Naive T lymphocytes and recent thymic emigrants are associated with HIV-1 disease history in French adolescents and young adults infected in the perinatal period: the ANRS-EP38-IMMIP Study

40-word summary: In youths infected with HIV during the perinatal period, CD4 N and CD4 RTE percentages were positively correlated with both CD4 T-cell count and past and current HIV replication, demonstrating the persistence of high-level thymic activity in long-term infection. linear


Introduction
The first generation of patients infected with HIV-1 during the perinatal period has now reached adolescence or adulthood [1][2]. Pediatric HIV infection differs from infection during adulthood in terms of clinical progression, pathophysiology, response to treatment, and the occurrence of viral replication during immune system development [2][3]. Adolescence may be associated with poorer compliance with treatment in some patients, and physiological changes during puberty may affect the immune system [4]. The current immune and virological status of youths perinatally infected with HIV and their covariates have been the subject of limited investigations. The restoration and maintenance of the naive T-lymphocyte compartment is of importance for the future of these individuals, as it is directly associated with the diversity of the T-cell receptor (TCR) repertoire and critical for immunity to foreign antigens [5]. Previous pediatric studies have shown that CD4 T-lymphocyte recovery is driven principally by the de novo production of naive T lymphocytes in the thymus, and that it declines with age and with HIV infection [6][7].
Our aim, within the ANRS-EP38-IMMIP study, was to provide more detailed knowledge of the pathophysiological characteristics of youths, 15 years or more after perinatal infection. We previously reported that blood cell-associated HIV-DNA levels in these youths, aged 15-24 years, were similar to those in infected adults. However, contrary to findings for adults, we found no negative correlation between HIV-DNA level and CD4 Tcell counts in patients with suppressed viral replication [8]. This pattern of immunovirological equilibrium was observed in children and may result from sustained naive CD4 T-lymphocyte production by the thymus. We now report on the levels of naive CD4 (CD4N) and CD8 (CD8N) T lymphocytes, and CD4 recent thymic emigrants (CD4RTE) in the HIV-infected youths included in the ANRS-EP38-IMMIP study, and on the HIV disease factors associated with these three T-lymphocyte subsets.

Patients
The characteristics of the 93 HIV-infected youths included in the ANRS-EP38-IMMIP are presented in Table 1 for the aviremic and viremic groups. The study was approved by the local ethics committees. All patients, and their legal guardians for those under 18 years of age, received written information and signed an informed consent form. The inclusion criteria were (1) HIV-1 infection following vertical transmission, (2) aged over 15 years, (3) no change in treatment status (treated or untreated) during the previous six months, and (4) introduction of care for HIV infection before 1996, to ensure that all patients had similar access to HAART. A single 25 ml blood sample was taken for biological evaluations.
CD4RTE levels were defined as the percentage of naive CD45RA + CCR7 + CD4 + T lymphocytes positive for CD31.

Virological assays
HIV-1 reverse transcriptase (RT) and V3 Env sequences were obtained from blood cell HIV-DNA as previously described (http://www.hivfrenchresistance.org/). HIV-1 B and non-B subtypes were defined by phylogenetic analysis (http://www.hiv-web.lanl.gov). HIV-1 coreceptor usage was determined with the SVMGeno2pheno algorithm, with a 5.75% false positive rate [9]. HIV-RNA was quantified at the clinical sites, whereas HIV-DNA was quantified at a central laboratory, by real-time PCR with the Generic HIV-DNA Cell kit (Biocentric, Bandol, France) [8]. Serological tests for CMV were carried out on frozen plasma, with the CMV IgG Liaison II kit (Diasorin, Antony, France).

Statistical analysis
A full description of study variables has been provided elsewhere [8]. We studied univariate associations between T-lymphocyte subsets and variables defining current and past HIV disease, using the Wilcoxon test for categorical variables, and Pearson's correlation coefficients for continuous variables. Analyses were stratified on the basis of plasma HIV-RNA detection, using a cutoff value of 80 HIV-RNA copies/ml to define aviremic and viremic patients. Multivariate analyses were performed by linear regression. Separate models were built for aviremic and viremic patients, due to interactions between plasma HIV-RNA detection and variables describing HIV disease history (data not shown). For each dependent variable, one full model was adjusted for non collinear variables associated with the dependent variable in univariate analysis at P <0.20. Age, as exposure factor of interest was systematically included in all models, as was current HIV-RNA level for viremic patients, whatever the P-value. Economical models including only variables with P values <0.05 gave similar results, supporting the stability of analyses (data not shown). Variables non included in model because of collinearity are indicated in tables footnotes. Analyses were conducted with SAS statistical software (version 9.2). P<0.05 defined statistical significance.
CD4N percentage was positively correlated with cumulative viremia over the last 10 years (r=0.335, P=0.01). It also tended to be positively correlated with HIV-DNA level in PBMCs, and negatively correlated with the duration of the last period during which plasma HIV-RNA level was below 500 copies/ml ( Table 2). Patients harboring X4R5 viruses had a significantly higher CD4N percentage than patients harboring R5 viruses (medians: 44% vs. 61%, P=0.001). CD4N percentage was not associated with sex, CMV seropositivity, nadir CD4 T-cell percentage, or the time for which CD4 T-cell percentage was below 15% during the lifetime of the patient ( Table 2).
There was a trend towards a higher CD8N percentage in patients with X4R5 viruses than in those with R5 viruses (medians: 45% vs. 35%, respectively P=0.10), and towards a negative correlation with the cumulative duration of HAART over the last 10 years (r=-0.263, P=0.06).
CD8N percentage was not correlated with past HIV-RNA levels or past immunosuppression ( In multivariate analysis, CD4N percentage was independently associated with CD4 Tcell count, X4 coreceptor usage and tended to be associated with cumulative viremia over the last 10 years, after adjustment for age and CDC stage ( Table 2). CD8N percentage was independently associated with coreceptor usage and sex, and tended to be associated with CD4 T-cell count after adjustment for age, CMV serostatus and cumulative duration of HAART over the last 10 years. CD4RTE percentage was independently associated with CD4 T-cell count, sex, and time for which CD4 T-cell percentage was below 15% during the patient's lifetime, after adjustment for age and CMV serostatus.  Figure 1E), but not with CD8N percentage (r=0.132, P=0.64, Figure 1F).
In multivariate analysis, both CD4N and the CD8N percentages were independently associated only with CD4 T-cell count (Table 3). In an alternative model, the CD8N percentage was associated only with CD4 T-cell count after adjustment for age at nadir CD4 T-cell %, cumulative viremia and CMV seropositivity (data not shown).

Discussion
We found that, 15 or more years after perinatal infection, naive CD4 T-lymphocyte levels and thymic activity were preserved and positively correlated with current CD4 T-cell count. High levels of viral replication and/or viral cytopathogenicity over the patient's lifetime were associated with higher levels of naive CD4 T lymphocytes at the time of the study.
The CD4N percentages of our study population were in the range reported for uninfected individuals, corresponding to half to two thirds of total CD4 T lymphocytes [11][12]. CD4RTE values were also similar to those reported for healthy young adults [13]. Our results extend those of a previous study on 20 perinatally infected youths on suppressive HAART, which reported normal levels of CD4 T-lymphocyte subsets and thymic activity [12]. CD8N T-lymphocyte percentages were slightly lower than those reported for healthy young adults in our aviremic patients and were lower still in viremic patients [14]. The different patterns of CD4 and CD8 T-lymphocyte restoration reflect the more robust expansion of memory CD8 than of memory CD4 T lymphocytes in response to HIV replication, chemotherapy-induced lymphodepletion and aging [6,[15][16].
CD4RTE percentage was directly correlated with total CD4 T-lymphocyte count, as were the CD4N and CD8N percentages. In patients infected during adulthood, immune restoration involves both thymus-driven T-lymphocyte production and homeostatic proliferative expansion, and the failure of CD4 T-cell restoration despite prolonged viral suppression is associated with defective thymus function [17][18]. Two of the 93 patients included in the ANRS-EP38-IMMIP study had current CD4 T-cell counts below 350 cells/µl, despite being aviremic. One patient had recently experienced viral blips, whereas the other had remained continuously aviremic for more than six years and was, thus, the only patient that could have been classified as a "low CD4 responder" [18]. Overall, our results highlight the active role of the thymus in both qualitative and quantitative immune restoration in the patients studied.
One key finding of this study was that, in patients in which HIV replication was suppressed at the time of the study, higher CD4N levels were associated with higher levels of viral replication in the previous 10 years. Furthermore, there was a trend towards higher CD4N percentages in aviremic than in viremic patients. The results are expressed as percentages, and this association may therefore reflect either an increase in the production of naive T lymphocytes or higher levels of memory CD4 T-lymphocyte destruction in the presence of viral replication. In this second case, higher levels of viral replication would be associated with a higher CD4N percentage and a lower CD4 T-cell count, and we would expect a negative correlation between CD4N and CD4 T-cell count to be observed. By Among patients with suppressed viral replication, CD4N and CD8N percentages were higher in patients harboring X4R5 viruses than in those with R5 viruses. This association is counterintuitive, as X4R5 viruses infect thymocytes and CD4N T lymphocytes more efficiently than R5 viruses, because of the more widespread expression of CXCR4 on these cells [17]. Given that these differences were observed only in aviremic patients, the detection of X4R5 viruses by cell-associated HIV-DNA sequencing of archived virus may reflect past events, such as deeper immunosuppression associated with higher levels of viral replication, and/or infection with more cytopathogenic viruses [22]. However, potent peripheral immune restoration has been observed in children with severe immunosuppression at treatment initiation [23][24][25], and the association between CD4N percentage and viral tropism is consistent with that between CD4N percentage and cumulative viremia.

Acknowledgments
We thank all the patients who agreed to participate in this study. We would also like to thank the nurses and staff members from the various clinical sites. We thank Sandrine   HIV-RNA (log10 copies/ml)