The role of the thymus in the regeneration of naive T cells has been suggested in T-cell depletion scenarios , including immunological recovery after highly active retroviral therapy (HAART) in HIV-infected patients [2–8]. Increments in naive T-cell absolute numbers have been observed in both children and adult patients after HAART , as well as increments in the number of cells carrying a molecular marker of a recent thymic emigrant, T-cell receptor excision circles (TREC) [2–4,6]. Thymic volume measurement using radiological methods [5–8] has also been useful to determine that thymic function might be involved in the repopulation of T cells. Using these tools, we have recently reported an increase in thymic function during 48 weeks under HAART in adult HIV-infected patients .
Factors involved in the thymic rebound in HIV-infected patients under HAART are still unknown and might be of relevance in HIV infection. IL-7 could be a good candidate for the following reasons: (i) IL-7 is a pleiotropic and non-redundant cytokine  that plays a critical role in the survival, proliferation and development of B and T cell precursors and naive T cells [11–17]. (ii) In animal models, it enhances the peripheral expansion of mature T cells after T-cell depletion in both thymus-deficient  and thymus-bearing hosts, although IL-7 was found to enhance preferentially the thymic-dependent pathways in T-cell regeneration . It has recently been observed that the administration of exogenous IL-7 in thymic organ cultures and mice enhances the generation of TREC-bearing cells . (iii) In humans, an inverse correlation between CD4 cell numbers and IL-7 serum levels has been observed in different T-cell depletion scenarios , including HIV infection [21–24]. These data provide evidence that IL-7 is a master regulator of T-cell homeostasis [21,25]. The aim of this work was to evaluate the role of IL-7 in thymic rebound in adult HIV-infected patients under HAART.
Patients and methods
In August 1998, the Viral Hepatitis and AIDS Study Group of Virgen del Rocio University Hospital began a study in order to determine the role of the thymus in the repopulation of T cells in adult patients under HAART. From that date, a total of 54 consecutive antiretroviral-naive HIV-infected patients starting HAART were asked to participate in the study. Up to February 2002, 49 patients had agreed to participate. HAART was defined as a combination of at least two nucleoside reverse transcriptase inhibitors plus a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. Patients were evaluated at baseline and at weeks 12, 24, 36 and 48 of treatment. All patients reported therapy compliance equal to or higher than 95%. Each timepoint was analysed for CD4, CD8, and CD3 T cell subsets and HIV-1-RNA levels in fresh samples. Isolated peripheral blood mononuclear cells (PBMC) were kept frozen in liquid nitrogen, and serum samples were kept at −20°C until further determinations. Forty-seven patients had available samples for TREC determination. Thoracic computed tomography (CT) for measuring thymic volume was taken in all patients at baseline and at weeks 12, 24 and 48 of treatment. Written informed consent was obtained from all patients, and the ethical committee of the Hospital approved the study.
The determination of IL-7 levels was performed using a high sensitivity colorimetric enzyme-linked immunosorbent assay (IL-7 immunoassay kit; R&D System QuantikineHS, Minneapolis, MN, USA) according to the manufacturer's recommendations.
Thoracic computed tomography
Mediastinic CT was performed using a modified previously described method . Briefly, coded samples were always measured by the same radiologist using a 3000 GE Sytec Scanner using 5 mm thick contiguous sections at 5 mm intervals. Thymic tissue was carefully delimited in all the slices between the first and the last in order to exclude mediastinic fat infiltration (a high density for soft tissue and a low density for surrounding fat). CT Sytec version 4 software (General Electrics Medical Systems, Milwaukee, WI, USA) integrated all the defined thymic areas along the slices (the first and last slice volumes being automatically halved to account for partial volume averaging) to calculate thymic volume.
At each timepoint, CD4, CD8, CD3, CD19 (B cells), and CD16+CD3− (natural killer cells) absolute numbers were determined in fresh samples, as previously described . Briefly, a frozen aliquot was used to determine the percentages of naive CD4 T cells (CD4+CD45RA+CD45RO−), memory CD4 T cells (CD4+CD45RA−CD45RO+), naive CD8 T cells (CD8+CD45RA+CD11alow), and memory CD8 T cells (CD8+CD45RA−CD11ahigh). Naive and memory CD4 and CD8 T-cell absolute numbers were calculated according to CD4 and CD8 T-cell counts obtained from fresh blood samples.
T-cell receptor excision circle-bearing cells quantification in peripheral blood mononuclear cells
One proposed molecular marker for the determination of recent thymic cell emigrants is the quantification of TREC generated during the rearrangement of T-cell receptor genes. A polymerase chain reaction (PCR)-based method for quantifying the δRec– ΨJα TREC number has been described by Douek et al. . We have adapted this method to a real-time PCR using a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) for the quantification of both the characteristic signal-joint sequences harboured in the generated TREC and the β-globin gene (in order to normalize by DNA content) . TREC levels in each PBMC sample were determined using a standard curve of a cloned signal-joint fragment of 375 base pairs (bp) obtained from a pediatric thymic sample . In addition to the TREC per 106 PBMC, the absolute counts of TREC/μl was derived for each sample. Because all the TREC in PBMC are included in the CD3 subpopulation, the TREC/106 PBMC proportion was corrected by the percentage of CD3 cells found by flow cytometry in the PBMC, yielding the number of TREC/106 CD3 cells. This proportion was then multiplied by the absolute CD3 cell counts obtained in fresh blood by flow cytometry.
Plasma HIV-1 RNA was measured by a quantitative PCR (HIV Monitor test kit version 1.5; Roche Molecular Systems, Hoffman-La Roche, Basel, Switzerland) according to the manufacturer's instructions. This assay has a detection limit of 50 HIV-1-RNA copies/ml.
Statistical analysis was performed using the Statistical Package for the Social Sciences software package (SPSS 10.0, Chicago, Illinois, USA). All continuous variables were expressed as mean ± standard error of the mean (SEM). Categorical variables were expressed as the number of cases (percentage). Pearson's correlation coefficient analysis was used to assess bivariate correlations between all the variables, with the exception of TREC-bearing cells, in which Spearman's test was used because this variable did not follow a normal distribution. An analysis of variance test for repeated measurements was used to denote significant changes during the follow-up, and to compare these values with baseline values corrected for multiple comparisons. Friedman and Wilcoxon tests were used for TREC-bearing cell measurements because a small number of patients had all the determinations during the follow-up (n = 11). Multiple linear regression was performed to assess independent associations between variables using a stepwise procedure. Predictor variables were considered for inclusion in the statistical model if the association with the parameter of interest (changes in thymic volume versus baseline) were observed in the univariate analysis (P < 0.1) or if the predictor variable had previously been related to thymopoiesis (i.e. age).
Patients’ baseline characteristics are summarized in Table 1. The correlation between baseline IL-7 levels and some parameters of interest was analysed. An inverse correlation was found with total, memory and naive CD4 T-cell counts, with naive CD8 T-cell counts, with total naive T-cell counts (naive CD4 plus naive CD8 T cells), with TREC-bearing cells, and with thymic volume. No correlation was found between IL-7 levels and the rest of the parameters studied (Table 2). The correlation between IL-7 levels and thymic function-associated parameters is shown in Fig. 1.
For a better understanding of the association between IL-7 levels and the thymic function of patients under HAART, changes in IL-7 levels, thymic volume, T-cell phenotype and TREC-bearing cells were analysed during follow-up. In all patients, HIV-1-plasma viremia decreased more than 1 log10 copies/ml during follow-up, reaching viral load levels below the limit of detection (≤ 50 copies/ml) in more than 90% of them. During follow-up, increased levels of thymic function-related markers along with a significant decrease in IL-7 levels were found. Memory T cells did not increase after 48 weeks under HAART. IL-7 levels were lower during follow-up compared with baseline levels at statistical significance (P < 0.05) (Fig. 2a). All thymic function-associated parameters showed a significant increase during follow-up compared with baseline values (P < 0.05), with the exception of TREC-bearing cells at week 48 of follow-up (P = 0.671) (Fig. 2b–d). On the other hand, statistical significance was not reached during follow-up when total memory T cells (memory CD4 T cells plus memory CD8 T cells) were analysed compared with baseline values (P > 0.05) (Fig. 2c). Total CD8 T-cell counts did not significantly increase during the follow-up (P > 0.05).
IL-7 is independently associated with changes in thymic volume
Multiple linear regression was performed to consider changes in thymic volume at each timepoint of follow-up versus baseline, as a dependent variable. Variables showing a univariate association level of less than 0.1 with changes in thymic volume were entered into the multivariate analysis as potential independently associated variables (Table 3). Only baseline IL-7 levels were independently associated with changes in thymic volume at weeks 12 (P = 0.019), 24 (P = 0.008) and 48 (P = 0.001) versus baseline. Fig. 3a–c shows the correlation between IL-7 levels at baseline and the changes in thymic volume. No variables were found to be independently associated with changes in total naive T cells or changes in TREC-bearing cell numbers at any timepoint of follow-up compare with baseline (data not shown).
The data presented here suggest that higher IL-7 levels produced in response to T-cell depletion before therapy may activate thymic rebound in adult HIV-infected patients under HAART.
Thymic tissue measurement is now an accepted biological marker for thymus function . We have recently reported an increase in thymic function, measuring thymic volume, as early as 12 weeks after HAART in HIV-infected patients. Also, a direct correlation between thymic volume and TREC- bearing cells and an inverse correlation between thymic volume and age has been found, supporting the reliability of the technique for measuring the thymus . We have also reported that thymic volume was an independent predictor for CD4 T-cell repopulation in HIV-infected adults under HAART .
The role of IL-7 in T-cell homeostasis in response to T-cell depletion has not been found for other cytokines and soluble factors [19,21,22]. In animal models, it has been shown that IL-7 enhances the peripheral expansion of mature T cells , and preferentially thymic-dependent pathways in T-cell generation after T-cell depletion . Recent studies have shown that the administration of exogenous IL-7 to in-vitro systems and mice increased the number of TREC-bearing cells produced by the thymus . These observations and others [28,29] strongly suggest an important role of this cytokine in the regeneration of T cells by the thymus, at least in murine models.
The inverse correlation between IL-7 levels and total CD4 T cells before therapy supports the important role of IL-7 in response to T-cell depletion. This is the first study in which an inverse correlation between IL-7 levels and thymic volume before therapy was observed. Consequently, IL-7 seems not to be necessarily produced in the thymus, but probably in the periphery. Patients with an absence of a radiological image of the thymus thus had higher levels of IL-7. These data support findings previously reported, in which IL-7 in untreated patients should preferentially be produced by peripheral lymphoid organs in response to T-cell depletion .
A significant decrease in IL-7 levels during follow-up compared with baseline levels was observed, probably in response to the significant increase in total CD4 T-cells during follow-up. These results suggest that the homeostatic response is fast and sensitive in HIV replication control after HAART. The increase in thymic function-associated parameters and the stability in the memory T-cell subset during follow-up suggest that repopulation of HIV-infected adults under HAART is preferentially led by the thymus, at least in the first 12 weeks of follow-up. We speculate that the difference between naive and memory T cells during follow up could be explained partly by the different mechanisms of homeostatic cell proliferation. Naive T cells proliferate relatively slowly and depend on the contact of self-major histocompatibility complex and peptide ligands. In T-cell depletion conditions, when the total number of naive T cells decreases below a certain level, IL-7 is increased because of a reduction in the number of T-cell receptors and MHC–peptide interactions . The fast MHC-independent homeostatic regulation of memory T cells could perhaps explain why IL-7 seems not to be involved in their expansion.
The IL-7 level was the only variable associated with changes in thymic volume by multiple linear regression analysis during the follow-up. These data strongly suggest that a baseline IL-7 level is a factor that might trigger thymic rebound in HIV-infected adults under HAART. Two conditions have to be considered, that there is no irreversible damage of the thymus and no irreversible damage of the peripheral lymphoid organs. No parameter was independently associated with changes in either naive T cells or TREC-bearing cells at each timepoint compared with the baseline. This fact could be explained because the naive T-cell number is not a true thymic-related marker in HIV infection. Therefore, a naive T-cell increase could be caused by peripheral expansion after HAART [30,31]. The small number of patients could explain the absence of a correlation between IL-7 levels and changes in TREC-bearing cell counts during follow-up. Also, it might be possible that thymic volume measurement could be more reliable and a better tool for the determination of thymic function compared with TREC-bearing cell determination .
In our opinion, IL-7 should not be considered to be an immunomodulatory therapy, at least in HIV infection, because patients with irreversible damage to the thymus can not produce new T cells independently of the levels of IL-7. Moreover, the administration of IL-7 in order to increase T-cell counts is also discouraged, because this cytokine has been shown to enhance HIV replication [22,32–34]. HIV infection could thus be considered a ‘soil immunodeficiency’ and not a ‘signal immunodeficiency'. Nevertheless, the potential use of IL-7 as a therapy in other types of immunocompromised patients might be indicated.
In summary, our data show for the first time in humans that IL-7 might be involved in the activation of increased thymic volume after T-cell depletion, and specifically in adult HIV-infected patients undergoing HAART. The absence of a radiological image of the thymus in some patients with higher levels of IL-7 also supports the theory that IL-7 is mainly produced in peripheral organs in untreated patients. New soluble factors involved in the modification of thymic function in patients under HAART and new therapeutic strategies in order to improve immune reconstitution in these patients are under research.
The authors would like to thank the individuals who participated in this study, as well as M. Olivera and A. Gayoso for excellent technical assistance.
Sponsorship: This work was supported by research grants from the Fondo para la Investigación y Prevención del SIDA en España (FIPSE 2132/00 and FIPSE 12304/02) integrated by the Ministerio de Sanidad y Consumo, Abbott Laboratories, Boehringer Ingelgeim, Bristol Myers Squibb, GlaxoSmithKline, Merck Sharp and Dohme and Roche, Fondo de Investigaciones Sanitarias (FIS 00/0521), and by the kind support of Fundación Wellcome, España. This work was also partially supported by Red de Investigación en SIDA (RIS) by Ministerio de Sanidad y Consumo (SPAIN).
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Keywords:© 2003 Lippincott Williams & Wilkins, Inc.
Highly active antiretroviral therapy; HIV; IL-7; immune reconstitution; thymus