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Pavel BostikFMHS
Charles University Medical SchoolUniversity Hospital
Hradec KraloveCzech Republic
DIFFERENTIAL EFFECTS OF (LENTI)VIRUS INFECTION ON AKT
SIGNALLING IN CD4+ T CELLS
Cercocebus atysSIVsm
Pan troglodytes)SIVcpz
HIV-1 (M,N, O)
HIV-2
ORIGIN OF HIV
Macacca mulata
Sooty mangabey
SIVsykSIVcol
L-Hoestova monkey
Mandrill
SIVlho SIVrcm
SIVmnd
SIVver
SIVsm
Sykes monkeyColobus monkey
Red-capped mangabeyVervet
SIVcpz
CHimpanzee
NATURALLY SIV INFECTED NHP
1. High levels of acute viremia and significant chronic viral loads2. Replication predominantly in short lived T cells; poorly controlled3. Significant antibody responses
LENTIVIRUS INFECTION IN DISEASE SUSCEPTIBLE AND DISEASE RESISTANT SPECIES
4. Apoptosis of bystander cells5. Chronic immune hyperactivation6. Acute depletion of CD4+ T cells in mucosal tissues7. CTL responses8. Progressive loss of CD4+ T cells9. Loss of CD4+ T cell help
4. Low levels of apoptosis 5. Normal immune activation6. Initial acute depletion followed by rebound7. Poorly detectable CTL responses8. Slight loss, preserved homeostasis9. Maintained CD4+ T cell help10. Low levels of CCR5 expression
HIV/SIV – DISEASE SUSCEPTIBLE SIV - DISEASE RESISTANT
0
2
4
6
8
10
12
14
16
18
SM+ RM+ SM- RM-
% A
nn
exi
n-V
Po
stiv
e **
APOPTOSIS OF CD4+ T CELLS
GSK3b is one of the kinases identified to be dysregulated in SIV+ RM Bostik et al.,J Virol 2001.
TCR/ CytokineCD28
CD4 CD3 Receptor
lck ZAP70
Cot
MAPK
GSK3
PI3K
Akt
NFkBPGE2 p53
Cell deathTranscription
Ser473
Thr308 p
COX2
PDK1
PIP3
PTEN
p
AKT INVOLVED IN MULTIPLE T CELL FUNCTIONS
APC
GSK-3b
NS Act LiCl
Act NS Act LiCl
Act
b-actin
1 32 4 5 6SM
GSK-3b
SIV+ SIV-
NS Act NS ActAct LiCl
Act LiCl
b-actin
7 98 10 11 12RM
EXPRESSION OF GSK3b
*pGSK-3b
NS Act LiCl
Act LY
ActLiClLY
SIV-
b-actin
NS Act LiCl
Act LY
ActLiClLY
SIV+
*pGSK-3b
b-actin
SM
RM
1 32 4 5 76 8
13
9 1110 12
1514 16 17 1918 20 21 2322 24
PHOSPHORYLATION OF GSK3b
pAkt Thr308 pAkt Ser473 Akt0%
10%
20%
30%
40%
50%
60%
70%
80% base RM-
base RM+
base SM-
base SM+
p<0,001
FRACTION OF CD4+ T CELLS EXPRESSING pAKT
pAkt Thr308 pAkt Ser473 Akt0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
RM CD4+ T cellsNEGNEG ACTPOSPOS ACT
pAkt Thr308 pAkt Ser473 Akt0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
SM CD4+ T cellsNEGNEG ACTPOSPOS ACT
p<0,005
p<0,005
EFFECT OF ACTIVATION ON pAKT
All cells CM EM N0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
BASE ACT ACT+LiCl
p<0,01p<0,01 p<0,01
Central memory CD28+CD95+, Effector memory CD28-CD95+ Naïve CD28+CD95-
AKT Thr308 phosphorylation in CD4+ T cells from SIV+ RM
p<0,01
p<0,01
p<0,01
All cells CM EM N0%
10%
20%
30%
40%
50%
60%
BASE ACT ACT+LiCl ACT+LY
p<0,02
AKT Thr308 phosphorylation in CD4+ T cells from SIV+ RM
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Act Act + LY Act + LiCl
Rel
ativ
e A
nn
exin
V P
ost
ive
SIV+SM (n=5)
SIV-SM (n=8)
SIV+RM (n=4)
SIV-RM (n=7)
**
***p<0.05
p<0.05 p<0.001
*p<0.05 **
No Change
AICD
GSK-3b
Monkey 1
U I
Monkey 2
U I
EFFECT OF IN-VITRO INFECTION
CONCLUSIONS
1) GSK-3b transcription IS markedly reduced not only at the message level but also at the level of protein expression in CD4+ T cells from SIV+RM
2) Baseline levels of Akt and p-AktThr308 are comparable, the levels of phosphorylated AktSer 473 are significantly lower in cells from SIV+ SM compared to the SIV+ RM
3) Stimulation of CD4+ T cells leads to a marked increase in both total Akt and *p-AktThr308 in the SIV+RM, which correlates to increased susceptibility for AICD
4) Phosphorylation differences of Akt at Ser473 and Thr308 in anti-CD3/CD28 activated CD4+ T cells are both species and CD4+ T cell sub-population specific
Thank you for your attention
Emory UniversityS. Stephenson
F. Villinger
FMHSM. Schmidt
V. Bostik
Support:
GACRNIH