N the Chronic Phase of Hiv Infection Cd4 Tcell Counts Decrease Continually
Abstract
We compared the number/percentages of naive and memory CD4+ T-cells, CD38+CD8+ T-cells, and CD28+CD4+ and CD28+CD8+ T-cells in patients with advanced HIV disease (baseline CD4+ count<100) with those with less advanced (baseline CD4+ cell count>100) HIV disease during 4 years of suppressive highly active antiretroviral therapy. This prospective, longitudinal study included 30 treatment-naive patients and 32 controls. Advanced HIV-infected patients (n=13) gained more CD4+ T-cells than less advanced patients (n=11) at 1 month (median: 60 vs. 36μL−1), 3 months (86 vs. 14), 6 months (111 vs. 23), 12 months (174 vs. 47), 24 months (162 vs. 72) and 48 months (257 vs. 123) (P=0.15, P<0.001, P=0.026, P=0.021, P=0.1 and P=0.06, respectively). Advanced patients gained more naive CD4+ T-cells at 48 months compared to less advanced patients (27.3 vs. 11.4%, P=0.05). The relative gain in memory CD4+ T-cells was greater in advanced vs. less advanced patients at 1 month (median: 6.4 vs. 1.4%), 3 months (4.3 vs. 2.0), 6 months (6.7 vs. 1.6), 12 months (6.9 vs. 2.4), 24 months (7.5 vs. 3.1) and 48 months (11.3 vs. 6.8) (P=0.002, P=0.013, P<0.001, P=0.004, P=0.001 and P=0.015, respectively). At 48 months, CD38+CD8+ T-cells and naive CD4+ T-cells reached normal values (9.2%, P=0.869 vs. controls and 47.5%, P=0.699, respectively) in less advanced patients, as did CD38+CD8+ T-cells in advanced patients (4.7%, P=0.309 vs. controls). The kinetics of naive and memory CD4+ T-cell reconstitution is different in less advanced compared to advanced HIV patients.
Introduction
Human immunodeficiency virus type 1 (HIV-1) infection induces a progressive loss in the number of CD4+ T-cells, which is parallel to the development of severe immunodeficiency, increased susceptibility to opportunistic infection, and subsequently death. The ability of highly active antiretroviral therapy (HAART) to reconstitute the immune system of HIV-infected patients is usually estimated by monitoring the increase in the number of CD4+ T-cells during viral suppression ( Weis et al., 2001)
It has been hypothesised that in HIV-infected patients with undetectable viremia the number of CD4+ T-cells will increase for a certain period of time before reaching a plateau. This concept has been supported by several studies which demonstrated a significantly slower increase in the number of CD4+ T-cells after the first or second year of treatment in virological responders ( Kaufmann et al., 2002; Valdez et al., 2002; Smith et al., 2003). Contrary to these findings, [Hunt (2003)] have recently provided evidence that CD4+ T-cell counts continue to rise after 4 years in people on continuously successful HAART, regardless of the baseline CD4+ T-cell value.
The complete assessment of the quality of immune reconstitution during HAART does not only depend on the increase in the number of CD4+ T-cells, but also includes monitoring the correction in virus-mediated changes in T-cell phenotype and function ( Autran et al., 1997; Li et al., 1998; Carcelain et al., 1999; Al-Harthi et al., 2000; Bart et al., 2000; Plana et al., 2000; Martin et al., 2001; Munoz-Calleja et al., 2001).
The aim of this study was to compare the effect of 4 years of undetectable plasma viremia on the percentage of naive CD45RA+CD62L+ and memory CD45RO+ CD4+ T-cells, activated CD38+CD8+ T-cells, and the expression of the costimulatory molecule CD28 on CD4+ and CD8+ T-cells in the peripheral blood of HIV-infected adults. We compared the kinetics of selected immune parameters between HIV-infected persons who began HAART with less than 100 CD4+ T-cells (advanced) and those who began therapy with higher CD4+ T-cell counts (moderately advanced). We limited our analysis to patients who become undetectable after 6 months of HAART and maintained <50 copies per mL during the follow-up period.
Materials and methods
Study design and patients
This prospective, 4-year longitudinal study was conducted at the University Hospital for Infectious Diseases 'Dr. Fran Mihaljević', Zagreb, Croatia, between December of 1998 and November of 2002. We enrolled 30 treatment-naive HIV-1 infected adults. The HIV patients were treated with a combination of zidovudine or stavudine, lamivudine and indinavir at standard doses. Included in this analysis were 24 patients who became undetectable (less than 50 copies of HIV-1 RNA mL−1 of plasma) at 6 months and stayed so thereafter.
Immunological (flow cytometry) and virological analysis (quantitative reverse transcriptase polymerase chain reaction, or RT-PCR, for HIV-1 RNA) were done before and after 1, 3, 6, 12, 24 and 48 months of treatment. Plasma samples for HIV-1 RNA quantification and peripheral blood samples for flow cytometry were collected as a part of routine diagnostic follow-up.
The study also included 32 apparently healthy controls. Informed consent was obtained from all patients and healthy controls. The study excluded children (<17 years of age) and pregnant women. The study was approved by the Ethics Committee of the hospital.
Viral load
The quantification of HIV-1 RNA in the plasma of HIV-1 infected persons was performed by using the Cobas Amplicor™ HIV-1 monitor Test, version 1.5, UltraSensitive specimen preparation (Roche Diagnostic Systems Inc., Branchburg, NJ) with a lower limit of detection of 50 copies of HIV-1 RNA per mL.
Flow cytometry
To assess the quantity of T-cell immune reconstitution, we compared several lymphocyte subpopulations in HIV-1 infected patients before and during treatment to healthy controls. The percentage of CD28+CD4+ and CD28+CD8+ T-cells, naive CD45RA+CD62L+ and memory CD45RO+ CD4+ T-cells and activated CD38+CD8+ T-cells were determined by three-colour flow cytometry by using a panel of monoclonal antibodies specific for CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD56, HLA-DR, CD38, CD28, CD45RA, CD45RO and CD62L (DAKO, Glostrup, Denmark) conjugated with FITC, PE or RPE-Cy5. Absolute counts of CD4+ T-lymphocytes were determined directly on the cytometer by using flow-count fluorospheres and a four-colour flow cytometry panel CYTO-STAT tetra CHROME (CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5, Beckman Coulter Inc., FL) as recommended (Centers for Disease Control, 2003).
Peripheral blood samples were prepared for flow cytometry by using a whole-blood nonwash method on Multi-Q-Prep System with ImmunoPrep Reagent System (Beckman Coulter Inc.). The samples were analysed on an Epics XL-MCL flow cytometer (Beckman Coulter, Inc.).
Statistical analysis
The changes in the immunological parameters from the baseline throughout the follow-up period were analysed by a Wilcoxon signed rank test (P<0.05 was considered significant). The comparison between the two independent groups was done by a Mann–Whitney test. All reported P-values are two-sided and exact. A generalized additive model was fitted and the trajectories of the T-cell counts were described by drawing a smooth curve using robust local regression ( Cleveland et al., 1998). Statistical analysis was performed with SAS software version 8.2 (SAS Institute Inc., 2001) and StatXact release 5.0.3. (Cytel Software Corporation, Cambridge, MA). The loess option of PROC GAM was used to plot T-cells over time (SAS Institute Inc., 2001).
Results
Patients
Out of the 30 patients enrolled, 24 met the inclusion criteria related to the undetectable viral load within 6 months of starting their first HAART regimen and completing all follow-ups. The pretreatment viremia in the 24 patients included in this analysis was 5.3 log10 copies of HIV-1 RNA per mL (range 3.4–6.7 log10 copies of HIV-1 RNA per mL). After 3 months of HAART, plasma viremia was undetectable in 14 patients. Ten out of 24 patients had detectable plasma viremia after 3 months of treatment (median 3.6 log10 copies of HIV-1 RNA per mL, range 3.0–4.8 log10 copies of HIV-1 RNA per mL) but subsequently achieved undetectable plasma viremia after 6 months of HAART.
The patients were divided into two groups, based on their initial CD4+ T-cell counts (< or >100 cells µL−1). HIV-infected patients with<100 CD4+ T-cells µL−1 before treatment (n=13) were considered advanced (Table 1). At baseline, median percentages of naive and memory CD4+ T-cells in advanced patients were 0.9% and 2.1%, respectively. Patients with initial CD4+ T-cell counts >100 cells per µL (n=11) were classified as moderately advanced. Median baseline percentages of naive and memory CD4+ T-cells in moderately advanced patients were 32.6% and 10.1%, respectively.
Table 1
Baseline demographic and immunological parameters of the study population
| Parameter | Healthy controls | Advanced HIV-patients (baseline CD4+ T-cell count <100 cells per µL) | Moderately advanced HIV-patients (baseline CD4+ T-cell count >100 cells per µL) |
| Number | 24 | 13 | 11 |
| Age (years) | 37.5 (31–44) | 36 (34–42) | 41 (28–44) |
| Sex ratio (male/female) | 20/4 | 10/3 | 10/1 |
| CD4+ T-cells per µL | 936.5 (785.5–1123) | 23 (4–55) | 189 (164–225) |
| CD4+ T-cells (%) | 47.3 (42.8–49.7) | 2.6 (0.7–6.1) | 15,6 (11.8–19.7) |
| CD45RA+CD62L+ CD4+ T-cells (%) | 47.5 (41.6–52.3) | 0.9 (0–15) | 32.6 (21.2–48.7) |
| CD45RO+CD4+ T-cells (%) | 23.3 (21.3–25.9) | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) |
| CD8+ T-cells (%) | 21.6 (16.7–25.8) | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) |
| CD38+CD8+ T-cells (%) | 9.2 (7.0–11.0) | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) |
| CD28+CD4+ T-cells (% of total CD4+ T-cells) | 95.6 (93.5–97.1) | 60.2 (56.0–75.0) | 59,1 (45.1–85.9) |
| CD28+CD8+ T-cells (% of total CD8+ T-cells) | 77.0 (75.0–85.6) | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) |
| Parameter | Healthy controls | Advanced HIV-patients (baseline CD4+ T-cell count <100 cells per µL) | Moderately advanced HIV-patients (baseline CD4+ T-cell count >100 cells per µL) |
| Number | 24 | 13 | 11 |
| Age (years) | 37.5 (31–44) | 36 (34–42) | 41 (28–44) |
| Sex ratio (male/female) | 20/4 | 10/3 | 10/1 |
| CD4+ T-cells per µL | 936.5 (785.5–1123) | 23 (4–55) | 189 (164–225) |
| CD4+ T-cells (%) | 47.3 (42.8–49.7) | 2.6 (0.7–6.1) | 15,6 (11.8–19.7) |
| CD45RA+CD62L+ CD4+ T-cells (%) | 47.5 (41.6–52.3) | 0.9 (0–15) | 32.6 (21.2–48.7) |
| CD45RO+CD4+ T-cells (%) | 23.3 (21.3–25.9) | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) |
| CD8+ T-cells (%) | 21.6 (16.7–25.8) | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) |
| CD38+CD8+ T-cells (%) | 9.2 (7.0–11.0) | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) |
| CD28+CD4+ T-cells (% of total CD4+ T-cells) | 95.6 (93.5–97.1) | 60.2 (56.0–75.0) | 59,1 (45.1–85.9) |
| CD28+CD8+ T-cells (% of total CD8+ T-cells) | 77.0 (75.0–85.6) | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) |
Values expressed as median. Values in parentheses are interquartile range.
Table 1
Baseline demographic and immunological parameters of the study population
| Parameter | Healthy controls | Advanced HIV-patients (baseline CD4+ T-cell count <100 cells per µL) | Moderately advanced HIV-patients (baseline CD4+ T-cell count >100 cells per µL) |
| Number | 24 | 13 | 11 |
| Age (years) | 37.5 (31–44) | 36 (34–42) | 41 (28–44) |
| Sex ratio (male/female) | 20/4 | 10/3 | 10/1 |
| CD4+ T-cells per µL | 936.5 (785.5–1123) | 23 (4–55) | 189 (164–225) |
| CD4+ T-cells (%) | 47.3 (42.8–49.7) | 2.6 (0.7–6.1) | 15,6 (11.8–19.7) |
| CD45RA+CD62L+ CD4+ T-cells (%) | 47.5 (41.6–52.3) | 0.9 (0–15) | 32.6 (21.2–48.7) |
| CD45RO+CD4+ T-cells (%) | 23.3 (21.3–25.9) | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) |
| CD8+ T-cells (%) | 21.6 (16.7–25.8) | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) |
| CD38+CD8+ T-cells (%) | 9.2 (7.0–11.0) | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) |
| CD28+CD4+ T-cells (% of total CD4+ T-cells) | 95.6 (93.5–97.1) | 60.2 (56.0–75.0) | 59,1 (45.1–85.9) |
| CD28+CD8+ T-cells (% of total CD8+ T-cells) | 77.0 (75.0–85.6) | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) |
| Parameter | Healthy controls | Advanced HIV-patients (baseline CD4+ T-cell count <100 cells per µL) | Moderately advanced HIV-patients (baseline CD4+ T-cell count >100 cells per µL) |
| Number | 24 | 13 | 11 |
| Age (years) | 37.5 (31–44) | 36 (34–42) | 41 (28–44) |
| Sex ratio (male/female) | 20/4 | 10/3 | 10/1 |
| CD4+ T-cells per µL | 936.5 (785.5–1123) | 23 (4–55) | 189 (164–225) |
| CD4+ T-cells (%) | 47.3 (42.8–49.7) | 2.6 (0.7–6.1) | 15,6 (11.8–19.7) |
| CD45RA+CD62L+ CD4+ T-cells (%) | 47.5 (41.6–52.3) | 0.9 (0–15) | 32.6 (21.2–48.7) |
| CD45RO+CD4+ T-cells (%) | 23.3 (21.3–25.9) | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) |
| CD8+ T-cells (%) | 21.6 (16.7–25.8) | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) |
| CD38+CD8+ T-cells (%) | 9.2 (7.0–11.0) | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) |
| CD28+CD4+ T-cells (% of total CD4+ T-cells) | 95.6 (93.5–97.1) | 60.2 (56.0–75.0) | 59,1 (45.1–85.9) |
| CD28+CD8+ T-cells (% of total CD8+ T-cells) | 77.0 (75.0–85.6) | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) |
Values expressed as median. Values in parentheses are interquartile range.
Absolute counts of CD4+ T-cells
Absolute counts of CD4+ T-cells in advanced HIV-infected patients increased from a median of 23 cells µL−1 (interquartile range, hereafter IQR, 4–55) before treatment to a median of 298 cells µL−1 (IQR 188–322) after 4 years of HAART (Table 2). Following an initial rapid increase in CD4+ T-cell counts in the first 3 months of treatment, there continued to be a less marked but sustained increase in median CD4+ T-cell count during the remainder of the first year. There were statistically significant increases in the CD4+ T-cell counts at the end of the first and fourth years of treatment.
Table 2
Number of CD4+ T-cells and percentages of CD8+ and CD38+ CD8+ T-cells in uninfected healthy controls and in advanced and moderately advanced HIV infected patients before and after 1, 3, 6, 12, 24 and 48 months of highly active antiretroviral therapy
| Advanced patients (N=13) | Moderately advanced patients (N=11) | |||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P |
| CD4+ T-cells (cells per µL) | ||||
| 0 | 23 (4–55) | 189 (164–225) | ||
| 1 | 72 (59–205) | 213 (200–256) | 0.15 | |
| 3 | 123 (82–201) | 216 (171–241) | <0.001 | |
| 6 | 125 (107–205) | 221 (200–299) | 0.026 | |
| 12 | 215 (104–235) | 242 (211–336) | 0.021 | |
| 24 | 237 (115–262) | 301 (326–387) | 0.1 | |
| 48 | 298 (188–322) | 314 (260–495) | 0.06 | |
| Controls | 936.5 (785.5–1123) | <0.0001 | ||
| CD8+ T-cells % | ||||
| 0 | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) | ||
| 1 | 48.7 (42.3–60.6) | 56.5 (43.6–59.7) | NS | |
| 3 | 50.4 (45.7–60.1) | 54.6 (42.9–60.4) | NS | |
| 6 | 54.4 (44.7–59.4) | 53.2 (45.5–61.3) | NS | |
| 12 | 55.2 (44.7–58.2) | 51.3 (46.9–60.1) | NS | |
| 24 | 51.2 (45.1–55.2) | 50.9 (48.3–55.6) | NS | |
| 48 | 51.0 (44.9–56.7) | 48.1 (43.1–50.5) | NS | |
| Controls | 21.6 (16.7–25.8) | <0.0001 | ||
| CD38+CD8+ T-cells % | ||||
| 0 | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) | ||
| 1 | 28.0 (21.4–36.9) | 32.2 (29.1–36.1) | NS | |
| 3 | 29.8 (17.0–32.0) | 21.9 (20.0–28.3) | NS | |
| 6 | 21.0 (18.5–25.3) | 19.5 (17.6–23.2) | NS | |
| 12 | 17.0 (14.5–22.2) | 17.3 (13.2–19.0) | NS | |
| 24 | 10.1 (8.6–16.9) | 11.0 (8.9–14.6) | NS | |
| 48 | 4.7 (3.1–11.7) | 9.2 (6.5–12.6) | NS | |
| Controls | 9.2 (7.0–11.0) | 0.44 (0.42–0.45) | ||
| Advanced patients (N=13) | Moderately advanced patients (N=11) | |||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P |
| CD4+ T-cells (cells per µL) | ||||
| 0 | 23 (4–55) | 189 (164–225) | ||
| 1 | 72 (59–205) | 213 (200–256) | 0.15 | |
| 3 | 123 (82–201) | 216 (171–241) | <0.001 | |
| 6 | 125 (107–205) | 221 (200–299) | 0.026 | |
| 12 | 215 (104–235) | 242 (211–336) | 0.021 | |
| 24 | 237 (115–262) | 301 (326–387) | 0.1 | |
| 48 | 298 (188–322) | 314 (260–495) | 0.06 | |
| Controls | 936.5 (785.5–1123) | <0.0001 | ||
| CD8+ T-cells % | ||||
| 0 | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) | ||
| 1 | 48.7 (42.3–60.6) | 56.5 (43.6–59.7) | NS | |
| 3 | 50.4 (45.7–60.1) | 54.6 (42.9–60.4) | NS | |
| 6 | 54.4 (44.7–59.4) | 53.2 (45.5–61.3) | NS | |
| 12 | 55.2 (44.7–58.2) | 51.3 (46.9–60.1) | NS | |
| 24 | 51.2 (45.1–55.2) | 50.9 (48.3–55.6) | NS | |
| 48 | 51.0 (44.9–56.7) | 48.1 (43.1–50.5) | NS | |
| Controls | 21.6 (16.7–25.8) | <0.0001 | ||
| CD38+CD8+ T-cells % | ||||
| 0 | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) | ||
| 1 | 28.0 (21.4–36.9) | 32.2 (29.1–36.1) | NS | |
| 3 | 29.8 (17.0–32.0) | 21.9 (20.0–28.3) | NS | |
| 6 | 21.0 (18.5–25.3) | 19.5 (17.6–23.2) | NS | |
| 12 | 17.0 (14.5–22.2) | 17.3 (13.2–19.0) | NS | |
| 24 | 10.1 (8.6–16.9) | 11.0 (8.9–14.6) | NS | |
| 48 | 4.7 (3.1–11.7) | 9.2 (6.5–12.6) | NS | |
| Controls | 9.2 (7.0–11.0) | 0.44 (0.42–0.45) | ||
Statistically significant increase from the previous measurement are marked.
P<0.05;
P<0.01;
P<0.001; Wicoxon signed ranked test.
Between group comparisons of the difference of values at particular time points and the baseline value.
Comparison between values after 48 months of treatment in moderately and advanced HIV-infected patients and healthy controls using the Kruskal–Wallis test with Monte Carlo estimations of exact P-values and their 99% CI.
IQR, interquartile range; NS, not significant.
Table 2
Number of CD4+ T-cells and percentages of CD8+ and CD38+ CD8+ T-cells in uninfected healthy controls and in advanced and moderately advanced HIV infected patients before and after 1, 3, 6, 12, 24 and 48 months of highly active antiretroviral therapy
| Advanced patients (N=13) | Moderately advanced patients (N=11) | |||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P |
| CD4+ T-cells (cells per µL) | ||||
| 0 | 23 (4–55) | 189 (164–225) | ||
| 1 | 72 (59–205) | 213 (200–256) | 0.15 | |
| 3 | 123 (82–201) | 216 (171–241) | <0.001 | |
| 6 | 125 (107–205) | 221 (200–299) | 0.026 | |
| 12 | 215 (104–235) | 242 (211–336) | 0.021 | |
| 24 | 237 (115–262) | 301 (326–387) | 0.1 | |
| 48 | 298 (188–322) | 314 (260–495) | 0.06 | |
| Controls | 936.5 (785.5–1123) | <0.0001 | ||
| CD8+ T-cells % | ||||
| 0 | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) | ||
| 1 | 48.7 (42.3–60.6) | 56.5 (43.6–59.7) | NS | |
| 3 | 50.4 (45.7–60.1) | 54.6 (42.9–60.4) | NS | |
| 6 | 54.4 (44.7–59.4) | 53.2 (45.5–61.3) | NS | |
| 12 | 55.2 (44.7–58.2) | 51.3 (46.9–60.1) | NS | |
| 24 | 51.2 (45.1–55.2) | 50.9 (48.3–55.6) | NS | |
| 48 | 51.0 (44.9–56.7) | 48.1 (43.1–50.5) | NS | |
| Controls | 21.6 (16.7–25.8) | <0.0001 | ||
| CD38+CD8+ T-cells % | ||||
| 0 | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) | ||
| 1 | 28.0 (21.4–36.9) | 32.2 (29.1–36.1) | NS | |
| 3 | 29.8 (17.0–32.0) | 21.9 (20.0–28.3) | NS | |
| 6 | 21.0 (18.5–25.3) | 19.5 (17.6–23.2) | NS | |
| 12 | 17.0 (14.5–22.2) | 17.3 (13.2–19.0) | NS | |
| 24 | 10.1 (8.6–16.9) | 11.0 (8.9–14.6) | NS | |
| 48 | 4.7 (3.1–11.7) | 9.2 (6.5–12.6) | NS | |
| Controls | 9.2 (7.0–11.0) | 0.44 (0.42–0.45) | ||
| Advanced patients (N=13) | Moderately advanced patients (N=11) | |||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P |
| CD4+ T-cells (cells per µL) | ||||
| 0 | 23 (4–55) | 189 (164–225) | ||
| 1 | 72 (59–205) | 213 (200–256) | 0.15 | |
| 3 | 123 (82–201) | 216 (171–241) | <0.001 | |
| 6 | 125 (107–205) | 221 (200–299) | 0.026 | |
| 12 | 215 (104–235) | 242 (211–336) | 0.021 | |
| 24 | 237 (115–262) | 301 (326–387) | 0.1 | |
| 48 | 298 (188–322) | 314 (260–495) | 0.06 | |
| Controls | 936.5 (785.5–1123) | <0.0001 | ||
| CD8+ T-cells % | ||||
| 0 | 57.1 (43.5–63.0) | 58.0 (44.5–63.7) | ||
| 1 | 48.7 (42.3–60.6) | 56.5 (43.6–59.7) | NS | |
| 3 | 50.4 (45.7–60.1) | 54.6 (42.9–60.4) | NS | |
| 6 | 54.4 (44.7–59.4) | 53.2 (45.5–61.3) | NS | |
| 12 | 55.2 (44.7–58.2) | 51.3 (46.9–60.1) | NS | |
| 24 | 51.2 (45.1–55.2) | 50.9 (48.3–55.6) | NS | |
| 48 | 51.0 (44.9–56.7) | 48.1 (43.1–50.5) | NS | |
| Controls | 21.6 (16.7–25.8) | <0.0001 | ||
| CD38+CD8+ T-cells % | ||||
| 0 | 50.0 (45.3–51.5) | 47.7 (40.2–51.0) | ||
| 1 | 28.0 (21.4–36.9) | 32.2 (29.1–36.1) | NS | |
| 3 | 29.8 (17.0–32.0) | 21.9 (20.0–28.3) | NS | |
| 6 | 21.0 (18.5–25.3) | 19.5 (17.6–23.2) | NS | |
| 12 | 17.0 (14.5–22.2) | 17.3 (13.2–19.0) | NS | |
| 24 | 10.1 (8.6–16.9) | 11.0 (8.9–14.6) | NS | |
| 48 | 4.7 (3.1–11.7) | 9.2 (6.5–12.6) | NS | |
| Controls | 9.2 (7.0–11.0) | 0.44 (0.42–0.45) | ||
Statistically significant increase from the previous measurement are marked.
P<0.05;
P<0.01;
P<0.001; Wicoxon signed ranked test.
Between group comparisons of the difference of values at particular time points and the baseline value.
Comparison between values after 48 months of treatment in moderately and advanced HIV-infected patients and healthy controls using the Kruskal–Wallis test with Monte Carlo estimations of exact P-values and their 99% CI.
IQR, interquartile range; NS, not significant.
In less advanced patients, CD4+ T-cell counts increased from a median of 189 cells µL−1 (IQR 164–225) before treatment to 341 cells µL−1 (IQR 260–495) after 4 years of treatment (Table 2). The changes in CD4+ T-cell counts in moderately advanced patients were more gradual compared with those in advanced patients.
Advanced HIV-infected patients gained more CD4+ T-cells than less advanced patients at 1 month (median: 60 vs. 36 cells µL−1), 3 months (86 vs. 14 cells µL−1), 6 months (111 vs. 23 cells µL−1), 12 months (174 vs. 47 cells µL−1), 24 months (162 vs. 72 cells µL−1) and 48 months (257 vs. 123 cells µL−1) (P=0.15, P<0.001, P=0.026, P=0.021, P=0.1 and P=0.06, respectively; Table 2, Fig. 1).
Figure 1
Loess estimate differences of CD4+ T-cell counts.
Naive CD4+ T-cells
Percentages of naive CD4+ T-cells increased in advanced patients increased from baseline (median 0.9%, IQR 0–15%) to a median of 40.2% (IQR 37.0–45.6%) after 48 months of treatment (Table 3). There was an early and statistically significant increase in naive CD4+ T-cells within 3 months of HAART followed by the period of little change until the end of the first year. Significant changes in naive CD4+ T-cells were also observed at the end of the second year of treatment but with little change thereafter.
Table 3
Percentages of CD28+ CD4+, CD28+ CD8+ T, CD45RO+CD4+ and CD45RA+CD62L+ T cells in uninfected healthy controls and, advanced and moderately advanced HIV infected patients before and after 1, 3, 6, 12, 24 and 48 months of highly active antiretroviral therapy
| Advanced patients (N=13) | Moderately advanced patients (N=11) | ||||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P | |
| CD28+CD4+ T-cells | |||||
| 0 | 60.2 (56.0–75.0) | 59.1 (45.1–85.9) | |||
| 1 | 74.0 (58.9–86.7) | 70.1 (60.2–85.6) | NS | ||
| 3 | 76.2 (60.1–85.0) | 73.4 (64.5–87.6) | NS | ||
| 6 | 77.7 (60.1–88.8) | 79.7 (60.3–82.3) | NS | ||
| 12 | 76.6 (64.5–81.3) | 85.2 (74.5–89.7) | NS | ||
| 24 | 83.2 (70.9–90.1) | 88.1 (77.8–89.6) | NS | ||
| 48 | 90.1 (80.9–93.6) | 86.9 (80.5–91.8) | NS | ||
| Controls | 95.6 (93.5–97.1) | <0.0001 | |||
| CD28+CD8+ T-cells | |||||
| 0 | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) | |||
| 1 | 40.6 (35.0–52.1) | 42.4 (30.1–52.3) | NS | ||
| 3 | 44.3 (37.7–47.8) | 43.6 (30.1–52.3) | NS | ||
| 6 | 45.4 (39.5–51.0) | 49.8 (39.1–60.1) | NS | ||
| 12 | 45.8 (41.4–88.1) | 57.6 (45.6–64.5) | NS | ||
| 24 | 59.6 (55.6–64.5) | 59.6 (55.6–65.6) | NS | ||
| 48 | 73.0 (70.6–75.6) | 69.5 (64.5–75.9) | NS | ||
| Controls | 77.0 (75.0-86.0) | <0.0001 | |||
| CD45RO+CD4+ T-cells | |||||
| 0 | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) | |||
| 1 | 9.5 (5.2–11.5) | 10.6 (8.1–15.2) | 0.002 | ||
| 3 | 8.3 (5.2–13.2) | 12.3 (7.9–15.3) | 0.013 | ||
| 6 | 9.1 (6.9–12.5) | 12.5 (7.6–14.0) | <0.001 | ||
| 12 | 10.2 (3.3–12.9) | 13.2 (9.5–13.6) | 0.004 | ||
| 24 | 10.4 (9.6–11.2) | 13.6 (11.0–14.5) | 0.001 | ||
| 48 | 15.6 (12.0–20.5) | 17.8 (16.5–20.1) | 0.015 | ||
| Controls | 23.3 (21.3–26.0) | <0.0001 | |||
| CD45RA+CD62L+ CD4+ T-cells | |||||
| 0 | 0.9 (0–15) | 32.6 (21.2–48.7) | |||
| 1 | 20.1 (18.5–20.6) | 35.2 (20.1–45.6) | NS | ||
| 3 | 22.3 (18.9–24.6) | 40.1 (22.6–50.2) | NS | ||
| 6 | 21,5 (19.2–24.6) | 38.0 (20.6–54.6) | NS | ||
| 12 | 25.9 (20.4–30.8) | 39.8 (22.3–54.2) | NS | ||
| 24 | 33.2 (27.7–36.8) | 40.5 (25.6–55.1) | NS | ||
| 48 | 40.2 (37.0–45.6) | 50.6 (38.9–56.9) | 0.05 | ||
| Controls | 47.5 (52.3–41.6) | 0.056 (0.05-0.06) | |||
| Advanced patients (N=13) | Moderately advanced patients (N=11) | ||||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P | |
| CD28+CD4+ T-cells | |||||
| 0 | 60.2 (56.0–75.0) | 59.1 (45.1–85.9) | |||
| 1 | 74.0 (58.9–86.7) | 70.1 (60.2–85.6) | NS | ||
| 3 | 76.2 (60.1–85.0) | 73.4 (64.5–87.6) | NS | ||
| 6 | 77.7 (60.1–88.8) | 79.7 (60.3–82.3) | NS | ||
| 12 | 76.6 (64.5–81.3) | 85.2 (74.5–89.7) | NS | ||
| 24 | 83.2 (70.9–90.1) | 88.1 (77.8–89.6) | NS | ||
| 48 | 90.1 (80.9–93.6) | 86.9 (80.5–91.8) | NS | ||
| Controls | 95.6 (93.5–97.1) | <0.0001 | |||
| CD28+CD8+ T-cells | |||||
| 0 | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) | |||
| 1 | 40.6 (35.0–52.1) | 42.4 (30.1–52.3) | NS | ||
| 3 | 44.3 (37.7–47.8) | 43.6 (30.1–52.3) | NS | ||
| 6 | 45.4 (39.5–51.0) | 49.8 (39.1–60.1) | NS | ||
| 12 | 45.8 (41.4–88.1) | 57.6 (45.6–64.5) | NS | ||
| 24 | 59.6 (55.6–64.5) | 59.6 (55.6–65.6) | NS | ||
| 48 | 73.0 (70.6–75.6) | 69.5 (64.5–75.9) | NS | ||
| Controls | 77.0 (75.0-86.0) | <0.0001 | |||
| CD45RO+CD4+ T-cells | |||||
| 0 | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) | |||
| 1 | 9.5 (5.2–11.5) | 10.6 (8.1–15.2) | 0.002 | ||
| 3 | 8.3 (5.2–13.2) | 12.3 (7.9–15.3) | 0.013 | ||
| 6 | 9.1 (6.9–12.5) | 12.5 (7.6–14.0) | <0.001 | ||
| 12 | 10.2 (3.3–12.9) | 13.2 (9.5–13.6) | 0.004 | ||
| 24 | 10.4 (9.6–11.2) | 13.6 (11.0–14.5) | 0.001 | ||
| 48 | 15.6 (12.0–20.5) | 17.8 (16.5–20.1) | 0.015 | ||
| Controls | 23.3 (21.3–26.0) | <0.0001 | |||
| CD45RA+CD62L+ CD4+ T-cells | |||||
| 0 | 0.9 (0–15) | 32.6 (21.2–48.7) | |||
| 1 | 20.1 (18.5–20.6) | 35.2 (20.1–45.6) | NS | ||
| 3 | 22.3 (18.9–24.6) | 40.1 (22.6–50.2) | NS | ||
| 6 | 21,5 (19.2–24.6) | 38.0 (20.6–54.6) | NS | ||
| 12 | 25.9 (20.4–30.8) | 39.8 (22.3–54.2) | NS | ||
| 24 | 33.2 (27.7–36.8) | 40.5 (25.6–55.1) | NS | ||
| 48 | 40.2 (37.0–45.6) | 50.6 (38.9–56.9) | 0.05 | ||
| Controls | 47.5 (52.3–41.6) | 0.056 (0.05-0.06) | |||
Statistically significant increase from the previous measurement are marked.
(P<0.05);
(P<0.01);
(P<0.001); Wicoxon signed ranked test.
Between group comparisons of the difference of values at particular time points and the baseline value.
Comparison between values after 48 months of treatment in moderately and advanced HIV infected patients and healthy controls using the Kruskal–Wallis test with Monte Carlo estimations of exact P-values and their 99% CI.
IQR, interquartile range; NS, not significant.
Table 3
Percentages of CD28+ CD4+, CD28+ CD8+ T, CD45RO+CD4+ and CD45RA+CD62L+ T cells in uninfected healthy controls and, advanced and moderately advanced HIV infected patients before and after 1, 3, 6, 12, 24 and 48 months of highly active antiretroviral therapy
| Advanced patients (N=13) | Moderately advanced patients (N=11) | ||||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P | |
| CD28+CD4+ T-cells | |||||
| 0 | 60.2 (56.0–75.0) | 59.1 (45.1–85.9) | |||
| 1 | 74.0 (58.9–86.7) | 70.1 (60.2–85.6) | NS | ||
| 3 | 76.2 (60.1–85.0) | 73.4 (64.5–87.6) | NS | ||
| 6 | 77.7 (60.1–88.8) | 79.7 (60.3–82.3) | NS | ||
| 12 | 76.6 (64.5–81.3) | 85.2 (74.5–89.7) | NS | ||
| 24 | 83.2 (70.9–90.1) | 88.1 (77.8–89.6) | NS | ||
| 48 | 90.1 (80.9–93.6) | 86.9 (80.5–91.8) | NS | ||
| Controls | 95.6 (93.5–97.1) | <0.0001 | |||
| CD28+CD8+ T-cells | |||||
| 0 | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) | |||
| 1 | 40.6 (35.0–52.1) | 42.4 (30.1–52.3) | NS | ||
| 3 | 44.3 (37.7–47.8) | 43.6 (30.1–52.3) | NS | ||
| 6 | 45.4 (39.5–51.0) | 49.8 (39.1–60.1) | NS | ||
| 12 | 45.8 (41.4–88.1) | 57.6 (45.6–64.5) | NS | ||
| 24 | 59.6 (55.6–64.5) | 59.6 (55.6–65.6) | NS | ||
| 48 | 73.0 (70.6–75.6) | 69.5 (64.5–75.9) | NS | ||
| Controls | 77.0 (75.0-86.0) | <0.0001 | |||
| CD45RO+CD4+ T-cells | |||||
| 0 | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) | |||
| 1 | 9.5 (5.2–11.5) | 10.6 (8.1–15.2) | 0.002 | ||
| 3 | 8.3 (5.2–13.2) | 12.3 (7.9–15.3) | 0.013 | ||
| 6 | 9.1 (6.9–12.5) | 12.5 (7.6–14.0) | <0.001 | ||
| 12 | 10.2 (3.3–12.9) | 13.2 (9.5–13.6) | 0.004 | ||
| 24 | 10.4 (9.6–11.2) | 13.6 (11.0–14.5) | 0.001 | ||
| 48 | 15.6 (12.0–20.5) | 17.8 (16.5–20.1) | 0.015 | ||
| Controls | 23.3 (21.3–26.0) | <0.0001 | |||
| CD45RA+CD62L+ CD4+ T-cells | |||||
| 0 | 0.9 (0–15) | 32.6 (21.2–48.7) | |||
| 1 | 20.1 (18.5–20.6) | 35.2 (20.1–45.6) | NS | ||
| 3 | 22.3 (18.9–24.6) | 40.1 (22.6–50.2) | NS | ||
| 6 | 21,5 (19.2–24.6) | 38.0 (20.6–54.6) | NS | ||
| 12 | 25.9 (20.4–30.8) | 39.8 (22.3–54.2) | NS | ||
| 24 | 33.2 (27.7–36.8) | 40.5 (25.6–55.1) | NS | ||
| 48 | 40.2 (37.0–45.6) | 50.6 (38.9–56.9) | 0.05 | ||
| Controls | 47.5 (52.3–41.6) | 0.056 (0.05-0.06) | |||
| Advanced patients (N=13) | Moderately advanced patients (N=11) | ||||
| Treatment time points (months) | Median (IQR) | Median (IQR) | P | P | |
| CD28+CD4+ T-cells | |||||
| 0 | 60.2 (56.0–75.0) | 59.1 (45.1–85.9) | |||
| 1 | 74.0 (58.9–86.7) | 70.1 (60.2–85.6) | NS | ||
| 3 | 76.2 (60.1–85.0) | 73.4 (64.5–87.6) | NS | ||
| 6 | 77.7 (60.1–88.8) | 79.7 (60.3–82.3) | NS | ||
| 12 | 76.6 (64.5–81.3) | 85.2 (74.5–89.7) | NS | ||
| 24 | 83.2 (70.9–90.1) | 88.1 (77.8–89.6) | NS | ||
| 48 | 90.1 (80.9–93.6) | 86.9 (80.5–91.8) | NS | ||
| Controls | 95.6 (93.5–97.1) | <0.0001 | |||
| CD28+CD8+ T-cells | |||||
| 0 | 39.6 (19.6–44.8) | 34.4 (25.1–50.1) | |||
| 1 | 40.6 (35.0–52.1) | 42.4 (30.1–52.3) | NS | ||
| 3 | 44.3 (37.7–47.8) | 43.6 (30.1–52.3) | NS | ||
| 6 | 45.4 (39.5–51.0) | 49.8 (39.1–60.1) | NS | ||
| 12 | 45.8 (41.4–88.1) | 57.6 (45.6–64.5) | NS | ||
| 24 | 59.6 (55.6–64.5) | 59.6 (55.6–65.6) | NS | ||
| 48 | 73.0 (70.6–75.6) | 69.5 (64.5–75.9) | NS | ||
| Controls | 77.0 (75.0-86.0) | <0.0001 | |||
| CD45RO+CD4+ T-cells | |||||
| 0 | 2.1 (5.0–6.9) | 10.1 (6.6–13.0) | |||
| 1 | 9.5 (5.2–11.5) | 10.6 (8.1–15.2) | 0.002 | ||
| 3 | 8.3 (5.2–13.2) | 12.3 (7.9–15.3) | 0.013 | ||
| 6 | 9.1 (6.9–12.5) | 12.5 (7.6–14.0) | <0.001 | ||
| 12 | 10.2 (3.3–12.9) | 13.2 (9.5–13.6) | 0.004 | ||
| 24 | 10.4 (9.6–11.2) | 13.6 (11.0–14.5) | 0.001 | ||
| 48 | 15.6 (12.0–20.5) | 17.8 (16.5–20.1) | 0.015 | ||
| Controls | 23.3 (21.3–26.0) | <0.0001 | |||
| CD45RA+CD62L+ CD4+ T-cells | |||||
| 0 | 0.9 (0–15) | 32.6 (21.2–48.7) | |||
| 1 | 20.1 (18.5–20.6) | 35.2 (20.1–45.6) | NS | ||
| 3 | 22.3 (18.9–24.6) | 40.1 (22.6–50.2) | NS | ||
| 6 | 21,5 (19.2–24.6) | 38.0 (20.6–54.6) | NS | ||
| 12 | 25.9 (20.4–30.8) | 39.8 (22.3–54.2) | NS | ||
| 24 | 33.2 (27.7–36.8) | 40.5 (25.6–55.1) | NS | ||
| 48 | 40.2 (37.0–45.6) | 50.6 (38.9–56.9) | 0.05 | ||
| Controls | 47.5 (52.3–41.6) | 0.056 (0.05-0.06) | |||
Statistically significant increase from the previous measurement are marked.
(P<0.05);
(P<0.01);
(P<0.001); Wicoxon signed ranked test.
Between group comparisons of the difference of values at particular time points and the baseline value.
Comparison between values after 48 months of treatment in moderately and advanced HIV infected patients and healthy controls using the Kruskal–Wallis test with Monte Carlo estimations of exact P-values and their 99% CI.
IQR, interquartile range; NS, not significant.
In less advanced patients, naive CD4+ T-cells increased from a median 32.6% (IQR 21.2–48.7%) at baseline to 50.6% (IQR 38.9–56.9%) after 48 months of HAART (Table 3). Unlike advanced HIV patients, there was no early (first 3 months) increase in naive CD4+ T-cells in moderately advanced patients. A significant increase in the percentage of naive CD4+ T-cells in moderately advanced patients was observed between the third and sixth months of treatment but we failed to observe any additional increase until the end of the first year. The percentage of naive CD4+ T-cells in moderately advanced patients increased significantly at the end of the second and third years of treatment. At 48 months, the percentages of naive CD4+ T-cells in less advanced patients (median 50.6%) reconstituted to the levels of healthy controls (median 47.5%, P=0.699).
Advanced patients gained relatively more naive CD4+ T-cells at 48 months compared to less advanced patients (27.3 vs. 11.4%, P=0.05; Fig. 2).
Figure 2
Loess estimates differences of the percentage of memory CD4+ T-cells.
Memory CD45RO+ T-cells
Suppressive HAART enabled an increase in memory CD4+ T-cells in advanced HIV-infected patients from baseline median 2.1% (IQR 5.0–6.9%) to a median of 15.6% (IQR 12.0–20.5%) after 48 months (Table 3). Following an initial rapid and significant increase within the first month of treatment, percentages of memory CD4+ T-cells slightly decreased during the next two months, with little change until the second significant increase observed at the end of the fourth year of HAART.
In less advanced patients, percentages of memory CD4+ T-cells increased from a baseline median 10.1% (IQR 6.6–13.0%) to a median 17.8% (IQR 16.5–20.1%) at the end of follow-up (Table 3). Similarly to advanced patients, an early and statistically significant increase in memory CD4+ T-cells occurred during the first month of HAART. There was a period of sustained but not significant increase in memory CD4+ T-cells during the remainder of the first year as well as during the second year of HAART followed by the significant increase at the end of the follow-up.
The relative gain in memory CD4+ T-cells was greater in advanced versus less advanced patients at 1 month (median: 6.4 vs. 1.4%), 3 months (4.3 vs. 2.0%), 6 months (6.7 vs. 1.6%), 12 months (6.9 vs. 2.4%), 24 months (7.5 vs. 3.1%) and 48 months (11.3 vs. 6.8%) (P=0.002, P=0.013, P<0.001, P=0.004, P=0.001 and P=0.015, respectively, Fig. 3).
Figure 3
Loess estimates differences of the percentage of naive CD4+ T-cells.
CD8+ T-cells
Four years of HAART had little influence on the percentages of CD8+ T-cells in both advanced and moderately advanced patients. Percentages of CD8+ T-cells in advanced HIV-infected patients slightly decreased from a median of 57.1% (IQR 43.5–63.0%) before treatment to a median of 51.0% (IQR 44.9–56.7%) after 4 years of treatment (Table 2). In less advanced HIV-infected patients, percentages of CD8+ T-cells also decreased from a pre-treatment median of 58.0 (IQR 44.5–63.7) to 48.1% (IQR 43.1–50.5%) after 48 months of HAART (Table 2).
Other lymphocyte subpopulations
There was no difference in the kinetics of CD38+CD8+ T-cells, CD28+CD4+ and CD28+CD8+ T-cells between advanced and less advanced patients during follow-up.
HAART-induced suppression of viral replication enabled a decrease in the percentage of activated CD38+CD8+ T-cells in advanced HIV-infected patients from a pre-treatment median of 50.0% (IQR 45.3–51.3%) to 4.7% (IQR 3.1–11.7%) after 48 months (Table 2). Percentages of CD38+CD8+ T-cells in less advanced patients decreased from baseline median of 47.7% (IQR 40.2–51.0%) to 9.2% (IQR 6.5–12.6%) (Table 2).
The most dramatic decrease in the percentage of CD38+CD8+ T-cells in both patient groups occurred within the first month of HAART. In moderately advanced patients, a statistically significant decrease in the percentages of these cells was observed during the first 6 months of treatment. The initial rapid decrease in the percentages of CD38+CD8+ T-cells was parallel to the treatment-induced reduction of plasma viremia to undetectable levels in 10 out of 24 patients after 3 months and all patients after 6 months of HAART (inclusion criteria). The percentages of CD38+CD8+ T-cells showed little change between the sixth month of HAART and the remainder of the first year. Interestingly, the percentages of CD38+CD8+ T-cells decreased even further at the end of the second and fourth year of suppressive HAART. At month 48, CD38+CD8+ T-cells reached normal values (median 9.2% for controls) in both advanced (4.7%, P=0.309 vs. controls) and less advanced patients (9.2, P=0.869 vs. controls).
The percentages of CD4+ T-cells expressing costimulatory molecule CD28 increased from the baseline median of 60.2% (IQR 56.0–75.0%) before treatment to a median of 90.1 (IQR 80.9–93.6%) after 48 months of HAART (Table 3). Antiretroviral treatment appeared to have little influence on the percentages of CD4+ T-cells expressing a costimulatory molecule CD28 during the first year in advanced patients. The majority of increase in the percentages of CD28+CD4+ T-cells in advanced patients occurred during the second and fourth year of treatment.
In less advanced patients, percentages of CD28+CD4+ T-cells increased from the pretreatment median of 59.1% (IQR 45.1–85.9%) to a median of 89.5% (IQR 80.5–91.8%) after 48 months of HAART (Table 3). Similarly to advanced patients, a period of little change during the first 6 months of treatment was followed by statistically significant increases in the percentage of these cells at the end of the first, second and fourth year of HAART.
The percentages of CD8+ T-cells expressing costimulatory molecule CD28 in advanced patients increased from the baseline median of 39.6% (IQR 19.6–44.8%) before treatment to a median of 73.0 (IQR 70.6–75.6%) after 48 months of HAART (Table 3). A statistically significant increase in the percentages of CD28+CD8+ T-cells was observed within the first month of treatment as well as at the end of the second and fourth year.
In less advanced patients, the percentages of CD28+CD8+ T-cells increased from the baseline median of 34.3 (IQR 25.1–50.1%) to a median of 69.5% (IQR 64.5–75.9%, Table 3). Similarly to advanced patients, the percentages of these cells significantly increase within the first month of treatment. After a 2-month period of little change, the percentages of CD28+CD8+ T-cells continued a statistically significant increase at all time intervals until the end of follow-up.
Discussion
Our results have shown that the kinetics of a HAART-induced increase in truly naive and memory subpopulations of CD4+ T-cells is different in moderately advanced compared to advanced HIV-infected patients. Advanced HIV-infected patients had a more pronounced gain in the percentage of memory CD4+ T-cells during the first and at the end of the second and fourth years of HAART compared to less advanced patients. In addition, the relative gain in naive CD4+ T-cells was also significantly higher in advanced compared to moderately advanced HIV-infected patients after 4 years of HAART. Four years of suppressive HAART enabled the complete reconstitution of naive CD4+ T-cells in less advanced but not in advanced patients. Additionally, the long-term control of viral replication enabled the decrease of activated CD38+CD8+ T-cells to the levels of healthy controls.
The reason for the observed difference in the gain of memory CD4+ T-cells in advanced vs. less advanced HIV-infected patients during suppressive HAART is not clear. A model of T-cell reconstitution as a composite of memory cell redistribution from lymphoid tissues to the periphery and a regeneration of naive T-cells from thymic origin provides a possible explanation for the observed difference (Autran, 1999). The kinetics of memory CD4+ T-cells observed in this study appeared to be triphasic: an early and rapid increase followed by the more gradual increase, and a subsequent significant increase at the end of follow-up. The initial increase in the percentage of these cells probably corresponds to the redistribution phase of memory T-cells from lymphoid tissues to the peripheral blood.
It is believed that the amount of memory T-cells 'entrapped' in the peripheral lymphoid tissues is much higher in patients with advanced HIV disease compared with asymptomatic patients or those with early HIV disease. Therefore, the number of memory T-cells migrating to the periphery upon HAART initiation is expected to be much higher in advanced patients and this could explain the observed difference in memory CD4+ T-cell gains between our two patient groups.
According to a different model of CD4+ T-cell depletion during HIV infection being a result of disturbed homeostasis, it is possible that the homeostatic pressure to compensate for the loss of this cellular subpopulation was more pronounced in patients with lower pre-treatment CD4+ T-cells count ( Grossman et al., 2002).
Naive CD4+ T-cells are often regarded as a measure of true immune reconstitution because they enable a diversification of the T-cell receptor repertoire in the infected host. Our study has shown a complete restoration of naive CD4+ T-cells after 4 years of successful virological control only in less advanced patients. Our study has also shown an early increase in the naive CD4+ T-cells of advanced patients, similar to that observed by Mezzaroma (1999). The importance of naive CD4+ T-cells in long-term immunological recovery during HAART was also confirmed by Notermans et al. showing that a long-term increase in total CD4+ T-cells was predicted using the baseline number of naive CD4+ T-cells ( Notermans et al., 1999).
Literature data on the gain in the number of CD4+ T-cells during suppressive HAART in longitudinal studies is quite variable ( Valdez et al., 2002; Hunt et al., 2003; Lederman et al., 2003; Smith et al., 2003; Viard et al., 2004). Lederman et al. monitored CD4+ T-cell increase in 643 HIV-infected patients during 144 weeks ( Lederman et al., 2003). According to their results, the number of CD4+ T-cells increased from the pretreatment median of 266 CD4+ T-cells per µL to a median of 358 cells per µL−1 at the end of follow-up, showing similar kinetics of CD4+ T-cell reconstitution to that observed in our study.
The ability of the immune system to continuously reconstitute CD4+ T-cells for longer periods of time is still a controversial issue. The results of several longitudinal studies suggested that the ability of the immune system to continuously reconstitute total CD4+ T-cells for more than one or two years was limited ( Kaufmann et al., 2002; Valdez et al., 2002; Smith et al., 2003). A recent study by Hunt (2003) showed that virological responders to HAART continue to increase their CD4+ T-cell count even after 4 years of treatment, regardless of the initial CD4+ T-cells count. However, this view was contradicted by Viard (2004) who failed to demonstrate any significant gain in CD4 T-cells by prolonging HAART for more than 3 years and questioned the value of life-long HAART.
The results of our study have show a continuous increase in the number of CD4+ T-cells between the second and fourth year of HAART in both advanced and moderately advanced HIV-infected patients. Unfortunately, we were not able to analyse the selected parameters after 3 years of HAART. Therefore, we do not know if there was a difference between the second and third or third and fourth years of HAART. Therefore, we can not claim that the immune reconstitution continues for 4 years without reaching a plateau.
The ability of HAART to correct virus-induced downregulation of a costimulatory molecule CD28 on CD4+ and CD8+ T-cells has been described previously ( Arno et al., 1998; Valdez et al., 2002). Arno (1998) have shown a normalisation in the expression of CD28 on CD8+ T-cells in nine HIV-1 infected asymptomatic persons treated with monotherapy or double antiretroviral therapy who maintained undetectable plasma viremia for 2 years. Valdez (2002) observed the complete reconstitution of CD28 on CD8+ T-cells but not on CD4+ T-cell in patients who began treatment in moderately advanced HIV disease after 3 years of HAART. However, our study has shown that 4 years of HAART were not sufficient to completely reconstitute the expression of CD28 on CD4+ and CD8+ T-cells in both less advanced and advanced HIV-infected patients.
Sousa et al. have proposed a concept concerning the role of immune hyperactivation as an inducer of CD4+ T-cell depletion in HIV disease. Therefore, a reduction in the expression of activation markers such as CD38 (on CD8+ T-cells) is considered to be an essential part of immune reconstitution. Valdez et al. have shown that 3 years of HAART did not completely abrogate abnormal CD8+ T-cell activation in moderately advanced HIV disease. However, our study has shown a complete normalisation of CD38 overexpression on CD8+ T-cell after 4 years of HAART in both moderately advanced and advanced HIV-infected persons.
Both groups of HIV-infected persons included in our study showed the highest reduction in the percentage of CD38+CD8+ T-cells within the first month of treatment, which is probably related to the marked decrease in the plasma viremia observed in that period of time. A HAART-mediated decrease in the expression of CD38 on CD8+ T-cells parallel to the reduction in the plasma viremia has been previously described ( Savarino et al., 2000). Additionally, both groups of HIV-infected persons included in the study (regardless of their initial CD4+ T-cell counts) demonstrated a significant decrease in the percentage of CD38+CD8+ T-cells after 2 and 4 years of HAART.
In summary, this study indicates that the kinetics of the reconstitution of CD4+ T-cells, naive and memory CD4+ T-cells induced by HAART might be different in patients with a more advanced HIV infection compared to those with a less advanced infection. The pattern was similar, however the magnitude of the response compared to the baseline value was different at various time points. There was no difference in the pattern or magnitude of CD38+CD8+, CD28+CD4+ or CD28+CD8+ T-cells between patients with fewer than 100 CD4+ T-cells compared to those with more than CD4+ T-cell per µL.
Acknowledgement
This study was supported by a grant from the Croatian Ministry of Science, Education and Sports to Dr J. Begovac (0108027).
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Author notes
Editor: Willem van Eden
© 2006 Federation of European Microbiological Societies
© 2006 Federation of European Microbiological Societies
Source: https://academic.oup.com/femspd/article/46/3/351/554901
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