To characterize the immune reconstitution in vertically HIV-1-infected category C3 children after 3 years’ antiretroviral therapy (ART), 22 children were studied longitudinally. Children who achieved high CD4 T cell counts and T cell receptor rearrangement excision circles and low viral loads after ART showed values of naive T cells very close to age-matched long-term asymptomatic children or healthy controls. Our data indicate that in C3 children who respond to ART, the immune system may recover fully in quantitative and functional terms.
HIV-1 causes a progressive and severe immunodeficiency from the early phase of the disease, before overt CD4 T cell depletion . The slow sustained increase in naive (CD4+CD45RA+) T cells reported after antiretroviral therapy (ART) may derive either from the redistribution of circulating naive lymphocytes or from lymphocytes newly generated by the thymus . Whereas in adults a high proportion of T cells co-expressing CD38 and HLA-DR has been associated with a poor prognosis , in children CD38 is a marker of immaturity, its expression on CD4 T cells being a good prognostic marker .
We retrospectively studied 22 vertically HIV-1-infected infants in a 3 year follow-up study according to the time-course of the percentage of CD4 T cells and viral load by classifying children into: (i) a long-term asymptomatic (LTA) group of eight children with CD4 T cell counts greater than 25% and 500 cells/ml who were clinically asymptomatic during the whole follow-up; (ii) a group of six children in category C3 who were responsive to ART (Res), who achieved CD4 cell counts greater than 25% and over 500 cells/ml after 3 years’ ART; (iii) a group of six children in category C3 who were non-responsive to ART (non-Res) throughout the follow-up period despite ART. As a control group, 12 age-matched HIV-uninfected children were studied. This 3 year period coincided with the initiation of ART in ART-naive children or with a switch to highly active antiretroviral therapy (HAART) in previously treated children.
Viral loads were determined using an ultrasensitive reverse transcriptase–polymerase chain reaction assay (Amplicor Monitor, Roche Diagnostic Systems, Basel, Switzerland). T cell subsets were analysed by three-colour multiparametric flow cytometry (FACScan, Becton-Dickinson, San Jose, CA, USA) . Thymic function was studied by quantifying the T cell receptor rearrangement excision circles (TREC) in peripheral blood mononuclear cells using real-time quantitative polymerase chain reaction (LightCycler System, Roche Molecular Biochemicals, Idaho Falls, USA) . Differences between the infants’ characteristics were analysed using non-parametric tests because of the small sample size or the free distribution of data. The Mann–Whitney U test was used to compare groups.
The baseline characteristics of the different groups are described in Table 1. The mean percentages of CD4 and CD8 T cells, and viral loads were compared among the groups during follow-up. Eight out of 14 children in the Res group (57%) had an increase in percentage of CD4 T cells and a decrease in viral load up to the values of the LTA group, whereas these two markers remained without variation during follow-up in the non-Res group. Four children in the Res group had viral loads of less than 50 copies/ml by the end of the study versus none in the non-Res group despite HAART.
The Res group reached similar values of naive (CD45RA+CD62L+) CD4 and CD8 T cells to the LTA group (Fig. 1a,b), suggesting that the control of viral replication avoids naive T cells becoming activated, memory or effector . Inversely, the non-Res group had lower naive CD4 and CD8 T cell counts and higher activated (%CD4+HLA-DR+CD38+) and memory activated (%CD4+CD45RO+HLA-DR+) T cell counts than the other three groups (Fig. 1c,d). All infected children had higher values of CD8+HLA-DR+CD38+ and CD8+CD45RO+CD38+ T cells than the control group, possibly in relation to the level of viral load, with the highest values in the non-Res group (Fig. 1c,d).
The Res group had similar percentages of CD38+ and HLA-DR+CD38− on CD4 and CD8 T cells to the LTA groups, and in both groups those percentages were significantly higher than in the control group (Fig. 1e,f). Moreover, the non-Res group had higher percentages of CD8+CD38+ T cells than the control and LTA groups (Fig. 1e,f). Chronic activation of the immune system is a hallmark of HIV infection, as shown by the increased expression of activation markers (CD38+ and HLA-DR+) . However, in children CD38+ is also a marker of immaturity and thus the percentage of CD4+CD38+ is a marker of preserved immune status rather than activation [4,5]. Decreased activated T cells may be a marker of an effective response to ART, probably because T cells are chronically activated by HIV and therefore their decrease follows the ART-induced decline in viral load. Indeed, the Res group had low values of activated T cells, whereas they were high in the non-Res group, in agreement with previous results , suggesting a therapeutic failure. During HIV infection, the switch of naive to memory T cells is probably favoured either by their chronic stimulation by HIV or other agents and through the antigen-independent peripheral expansion of CD4+CD45RO+ T cells after thymical dysfunction caused by HIV. Memory CD4 T cells are productively infected by HIV, and they may also suffer apoptosis induced by an abnormal cellular activation, leading to the decline in CD4 T cells. Our data indicate that children with a good virological response to ART restore the normal pool of CD4 T cells.
Finally, TREC, the percentage of CD4 T cells and viral loads were compared longitudinally in two children. The start of ART or the switch to HAART in both children led to a marked increase in both markers (Fig. 1g,h), indicating that the CD4 increase was probably caused by the thymic production of new CD4 T cells, in agreement with previous results . Inversely, the decrease in the percentage of CD4 cells correlated with a decrease in TREC levels and with high viral loads.
We have found that those children with AIDS and profound immunodeficiency who responded well to ART in terms of the percentage of CD4 cells and viral loads were those with higher naive, and lower memory and activated CD4 and CD8 T cells, further supporting a functional reconstitution of the immune system. In addition, the restoration of CD4 T cells induced by ART in children late in the disease seems to be a consequence of the naive T cell reconstitution and decreased chronic activation of the immune system.
Ma José Bellón
Ma Luisa Abad
Ma Ángeles Muñoz-Fernández
The authors would like to thank to Dolores García Alonso and Consuelo Muñoz for their excellent technical assistance. They also wish to thank D. Douek for kindly supplying a 375 pb plasmid for TREC quantifying.
1. Resino S, Bellón J, Jiménez JL, Gurbindo D, Muñoz-Fernández MA. Impaired IL-5 production by T cells as prognostic marker of disease progression in HIV-1-infected children. Eur Cytokine Netw 2001, 12: 253–259.
2. Pakker NG, Notermans DW, de Boer RJ. et al
. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat Med 1998, 4: 208–214.
3. Liu Z, Cumberland WG, Hultin LE, Prince HE, Detels R, Giorgi JV. Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression. J Acquir Immune Defic Syndr Hum Retrovirol 1997, 16: 83–92.
4. de Martino M, Rossi ME, Azzari C, Gelli MG, Galli L, Vierucci A. Different meaning of CD38 molecule expression on CD4+ and CD8+ cells of children perinatally infected with human immunodeficiency virus type 1 infection surviving longer than five years. Pediatr Res 1998, 43: 752–758.
5. Resino S, Navarro J, Bellon JM, Gurbindo D, Leon JA, Muñoz-Fernández MA. Relationship between CD4+ T-lymphocyte subsets and the laboratory markers used to monitor the HIV-1 infection in children with potent antirretroviral therapy. Clin Exp Immunol 2001, 125: 266–273.
6. Correa R, Muñoz-Fernández MA. Viral phenotype affects the thymical production of new T-cells in HIV-1 infected children. AIDS 2001, 15: 1959–1963.
7. Brinchmann JE. Differential responses of T cell subsets: possible role in the immunopathogenesis of AIDS. AIDS 2000, 14: 1689–1700.
8. Vigano A, Saresella M, Rusconi S, Ferrante P, Clerici M. Expression of CD38 on CD8 T cells predicts maintenance of high viraemia in HAART-treated HIV-1-infected children. Highly active antiretroviral therapy.
Lancet 1998, 352: 1905–1906.
9. Douek DC, Koup RA, McFarland RD, Sullivan JL, Luzuriaga K. Effect of HIV on thymic function before and after antiretroviral therapy in children. J Infect Dis 2000, 181: 1479–1482.