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

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