Thymic involvement in recovery of immunity among HIV-infected adults on highly active antiretroviral therapy

Human immunodeficiency virus (HIV) infection is characterized by a decrease in both function and number of CD4 T cells, associated with viral replication and failure of homeostatic mechanisms involved in regeneration of immunity. The objective of highly active antiretroviral therapy (HAART) is to suppress HIV replication and achieve recovery of immunity in HIV-infected patients. This potent antiretroviral therapy allows successful suppression of HIV replication, with undetectable plasma HIV RNA levels being reached in the vast majority of patients on treatment. However, the degree of CD4 T cell repopulation induced by HAART is very heterogeneous between different patients,1 and depends on several host and virological factors.2 Previous studies and reviews have described broadly the characteristics of recovery of immunity in HIV-infected adults on HAART.1,2 Biphasic kinetics of CD4 cell repopulation following HAART have been proposed: an early increase phase associated with cellular redistribution from lymph nodes, and a later phase characterized by an increase in naive CD4 T cell counts associated with cellular de novo generation. During the last few years, thymopoiesis has been suggested as one of the mechanisms of synthesis involved in T cell recovery following HAART. In the present review, we have summarized most of the recent and significant evidence that supports the involvement of the thymus in the recovery of immunity in HIV-infected adults on HAART. The role of the thymus in the regeneration of naive T cells has been suggested previously in T cell depletion scenarios other than HIV disease, such as in intensive chemotherapy treatment received by patients diagnosed with cancer.3 In recent years, several studies have reported indirect evidence suggesting that thymic output might also be involved in recovery of immunity observed among HIV-infected patients on HAART.4–13 The use of the mediastinal imaging procedure has demonstrated the presence of thymic tissue in HIV-infected patients. By means of a single image, and representative mediastinal images, obtained by a thorax computerized tomography (CT) scan-based procedure, McCune et al.4 were the first to report the presence of abundant thymic tissue in HIV-infected adults. Later studies, using other imaging approaches, have also shown that increases in CD4 T cell counts induced by HAART were correlated with augmentation in thymic mass in both HIV-infected children5 and adults6 on HAART. Using a more reliable CT scan procedure, combined with the study of other molecular, cytometric and phenotypical thymic function-related markers, our group has observed recently that there is strong evidence for the involvement of thymic output in the recovery of immunity in antiretroviral-naive HIV-infected patients following HAART (Figure 1).7 Moreover, evidence of thymic function has also been reported among heavily-treated HIV-infected adults in whom virology has failed despite HAART.8 In the last few years, age has been strongly related to T cell repopulation in individuals receiving HAART.14 Thus, younger individuals have been reported to show higher CD4 T cell repopulation levels under HAART than older patients. Some authors have suggested that this response might be because younger patients have a more preserved thymic function and mass,14 which has been associated with immunological homeostasis produced mainly during childhood or young adulthood.15,16 Later studies, which analysed the influence of age on recovery of immunity among HIV-infected adults on HAART, have confirmed these findings.17,18 Several procedures to assess thymic output in HIV-infected patients have been proposed. The markers used most frequently include thymic volume, measured by means of different thorax imaging procedures, quantification of T cell receptor excision circle (TREC)-bearing cells, and determination of naive T cell subsets. All these tools have advantages and limitations for the accurate determination of thymic function. Thymic size has been proposed as a useful and accessible way of assessing the impact of thymus on immunological recovery in HIV-infected individuals on HAART. Several thorax imaging approaches have been developed,4–10 the most widely used being the mediastinal CT scan. Using this method, the presence of abundant thymic tissue in HIV-infected patients was demonstrated several years ago.4 Later, a relationship was identified between T cell repopulation and the increase in thymic size induced by HAART in both HIV-infected children5 and adults.6 However, this procedure, based on measuring the size of the thymus from a single representative CT image, had an important limitation: mediastinal fat could be mistaken for thymic tissue. Recently, we described an improved CT scan-based procedure, using detailed delineation of thymic shadows along several thin sections, which has allowed the accurate differentiation of thymic volume, minimizing the possible inclusion of mediastinal fat.7 Using this method, our group demonstrated that before implementation of HAART, thymic volume could be considered an independent predictor for an early successful immunological response induced by HAART in HIV-infected adults.9 Therefore, antiretroviral therapy-naive patients, who have a higher thymic volume at baseline, are more likely to be repopulated earlier with CD4 T cells than those patients who have lower thymic volume at that time (Figure 2). These results have been confirmed recently in a cohort of antiretroviral-naive HIV-infected children.10 TRECs are products of T cell receptor gene rearrangements that are generated during lymphocyte maturation in the thymus.11,12 TRECs have been proposed as reliable thymic function-related markers because of their inherent characteristics: they are stable, they do not duplicate during the mitosis phase, and they become diluted rapidly during T cell proliferation. TREC levels are reported to be increased in HIV-infected adults following HAART, suggesting that renewed thymic function occurs in this setting.11 However, several limitations associated with this thymic function-related marker have been proposed. The increase in TREC-bearing cells might also be the result of concomitant reduction in T cell proliferation and immune activation following HAART.12 Mechanisms of redistribution of TREC-bearing cells from lymph nodes to the peripheral blood have not been excluded completely.13 Despite these limitations, the association between the increase in TREC-bearing cells and thymic volume following HAART in an HIV-infected adult cohort, which has been described recently,7 and evidence from animal models,19 support the use of this molecular tool as a thymic function-related marker. Recent evidence supports the view that immune reconstitution via the thymus is characterized by an increase in TREC+ naive T cells. Since this new T cell repertoire is polyclonal, it is able to mediate immune responses to new antigens. In contrast, reconstitution via peripheral expansion of pre-existing T cells may limit the T cell repertoire to pre-existing memory T cells that have a reduced capacity to respond to new antigens.20 Thus, T cell reconstitution via thymopoiesis is associated with the presence of naive-phenotype T cells and high TREC levels, whereas T cell regeneration via peripheral expansion is associated with memory-phenotype T cells and low TREC levels.5,21 According to this, the memory T cell count increase takes place within the first weeks or months after HAART.1,2 However, naive T cell subset repopulation tends to occur more slowly during the first year on potent antiretroviral therapy.1,2,11 Therefore, determining the phenotype of peripheral blood T cells can be helpful in evaluating the involvement of thymic function in the reconstitution of immunity in HIV-infected patients on HAART. In the setting of HIV disease in children, naive T cell repopulation has been reported to occur within the first weeks on HAART,22,23 and to be correlated with the enlargement of thymic volume.5 In contrast, in the setting of adult HIV infection, the association between thymic function and recovery of naive T cell counts has been a controversial topic for several years. HIV-infected adults with abundant thymic tissue at baseline have been reported to achieve higher initial increases in naive T cell counts following HAART than patients whose thymic volume was less than abundant before starting potent therapy.7 However, a significant improvement in naive T cell counts has also been demonstrated among thymectomized HIV-infected patients undergoing HAART.24 Thus, this recovery of immunity has been attributed to mechanisms other than thymopoiesis, such as peripheral expansion25 and/or a rapid conversion into memory/ effector-phenotype T cells.26 In contrast, new evidence supports the use of naive T cell counts as reliable thymic function-related markers in adult patients. Franco et al.7 demonstrated that naive CD4 cell count increases, achieved in a population of antiretroviral-naive HIV-infected adults, were strongly correlated with increases in TREC-bearing cell counts following HAART. Moreover, repopulation of naive cell subsets is more likely to be higher in younger than older patients,18 regardless of their naive T cell count at the start of antiretroviral therapy. It has been suggested that thymus-dependent pathways are involved in this immune recovery. Several immunological strategies have been designed following knowledge of thymic involvement in the recovery of immunity in HIV-infected patients on HAART. These strategies should enhance CD4 T cell repopulation in adult HIV-infected patients who do not achieve significant immunological recovery despite HAART. Thus, the function and use of several endogenous cytokines related to thymic output, such as interleukin (IL)-2 and IL-7, have been analysed. Combination therapies, including IL-2 plus HAART aimed at boosting T cell activity and maturation, have contributed significantly to the restoration of immune function in HIV-infected patients. HIV-infected adults on this regimen have been reported to achieve significantly higher increases in CD4 T cell numbers than patients treated only with HAART.27,28 This immunological approach might be a feasible therapeutic tool in virologically suppressed HIV-infected patients with CD4 cell counts lower than 200 cells/mm3 despite an appropriate HAART regimen. However, recent studies have suggested that the repopulation of the immune system, achieved by IL-2 treatment, may not be associated with a thymopoietic mechanism. This recovery of immunity is more likely to be the result of peripheral expansion of existing naive CD4 T cells, rather than an increase in thymic output.29,30 During the last few years, several lines of evidence have demonstrated the involvement of IL-7 levels in the restoration of homeostasis following T cell depletion scenarios,31 mainly through thymus-dependent pathways.32 Furthermore, a recent study has suggested that high levels of endogenous IL-7, produced in response to T cell depletion before HAART, may activate thymic rebound in adult HIV-infected patients on HAART.33 Unfortunately, exogenous administration of IL-7 should be approached with caution, since this cytokine has also been shown to increase naive T cell susceptibility to HIV infection.34 All these immunological approaches described herein, including growth hormone administration,35 have an important limitation. HIV-infected patients showing irreversible thymus damage will not be able to produce new T cells despite the exogenous administration of these substances. However, the application of these immunological findings in the setting of other immunodeficiency diseases might be indicated. Furthermore, thymic transplantation in HIV-infected adults with long-term viral suppression could be an alternative approach to explore in future studies. The combined use of thymic function-related markers shows that thymic tissue plays a role in CD4 T cell recovery reached under potent antiretroviral therapy in both HIV-infected children and adults. Evidence supporting this hypothesis is described above. However, short-term application of this immunological evidence cannot be considered at the present time. In the future, this insight may improve our understanding of the pathogenesis of HIV infection and enable the design of new approaches to HIV infection management. This work has been supported by research grants from Fondo Para la Investigación y Prevención del SIDA en España (FIPSE 2132/00 and FIPSE 12304/02, integrated by Ministerio de Sanidad y Consumo, Abbott Laboratories, Boehringer Ingelheim, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp and Dohme and Roche Laboratories). This work has also been partially supported by Red de Investigación en SIDA (RIS) by Ministerio de Sanidad y Consumo (Spain). Corresponding author. Tel: +34-955-012-391; Fax: +34-955-012-390; E-mail: mleal@cica.es Figure 1. Response of thymic function-related markers to HAART. Mean ± s.e.m. (a) naive CD4 T cells/mm3, (b) TRECs/mm3, and (c) thymic volume (cm3) are shown for 15 HIV-1 infected subjects starting HAART (*Wilcoxon signed rank test, P < 0.05). Adapted from reference 7. Figure 2. Kaplan–Meier estimates of the probability of CD4 T cell repopulation among 37 untreated HIV-infected patients studied, depending on thymic volume (TV) at baseline. After 189 days (median of follow-up), eight out of 19 (42.5%) patients with TV ³3.22 cm3 had reached the required CD4 T cell repopulation, compared with only three out of 18 (16.6%) patients with TV < 3.22 cm3 (χ2 test, P = 0.003). Adapted from reference 9.