Regulatory T cell abnormalities associated with aberrant CD4+ T-cell

responses in HIV+ patients with immune reconstitution disease (IRD)

Nabila SEDDIKI

National Centre in HIV Epidemiology and Clinical Research,

The University of NSW,

Centre For Immunology

• Anti-retroviral therapy (ART) may have adverse effects in HIV patients who start when very

immunodeficient (usually within two months); this results in an excessive inflammatory response

referred to immune reconstitution disease (IRD)

• IRD occurs more frequently in HIV-infected patients :

• Who start ART with less than 100 CD4 (cells/L)

• Who have more than 1 log10 viral load decline from baseline (< 2 months)

• With prior opportunistic infection history (some patients with NO OI have been reported)

• IRD has been associated with bacteria, viruses and fungi but mycobacterial IRD has been defined

as an important cause of mortality

• We postulated that deficits in T regulatory cell (Treg) numbers/function may be

defective in these patients and are not able to turn off the excessive T cell responses

Immune Reconstitution Disease

Patients characteristics

In a cross-sectional study including HIV+ patients with less than 50 CD4 (cells/l) before ART: 8 with mycobacterial IRD and 6 without IRD at wk0, wk4 and wk48 after starting ART

Patient ID age/sex nadir CD4 at presentation MAI(count/ l) CD4 (count/ l) ( cpy/ml) detection method

IRD

1 50/F 20 108 240 PCR + culture

2 35/M 48 285 600 PCR

3 43/M 45 92 <50 culture

4 43/M 30 620 <400 PCR + culture

5 34/M 20 57 116,00 culture

6 38/M 21 144 6200 PCR + culture

7 39/M 8 189 7600 not detected

8 38/M 56 100 23,500 PCR

HIV+ w/o IRD(mean)

HIV+ w0 (49) 22.6 23.67 582,429 no

HIV+ w4 (49) 22.6 93.88 83,658 no

HIV+ w48 (49) 22.6 175.2 20,20 no

Healthy (36) NA 1027 NA NAcontrol

IRD CD4+ T cells secrete high levels of IL-2 and IFN-

0 CMV MAI0

500

1000

1500IRD+ HIV+

HIV-

HIV+ (w0)

HIV+ (w48)

HIV+ (w4)

A n t ig e n ( 5 g /m l )

Intra cellular staining for cytokine ELISpot

0.0

0.4

0.8

1.2

1.6

2.0

0 C M V M A I

IL-2/IFN-

A n tig e n ( 5 g /m l)

p a t ie n tc o n t ro l

IFN-

High % of Foxp3+CD25+CD127lo CD4+ Treg in IRD vs healthy

IRD

0

10

20

30P < 0.05

P < 0.01P < 0.01

HIV+before ART)

HIV+(w4post ART)

controlHIV-

C D 1 2 71 0 2 1 0 3 1 0 4 1 0 5

1 0 2

1 0 3

1 0 4

1 0 5

6 .4 6

control HIV-

1 02 1 0 3 1 04 1 0 5

1 02

1 03

1 04

1 05

2 3 . 4

HIV+IRD+

1 02

1 03

1 04

1 05

C D 1 2 7

1 02

1 0 3

1 0 4

1 0 5

2 3 .4

7 0 .6

1 02

1 03

1 04

1 05

1 0 2

1 03

1 04

1 0 5

9 6 .7

1 .5 3

F o x p 3

Treg in IRD proliferate extensively

10 20 30

10

20

30

r=0 .6 5

P =0 .0 0 5

C D 25+ C D 127 lo as % C D 4+ T ce ll

c o n tro l

p a t ie n t

40

*High IL-2 secretion in IRD may help Treg survival

ex vivo

0 M T B M A I G A G

A n t ig e n ( 5 g / m l)

0

10

20

30

40

50

In vitro

0 102 103 104 105

CD127

0

102

103

104

105

CD

25

CD4+ CD45RO+ CD25+ CD127lo

CD4+ CD45RO+ CD25+ CD127hi

0

1

2

3

4

5

IRDHIV+before ART)

HIV+(w4postART)

P < 0.001P < 0.001

P > 0.05

controlHIV-

0 102 103 104 105

CD127

0

102

103

104

105

21.3

22.3

CD4+ CD25+ CD127lo

CD4+ CD25- CD127lo

CD

25

0

5

10

15

HIV+(w4post ART)

controlHIV-

IRD

p<0.05p<0.001

High proportion of CD4+ CD127lo cells in IRD vs HIV+ patients w/o IRD

High IL-7, IL-6 and IL-4 cytokine level in IRD vs controls

Healthy (n=13) IRD (n=7) HIV+ (w4) (n=6) HIV+ (w48) (n=6)

IL-2 7.80.2 8.30.4 7.320.14 7.480.3

IL-4 7.980.31 9.520.4* 8.020.24 8.330.34

IL-6 4.840.09 28.039.2** 8.711.7 5.610.38

IL-7 0.740.09 5.62 1.08*** 4.3410.69 2.9330.66

IL-10 5.730.2 9.271.77* 8.881.55 6.380.41

TNF- 5.30.22 5.770.23 4.960.11 5.140.22

IFN- 83.313.1 169.141.3 92.635.86 88.264.01

IL-21 3.210.17 4.390.21 4.750.91 3.5 0.458

BAFF 3.624.29 4.490.67 2.940.32 2.820.27

(pg/ml)

(pg/ml)

(pg/ml)

(pg/ml )

(pg/ml)

(pg/ml)

(pg/ml)

(ng/ml )

(ng/ml)

- High IL-7 level might be due to the absence or decrease of its receptor IL-7R (CD127)

IL-7 level remains high in IRD after immune reconstitution

IRD+ HIV+(w4post ART)

control HIV- HIV+(w48post ART)

HIV+ (before ART)

0

5

10

15

20

25

P < 0.001

P > 0.05

P > 0.05

P > 0.05

0

500

1000

1500

IRD HIV+(w4post ART)

control HIV- HIV+(w48post ART)

HIV+ (before ART)

P < 0.001

P > 0.05P > 0.05

P < 0.01

2000

HIV+ (w48 post ART)IRD

P > 0.05

0

200

400

600

800

HIV+ (w48 post ART)IRD

P < 0.01

0

3

6

9

12

15

Treg in IRD do not suppress efficiently CD4+ T cell proliferation

0

5

10

15

0

5

10

15 patient#3

0

5

10

15patient#1 patient#2

98.4%

51.9%

78.8%

49%

73.2%

98.9%

49.3%

47%

67%

83%

98.4% 98.4%

- Treg from 3 patients have a defective function

- CD4+ effectors from patient #3 are resistant to suppression

CD

25

102 10 3 104 105

102

103

104

105

102 10 3 104 105

102

103

104

105

Healthy control Patient

Sc:Rc Sc:RpSp:Rp Sp:Rc

CD127

- Irr APCs- anti-CD3+ 72h culture

3HTdR

T cell proliferation

Treg in IRD do not suppress efficiently inflammatory cytokine secretion

In accordance with suppression of proliferation data …

IFN-

0

5000

10000

15000

20000

25000

1 2 3 4 2 3 4 2 3 4

p a t ie n t # 1 p a t ie n t # 2 p a t ie n t # 3

IL-6

0

1000

2000

3000

1 2 3 4 2 3 4 2 3 4

p a t ie n t # 1 p a t ie n t # 2 p a t ie n t # 3

TNF-

0

500

1000

1500

2000

1 2 3 4 2 3 4 2 3 4

p a t ie n t # 1 p a t ie n t # 2 p a t ie n t # 3

resi

du

al c

yto

kin

e s

ecr

etio

n (

pg

/ml)

Abstract #: MOPEA020

- Treg from 3 patients have a defective function

- CD4+ effectors from patient #3 are resistant to

suppression

Treg in IRD do not produce sufficient IL-10 to suppress high inflammatory cytokine secretion

resi

dua

l cyt

okin

e se

cre

tion

(pg/

ml)

Sufficient IL-10, regulatory cytokine, production is compromised in IRD patients probably due

to the presence of high effector/inflammatory cytokine (IFN-, IL-6 and TNF-)

0

100

200

300 IL-10

1 2 3 4 2 3 4 2 3 4

p a tie n t# 1 p a tie n t# 2 p a tie n t# 3

IL-7, IL-4 and IL-15 inhibit suppressive Treg function

IL-2 IL-4 IL-6 IL-7 IL-15 IL-21 cont (no cytk)0

20

40

60

80

100

IL-2=100U/ml

IL-4=100U/ml

IL-6=100U/ml

IL-7=10ng/ml

IL-15=10ng/ml

IL-21=20ng/ml

Increased levels of IL-2, IL-4 and IL-7 found in the plasma of patients with IRD may help the

pathogenic high proliferation of CD4+ T cell observed in these patients

Summary

• Significant expansion of CD127loFoxp3+CD25+Treg in HIV+ patients with IRD compared

to HIV+ w/o IRD

• higher ratio Treg to effector/memory subsets in IRD compared to patients w/o IRD

• High proportion of CD4+ T cells with low IL-7R expression explaining probably the high

IL-7 level in the plasma of these patients

• In vitro suppression assays demonstrated abnormalities in the functional capacity of both

suppressors and responders from these patients, and interestingly IL-10 secretion was

diminished in patients

• Increased levels of IL-4, IL-6 and IL-7 in plasma. Some of these cytokines strongly

inhibited Treg suppression in vitro

Despite the high Treg expansion in IRD, their ability to induce suppression and down regulate aberrant immune responses is compromised, probably due to an imbalance in regulatory versus effector cytokine (IFN- to IL-10)

Abstract #: MOPEA020

Acknowledgements-Centre For Immunology and NCHECR

Sarah Sasson

John Zaunders

Mee Ling Munier

David van Bockel

Suzanna Ip

David Cooper

Anthony Kelleher

- St Vincents Hospital

Patients

Sarah Pett

Debbie Mariott

- Nepean Hospital

Brigitte Nanan

NHMRC