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Functional patterns of HIV-1-specific CD4 T-cell responses in children are influenced by the extent of virus suppression and exposure

Correa, Rafaela; Harari, Alexandrea; Vallelian, Florencea; Resino, Salvadorb; Munoz-Fernandez, M Angelesb; Pantaleo, Giuseppea

Author Information

From the aLaboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy, Department of Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011 Lausanne, Switzerland

bLaboratorio de Immuno-Biologia Molecular, Hospital General Universitario Gregorio Maranon, Madrid, Spain.

Received 5 May, 2006

Accepted 14 September, 2006

Correspondence to Giuseppe Pantaleo, MD, Laboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue Bugnon, 1011 Lausanne, Switzerland. Tel: +41 21 3141071; fax: +41 21 3141070; e-mail: [email protected]

doi: 10.1097/QAD.0b013e32801120bc
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Abstract

Introduction

Virus-specific CD4 T cells play a critical role in antiviral immunity and particularly in maintaining effective cytotoxic T lymphocyte responses [1–7]. With regard to HIV-1 infection, the early and massive depletion of HIV-1-specific CD4 T cells [8–10] can certainly contribute to the lack of effective immune control of virus replication and to the progression of HIV-1-associated disease. Recent studies in chronic HIV-1-infected and healthy HIV-negative adults have investigated virus-specific, e.g. HIV-1, cytomegalovirus, Epstein–Barr virus, and herpes simplex virus, CD4 T-cell responses on the basis of the ability to secrete IFN-γ and IL-2 after specific antigen stimulation. Three functionally distinct populations of antigen-specific CD4 T cells were identified [11,12]: (i) single IFN-γ-secreting cells; (ii) dual IL-2/IFN-γ-secreting cells; and (iii) single IL-2-secreting cells. The single IFN-γ and IFN-γ/IL-2-secreting CD4 T cells were contained within CCR7− memory cells [i.e. memory T cells with potential effector function (TEM)] [13], whereas single IL-2-secreting cells were contained within the CCR7+ cell population, i.e. typical central memory cells (TCM) [11,14,15]. Those studies demonstrated: (i) the selective loss in the frequency of HIV-1-specific IL-2-secreting CD4 T cells [11]; (ii) the association between different patterns of virus-specific CD4 T-cell responses and antigen load and persistence; (iii) the association between the dominant IL-2 and poly-functional (IL-2 plus IFN-γ) virus-specific CD4 T-cell responses and effective control of virus replication; and (iv) the lack of full recovery of the loss of HIV-1-specific IL-2-secreting CD4 T cells even after long-term successful antiviral therapy (ART).

Variables percentages of IFN-γ-secreting CD4 T cells have consistently been found in both adults and children [11,16–20]. Two studies have analysed HIV-1-specific CD4 T-cell responses in children and have shown that IFN-γ-secreting cells were present in individuals with ongoing HIV-1 replication, and that they were at a very low frequency in children with controlled viraemia or after ART [19,20]. The presence of HIV-1-specific IL-2-secreting CD4 T cells was not examined in those studies. The investigation of HIV-1-specific CD4 T-cell responses in children based on the recent advances in adults is important to determine the extent of HIV-1 CD4 T-cell dysfunction in paediatric HIV-1 infection. For these purposes, HIV-1-specific CD4 T-cell responses were analysed in the blood of 23 HIV-1-infected children on the basis of the ability of CD4 T cells to secrete IFN-γ and IL-2 after antigen-specific stimulation.

We provide evidence that substantial differences in the patterns of CD4 T-cell responses were associated with different conditions of response to ART. Interestingly, children successfully treated with ART showed dominant IL-2 HIV-1-specific CD4 T-cell responses, thus indicating a greater capacity of immune restoration in children compared with adults.

Patients, materials and methods

Study groups

Twenty-three children born to HIV-infected mothers were enrolled in the present study (mean ± SE age 10.7 ± 0.7 years). The children were followed at the Department of Immunopediatry in the Hospital General Universitario Gregorio Marañon (Madrid, Spain). Children were monitored every 2–3 months for viral load and the percentage of CD4 and CD8 T cells. All children were treated with ART consisting of at least two nucleoside analogue reverse transcriptase inhibitors plus one or more HIV protease inhibitors. Drugs were prescribed by the attending paediatrician according to Centers for Disease Control and Prevention guidelines [21] upon obtaining written informed consent from the legal guardians. The study was approved by the local hospital Institution Review Board.

Analysis of viral load and of T-cell populations

Blood samples were collected in ethylenediammine tetraacetic acid-containing tubes and the plasma was isolated within 4 h and stored at −70°C. The viral load was measured in 200 μl plasma using a quantitative reverse polymerase chain reaction assay (Amplicor Monitor; Roche Diagnostics System, Basel, Switzerland), with a sensitivity of 400 RNA copies/ml. CD4 and CD8 T-cell populations in peripheral blood were quantified by direct immunofluorescence using monoclonal antibodies specific for CD4 and CD8 cells and flow cytometry (fluorescence-activated cell sorter scan; Becton Dickinson, Immunocytometry Systems, San Jose, California, USA) as previously described [22]. In contrast to the absolute number of CD4 T cells, the percentage does not change with age [23] and is therefore considered the best progression marker of HIV infection in children [24].

Intracellular cytokine staining

Intracellular IFN-γ and IL-2 production was assessed as previously described [11]. Blood mononuclear cells (2–4 × 106 cells in 2 ml RPMI 1640 Gutamax-1 medium containing 10% inactivated fetal calf serum) were stimulated with 5 μg/ml HIV-p55 gag (Protein Sciences, Meriden, Connecticut, USA) or 200 ng/ml staphylococcal enterotoxin B (Calbiochem, La Jolla, California, USA; positive control) for 14 h at 37°C, in the presence of 0.5 μg/ml purified anti-CD28 antibody (Becton Dickinson) and 1 μg/ml GolgiPlug (Becton Dickinson). Cell surface staining was completed as described after 14 h of activation in vitro[11]. Cells were then permeabilised and fixed with FACS permeabilising solution (Becton Dickinson) and labeled with anti-human IFN-γ-allophycocyanin and IL-2-phycoerythrin antibodies (Pharmingen, San Diego, California, USA). Simultaneously, T-cell activation was assessed by staining with anti-CD69-fluorescein isothiocyanate antibody (Becton Dickinson). The number of non-gated events ranged between 105 and 106 events. An intracellular cytokine staining to be considered positive had to be at least threefold higher than the background levels (unstimulated cells) and to have a percentage of cytokine-secreting cells above 0.03%.

Statistical analysis

All statistical tests were interpreted at the 5% significance level (P < 0.05). Analysis of variance was performed to compare the variables between the three groups of HIV-infected children defined by the pattern of HIV-1-specific CD4 T cells. Pearson's correlation test was used to determine the correlation between the different variables. In order to compare the differences between each group of HIV-infected children, Levene's test was performed to assess the homogeneity of the variances. The independent samples Student's t-test was used when variances of compared variables were homogeneous, and the Welch's test was used when variances were not homogeneous.

Results

The frequency of HIV-1-specific CD4 T cells in 23 HIV vertically infected children was evaluated on the basis of their ability to secrete IL-2 and IFN-γ after stimulation with HIV-1 p55 gag protein. All the children were treated with ART including two nucleoside inhibitors of reverse transcriptase and one protease inhibitor for an extended period of time (mean 7 years, Table 1). Interestingly, on the basis of the differences in the percentage of CD4 T-cell populations secreting IL-2 or IFN-γ, we observed three distinct patterns of HIV-1-specific CD4 T-cell responses in the 23 children studied. Two examples for each pattern are shown in Fig. 1a–c. The majority (more than 70%) of the HIV-1-specific CD4 T cells were single IL-2-secreting cells in 10 out of 23 children (Fig. 1a). The three cytokines secreting CD4 T-cell populations, i.e. single IFN-γ, dual IL-2/IFN-γ, and single IL-2-secreting cells were well represented in eight out of 23 children (Fig. 1b). The majority (more than 60%) of HIV-1-specific CD4 T cells were single IFN-γ-secreting cells in five out of 23 children (Fig. 1c). Therefore, three groups were defined: (i) a group with a dominant IL-2 CD4 T-cell response; (ii) a group with a polyfunctional (IL-2 plus IL-2/IFN-γ plus IFN-γ) CD4 T-cell response; and (iii) a group with a dominant IFN-γ CD4 T-cell response. Cumulative data and the mean percentage ± SE of the three CD4 T-cell populations in the three groups of children are also shown in Fig. 1a–c.

T1-3
Table 1:
Clinical and immunological parameters in the three groups of HIV infected children identified on the basis of the patterns of IL-2 and IFN-γ secreting CD4 T cells.
F1-3
Fig. 1:
Analysis of HIV-specific CD4 T-cell responses in HIV-infected children. Flow cytometry profiles of the distribution of HIV-1-specific IL-2, IL-2/IFN-γ, and IFN-γ-secreting cells gated on CD4 T-cell populations and mean ± SE percentage of these cell populations are shown. Patients were classified in three groups based on the functional patterns of HIV-1-specific CD4 T cells: group IL-2 (a), polyfunctional group (b), and group IFN-γ (c). Profiles of two representative children for each group are shown. The cluster of events shown in red corresponds to the responder CD4 T cells, i.e. expressing IL-2 or IFN-γ, whereas the cluster of events in blue corresponds to the non-responder CD4 T cells.
Table 1
IL-2;
Table 1
IL-2/IFN-γ;
Table 1
IFN-γ.

In order to identify the differences between the three groups that could help to explain the patterns of CD4 T-cell responses, a series of parameters including age, percentage of CD4 and CD8 T cells, viral load at the time of the assessment of CD4 T-cell functions, and time of ART were analysed (Table 1). The statistical analysis of variance showed that among the different variables analysed, viral load was the only variable significantly different between the three groups. Viral load was significantly higher in the IFN-γ group (71 880 ± 22 727 HIV-1-RNA copies/ml of plasma) compared with the IL-2 group (798 ± 296 copies/ml, P = 0.019) and to the polyfunctional group (988 ± 356 copies/ml, P = 0.024). On the basis of the analyses shown in Table 1 and on previous studies performed in HIV-1-infected adults [11,14,15], higher levels of viral load may well explain the differences in CD4 T-cell functions between the IFN-γ group and the IL-2 and the polyfunctional groups. However, the viral load levels at the time of the evaluation of CD4 T-cell functions were not able to explain the functional patterns between the IL-2 and the polyfunctional groups. We then performed a retrospective analysis of viral load levels within the IL-2 and polyfunctional groups during the past 2 years. As indicated by the kinetics of the viral load of two representative children for each group, all the children included in the IL-2 group had stable, low levels of viraemia during the past 2 years (mean 3187 ± 1277; two representative examples are shown in Fig. 2a). The children belonging to the polyfunctional group had low viral loads at the time of the study but had experienced transient increases in viraemia levels (Fig. 2a) during the past 2 years and the mean viraemia in the eight children belonging to this group was 25 580 ± 23 143. Finally, the children in the IFN-γ group had an extended period of uncontrolled high levels of viraemia and the mean viral load during the past 2 years was 21 132 ± 6166 (Fig. 2a). In addition, we also analysed within the three groups the number of blips experienced during the past 2 years above a cut-off of 5000 HIV-1-RNA copies per millilitre of plasma. As shown in Fig. 2b, the median number of blips was 10 in the single IFN-γ group, two in the polyfunctional group and none in the single IL-2 group. Taken together, these results suggested that the functional heterogeneity of HIV-1-specific CD4 T-cell responses observed in children was influenced by the levels of viral load and antigen exposure.

F2-3
Fig. 2:
Follow-up of viral load and percentage of CD4 T cells during the past 3 years preceding the functional analysis. Two representative children of the IL-2, polyfunctional, and IFN-γ groups are shown (a). The median number of blips experienced during the past 2 years above a cut-off of 5000 HIV-1-RNA copies/ml of plasma in each group (b).
Table 1
IL-2;
Table 1
IL-2/IFN-γ;
Table 1
IFN-γ.

To confirm this hypothesis further, we assessed the correlation between the frequency of the different functional populations of HIV-1-specific CD4 T cells and the levels of viraemia. We observed that the viral load was the only parameter that correlated with the frequency of the different functional populations of HIV-1-specific CD4 T cells. We found a significant negative correlation between the frequency of HIV-1-specific IL-2 secreting CD4 T cells and viraemia levels (Fig. 3a), but a positive correlation between viraemia levels and the frequency of IFN-γ-secreting cells (Fig. 3b). These results confirmed that viral load was a strong modulator of HIV-1-specific CD4 T-cell responses.

F3-3
Fig. 3:
Correlation between viral load and the proportion of IL-2 (a) and IFN-γ (b) secreting CD4 T cells. Viral load correlated negatively with the proportion of IL-2-secreting cells (P = 0.028) and positively with the proportion of IFN-γ-secreting cells (P = 0.003).

We then compared the magnitude of HIV-1-specific IL-2 and IFN-γ CD4 T-cell responses observed in children with those in three cohorts of adults [11], including subjects with chronic infection naive to ART, those successfully treated with ART, and long-term non-progressors (LTNP). First, we compared the CD4 T-cell responses between all children and the three cohorts of adults. The frequency of HIV-1-specific single IL-2-secreting CD4 T cells in children was significantly higher compared with untreated and ART-treated adults but not LTNP (Fig. 4a). The frequency of single IFN-γ-secreting cells was significantly higher in the children compared with the ART-treated adults, whereas no significant differences were observed between the children and the untreated and LTNP groups (Fig. 4a). Finally, the frequency of dual IL-2/IFN-γ-secreting CD4 T cells was significantly higher in LTNP compared with the children and the other adult groups (Fig. 4a). Second, we compared the CD4 T-cell responses observed in the IL-2 and IFN-γ children groups and those within the adult groups. The frequencies of both single IL-2 and single IFN-γ-secreting cells were significantly higher compared with those observed in the adult groups, i.e. untreated, treated and LTNP (Fig. 4b).

F4-3
Fig. 4:
Comparison of HIV-1-specific CD4 T-cell responses between HIV-infected children and adults. (a) Mean ± SE percentage of cytokine-secreting HIV-1-specific CD4 T-cell populations in the 23 children compared with different groups of HIV-infected adults. The percentage of single IL-2-secreting cells in children was significantly higher compared with untreated and antiretroviral therapy (ART)-treated adults but not to long-term non-progressors (LTNP). The frequency of single IFN-γ-secreting cells was significantly higher in the children compared with the ART-treated adults, and the frequency of dual IL-2/IFN-γ-secreting CD4 T cells was significantly higher in LTNP compared with the children and other adult groups.
Table 1
IL-2;
Table 1
IL-2/IFN-γ;
Table 1
IFN-γ. (b) Mean ± SE percentage of IFN-γ and IL-2-secreting CD4 T cells in the IFN-γ and the IL-2 groups in children and adults. The frequencies of both single IL-2 and single IFN-γ-secreting cells were significantly higher compared with those observed in the adult groups, i.e. untreated, treated and LTNP.
Table 1
Group IFN-γ children;
Table 1
group IL-2 children;
Table 1
untreated adults;
Table 1
treated adults;
Table 1
LTNP adults. *Significant differences between the frequencies of cytokine-secreting HIV-1-specific CD4 T cells in adults and the corresponding population in children: *** P < 0.001; ** P < 0.01; * P < 0.05.

Discussion

The present study has investigated HIV-1-specific CD4 T-cell responses in 23 children by assessing the ability of blood mononuclear cells to secrete IL-2 and IFN-γ after antigen-specific stimulation. These analyses have shown different patterns of IL-2 and IFN-γ HIV-1-specific CD4 T-cell responses associated with different levels of viral load. In this regard, recent studies have shown that the selective defect in HIV-1-specific IL-2-secreting CD4 T cells distinguishes between progressive and non-progressive infection in adults [11,18,25,26]. The selective defect in IL-2-secreting CD4 T cells has also been observed in other viral infections such as hepatitis C virus [27].

The three different patterns of HIV-specific CD4 T-cell responses observed in HIV-infected children include a dominant IFN-γ response, a polyfunctional (IFN-γ plus IL-2) response and a dominant IL-2 response. These functional patterns correlated with the viral load levels at the time of the functional analysis and over the past 2 years. The group with a dominant IFN-γ response had high viraemia levels at the time of the functional analysis and over the past 2 years. This functional pattern was similar to that observed in virus infections with uncontrolled viral replication [11,12,14,15]. The group with a polyfunctional response had low viral load at the time of the analysis, and had experienced blips of viraemia over the past 2 years. This functional pattern was similar to that observed in LTNP, in progressors with ART-mediated suppression of virus replication and in chronic viral infections such as cytomegalovirus or Epstein–Barr virus, generally associated with low levels/controlled viral load [11,12,14,15,28]. Finally, the group with a dominant IL-2 response had very low viraemia levels at the time of the analysis, and did not experience blips of viraemia over the past 2 years. The latter functional pattern was similar to that generally observed in the case of antigen clearance, such as after tetanus toxoid immunization [12,14,15]. This pattern of HIV-1-specific CD4 T-cell response has never been observed in HIV-infected adults. As mentioned above, among the different parameters investigated (Table 1) the viral load was the only one correlated with the functional profile but also with the magnitude of the response. In this regard, in agreement with a previous study [19], a positive correlation was found between the viral load and the frequency of IFN-γ-secreting CD4 T cells. Interestingly, we also showed that the viral load negatively correlated with the frequency of IL-2-secreting CD4 T cells.

We also observed that the frequencies of HIV-1-specific CD4 T cells were significantly higher in children compared with adults. The percentage of IFN-γ-secreting CD4 T cells in children belonging to the IFN-γ group was 10-fold higher than ART-treated adults [11,16–18]. Similarly, the percentage of IL-2-secreting CD4 T cells in children belonging to the IL-2 group was three to 10-fold higher than in LTNP and progressors [11]. In contrast to adults in whom thymic function is diminished, children have an adequate thymic repopulation [29–33], which supplies a greater pool of naive T cells with a broad T-cell receptor repertoire of specificities. This broad T-cell receptor repertoire may supply a multipotential capacity to generate specific T cells against a variety of antigens including HIV [34]. Greater thymic function might explain the higher frequencies of HIV-1-specific CD4 T cells in children and the presence of the group of children with a predominant IL-2-secreting CD4 T-cell response. In support of this hypothesis, it has been shown that after ART the thymus may recover the pool of naive T cells [29,30,35,36].

The few studies that have analysed HIV-1-specific CD4 T cells in children concluded that HIV-1-specific IFN-γ-secreting cells were present in HIV-infected children with ongoing HIV replication, whereas their frequency was very low in children with controlled viraemia or after ART [19,20]. Feeney et al. [20] reported that despite robust Gag-specific proliferation capacity, the frequency of IFN-γ-secreting Gag-specific CD4 T cells was low in the majority of ART-treated children. In the light of these results, the observed vigorous gag-specific CD4 T-cell proliferative responses, may be explained on the basis of the presence of HIV-1-specific IL-2-secreting CD4 T cells.

In conclusion, the present study demonstrates that HIV-1-infected children have significantly greater numbers of HIV-1-specific CD4 T cells compared with HIV-1-infected adults. More importantly, ART-mediated suppression of virus replication is associated with the development of both dominant IL-2 and polyfunctional (IFN-γ plus IL-2) CD4 T-cell responses. These results are consistent with the original observation of Connors on the influence of viral load on HIV-1-specific T-cell functions [37]. Whether these polyfunctional responses may contribute to the effective control of viral replication is unclear. However, the development of HIV-1-specific IL-2-secreting CD4 T cells and the presence of polyfunctional T-cell responses represent a valuable immunological marker of more effective control of virus replication.

Sponsorship: Financial support was received from Fundación para la Investigación y la Prevención del SIDA en España, FIPSE (grant 36514/05), Fundación para la Investigación Sanitaria (FIS) (PI040883), (PI052411), Plan Nacional de Salud (SAF 2003-09209, SAF-2004-06778), Red Temática Cooperativa de investigación en SIDA (RIS G03/173) of FIS, Swiss National Foundation (FN 3100-66788). S.R. is a staff researcher with FIS (CP04/00090).

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Keywords:

children; functional patterns; HIV-1-specific CD4 T cells; immune reconstitution

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