Human immunodeficiency virus type 1 (HIV-1) infection induces a severe CD4+ T-cell (CD4+) depletion, alterations in T-cells subsets, and a progressive impairment of the immune system.1,2 HIV infection also induces an increase in T-cell activation-associated cell surface markers such as CD38 and HLA-DR.3-5 These markers change in parallel with the progression of the HIV disease.3
In the classic model of AIDS pathogenesis, HIV infection is associated with a high rate of virus turnover and a short life span of virus-infected cells, and HIV-mediated killing of CD4+ is the central event in AIDS pathogenesis.6,7 An alternative theory is that chronic immune activation after HIV infection drives the progressive decline in CD4+ numbers and progression to AIDS.8-10 Thus, CD4+ depletion is related to virus-mediated death of infected CD4+ and apoptosis of uninfected "bystander" T-cells by activation-induced cell death. In a simian immunodeficiency virus-infected monkey model, viral infection induces minimal levels of immune activation and bystander immunopathology. However, this does not result in CD4+ depletion despite persistently high levels of virus replication, and they did not develop AIDS.11
The aim of this study was to analyze the association between CD4+ depletion and immune activation in HIV-1-infected children on highly active antiretroviral therapy (HAART).
One hundred forty-three vertically HIV-infected children were studied in a cross-sectional study. Inclusion criteria were (a) children on HAART with a protease inhibitor (PI) or nonnucleoside analogue HIV-1 reverse transcriptase inhibitor (NNRTI), (b) HAART for at least 24 weeks before the study, and (c) ≥5 years old.
Drugs were prescribed by the attending pediatrician according to the Centers for Disease Control and Prevention (CDC) guidelines.12 The study was conducted according to the Declaration of Helsinki and approved by the Ethical Committee of our hospital HGU "Gregorio Marañon." Clinical and immunologic classification was based on the 1994 revised guidelines of the CDC.13 We also studied 23 uninfected children as a healthy control group. The characteristics of the patients are shown in Table 1. We grouped children according to different immunologic degree of CD4+ depletion, immunologic status (CD4+ ≤15%, 15%-25%, and ≥25%) and virologic status [viral load (VL) <400 copies/mL; 400-10,000 copies/mL; and >10,000 copies/mL].
HIV-1 Infection Laboratory Markers
All infants were diagnosed as HIV-1-infected on the basis of positive results in both DNA polymerase chain reaction and virus culture assays.14 T lymphocytes in peripheral blood were quantified by flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems, San Jose, CA),14 and VL was measured in 200 μL of plasma using a quantitative assay (Amplicor monitor, Roche Diagnostic Systems, Brandenburg, NJ).14
Quantification of CD4+ or CD8+ T-Cell Subsets
The monoclonal antibodies used for the analysis of T-cell subsets were conjugated with fluorescein isothyocyanate (anti-IgG1, anti-HLA-DR, anti-CD45RA, anti-CD38, and anti-CD57), phycoerytherin (anti-IgG1, anti-CD45RO, anti-CD62L, anti-CD28, and anti-HLA-DR), and peridinin chlorophyll protein (anti-CD4 and anti-CD8). The monoclonal antibodies were obtained from Becton-Dickinson Immunocytometry Systems, except anti-CD38 (Immunotech, Marseilles, France). T-cell subsets were analyzed by 3-color multiparametric flow cytometry in total blood that had been lysed and washed.15 Naive T-cells were defined as cells with bright expression of CD45RA and positive for CD62L (CD45RAhi+CD62L+).15 Memory T-cells were defined as CD45RO+. Activated T-cells were defined as HLA-DR+, CD38+, and HLA-DR+CD38+. Memory-activated T-cells were defined as CD4+CD45RO+HLA-DR+ or CD8+CD45RO+CD38+. Naive plus central memory CD8+ cells were defined as CD8+CD28+CD57−, pre-effectors were defined as CD8+CD28−CD57−, and effectors were defined as CD8+CD28−CD57+. Acquisition was performed in a FACScan (Becton-Dickinson Immunocytometry Systems) cytometer using the Lysis II software (Becton-Dickinson) within 2 hours of cell staining as previously described.15 Data were analyzed using Lysis II analysis program (Becton-Dickinson). Appropriate isotypic controls (IgG1-FITC, IgG1-PE) were used to evaluate the nonspecific staining, which was deducted from the remaining results. We measured HLA-DR and CD38 relative fluorescence intensities as the mean of fluorescence intensities (MFI) using single-parameter histograms with no cursor sets.
We stratified HIV-infected children into groups, according to the degree of CD4+ depletion or current immunologic status (CD4+ ≤15%; 15%-25%, and ≥25%) and VL levels (VL <400 copies/mL; 400-10,000 copies/mL; and >10,000 copies/mL) (Table 1).
First, we analyzed the relationship between the percentage of T-cell subsets and percentage of CD4+ inside of each VL strata using the Pearson correlation coefficient. This analysis was carried out inside each current immunologic status or inside each VL level separately. Values for both range between −1 (a perfect negative relationship) and +1 (a perfect positive relationship). A value of 0 indicates no linear relationship.
Next, we carried out a crossed analysis for the differences among groups of current immunologic status or VL levels. Differences in immunologic markers among groups of children were analyzed using 1-way analysis of variance or H-Kruskal-Wallis according to sample size. Next, a nonparametric test (Mann-Whitney U test) was used. All P values were 2-tailed, and the threshold of significance was set at 0.05.
At the entry of study, all children were more than 5 years old, and they were on HAART for at least 24 weeks before the study. At the entry of study, HAART included a median of 2 (min; max: 0; 3) nucleoside analogue HIV reverse transcriptase inhibitor (NRTI), 1 (0; 3) PI, and 0 (0; 1) NNRTI. Inside each current immunologic status, children with VL <400 copies/mL; 400 to 10,000 copies/mL; and >10,000 copies/mL had similar age values and CD4+ or CD8+ percentages (Table 1).
Relationship Between the Percentage of T-Cell Subsets and Percentage of CD4+
We analyzed the correlation between the percentage of T-cell subsets and percentage of CD4+ inside each VL level group (VL <400 copies/mL; 400-10,000 copies/mL; and >10,000 copies/mL) (Table 2). Overall, we found a positive correlation of CD4+ with naive (CD45RAhi+CD62L+) T-cells and CD4+CD38+ cells (most of them naive cells). Conversely, a negative correlation of CD4+ with memory (CD45RO+) T-cells, activated (HLA-DR+, HLA-DR+CD38+) T-cells, and effector CD8+CD28−CD57+ T-cells were found. We also found a negative correlation between CD4+ and the level of HLA-DR expressed on T-cell surface measured as MFI.
T-Cell Subsets According to Immunologic Status Inside Each VL Group
We found in HIV infection an imbalance in naive/memory-effector distribution. HIV-infected children with low percentage of CD4+ had high values of memory (CD45RO+) and low values of naive (CD45RAhi+CD62L+) CD4+ or CD8+ (Fig. 1A, B). Furthermore, HIV-infected children with CD4+ >25% had similar values of memory and naive CD4+ as healthy controls, whereas memory and naive CD8+ values were different from healthy control group values (Fig. 1A, B).
We found an evident relationship between CD8+ expansion and low percentage of CD4+. Therefore, children with low percentage of CD4+ had diminished values of naive plus central memory CD8+ cells (CD8+CD28+CD57−), and these values were always lower than healthy control values. On the other hand, effector CD8+ (CD8+CD28−CD57+) and pre-effectors (CD8+CD28−CD57−) were upregulated in HIV-infected children with low percentage of CD4+, and these values were always higher than healthy control values (Fig. 2).
In HIV-infected children, an elevated proportion of T-cells (CD4+ or CD8+) that express CD38 and HLA-DR was directly associated with low percentage of CD4+ (Fig. 3A, B). Furthermore, we found elevated values of memory-activated T-cells (CD4+CD45RO+HLA-DR+ and CD8+CD45RO+CD38+) (Fig. 3A, B) in HIV-infected children with low percentage of CD4+. However, we found a different pattern of CD38 expression on T-cells (CD4+ or CD8+) (Figs. 4C and 2D). CD4+CD38+ percentage and MFI values were associated with high CD4+ values in children with VL <400 copies/mL. Furthermore, CD8+CD38+ values were not associated with low percentage of CD4+ (Table 2).
The most significant finding of the study was that independent of VL levels, chronically HIV-infected patients on HAART had persistently high expression of T-cell activation markers when their CD4+ values were low. We grouped the children in stratum of VL levels: VL <400 copies/mL could be considered as undetectable VL; the range of 400 to 10,000 copies/mL represents children on HAART who have some oscillations of VL during their HIV disease; and the range >10,000 copies/mL represents children who have wrong control of VL. Overall, inside each group of VL, HIV-infected children with the lowest percentage of CD4+ had the highest values of memory and activated CD4+ and CD8+ T-cells, and effector CD8+. At the same time, they had the lowest values of naive CD4+ and CD8+ T-cells and central memory CD8+.
The rates of cell death are in vivo increased in T-cells.16,17 Specifically, 30% to 60% of memory CD4+ are infected by simian immunodeficiency virus at the peak of infection, and most of these infected cells disappear within 4 days.18 However, during HIV chronic infection, other mechanisms could have an important role in CD4+ depletion.16,17 Direct killing of uninfected cells by HIV could be related to the chronic high level of T-cell activation that is induced by continuous HIV replication.19 Therefore, HIV-induced chronic immune activation is involved in the loss of CD4+ in HIV-infected patients, and the rate of CD4+ depletion correlates more with markers of immune activation than with VL. On the other hand, VL predicts the rate of CD4+ depletion, which could also be explained by a process wherein the level of viral replication either determines or reflects the prevailing level of T-cell activation, and thus only indirectly affects the CD4+ count.20
A mechanism of CD4+ depletion is the failure of T-cell production by the thymus of HIV-infected children. It is well established that HIV infection adversely affects the thymus in both children and adults.21 Yet the consequences of this thymic inhibition are worse in children, and this has been proposed as one cause of the rapid progression of the disease in them.22-24 In this study, HIV-infected children with low percentage of CD4+ had lower percentages of naive CD4+ and CD8+ than children with CD4+ >25%, when grouped by VL levels. Moreover, when we stratified all children according to immunologic status, we did not find association among VL and naive CD4+ or CD8+ cells (data not shown). However, when we stratified all children according to VL levels, we found a strong negative association among CD4+ and memory and activated T-cells in each VL strata. This fact apparently contradicts the previous data in HIV-infected children that indicated an inverse association between VL and thymic function.25,26 Our results indicate that naive T-cell depletion could be likely due to immune activation status rather than HIV replication.
Our results could explain the so-called immunologically discordant response to HAART, in which there is low percentage of CD4+ but undetectable VL. These patients maintain a high level of T-cell activation when compared with patients with both virologic and immunologic responses.27 However, virologically discordant patients with significant increases in their CD4+ counts despite incomplete viral suppression display decreased levels of immune activation.28 Moreover, our patients who optimally suppress viral replication showed high levels of T-cell activation when their percentages of CD4+ were low. This finding could contradict other studies that indicate a marked reduction in expression of T-cell activation markers as viral load is suppressed,28,29 but the Deeks and Hellerstein study28 showed T-cell activation decreases in patients failing therapy but who exhibit CD4+ reconstitution. Therefore, T-cell activation and CD4+ depletion seem to have a positive association, and T-cell activation could be a decisive factor in depletion and recovery of CD4+.
The similar association between CD4+ depletion and immune activation in each VL group supports the hypothesis that immune activation rather than viral replication could lead to the destruction and depletion of CD4+.10 The generalized immune activation not only provides a suitable target for viral replication but also may change T-cell subset homeostasis, and it is also probable that a strong immune activation can contribute to CD4+ depletion.8,15 In fact, the generation of activated CD4+ (T-cells preferentially infected by HIV) provides a fertile substrate for viral replication, thus creating the conditions for a vicious cycle in which more viral replication induces more immune activation, which in turn allows more viral replication, and so on.30 This immune activation may drive the progression of HIV disease by disrupting the homeostatic equilibrium of resting cell populations.30 Moreover, the burst of T-cell proliferation occurs continuously by stimulation of proinflammatory factors and antigens, and it causes the switch of resting to effector CD8+. In our work, we did not find an association between VL and percentage of CD8+CD28−CD57+, but we found a positive association between percentage of CD4+ and effector CD8+ inside each VL strata.
The CD38 molecule is expressed in immature lymphocytes,31 but is also induced after an acute active viral infection.32 In children, CD4+CD38+ is a marker of immaturity that is expressed in cells with a naive phenotype.3 This way, we found positive association between CD4+ and CD4+CD38+. By contrast, CD8+CD38+ is associated with a worse prognosis, a decline of CD4+ and an increase in VL.29,33 In this study, only HIV-infected children with CD4+ <15% had a slight increase in frequency of activated CD8+, but not in CD4+, when VL was high (data not shown). Therefore, the effect of HIV replication was only evident in HIV-infected children with severe immunodeficiency because they had an elevated immune activation.3,8 These findings would support assessing the expression of CD38 as a prognostic marker to monitor the response to HAART.29,34
Our study has several limitations: (a) The study used percentage of CD4+ as a parameter for CD4+ T-cell depletion. However, the number of CD8+ T-cells, which increases by inflammation, influences the percentage of CD4+. Thus, the association between CD4+ depression (low percentage of CD4+) and the levels of activation may be overly detected. However, we could not carry out this analysis because the groups of children with CD4+ <200 were too small to validate the statistical analysis. (b) The interpretation of the association of CD4+ depletion with T-cell phenotypes and VL may be more complicated when patients are on treatment. The source of immune activation in the patients with undetectable plasma viremia is unknown. It might be a result of residual viral replication within the tissues, drug side effects, or even a secondary response to immune reconstitution. In addition, protease inhibitor may have some inhibitory effect on apoptotic process and thus increase CD4+ count.
In this study, we report that HIV-infected children on HAART with different VL levels having similar degrees of CD4+ depletion showed approximately similar levels of immune activation and imbalance of naive, memory, and effector T-cells. In summary, our data suggest that elevated immune activation could be responsible for CD4+ depletion rather than HIV replication because immunologic status is associated directly to immune activation and not to VL levels in HIV-infected children on HAART.
We thank Jose Maria Bellón Cano and Alejandro Alvaro-Meca for their statistical assistance and Nicholas Weber for his language corrections.
1. Clerici M, Stocks NI, Zajac RA, et al. Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging. J Clin Invest
2. Sleasman JW, Aleixo LF, Morton A, et al. CD4+
memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children. AIDS
3. de Martino M, Rossi ME, Azzari C, et al. Different meaning of CD38 molecule expression on CD4+
cells of children perinatally infected with human immunodeficiency virus type 1 infection surviving longer than five years. Pediatr Res
4. Navarro J, Resino S, Bellón JM, et al. Association of CD8+
T lymphocyte subsets with the most commonly used markers to monitor HIV-1 infection in children treated with highly active antiretroviral therapy. AIDS Res Hum Retroviruses
5. Schlesinger M, Peters V, Jiang JD, et al. Increased expression of activation markers on CD8 lymphocytes in children with human immunodeficiency virus-1 infection. Pediatr Res
6. Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature
7. Perelson AS, Neumann AU, Markowitz M, et al. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science
8. Sousa AE, Carneiro J, Meier-Schellersheim M, et al. CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load. J Immunol
9. McCune JM. The dynamics of CD4+
T-cell depletion in HIV disease. Nature
10. Grossman Z, Meier-Schellersheim M, Sousa AE, et al. CD4+
T-cell depletion in HIV infection: are we closer to understanding the cause? Nat Med
11. Silvestri G, Sodora DL, Koup RA, et al. Nonpathogenic SIV infection of sooty mangabeys is characterized by limited bystander immunopathology despite chronic high-level viremia. Immunity
12. CDCP. Guidelines for use of antiretroviral agents in pediatric HIV infection. MMWR Morb Mortal Wkly Rep
13. CDCP. Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR CDC Surveill Summ
14. Munoz-Fernandez MA, Obregon E, Navarro J, et al. Relationship of virologic, immunologic, and clinical parameters in infants with vertically acquired human immunodeficiency virus type 1 infection. Pediatr Res
15. Resino S, Navarro J, Bellón JM, et al. Naïve and memory CD4+ T-cells and T-cell activation markers in HIV-1 infected children on HAART. Clin Exp Immunol
16. Finkel TH, Tudor-Williams G, Banda NK, et al. Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes [see comments]. Nat Med
17. Hellerstein MK, Hoh RA, Hanley MB, et al. Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection. J Clin Invest
18. Mattapallil JJ, Douek DC, Hill B, et al. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature
19. Muro-Cacho CA, Pantaleo G, Fauci AS. Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden. J Immunol
20. Moanna A, Dunham R, Paiardini M, et al. CD4+
T-cell depletion in HIV infection: killed by friendly fire? Curr HIV/AIDS Rep
21. Haynes BF, Markert ML, Sempowski GD, et al. The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection. Annu Rev Immunol
22. Kourtis AP, Ibegbu C, Nahmias AJ, et al. Early progression of disease in HIV-infected infants with thymus dysfunction. N Engl J Med
23. Nahmias AJ, Clark WS, Kourtis AP, et al. Thymic dysfunction and time of infection predict mortality in human immunodeficiency virus-infected infants. CDC Perinatal AIDS Collaborative Transmission Study Group. J Infect Dis
24. Meyers A, Shah A, Cleveland RH, et al. Thymic size on chest radiograph and rapid disease progression in human immunodeficiency virus 1-infected children. Pediatr Infect Dis J
25. Gibb DM, Newberry A, Klein N, et al. Immune repopulation after HAART in previously untreated HIV-1-infected children. Paediatric European Network for Treatment of AIDS (PENTA) Steering Committee. Lancet
26. Correa R, Munoz-Fernandez MA. Production of new T cells by thymus in children: effect of HIV infection and antiretroviral therapy. Pediatr Res
27. Paiardini M, Cervasi B, Galati D, et al. Early correction of cell cycle perturbations predicts the immunological response to therapy in HIV-infected patients. AIDS
28. Deeks SG, Hoh R, Grant RM, et al. CD4+
T cell kinetics and activation in human immunodeficiency virus-infected patients who remain viremic despite long-term treatment with protease inhibitor-based therapy. J Infect Dis
29. Resino S, Bellón J, Gurbindo D, et al. CD38 in CD8+
T cells predict virological failure in HIV-infected children receiving antiretroviral therapy. Clin Infect Dis
30. Silvestri G, Feinberg MB. Turnover of lymphocytes and conceptual paradigms in HIV infection. J Clin Invest
31. Jackson DG, Bell JI. Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation. J Immunol
32. Akbar AN, Salmon M, Janossy G. The synergy between naive and memory T cells during activation. Immunol Today
33. Mocroft A, Bofill M, Lipman M, et al. CD8+
lymphocyte percent: a useful immunological marker for monitoring HIV-1-infected patients. J Acquir Immune Defic Syndr Hum Retrovirol
34. Tilling R, Kinloch S, Goh LE, et al. Parallel decline of CD8+
T cells and viraemia in response to quadruple highly active antiretroviral therapy in primary HIV infection. AIDS
Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
CD4+; CD8+; T-cell subsets; naive; memory; effector; viral load