#P-U5

Premature Aging and Immune Senescence in HIV-1-Infected Children1 2 3 4 2 5 4 1Ketty Gianesin , Antoni Noguera-Julian , Marisa Zanchetta , Osvalda Rampon , Claudià Fortuny , Mireia Camós , Carlo Giaquinto , Anita De Rossi

1 Section of Oncology and Immunology, Unit of Viral Oncology and AIDS Reference Center, Department of Surgery, Oncology and Gastroenterology-DiSCOG, University of Padova, Padova, Italy2 Unitat d'Infectologia, Servei de Pediatria; Hospital Sant Joan de Déu-Universitat de Barcelona, Barcelona, Spain3 Istituto Oncologico Veneto (IOV)-IRCCS, Padova, Italy4 Department of Mother and Child Health, University of Padova, Padova, Italy5 Servei d'Hematologia; Hospital Sant Joan de Déu-Universitat de Barcelona, Barcelona, Spain

1. Introduction

Antiretroviral Therapy (ART) has significantly improved the prognosis of HIV-1-infected subjects,

resulting in the prevention of AIDS-related complications and significantly increased life expectancy [1, 2].

However, ART treatment does not restore full functionality of the immune system, and health status

remains characterized by a number of non-AIDS defining complications associated with aging, including

malignancies [3, 4], even among long-term ART-treated patients. Therefore, HIV-1-infected subjects may

suffer of premature and accelerated aging; chronic immune activation, due to persistence of HIV-1 virions,

may play a key role in this senescent pathway [5].

Little data are available about immune senescence and premature aging in pediatric HIV-1 infection, in

which the effects of chronic immune activation and immune senescence together with ART toxicity during

children's immune system maturation might be even more deleterious.

3. Results

Poster n.°: 923

3.1 Characteristics of the Study Population

HIV+(n=71)

HEU(n=65)

HUU(n=56)

Age, median [IQR]

Gender, n (%)

Exposed to prophylaxis, n (%)

Exposed to ART, n (%)

Duration of ART exposure, median [IQR] (weeks)

Percentage of lifetime on ART

Detectable plasmaviremia, n (%)

Plasmaviremia at sample collection, (log copies/ml)10

M/F

3.11 [1.40-4.48] 1.74 [0.99-3.31] 1.85 [0.84-3.46]

39 (55%)/32 (45%) 34 (52%)/31 (48%) 29 (52%)/27 (48%)

5 (7%) 61 (93.8%) ---

41 (58%)

48 [72-144]

57.5 [42.6-84.5]

55 (77%)

5.25 [4.75-5.60]

1.70 [2.54-4.28]

ART-naive

on ART

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3.2 Telomere Lenght is Shorter in HIV-1-Infected Children

The median TL in PBMC was significantly lower in

HIV+ children than in HEU and HUU children,

being the median TL of 2.21 [1.94-2.58], 2.63 [2.25-

3.21], 2.88 [2.49-3.1], respectively (overall,

p<0.0001 adjusted for age). TL inversely correlated

with age in HEU and HUU groups but not in HIV+

children (Figure 1A).

Among HIV+ group, ART-naïve children had

shorter TL compared with children on ART (2.11

[1.75-2.37] vs 2.46 [2.07-2.68]; p=0.0029 adjusted

for age). For neither of the two groups, TL was

associated with age (Figure 1B).

0 20 40 60 800

1.0

2.0

3.0

4.0

Age (months)

Telo

me

re L

en

gth

(T

/S r

atio

)

B.

3.3 Thymic Output is Lower in HIV-1-Infected Children

HEU and HUU children had higher levels of TREC than

HIV+ children did (5409 [3411-6712], 5370 [2380-8101], 53498 [2051-6780] TREC copies/10 PBMC; overall

p=0.0249 adjusted for age). TREC levels decreased

significantly with increasing age in HEU and HUU groups,

but not in HIV+ children (Figure 2A).

5No significant differences in TREC levels/10 PBMC were

found between ART-treated children and ART-naive

children (p=0.2995) (Figure 2B).

0 20 40 60 80

0

5000

10000

15000

20000A.

5T

RE

C c

op

ies/

10

PB

MC

Age (months)0 20 40 60 80

0

2000

4000

6000

8000

10000

12000

14000B.

5T

RE

C c

op

ies/

10

PB

MC

Age (months)

3.4 Phenotypic T-cell Alterations Occur Early in HIV-1-Infected Children

HIV+ chi ldren had a lower

percentage of CD4+ cells than HEU

and HUU children, but percentage of

CD4+ ce l l subsets d id not

significantly differ, with the exception

of exhausted PD-1+ cell subset,

which was more expanded in HIV+

children than in controls (p=0.050).

In addition, among HIV+ children,

both CD4+CD38+HLA-DR+ and

CD4+PD-1+ subsets were higher in

children with detectable viral load

than in children with undetectable

HIV-1 plasmaviremia (p=0.056 and

p=0.037, respectively).

HIV+ children had a lower frequency

of CD8+ recent thymic emigrant

(RTE) (overall, p=0.005) (Figure 3A)

and a higher percentage of

peripheral expanded cells (PEC;

CD45RA+CD31-) than control

g r o u p s ( o v e r a l l , p = 0 . 0 4 0 ) ,

suggesting a strong peripheral

proliferation (Figure 3B).

Interestingly, CD8+ RTE cells were lower in children with detectable viral

load than in children with undetectable plasmaviremia (p=0.039), and

tended to inversely correlate with HIV-1 RNA levels(r=-0.363, p=0.080)

(Figure 4).

A total of 71 perinatally HIV-1-infected (HIV+) children, aged 0-5 years,

65 exposed-uninfected (HEU) and 56 unexposed-uninfected (HUU) age-

matched children were included in the study. 30/71 (42%) HIV+ children

were ART-naïve, while the remaining were on ART (median time of 18

[11.0-36.5] months).

A subset of 24 HIV+ (15 with undetectable plasmaviremia (<50

copies/ml) and 9 with detectable plasmaviremia (median 3.64 [2.75-5.16]

log copies/ml)), 21 HEU and 18 HUU children were studied for immune 10

phenotype.

A. Peripheral blood mononuclear cells (PBMC) were isolated from

EDTA-treated peripheral blood by centrifugation on a Ficoll-Paque

gradient and studied for:

- Telomere length (TL) by monochrome quantitative real-time

PCR [6];

- Thymic output by T-cell receptor rearrangement excision circle

(TREC) levels quantification by quantitative real-time PCR [7];

- T-cell phenotyping by multicolor flow cytometry. Within the

CD3+CD4+ and CD3+CD8+ T-cell populations, the following

subsets were identified:

B. Plasma HIV-1 RNA levels were determined in HIV-1-infected

children using the COBAS Taqman HIV-1 test (Roche, Branchburg, NJ,

USA). The lower limit of detection was 50 HIV-1 RNA copies/ml.

C. Statistical analysis were performed using SPSS software v.22. All P-

values were two-tailed, and were considered significant when lower

than 0·05.

2. Patients & Methods

2.1 Study Population 2.2 Methods

Marker(s)

CD45RA+CD27+

CD45RA-CD27+

CD45RA-CD27-

CD45RA+CD27-

CD45RA+CD31+

CD45RA+CD31-

CD28-CD57+

CD38+HLA-DR+

PD-1+

Cell Subset

naive

central memory

effector memory

terminally differentiated

recent thymic emigrant (RTE)

peripheral expanded (PEC)

senescent

activated

exhausted

Figure 2. Correlation between age and TREC levels in HIV+, HEU and HUU children (A), and in ART-naïve and ART-treated

children (B). Panel A: TREC levels as function of age in HIV+ (in black; regression coefficient (b)=-17, p=0.3526), in HEU (in grey; b=-61, p=0.0088) and HUU (in

white; b=-86, p=0.0003) children. Panel B: TREC levels as function of age in ART-naive (in grey, b=-11, p=0.6796) and in ART-treated children (in black; b=-8,

p=0.7536).

Figure 1. Correlation between age and telomere length in HIV+, HEU and HUU children (A), and in ART-naïve and ART-

treated children (B). Panel A: telomere length as function of age in HIV+ (in black; regression coefficient (b)=-0.00175, p=0.5870), in HEU (in grey;

0.01024, p=0.0079) and HUU (in white; b=-0.01001, p=0.0112) children. Panel B: telomere length as function of age in ART-naive (in grey, b=-0.00540,

p=0.2269) and in ART-treated children (in black; b=-0.00455, p=0.2579).

b=-

r=-0.363p=0.080

HIV-1 RNA (copies/ml)101 102 103 104 105 106

20

40

60

80

100

% C

D8

+ R

TE

0

Figure 3. Percentage of recent thymic emigrant (RTE) CD8+ cells (A) and peripheral expanded (PEC) th thCD8+ cells (B) in HIV+, HEU and HUU children. Boxes and whiskers represent the 25-75 and 10-90 percentiles,

respectively; the median is the central line in each box.

Figure 4. Relationship between HIV-1 viral load and %CD8+ RTE cells in HIV+ children.

4. Conclusions

B.

The proportion of CD8+

cells with a senescent

phenotype (CD28-CD57+)

was higher in HIV+ children

than in HEU and HUU groups

(25.8% [12.4-43.2] vs 8.5%

[6.8-16.7] and 9.7% [3.3-

27.3]; p=0.004) (Figure 5A).

Figure 5. Percentage of senescent CD8+ cells (A), activated CD8+ cells (B) and exhausted CD8+ cells (C) in HIV+, HEU and HUU children. Boxes and whiskers

th threpresent the 25-75 and 10-90 percentiles, respectively; the median is the central line in each box.

PD-1 expression in viremic

subjects was significantly higher

than in those with undetectable

plasmaviremia (p=0.002), and

correlated with HIV-1 RNA levels

(r=0.471, p=0.021) and immune

activated cells (CD8+CD38+HLA-

DR+) markers (r=0.528, p=0.009).

Telomere length was inversely

correlated with percentages of

CD8+ senescent, activated and

exhausted cells in HIV+ children,

but not in HEU and HUU children

(Figure 6 A-I). 0 5 10 15 20 25 30Te

lom

ere

Le

ng

th (

T/S

ra

tio

)% CD8+ CD38+ HLA-DR+

% CD8+ CD28- CD57+

% CD8+ PD-1+

r=-0.458p=0.054

0 10 20 30 40 50 600

1.0

2.0

3.0

4.0

5.0

0

1.0

2.0

3.0

4.0

5.0r=-0.515p=0.019

0

1.0

2.0

3.0

4.0

5.0r=-0.637p=0.002

0 5 10 15 20

% CD8+ CD38+ HLA-DR+

0

1.0

2.0

3.0

4.0

5.0r=0.026p=0.911

0 105 15

r=0.112p=0.641

% CD8+ PD-1+

0

1.0

2.0

3.0

4.0

5.0

0 5 10 15 20 25 30

0 10 20 30 40 50% CD8+ CD28- CD57+

0

1.0

2.0

3.0

4.0

5.0

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)Te

lom

ere

Le

ng

th (

T/S

ra

tio

)

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)Te

lom

ere

Le

ng

th (

T/S

ra

tio

)

r=-0.036p=0.882

% CD8+ CD38+ HLA-DR+0 105 15

0

1.0

2.0

3.0

4.0

5.0

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)

r=0.110p=0.656

0 2 4 6 8 100

1.0

2.0

3.0

4.0

5.0

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)

r=-0.018p=0.934

0 10 20 30 40

r=-0.186p=0.498

0

1.0

2.0

3.0

4.0

5.0

Te

lom

ere

Le

ng

th (

T/S

ra

tio

)A. B. C.

D. E. F.

G. H. I.

% CD8+ PD-1+

% CD8+ CD28- CD57+

Figure 6. Correlation between telomere length and percentages of senescent (A-C), activated (D-F) and exhausted CD8+ cells (G-I). Telomere length correlates with senescent cells in HIV+ (A) children but not in HEU (B) and HUU (C) children. In addition, in HIV+ children, telomere length was inversely correlated with activated (D) and exhausted (G) cells; conversely, these relationships were not found in HEU (E, H) and HUU (F, I) children.

- HIV-1-infected children had a lower telomere length compared to age- and gender- matched controls, suggesting an accelerated biological aging;

- age-adjusted telomere length is shorter in ART-untreated than in ART-treated children, thus suggesting that HIV-1 itself, rather than exposure to antiretroviral drugs, influences the senescence process;

- immune senescence is worse more evident in CD8+ than in CD4+ cell compartment;

- immune senescence is more important in children with detectable HIV-1 viremia than those with undetectable viremia;

- telomere length inversely correlated with activated CD8+ cells suggesting that chronic immune activation affects biological aging and senescence.

5. References1. The European Collaborative Study. Mother-to-child transmission of HIV infection in the era of highly active antiretroviral therapy. Clin Infect

Dis 2005; 40:458-65

2. The Antiretroviral Therapy Cohort Collaboration. Life expectancy of individuals on combination antiretroviral therapy in high-income countries: a collaborative analysis of 14 cohort studies. Lancet 2008; 372:293-299

3. Guaraldi G, Orlando G, Zona S, et al. Premature age-related comorbidities among HIV-infected persons compared with the general population. Clin Infect Dis 2011; 53:1120-6

4. Chiappini E, Berti E, Gianesin K, et al. Pediatric human immunodeficiency virus infection and cancer in the highly active antiretroviral treatment (HAART) era. Cancer Lett 2014; 347:38-4

5. Desai S and Landay A. Early Immune Senescence in HIV Disease. Curr HIV/AIDS Rep 2010; 7:4-10

6. Rampazzo E, Bertorelle R, Serra L, et al. Relationship between telomere shortening, genetic instability, and site of tumour origin in colorectal cancers. Br J Cancer 2010; 102:1300-5

7. Ometto L, De Forni D, Patiri F, et al. Immune reconstitution in HIV-1-infected children on antiretroviral therapy: role of thymic output and viral fitness. AIDS 2002; 16:839-49

Percentage of CD8+ CD38+

HLA-DR+ and CD8+ PD-1+

cells were significantly

higher in HIV+ children than

in HEU and HUU groups

(overa l l p<0 .001 and

p<0.001, respect ive ly)

(Figure 5 B, C).

0

20

40

60

80

100

HIV+ HEU HUU

% C

D8

+ R

TE

overall p=0.005

p=0.635p=0.005

p=0.023

A.

0

20

40

60

80

100

HIV+ HEU HUU

% C

D8

+ P

EC

overall p=0.040

p=0.104

p=0.453

p=0.014

B.overall p=0.004

p=0.934

p=0.005

p<0.001

0

20

40

60

80

100

HIV+ HEU HUU

% C

D8

+C

D2

8-C

D5

7+A.

overall p<0.001

p=0.083

p=0.013

p<0.001

% C

D8

+C

D3

8+

HL

A-D

R+

0

10

20

30

40

50

HIV+ HEU HUU

C.overall p<0.001

p=0.921p<0.001

p<0.001

HIV+ HEU HUU

0

5

10

15

20

25

30

% C

D8

+P

D-1

+