Table S1. Characteristics of participants

Table S1. Characteristics of participants

P values^a

HIV-neg ART-naive ART HIV-neg vs

ART-naive HIV-neg vs

ART ART-naive

vs ART

Number of individuals 26 21 40

Age [median years (IQR)] 32 (28-40) 33 (29-48) 37.5 (30-46) NS NS NS

Gender (male,%) 22 87 69 <0.0001 0.0002 NS

CD4⁺ cell count [median cells/l (IQR)] NA 482 (362-548) 629 (503-787) NA NA <0.0001

Viral load [median log10 copies/ml (IQR)] NA 4.7 (4.2-4.8) <1.3 NA NA <0.0001

Plasma sCD14 [median pg/ml (IQR)] 1803 (1485-2062) 1894 (1471-2212) 1949 (1523-2527) NS NS NS

Duration of ART [median months (IQR)] 71 (35-172)

Type of ART at time of analysis

NRTI + PI 26/40

NRTI + NNRTI 14/40

IQR, interquartlile range; NS, not significant (P>0.05); NA, not available; NRTI, nucleoside and nucleotide reverse transcriptase inhibitors; PI, protease inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitors

aStatistical analysis performed by 1-Way ANOVA and, for categorical variables, by X²-test

Supplementary Fig. S1. Comparative analysis of fresh and frozen/thawed PBMCs for CD62L expression on T lymphocytes.

Ficoll-isolated PBMCs were stained with anti-CD3 and anti-CD62L antibodies and analyzed by flow cytometry immediately after isolation (fresh; filled gray histogram) and after standard freezing/thawing procedure (frozen/thawed;

solid lines). Histograms show the CD62L fluorescence distribution on CD3

⁺

gated live lymphocytes with specimens from a HIV-1-negative individual (right) and a HIV-1-infected ART patient (left). A representative set of data out of 5 is shown.

CD62L

⁺

cells were gated by means of isotype control (dashed lines; shown only for fresh samples) allowing less than 1%

events to be outside. The frequencies of CD62L

⁺

T cells are indicated (gray for fresh sample; black for frozen/thawed).

 

   

Supplementary Fig. S2. Gating strategy for CD3

⁺

CD4

⁺

(A) and CD3

⁺

CD8

⁺

(B) T-cell subsets. CD3

⁺

CD4

⁺

and CD3

⁺

CD8

⁺

T cells were identified on a dot plot of live lymphocytes previously gated on a forward/side scatter (FSC vs SSC) plot. Subsequently, naive and memory T-cell subsets were identified through a combination of cell surface markers (CD45RA, CCR7, CD27) as follows: naive (T

_N

, CD45RA

⁺

CCR7

⁺

CD27

⁺

), central memory (T

_CM

, CD45RA

^-

CCR7

⁺

CD27

⁺

), transitional memory (T

TM

, CD45RA

^-

CCR7

^-

CD27

⁺

), effector memory (T

EM

, CD45RA

^-

CCR7

^-

CD27

^-

) and terminally differentiated (T

_TD

, CD45RA

⁺

CCR7

^-

CD27

^-

). All subsets were gated by means of isotype control (dashed line) allowing ≤ 1% of events to be outside. The analysis of a representative HIV-neg individual is shown.

CD4

CD3 CD3⁺CD4⁺

CCR7

CD45RA

CD27

Central Memory (T_CM) CD45RA^-CCR7⁺CD27⁺

Naïve (T_n) CD45RA⁺CCR7⁺CD27⁺

Terminally Differentitated (T_TD)

CD45RA⁺CCR7^-CD27^-

Transitional Memory (T_TM) CD45RA^-CCR7^-CD27⁺ Effector Memory (T_EM)

CD45RA^-CCR7^-CD27^-

CD62L

% of Max

CD62L

% of Max

% of Max

FSC

SSC

A

CD8

CD3 CD3⁺CD8⁺

CCR7

CD45RA

Central Memory (T_CM) CD45RA^-CCR7⁺CD27⁺

Naïve (T_n) CD45RA⁺CCR7⁺CD27⁺

Terminally Differentitated (T_TD)

CD45RA⁺CCR7^-CD27^-

Transitional Memory (T_TM) CD45RA^-CCR7^-CD27⁺ Effector Memory (T_EM)

CD45RA^-CCR7^-CD27^-

CD27 CD62L

% of Max

CD62L

% of Max

% of Max

FSC

SSC

B

Supplementary Table S2. CD4

⁺

and CD8

⁺

T-cell subsets and their CD62L expression

P values

HIV-neg ART-naive ART HIV-neg vs

ART-naive HIV-neg vs

ART ART-naive vs ART Subset (% of CD4+ T cells)

naive (TN) 33 (29.1-40.3) 35 (24-39.9) 36.4 (30.8-41.1) NS NS NS

central memory (TCM) 27.3 (23.8-34) 23 (19.8-30.9) 27.9 (22.7-32.7) (0.082) NS NS

transitional memory (TTM) 20.4 (16.8-26.2) 19.1 (15-24.9) 20.5 (16.4-25.3) NS NS NS

effector memory (TEM) 5.9 (4.2-9.4) 7.8 (5.7-12.1) 5.6 (4.2-9.8) NS NS (0.059)

terminally differentiated (TTD) 0.4 (0.2-1) 1.1 (0.5-2) 0.5 (0.3-0.9) 0.006 NS 0.010

CD62L+ (% of CD4+ T cells)

total CD4 51.6 (46.7-60.6) 41.9 (15.6-57.4) 60.2 (45.4-64.5) 0.015 NS 0.001

naive (TN) 78.8 (60.3-84.4) 58.9 (27.1-80.4) 81.7 (67.4-87.1) 0.011 NS 0.002

central memory (TCM) 59 (46.3-67.8) 36.4 (14-65.8) 62.8 (52.1-70.3) 0.037 NS 0.006

transitional memory (TTM) 44.9 (37.8-50.5) 37.8 (17-59.9) 53.6 (40.8-60.6) NS (0.067) 0.043

effector memory (TEM) 23.7 (18-30.5) 18.8 (8.4-30.7) 22.4 (16.1-29.6) NS NS NS

terminally differentiated (TTD) 28.5 (17.8-39.1) 20.2 (9.8-31) 20.4 (11.8-30.3) NS NS NS CD62L (MFI on CD4+ T cells)

naive (TN) 2399 (1844-2924) 2202 (1355-2486) 2673 (1846-3362) NS NS 0.027

central memory (TCM) 1469 (1192-2349) 1422 (786-1598) 1727 (1367-2233) (0.053) NS 0.016 transitional memory (TTM) 1226 (1084-1690) 1165 (783-1478) 1473 (1127-1844) NS NS 0.027

effector memory (TEM) 1106 (936-1509) 1044 (815-1200) 1188 (908-1669) NS NS 0.003

terminally differentiated (TTD) 1212 (908-1655) 1052 (907-1216) 1200 (934-1638) (0.078) NS 0.021 Subset (% of CD8+ T cells)

naive (TN) 31.1 (25.8-36.7) 9.9 (4.3-18) 30.6 (17.7-40.9) <0.0001 NS <0.0001

central memory (TCM) 3.5 (2.8-5.4) 2.2 (1.2-3.4) 3.3 (2.5-5.3) NS NS NS

transitional memory (TTM) 11.5 (8-15.3) 22.6 (15.5-28.5) 13.8 (9.7-19.8) 0.0003 (0.074) 0.025

effector memory (TEM) 23.9 (18.1-28.4) 20.3 (14.8-31.4) 19 (14.1-28) NS NS NS

terminally differentiated (TTD) 10.7 (6.7-14.9) 23.1 (15.6-34.8) 11.8 (5.6-16.3) <0.0001 NS <0.0001 CD62L+ (% of CD8+ T cells)

total CD8 29.4 (24.6-35.4) 17.3 (6.6-24.2) 26.5 (20.3-37.9) <0.0001 NS 0.0007

naive (TN) 66.6 (52.9-77.8) 47.6 (20.7-75.2) 71.1 (55-79.3) 0.044 NS 0.011

central memory (TCM) 39.5 (27.4-46.8) 28.8 (13.8-39.6) 39 (29-45.5) 0.044 NS 0.036

transitional memory (TTM) 10.1 (5.6-15.3) 8.4 (4.1-14) 9 (4.8-14.1) NS NS NS

effector memory (TEM) 20.1 (14.1-25.4) 21.4 (11.4-28.9) 24.6 (18.9-31.2) NS NS NS

terminally differentiated (TTD) 20.6 (13-31.2) 15.7 (6.2-20.3) 14.1 (9.6-21.9) 0.006 0.016 NS CD62L (MFI on CD8+ T cells)

naive (TN) 2021 (1360-2788) 1629 (1176-2142) 2080 (1506-2519) NS NS (0.069)

central memory (TCM) 1517 (1119-2156) 1337 (1026-1802) 1493 (1056-1922) NS NS NS

transitional memory (TTM) 1129 (827-1326) 948 (845-1336) 983 (775-1288) NS NS NS

effector memory (TEM) 1274 (1056-1616) 1176 (935-1472) 1333 (971-1693) NS NS NS

terminally differentiated (TTD) 799 (693-982) 732 (593-954) 764 (670-837) NS NS NS

Values are indicated as median (IQR, interquartlile range); NS, not significant (P>0.05), however P values between 0.05 and 0.08 are also shown in parenthesis.

   

Supplementary Fig. S3. Spearman rank-correlations between plasma sCD62L levels and percentages of CD62L

⁺

cells among T

_N

and T

_CM

CD4

⁺

and CD8

⁺

T-cell subsets in HIV-1-infected patients analyzed all together (all HIV+, left panels) or separating ART-naïve (central panels) and ART subjects (right panels).

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.088

P = 0.496

sCD62L ng/ml

% CD62L+ (of CD4+ TN cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.095

P = 0.464

sCD62L ng/ml

% CD62L+ (of CD8+ TN cells)

0 500 1000 1500

0 20 40 60

80 ρ = -0.090

P = 0.697

sCD62L ng/ml

% CD62L+ (of CD4+ TCM cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.068

P = 0.769

sCD62L ng/ml

% CD62L+ (of CD8+ TN cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.113

P = 0.483

sCD62L ng/ml

% CD62L+ (of CD4+ TCM cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.111

P = 0.490

sCD62L ng/ml

% CD62L+ (of CD8+ TN cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.119

P = 0.608

sCD62L ng/ml

% CD62L+ (of CD4+ TN cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.084

P = 0.601

sCD62L ng/ml

% CD62L+ (of CD4+ TN cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = -0.103

P = 0.426

sCD62L ng/ml

% CD62L+ (of CD4+ TCM cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = 0.001

P = 0.995

sCD62L ng/ml

% CD62L+ (of CD8+ TCM cells)

0 500 1000 1500

0 20 40 60

80 ρ = 0.124

P = 0.593

sCD62L ng/ml

% CD62L+ (of CD8+ TCM cells)

0 500 1000 1500

0 20 40 60 80

100 ρ = 0.028

P = 0.862

sCD62L ng/ml

% CD62L+ (of CD8+ TCM cells)

all HIV+ ART-naive ART