1111

Clovis Palmer1,2, Anthony Jaworowski1, Joseph McCune3, Suzanne Crowe1

1Centre for Virology, Burnet Institute, Melbourne, Australia

2 University of New South wales, Sydney, Australia

3 Dpt. Medicine, University of California, San Francisco, USA

Glut1: establishing a new paradigm for HIV-1 infection by regulating glucose metabolism

and activation in CD4+ T cells in HIV-1-positive subjects

No stimulation

CD3

CD3 + 5ug/ml CD28

CD3 + 1ug/ml CD28

CD3 + 1ug/ml CD28

CD3 + 5ug/ml CD28

GLUT1

Glucose Transporter-1 (Glut1) belongs to a family of glucose transporters

Fu et al, 2004; Barata et al, 2004; Maratou et al, 2007

Glut1 and is the main glucose transporter on T cells and is regulated by

immunological signals

Glucose

Hx

G-6-P

Intracellular retention of glucose is facilitated by hexokinase (Hx) activity

Glycolysis (Lactate + 2 ATP)

Glucose is metabolized via glycolysis or oxidative phosphorylation depending on

the T cell activation status Powel and Delgoffe, 2010; Finlay and Cantrell, 2011

Oxidative Phos. (36 ATP)

Glucose metabolism in T cells

T cell

Oxidative phosphorylation

36 ATPs

Protein

DNA

Cell membrane

Cell growth

Proliferation

Antiviral response

Migration Viral replication

Resting T cell

Aerobic Glycolysis

2 ATPs

Amino Acids

Nucleotides

Lipids

Activated T cell

Hypothesis: HIV-1 infection increases glucose metabolism in T cells

Glut1

T cells in HIV- subjects

Rat

e o

f G

lyco

lyti

c M

etab

oli

sm

HIV

T cells in HIV+/cART subjects

Glut1

Glut1

T cellsin HIV+ subjects

Activated T cells

Glucose

Evaluate cell surface expression of glucose transporter 1 (Glut1) on T cells from

HIV-, HIV+ and HIV+/cART subjects

Aim 1

HIV- HIV+ HIV+/cART

Subjects (n) 25 45 35

Male (%) 96 97 100

Viral load(copies/ml)

- 79,1740 ± 78,094

<50

CD4 cell(cells/ul)

397 ± 193 496 ± 256

Subject groups

Fresh blood (The Alfred Hospital, Melbourne)

Lysed red blood cells

Method: Glut1 expression on T cells

Frozen PBMCs(UWA, USA)

Surface markers: CD3, CD4, CD8Metabolic marker: Glut1

Flow cytometry

HIV-1 infection is associated with increased Glut1 on CD4+T cells

% C

D4+

Glu

t1+

T c

ells

0

10

20

30

40

60

80

100 p<0.0001 p=0.0009

p=0.0001

n= 25 45 35

Glut1 on CD4+ T cells

% C

D4+

Glu

t1+

cel

ls

%C

D8+

Glu

t1+

T c

ells

0

40

8090

95

100

105

n= 25 45 35

Glut1 on CD8+ T cells

% C

D8+

Glu

t1+

cel

ls

Inverse relationship between CD4+Glut1+ T cells and absolute CD4 cell count in HIV+ subjects

BA

% CD4 vs %CD4+Glut1+ cells CD4 count vs %CD4+Glut1+ cells

% CD4+Glut1+ cells

Ab

solu

te C

D4

ce

ll co

unt

% CD4+Glut1+ cells

% C

D4+

cel

ls

Annexin V+ (apoptosis) and 7AAD+ (cell death) cells have high Glut1 expression

Increased glucose metabolic activity may contribute to CD4+ T cell loss during HIV-1

infection

Aim 2

To access the functional significance of Glut1

expression on CD4+ T cells

Glycolysis pathway

Time (min)

MF

I of

2-N

BD

G o

n C

D4+

T c

ells

0 30 60 90 1200

20

40

60

80

100

n=4

The intracellular concentrations of glucose-6-phosphate and L-lactate are elevated in CD4+ T cells from HIV+ subjects

Palmer et al., AIDS 2012, TUAA0104

HIV-1 infection increases aerobic glycolysis in CD4+ T cells in vivo

CD4+ T cells in HIV+ subjects have increased glucose uptake

Kinetics of glucose uptake in total CD4+ T

cells

MF

I (2-

NB

DG

)

2-NBDG

SS

C

CD

4+

Glu

t1-

CD

4+

Glu

t1+

Glut1+ T cells take up more

glucose

MF

I 2-N

BD

G in

CD

4+

T c

ells

0

20

40

60

p=0.001 p= 0.04

p=0.03

n= 8 8 8

Glucose uptake in total CD4+ T

cells

MF

I (2-

NB

DG

)

Human cytomegalovirus requires a glycolytic environment for replication

Yu et al., Trends Microbiol, 2011

Glut1-mediated metabolic pathway regulates HIV-1 infection in naïve CD4+Loisel-Meyer et al., PNAS, 2012

Host metabolism and viral infection

Are CD4+Glut1+ T cells more permissible to HIV-1 infection?

Flow cytometry

Glut1OX40 (PI3K)

PBMCs from study participants (n=4)

Time on cART: 6 ± 4.3 years

Plasma viral load: <50

CD4 T cell count: 274 ± 98

CD4+Glut1+ : 57.0 ± 18.9%

Incubate with HIV-1-GFP (72h)(Absence of exogenous stimuli)

CD4+Glut1+ T cells are more permissive to HIV-1 infection in vitro

Glut1

PI3K

Glut1

PI3K

Glut1

PI3K

HIV-1 infection in CD4+Glut1+ T cell subsets

% G

FP

+ c

ells

Glut1+ OX40+ Glut1+ OX40- Glut1-OX40-0

10

20

30

40

PI3K activity and Glut1 expressionare both required for efficient HIV-1 infection in vitro

n= 4

1. Circulating CD4+Glut1+ T are increased in HIV-infected subjects are not restored to normal levels

during cART and have high glycolytic activity,

2. The frequency of CD4+Glut1+ T cells correlates inversely with absolute CD4 count and a proportion

of these cells are highly apoptotic

3. CD4+Glut1+ T cells with high PI3K activity are more permissive to HIV-1 infection in vitro

Summary

CD4+Glut1+ T cells (Targets)

HIV-1

Vicious cycle

PI3KᵧmTORC1

Glut1 Glycolysis

CD4+T cell loss

Speculations: role of Glut1 in HIV-1 pathogenesis

HIV-1

Inhibition may limit glucose metabolic activity and activation

Baker IDI, Melbourne, AustraliaDarren Henstridge

Creative and Novel Ideas in HIV Research (CNIHR) Administrative Team

Centre for Virology (Pathogenesis lab), Burnet Institute,

Melbourne, Australia Mentor: Suzanne Crowe

Anthony JaworowskiJingling Zhou

Linda LamMaelenn Gouillou

Anna HearpsAnna Maisa

Wan-Jung ChengTom Angelovich

Division of Experimental Medicine at the University of California, San Francisco (UCSF)

Mentor: Joseph Mike McCune

AcknowledgementContact: cpalmer@burnet.edu.au

Centre for Virology(Virology/latency lab) Burnet Institute,

Melbourne, Australia Sharon Lewin

Suha SalehGabriela Khoury

Candida da Fonseca Pereira

Volunteers !!!!!

Center for AIDS ResearchClinical Research Core

University of Washington, USARobin Mohr

Centre for Infection and Inflammation Research, School of Medical Sciences, UNSW

Andrew Lloyd- Amany Zekry LabDavid Simar

Benny Baharuddin

Department of Chemistry, University of the West Indies, Kingston, Jamaica

Willem Mulder

Distribution of CD4+Glut1+ T cells

Ki67 and Glut1 expression

Glut1 and proliferation

Figure S4

A B

Glucose Oligomycin

2-Deoxyglucose

C

22222222

Clinical characteristics of study groups (Cross-sectional)

Variables Groups P value

n HIV-1

(A) HIV+ (B)

HIV/cART (C)

A vs B B vs C A vs C

Sex (M/F)

105 24/1 44/1 35/0 - - -

BMI (Kg/m2) 82 23.0 ± 2.5 24.7 ± 5.1 24.6 ± 5.9 0.17 0.40 0.26 Age (years)

93

45.2 ± 20.5

44.0 ± 10.7

44.6 ± 11.5

0.31

0.46

0.28

MSM (self-reported)

-

11/25

-

-

-

-

-

CD4+ T cell count (cells/ul)

63

-

397 ± 193

496 ± 256

-

0.06

-

% CD3+CD4+ T cells

105

54 ± 1

28 ± 14

38.7 ± 15

<0.0001

0.001

0.0001

Viral load (copies/ml)

72

-

79,174 ± 78,094

<50

-

-

-

Plasma Glucose (mmol/L)

80

4.8 ± 0.4

5.1± 1.0

5.1 ± 0.9

0.11

0.41

0.13

Plasma Insulin (μIU/ml)

48

9.2± 8.9

6.3 ± 7.6

14.5 ± 19.3

0.26

0.50

0.44

TNF (pg/ml)

48

6.0± 3.0

10.5 ± 6.0

10.6 ± 8.7

0.005

0.28

0.02

Alfred Hospital/Burnet Institute, Australia

CFAR, University of Washington, USA