880660.472
1
14880660.472
4.735
.034
HIV * Gender
12224946.991
2
6112473.495
1.945
.152
Error
185419987.176
59
3142711.647
Total
822902020.000
68
Corrected Total
317501616.235
67
Corrected Model
Log_VL
Squares
a. R Squared = .416 (Adjusted R Squared = .337)
6
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Tests of Between-Subjects Effects
Dependent Variable: CD38 MFI on CD56 bright NK
Type III Sum of
Source
df
Mean Square
F
Sig.
112223198.456a
8
14027899.807
8.853
.000
13079012.776
1
13079012.776
8.254
.006
Age
660866.287
1
660866.287
.417
.521
Log_VL
363600.400
1
363600.400
.229
.634
Log_CD4%
22767783.748
1
22767783.748
14.369
.000
HIV
3079391.111
2
1539695.556
.972
.385
Gender
1783878.616
1
1783878.616
1.126
.293
HIV * Gender
9050416.465
2
4525208.232
2.856
.066
Error
91901702.708
58
1584512.116
Total
1288343521.000
67
Corrected Total
204124901.164
66
Corrected Model
Intercept
Squares
a. R Squared = .550 (Adjusted R Squared = .488)
7
51.4
0.268
81.7
3.66
CD4
Va24
98.1
CD56
SSC-A
Supplementary Figures
49.9
37.7
FSC-A
CD14
CD3
Vb11
Internal negative
% of Max
SSC-A
0.282
Bulk CD4 T cells
CD56dim NK cells
CD38
PLZF
T-bet
Figure S1) Measurement of CD38, PLZF and T-bet
This figure illustrates an example of histograms generated from the acquisition of CD38,
PLZF and T-bet data of Bulk CD4+ T cells and CD56dim NK cells.
CD8
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
CD3+ cells
(a)
NK cells (CD56dim/bright)
iNKT cells
250K
10
5
10
5
200K
10
4
10
4
150K
10
3
10
3
10
2
10
2
3.9
0.423
100K
63.8
0
0
(b)
10
2
10
3
10
4
10
5
CD3+ cells
250K
200K
150K
0
9.52
0
10
3
10
4
10
5
CD3
SSC
0
TCR Valpha24
50K
0
10
10
4
10
3
10 2
3
10
4
10
5
NK cells (CD56dim/bright)
iNKT cells
5
10
10
5
10
4
10
3
10
2
3.47
0.514
100K
57.9
0
0
50K
7.54
0
0
10
2
10
3
10
4
10
5
0
CD3
(c)
3
10
4
10
5
0
TCR Vbeta11
CD56dim NK cells
% of Max
10
CD56bright NK cells
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
10 3
10 4
10 5
3
10
4
10
5
iNKT cells
100
0
10
CD56
0
0
10 3
10 4
10 5
0
10 3
10 4
10 5
T-bet
(d)
CD56dim NK cells
CD56bright NK cells
iNKT cells
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
10
3
10
4
10
5
0
0
10
3
10
4
10
5
0
10
3
10
4
10
5
T-bet
Figure S2) Comparison of NK, iNKT and intracellular transcription factor T-bet
staining using fresh and Cyto-chex stabilized whole blood. This figure illustrates examples
on dot-plots generated during pre-testing of protocols for staining and flow cytometry
analysis of T, iNKT and NK cell surface markers comparing (a) fresh blood and (b) Cytochex stabilized blood. The histograms are illustrating T-bet stainings of NK cells and iNKT
cells comparing (c) fresh blood and (d) Cyto-chex stabilized blood. Grey filled histograms
represent control samples without intracellular staining. Black lines indicate fully stained
samples
9
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
Figure S2
%CD56bright NK cells
%CD56dim NK cells
% iNKT cells
%CD4+ iNKT of iNKT
(a)
%CD56bright NK cells
%CD56dim NK cells
%CD4+ iNKT of iNKT
% iNKT cells
(b)
Figure S3: iNKT and NK cell subsets as percentage of lymphocytes.
Percentages of iNKT and NK cell subsets assessed in blood samples of HIV-1, HIV-2, HIVD and non-infected control individuals. (a) Percentage of iNKT cells (filled squares), CD4+
iNKT cells (open squares) CD56dim NK cells (open circles), and CD56bright NK cells (filled
circles) of peripheral blood lymphocytes. Comparisons of percentages of (b) total iNKT,
CD4+ iNKT, CD56dim NK cell and CD56bright NK cell subsets (labelled as described above)
in HIV-2 aviremic (viral load  75 RNA copies/mL blood) and viremic individuals (viral
load > 75 RNA copies/mL). The dashed line represents the median absolute number of the
respective cell type in HIV negative individuals. Median is depicted as solid bars. KruskalWallis test with Dunn´s post test, and Mann-Whitney test were used where appropriate.
*p<0.05.
10
Bächle SM, Malone DFG, et al
iNKT and NK cell activation in HIV infections
iNKT
800
CD4+ iNKT
600
CD38 MFI
CD38 MFI
600
400
200
0
400
200
0
-200
-200
non-HIV
HIV-2 aviremic
non-HIV
CD56dim NK
**
CD38 MFI
6000
4000
2000
0
HIV-2 aviremic
CD56bright NK
6000
CD38 MFI
**
800
**
4000
2000
0
non-HIV
HIV-2 aviremic
non-HIV
HIV-2 aviremic
Figure S4: Subsets of iNKT and NK cells in aviremic HIV-2 infection display enhanced
activation compared with HIV seronegative individuals. Degree of activation represented
by the median fluorescence intensity (MFI) level of CD38 surface expression was assessed.
Comparison of CD38 MFI expression levels on total iNKT (filled squares), CD4+ iNKT
(open squares), CD56dim (open circles) and CD56bright NK cells (filled circles) between HIV
seronegative (non-HIV) and aviremic HIV-2 infected individuals. The dashed line represents
the median CD38 MFI level of non-infected individuals. Mann-Whitney U-test was used for
the comparison.**p<0.01.
11
Bächle SM, Malone DFG, et al
T-bet in iNKT
PLZF in CD4+iNKT
***
3
15
2
10
1
5
0
0
HIV-1
HIV-2
HIV-D
HIV-1
HIV-2
HIV-D
*
15
3
10
2
1
5
0
non-HIV
T-bet in CD56bright NK
20
4
20
non-HIV
PLZF in CD56bright NK
25
4
relative MFI
T-bet in CD56dim NK
relative
relativeMFI
MFI
relative MFI
5
T-bet in CD4+iNKT
relative MFI
PLZF in CD56dim NK
(b)
relative MFI
relative MFI
(b)
relative MFI
relative MFI
relative MFI
PLZF in iNKT
relative
MFI
relative MFI
(a)
(a)
iNKT and NK cell activation in HIV infections
0
non-HIV
HIV-1
HIV-2
HIV-D
non-HIV
HIV-1
HIV-2
HIV-D
Figure S5: PLZF and T-bet expression in HIV infection
Expression of transcription factors PLZF and T-bet in iNKT and NK cells subsets of HIV-1,
HIV-2, HIV-D and controls were assessed by intracellular flow cytometry. All transcription
factors are expressed as a relative expression, MFI ratio, to an internal baseline control as
previously described. The dashed line at 1 represents the baseline level. A) Expression of
PLZF and T-bet in total iNKT (filled squares) and CD4+ iNKT (open squares) cell subsets in
HIV-infected and HIV-negative individuals. B) Expression of PLZF and T-bet in CD56dim
(open circles) and CD56bright (closed circles) NK cell subsets. *p<0.05, ***p<0.001
12