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Ruffin N et al. Low SAMHD1 expression following T cell activation and differentiation
renders CD4+ T cell susceptible to HIV-1
Supplemental Digital Content
Table, SDC 1. Patient characteristics PBMCs.
CD4 count in Copies HIVcells/ml of
1 RNA/ml of
n
Gender
plasma
plasma
median
median
M/F
(range)
(range)
49
14/9
522
<50
HIV-1+
23
13 (2-26)
10 (1-18)
(36-69)
61%/39%
(129-1080)
(0-74042)
cART+
32
4/2
255
54047
HIV-1+
6
1.5 (0-6)
NA
(18-39)
67%/33%
(145-805)
(982-158169)
cART47
2/4
900
94
HIV-1+
6
25 (0-28)
NA
(26-56)
33%/67%
(737-1483)
(0-308)
EC
HIV-1 infected individuals under treatment (HIV-1+ cART+), non-treated (HIV-1+ cART-) and elite-controllers
Age in
years
median
(range)
Median time
since HIV-1
diagnostic
years (range)
Median
treatment
duration
years (range)
(HIV-1+ EC). Gender M: male, F: female. NA: non-attributable.
Table, SDC 2. Patient characteristics Lymph Nodes.
ID
Age
years
Gender
1
2
3
4
5
6
7
8
9
10
11
53
38
86
76
86
66
57
64
33
52
72
F
M
M
M
M
M
M
M
M
M
M
12
36
M
HIV-1
status
Time since
HIV-1
diagnostic
years
Treatment
duration
years
negative
positive
CD4
count
cells/ml of
blood
Copies HIV-1
RNA/ml of
plasma
NA
2
4
1,5
3
2
2
4
1,5
3
0.35
186
534
176
122
573
2 (<50)
3(<50)
3(<50)
14(<50)
14(<50)
5
5
not
available
<50
Gender M: male, F: female. NA: non-attributable.
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Figure, SDC 3. SAMHD1 expression following PHA/IL-2 stilmulation. Purified CD4+ T cells
from healthy individuals (n=3) were stained with Cell Trace Violet and cultured in the presence
of phytohemagglutinin (PHA, 1μg/ml) and Interleukin- 2 (IL-2, 100U/ml). At day 4, cells were
stained with anti-CD3 and anti-CD4. FoxP3 permeabilization buffer was used for the
intracellular staining of SAMHD1. Aqua Vivid was included in the procedure to remove dead
cells from the analysis. (A) Representative dot plot of SAMHD1 and Cell Trace expression
among gated live lymphocytes cultured in medium or in the presence of PHA/IL-2. (B) Total
SAMHD1 expression measured by mean fluorescent intensity (MFI) in cells gated according to
the number of cell division after PHA/IL-2 stimulation. Data represent mean with SD of the 3
donors. **p<0.01, 1-way ANNOVA tests followed by Dunn’s multiple comparison test.
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Figure, SDC 4. SAMHD1 expression and phosphorylation after TCR triggering. Purified
CD4+ T cells from healthy individuals (n=4) were stained with Cell Trace Violet and cultured in
the presence of coated anti-CD3 (2μg/ml) and anti-CD28 (2μg/ml) mAbs. At day 3, CD3+CD4+
T cells were fixed and permeabilized with methanol before the staining for total SAMHD1 and
phosphorylated SAMHD1 (p-SAMHD1, a kind gift from Monsef Benkirane). (A) Representative
histogram of p-SAMHD1 staining on CD4+ T cells stimulated (anti-CD3/anti-CD28) or cultured
in medium alone. Isotype control is also displayed. (B) Representative dot-plots of p-SAMHD1
and SAMHD1 staining with cell trace on CD4+ T cells stimulated (anti-CD3/anti-CD28) or
cultured in medium alone. (C) Phosphorylated SAMHD1 and (D) total SAMHD1 expression
measured by mean fluorescent intensity (MFI) in cells gated according to the number of cell
division. Data represent mean with SD.
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Figure, SDC 5. SAMHD1 expression in peripheral lymphocytes. Fresh PBMCs from HIV-1
negative (n=13) or HIV-1 infected subjects (n=35), were stained with anti-CD3, -CD4. FoxP3
permeabilization buffer was used for the intracellular staining of SAMHD1. Aqua Vivid was
included in the procedure to remove dead cells from the analysis. (A) Representative dot plot of
SAMHD1 and CD3 expression among gated live lymphocytes. (B) Percentages of SAMHD1+
cells among CD3+ and CD3- lymphocytes (left panel) and among CD4+ and CD4- T cells from
HIV-1- and HIV-1+ individuals. *** p<0.001, Wilcoxon matched-pairs signed rank test.
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CD4+ T cells
Count
SAMHD1
HIV-
SAMHD1
Lymphocytes
HIV+
cART+
HIV+
cART-
HIV+
EC
CD45RO
CD45RO
CD45RO
Figure, SDC 6. Representative dot-plots and histograms for SAMHD1/CD45RO and Ki67
expressions.
Representative dot-plots of SAMHD1 and CD45RO expression among live lymphocytes and
CD4+ T cells showing the gating of SAMHD1+ and SAMHD1low cells (left panel), and
representative histogram of Ki67 expression in gated SAMHD1+ and SAMHD1low cells. PBMCs
were isolated from healthy controls and HIV-infected patients.
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Figure, SDC 7. Activated phenotype of peripheral T cells in HD and HIV-1+ subjects. Fresh
PBMCs from HIV-1 negative (HIV-, n=13) or HIV-1 infected subjects treated (cART+ HIV+,
n=23), non-treated (cART- HIV+, n=6) or elite-controllers (HIV+ EC, n=6) were stained with
anti-CD3, -CD4, -CD45RO and alternatively with anti-PD1, or anti-HLA-DR and -CD38. FoxP3
permeabilization buffer was used for the intracellular staining of SAMHD1. Aqua Vivid was
included in the procedure to remove dead cells from the analysis. (A), (C) and (E) Data represent
percentages of positive cells among total, CD45RO+ and CD45RO- CD4+ T cells. (B), (D) and
(F) Percentages of positive cells among SAMHD1+ and SAMHD1low gated on total, CD45RO+
and CD45RO- CD4+ T cells. *p<0.05, **p<0.01, ***p<0.001, 2-way ANNOVA tests followed
by Bonferroni post-tests.
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Figure, SDC 8. Cytokine production of LNMC CD4+ T cells during HIV-1 infection.
LNMCs from HIV-negative (HIV-, n=3) and treated HIV-1 infected individuals (HIV1+cART+, n=4) were stimulated with PMA/Ionomycin in the presence of momensin for 5 hours.
Cells were stained for intracellular cytokine (IL21, IL17-A, TNFα, IFNγ and IL2) using the
FoxP3 permeabilization buffer kit. Aqua Vivid was included in the procedure to remove dead
cells from the analysis. Data represent individual percentages of positive cells among CD4+ Tcells from HIV- and treated-HIV-1+ individuals, with mean±SD. Data were analyzed using the
Mann-Whitney test.
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Figure, SDC 9. Tfh from tonsils display similar phenotype as from LN. Cells were
mechanically isolated from human tonsils (n=10) and stained for CXCR5, PD1, SAMHD1 and
Bcl-6. For the qPCR experiments, purified CD4+ T cells from tonsils (n=5) were separated using
the CD4 T cell Isolation Kit II (Miltenyi) and stained with anti-CD3, -CD4, -CXCR5 and -PD1.
CD4+ T cells were then sorted according to CXCR5 and PD1 expression. Total RNA was
isolated using RNeasy kit (Qiagen) and reverse transcription was performed using AffinityScript
qPCR cDNA Synthesis kit (Agilent, Stratagene) and random primers, according to the
manufacturer’s instructions. qPCR was performed using an ABI Prism 7500 Sequence Detection
System (Applied Biosystems) in 20 µL reaction with KAPA SYBR Green (Kapa Biosystems)
and 0.2 µM of each primer. Sequences for the specific primers were as followed: SAMHD1
forward: CCCAACAGAGCAATCAGGA, reverse: TGTCTGCACACCACTGAACA; Bcl-6
forward:
ATGGAGCATGTTGTGGACACT,
reverse:
GGCTGTTGAGGAACTCTTCAC;
FoxP3 forward: CCATGCCTCCTCTTCTTCCT, reverse: ACCATGACTAGGGGCAGTGT;
PRDM1 forward: TCCAGCACTGTGAGGTTTCA, reverse: TCAAACTCAGCCTCTGTCCA;
and
S14
forward:
GGCAGACCGAGATGAATCCTC,
reverse:
CAGGTCCAGGGGTCTTGGTCC respectively. The relative levels of SAMHD1 mRNA were
calculated using the 2−ΔΔCT method. (A) Representative dotplot of CXCR5 and PD1 expression
on gated live CD4+CD3+ lymphocytes from tonsils showing the gating strategy used for the
analysis and the sorting of the cells. (B) Bcl-6 mRNA relative expression in CD4+ T-cell
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subpopulations. (C) SAMHD1 mRNA relative expression. (D) PRDM1 mRNA relative
expression. (E) Representative histogram of and individual percentages of FoxP3 expression
among the gated populations. (F) FoxP3 mRNA relative expression. *p<0.05, **p<0.01,
***p<0.001, 1-way ANNOVA tests followed by Dunn’s multiple comparison test.
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Figure, SDC 10. Phenotype of tonsilar CD4+ T cells. Cells were mechanically isolated from
human tonsils (n=5) and stained for CXCR5, PD1 together with SAMHD1, CTLA-4, ICOS,
CD38, Ki67, CD161, CD26, CXCR4 and CCR5 using the FoxP3 permeabilization buffer. Aqua
Vivid was included in the procedure to remove dead cells from the analysis. For CXCR4 and
CCR5 staining, fresh tonsils were used. (A) Representative histogram of and individual
percentages of CTLA-4, ICOS, CD38, Ki67, CD161 and CD26 expression among the CXCR5PD1-, CXCR5+PD1int, CXCR5-PD1+ and CXCR5hiPD1hi CD4+ T cells. (B) Representative
histogram of and individual percentages of CXCR4 and CCR5 expression among the gated
populations. *p<0.05, **p<0.01, ***p<0.001, 1-way ANNOVA tests followed by Dunn’s
multiple comparison test.
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Figure, SDC 11. Tfh do not proliferate in vitro. Cells were mechanically isolated from human
tonsils (n=4) and CD4+ T cells were separated using the CD4 T cell Isolation Kit II (Miltenyi).
Cells were then stained with Cell trace Violet before further staining for CD3, CD4, CXCR5 and
PD1. CD4+ T cells were then sorted according to CXCR5 and PD1 expression. Cells were
cultured for 3-6 days with anti-CD3 and anti-CD28 antibodies (2ug/ml) and stained
intracellularly for SAMHD1 using the FoxP3 permeabilization buffer. Aqua Vivid was included
in the procedure to remove dead cells from the analysis. (A) Data represents cell division
measured by mean of cell trace dilution with SAMHD1 expression. (B) Percentages of
proliferative cells (n=4). *p<0.05, 1-way ANNOVA tests followed by Dunn’s multiple
comparison test.
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