Supplemental Table 1. Clinical and epidemiological characteristics of the

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1 Tendeiro et al. Supplemental Digital Content 1_ Page 2 of 1 Supplemental Table 1. Clinical and epidemiological characteristics of the therapy naïve and treated infected individuals. a Number [male/female] 16 [6/1] 28 [9/19] 1 [5/5] ge, years 44 (27-57) 54 (19-78) 57 (34-62)** Caucasian/black 15/1 15/13 6/4 CD4 + T-cells, % 59 (4-77) 4 (1-66) ** 26 (4-4) ***/## CD4 + T-cells/μl 818 ( ) 553 ( ) * 288 (84-554) ***/## Viremia, HIV RN cp/ml - 2 ( ) 2 ( ) Proviral DN, cp/1 6 PMC - 5 (5-133) 13 (5-726) -cells, % 6 (3-13) 6 (3-2) 1 (4-17) * -cells/μl 12 (63-369) 131 (3-369) 135 (63-313) Data are expressed as median, with limits in brackets. Statistical analysis was performed with Mann-Whitney tests. * P<.5; ** P<.1; *** P<.1 in comparison with seronegatives. ## P<.1 for comparisons between infected cohorts. a RT regimens were as follows (number of patients): 3TC+ZT+LPV/r (3); 3TC+d4T+SQV/r (2); 3TC/ZT+IDV/r (1); 3TC+ZT+IDV (1); C+3TC+ZT (3). Nucleoside analog reverse transcriptase inhibitors (NRTIs): ZT Zidovudine, 3TC- Lamivudine, C- abacavir, d4t- Stavudine, r- ribavirine. Protease inhibitors (PIs)- LPV- Lopinavir, IDV- Indinavir, SQV- Saquinavir.

2 Tendeiro et al. Supplemental Digital Content 1_ Page 3 of 1 Supplemental Table 2. Relationship between absolute counts of circulating -cell populations and markers of disease progression in the and cohorts. -cells/μl Total -cells/μl CD27 neg IgD + CD27 + CD27 + IgD + CD27 + IgD neg (n=38) CD4 + T-cells/µl.3522; ; ;<.1.783;< ;<.1 Viremia, HIV RN cp/ml -.754; ; ; ; ;.74 %HL-DR + CD38 + within CD4 a.153; ; ; ; ;.36 %HL-DR + CD38 + within CD8 a -.39; ; ; ; ;.93 β2-microglobulin (mg/l) b -.211; ; ;< ;< ;.2 (n=2) CD4 + T-cells/µl.2144; ; ; ; ;.362 Viremia, HIV RN cp/ml.98; ; ; ; ;.3914 %HL-DR + CD38 + within CD4 a -.256; ; ; ; ;.6932 %HL-DR + CD38 + within CD8 a -.669; ; ; ; ;.1736 β2-microglobulin (mg/l) b -.374; ; ; ; ;.9286 Spearman s correlation coefficient was used and results are expressed as R;P, with significant correlations in bold. a Frequency of cells co-expressing the activation markers HL-DR and CD38 within CD4 + and CD8 + T-cells (median, range) : 3.4%, % (CD4), and 15.1%, % (CD8); untreated : 5.5%, % (CD4), and 23.7%, % (CD8). No significant differences were found between infected cohorts, which exhibited significantly higher levels than seronegative controls (CD4: 1.1%,.7-2.%; CD8: 2.7%, %). b β2-microglobulin serum levels were assessed for 3 (median: 2.6 mg/l, range: mg/l) and 14 (median: 2.4 mg/l, range: mg/l) infected individuals, with no statistically significant differences being observed between cohorts.

3 Tendeiro et al. Supplemental Digital Content 1_ Page 4 of 1 Supplemental Table 3. Clinical and epidemiological characteristics of the and cohorts stratified according to degree of CD4 + T-cell depletion. >35 CD4 + /µl <35 CD4 + /µl >35 CD4 + /µl <35 CD4 + /µl Number [male/female] 16 [6/1] 22 [7/15] 16 [7/9] 1 [7/3] 1 [8/2] ge, years 44 (27-57) 56 (28-78) * 51 (19-63) 41 (27-61) 35 (23-49) # Caucasian/black 15/1 13/9 8/8 1/ 5/5 CD4 + T-cells, % 59 (4-77) 45 (22-66) **/ (4-32) *** 46 (17-74) * 14 (2-32) ***/+++ CD4 + T-cells/μl 818 ( ) 661 ( ) (52-344) *** 885 ( ) 298 (18-344) ***/+++ Viremia (HIV RN cp/ml) - 2 ( ) 2 ( ) 5x1 3 (4-19x1 3 ) 5x1 5 (71-447x1 3 ) ++/## Proviral DN (cp/1 6 PMC) - 78 (5-133) 57 (5-12) 81 (5-573) 41 (5-975) -cells, % 6 (3-13) 7 (3-17) 6 (3-2) 5 (2-11) 6 (2-16) -cells/μl 12 (63-369) 141 (52-357) 1 (3-369) 118 (56-46) 117 (33-267) Data are expressed as median, with limits in brackets. Statistical analysis was performed with Mann-Whitney tests. * P<.5; ** P<.1; *** P<.1 in comparison with seronegatives. ++ P<.1; +++ P<.1 in comparison with the reciprocal subgroup (>35 versus <35). # P<.5; ## P<.1 for comparisons between infected cohorts.

4 Tendeiro et al. Supplemental Digital Content 1_ Page 5 of 1 Supplemental Table 4. Clinical and epidemiological characteristics of the and cohorts stratified according to viremia status. Number [male/female] 16 [6/1] 28 [9/19] 1 [5/5] 3 [2/1] 17 [13/4] ge, years 44 (27-57) 56 (28-78) 52 (19-62) 47 (3-61) 38 (23-59) Caucasian/black 15/1 17/11 4/6 3/ 12/5 CD4 + T-cells, % 59 (4-77) 37 (13-66) ***/+ 24 (4-53) *** 6 (59-74) # 27 (2-56) ***/++ CD T-cells/μl ( ) ( ) */+ (84-596) *** ( ) ( ) ** Viremia (HIV RN cp/ml) - <2 7.6x1 3 ( ) <4 1.9x1 4 (71-447x1 3 ) Proviral DN (cp/1 6 PMC) - 5 (5-133) (5-12) 14 (5-324) 53 (5-975) -cells, % 6 (3-13) 7 (3-17) 7 (3-2) 1 (4-11) 6 (2-16) -cells/μl 12 (63-369) 135 (37-357) 131 (3-369) 125 (92-324) 111 (33-46) Data are expressed as median, with limits in brackets. Statistical analysis was performed with Mann-Whitney tests. * P<.5; ** P<.1; *** P<.1 in comparison with seronegatives. + P<.5; ++ P<.1 in comparison with the reciprocal subgroup ( versus ). # P<.5 for comparisons between infected cohorts.

5 CD27 + IgD + -cells/ l CD27 + IgD + -cells/ l CD27 + IgD neg -cells/ l CD27 + IgD neg -cells/ l CD27 + -cells/ l CD27 + -cells/ l Tendeiro et al. Supplemental Digital Content 1_ Page 6 of <.1 < <.1 < <.1 < Seroneg Seroneg >35 <35 Supplemental Figure 1. bsolute numbers of memory -cell subsets in infected individuals. () Circulating numbers of total memory cells (CD27 + ), switched memory cells (CD27 + IgD neg ) and unswitched memory cells (CD27 + IgD + ) in infected, as well as seronegative (Seroneg) and + individuals. () + patients were further subdivided according to disease stage (early: >35 CD4 + T-cells/µl; late: <35 CD4 + T-cells/µl) and levels of plasma viral load (aviremic: undetectable plasma viral load; viremic: detectable) and the absolute counts of memory cell populations are shown. Each dot represents one individual and bars indicate median. Statistical analysis was performed using the Mann-Whitney test and significant P values are shown.

6 Tendeiro et al. Supplemental Digital Content 1_ Page 7 of <.1 < % CD % CD27 + IgD neg < C <.1 < % CD27 + IgD + 3 < >35 <35 >35 <35 Supplemental Figure 2. -cell disturbances in as compared to groups of infected patients stratified to disease stage and viremia status. Patients were divided according to disease stage (early: >35 CD4 + T-cells/µl; late: <35 CD4 + T-cells/µl) and levels of plasma viral load (aviremic: undetectable plasma viral load; viremic: detectable). Relative frequencies of () total memory cells (CD27 + ), () switched memory cells (CD27 + IgD neg ) and (C) unswitched memory cells (CD27 + IgD + ) within total -cells. Comparisons were made between seronegatives and all groups of infected individuals. Each dot represents one individual and bars indicate median. Statistical analysis was performed using the Mann-Whitney test and significant P values are shown.

7 Tendeiro et al. Supplemental Digital Content 1_ Page 8 of 1 8 < % CD % CD27 + IgD neg 8 6 < % CD27 + IgD + C 4 < < < >35 <35 >35 <35 Supplemental Figure 3. -cell disturbances in treated as compared to untreated infected patients stratified to disease stage and viremia status. Patients were divided according to disease stage (early: >35 CD4 + T-cells/µl; late: <35 CD4 + T-cells/µl) and levels of plasma viral load (aviremic: undetectable plasma viral load; viremic: detectable). Relative frequencies of () total memory cells (CD27 + ), () switched memory cells (CD27 + IgD neg ) and (C) unswitched memory cells (CD27 + IgD + ) within total -cells. Comparisons were made between seronegatives and all groups of infected individuals. Each dot represents one individual and bars indicate median. Statistical analysis was performed using the Mann-Whitney test and significant P values are shown.

8 rgp36 (S/CO) rpc2-c3 (S/CO) IgG (mg/dl) IgM (mg/dl) Ig (mg/dl) Tendeiro et al. Supplemental Digital Content 1_ Page 9 of Untreated Untreated Untreated Supplemental Figure 4. Serum levels of immunoglobulin and of specific antibodies against envelope glycoproteins gp125 and gp36. () Serum levels of total IgG, total IgM, and total Ig in untreated HIV+ and in RT-treated + patients. () Serum levels of specific antibodies against gp36 and gp125 expressed as OD clinical sample /OD cut-off (S/CO) ratios in + individuals. Each dot represents one individual. Patients with viremia below cut-off are shown as open dots and viremic patients as filled dots. Normal range of immunoglobulin values is represented by grey shading. Statistical analysis was performed using the Mann-Whitney test and significant P values are shown. ars represent median.

9 FF (pg/ml) FF (pg/ml) Tendeiro et al. Supplemental Digital Content 1_ Page 1 of >35 <35 >35 < >35 <35 >35 <35 Supplemental Figure 5. Serum FF levels in HIV infected individuals stratified to disease stage and viremia status. Patients were divided according to disease stage (early: >35 CD4 + T- cells/µl; late: <35 CD4 + T-cells/µl) and levels of plasma viral load (aviremic: undetectable plasma viral load; viremic: detectable). Comparisons were made between seronegatives and () groups of and infected individuals; () groups of untreated and treated infected individuals. Each dot represents one individual and bars indicate median. Statistical analysis was performed using the Mann-Whitney test and significant P values are shown.