Supplemental Figure 1

Piceatannol 1 5 10 30 50 100

SP600125 0,1 1 2 5 10 20

ER-27319 1 5 10 30 50 100

Inhibitors concentration (µM)

A

B

NI HIV0

20

40

60

80

100

120

140

160MockPiceatannolSP600125ER-27319

% o

f cel

l via

bilit

y

NT

AMD3100

GF109203

wortmannin

PD98059 PDTC

SP600125

SB203580

ER-273190

20

40

60

80

100

120MockHIV

% o

f cel

l via

bilit

y

Supplemental Figure 1: B cell survival (A) B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM) and ER-27319 (SYK, 50µM)) or with AMD3100 (100µM) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. One representative experiment from 4 is represented (average+SEM). (B) B cells were pre-treated with increasing concentrations of JNK and SYK inhibitors (SP600125 (JNK, 0,1µM to 20µM); ER-27319 and Piceatannol (SYK 1µM to 100µM)) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. Results from one representative experiment from two. Doted line represent the threshold of cell viability determined at 80% in comparison to Mock-NI condition.

Supplemental Figure 2

Supplemental Figure 2: Cell activation and inhibition of activation by kinases inhibitors. B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM); ER-27319 (SYK, 50µM); Piceatannol (SYK, 50µM)) and AMD3100 (100µM) for 4h before CD40L/IL-4 stimulation. 24h post-stimulation, iMFI of cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Average of iMFI + SEM of minimum 4 experiments for each conditions (4 to 6 different donors for the NA (non-activated) conditions and 6 to 10 donors for the CD40L/IL-4 conditions). NA; Non-activated (*=p<0,05 when comparing NT-NA condition with kinase inhibitor-NA and #=p<0,05 when comparing NT-CD40L/IL-4 condition and kinases inhibitor-CD40L/IL-4). NT: non-treated.

01020304050607080 NA

CD40L/IL-4iM

FI (C

D71

)

010002000300040005000600070008000 NA

CD40L/IL-4

iMFI

(CD

69)

NT

GF109203

PD98059

SP600125

ER-27319 0

100200300400500600 NA

CD40L/IL-4

iMFI

(CD

86)

*

#

#

#

*

* *

##

# #

#

**

*

Supplemental Figure 3

0.1

1

10Non-activated

AID

mRN

A (F

old

chan

ge, l

og)

0.1

1

10

100CD40L/IL-4

AID

mRN

A (F

old

chan

ge, l

og)

*

**

NT

GF1

0920

3

wor

tman

nin

PD98

059

PDTC

SP60

0125

SB20

3580

ER-2

7319

Pice

atan

nol

CD71

CD69

CD86

0

2

4

6

8

10

12

NT-NAHIVCD40L/IL-4HIV/CD40L/IL-4

iMFI

(fol

d ch

ange

)

CB

*##

*#

A

NT-NA

CD40L/IL-4

0

5

10

15

20

AID

mRN

A (F

old

chan

ge)

Supplemental Figure 3: AID mRNA expression in CD40L/IL-4 treated B cells. (A) B cells were pre-treated with kinase inhibitors; GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM), ER-27319 (SYK, 50µM) or Piceatannol (SYK, 50µM) for 4h before CD40L/IL-4 stimulation or cells were kept non-activated as control. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to non treated (NT) condition that was considered a fold change of 1. Each black (Non-activated conditions) or grey (CD40L/IL-4 stimulated conditions) plot represents one donor. Black bar represents the average of AID mRNA fold change . (B) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4 for 24h. iMFI of cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Fold changes were calculated in comparison to NT-NA (Non-Activated) condition that is considered a fold change of 1. Average of iMFI + SEM of minimum 4 independent experiments for each conditions (4 or 5 different donors regarding the condition). #=p<0,05 when comparing HIV condition with HIV/CD40L/IL-4 and *=p<0,05 when comparing CD40L/IL-4 condition with HIV/CD40L/IL-4 (C) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to NT-NA condition that is considered a fold change of 1. Average + standard deviation of 2 independent experiments is shown.

Supplemental Figure 4

V(D)Jµ δ γ2

IgMGermline IH-CH transcription

AID

V(D)Jγ2

IgG2Productive (VDJ-CH) Ig transcript

Excised DNA segment=circle

transcript

CSR

δµ

Supplemental Figure 4: Class Switch Recombination (CSR). Mechanism of class switch recombination that allows isotype selection in mature activated B cells. This mechanism produces a DNA recombinant of the constant region of the immunoglobulin heavy chain and an excised DNA segment.