Age-associated decreases in thymic function significantly hinder immune reconstitution in immunodeficient adults. We have hypothesized that thymic function can be pharmacologically enhanced in humans and herein present strong support for this hypothesis. In this prospective randomized analysis of HIV-1–infected adults, GH treatment was associated with robust increases in thymic density, frequency of circulating TRECs within PBMCs, and the number of circulating naive CD4+ T cells. Similar changes were not observed in those who did not receive GH. These data offer compelling evidence that GH enhances human thymopoiesis. Additionally, increased PBMC TREC frequency and T cell gains were most pronounced in GH recipients with higher gains in circulating IGF-1 levels. These findings suggest that IGF-1 occupies an important role in GH-mediated enhancement of T cell production and offer fundamental insight into the mechanism of GH effects on the human immune system.
Further, these findings demonstrate that declines in thymic function are reversible in human adults. Beyond the implications for the development of therapies for HIV-1 disease and bone marrow transplantation, this work also establishes a basis for additional mechanistic and functional studies of thymic recovery.
GH treatment induced striking radiographic changes in the thymus by 6 months, along with sizeable increases in PBMC TREC frequency and circulating naive T cells, indicating enhanced thymic function. Given the close proximity of the thymus to the heart and lungs, histologic confirmation of thymic hyperplasia could not be obtained due to high biopsy risks. Nevertheless, animal studies reporting histologic evidence of thymic hyperplasia with GH treatment (26
), in addition to other corroborating data in this study, suggest that the observed radiographic changes represent functional thymus.
Recent thymic emigrants (RTEs) and naive T cells initially traffic to lymphoid tissues. Therefore, GH-associated increases in peripheral lymphocytes may have begun before such changes were detected in peripheral blood, and increases in TREC frequency and naive T cells may have been underestimated. Nevertheless, the number of circulating CD4+
naive T cells increased markedly with GH treatment. Small increases were also noted in the absence of GH that were comparable to those reported in other HIV-1–infected individuals with good virologic suppression (6
). Despite enhancing thymopoiesis, GH treatment did not substantially increase the number of CD8+
naive T cells. However, after non-naive CD8+
cells were excluded, we did detect considerable gains in naive CD8+
cells. CD11a is expressed on CD8+
T cells in HIV-1–seronegative and untreated HIV-1–seropositive individuals and is associated with functional characteristics of memory effector cells (33
). In our ARV-treated subjects, CD11a was expressed on nearly a quarter of CD8+
cells. This finding adds to evidence (38
) supporting the use of multiple phenotypic markers to identify naive CD8+
T cells in HIV-1 disease.
GH treatment significantly decreased the percentage and number of T cells bearing activation markers. Although the mechanisms of this effect are unclear, GH or IGF-1 may diminish immune activation, as STAT5, a major GH signaling molecule, is necessary for regulatory T cell function (40
) and IGF-1 has been shown to increase T cell production of the regulatory cytokine IL-10 (42
Although GH and IGF-1 enhance multilineage hematopoiesis (26
), GH treatment did not appear to be associated with substantial changes in circulating hematopoietic cells. The number of circulating CD34+
cells (a population that presumably includes prethymic progenitor cells) was not altered. This finding suggests that GH does not enhance thymopoiesis by enhancing bone marrow export of prethymic progenitor cells. We cannot, however, rule out potential effects of GH or IGF-1 on the ability of progenitor cells to successfully engraft and develop in the thymic microenvironment (47
IGF-1, a proximal mediator of the metabolic action of GH, had a key role in the immunologic effects of GH treatment. After 1 year, subjects with higher gains in circulating IGF-1 had 93% more naive CD4+
T cells, 24% more total CD4+
T cells, and a 235% increase in PBMC TREC frequency compared with subjects not receiving GH. However, GH treatment increased thymic density regardless of IGF-1 gains. This may indicate that GH exerts direct effects on the thymus or that its thymic effects are mediated by IGF-1 in a primarily paracrine manner (48
). Nevertheless, baseline levels of IGF-1 and GH secretion were strong and independent predictors of the thymic response to GH, and it is possible that higher basal somatotropic tone may preserve the ability of the thymus to recover function.
While GH-associated gains in thymic density, PBMC TREC frequency, and naive T cells point strongly to enhancement of thymopoiesis, there are alternative interpretations of these data. For instance, since an increased frequency of naive T cell TRECs has been reported in association with increased thymic function in humans (22
), it is not clear why an increase in naive T cell TREC frequency was not observed in our GH recipients. This observation raises the possibility that gains in T cells and PBMC TREC frequency may be due to other GH effects, such as decreased T cell activation. Although analysis of mechanisms was not a primary goal of the study, we explored this possibility further by using repeated-measures regression models to estimate how much of the effect of GH on major outcomes of interest (NCD4 count, total CD4+
T cell count, and PBMC TREC frequency) might be mediated by changes in 3 key variables: thymic density, circulating IGF-1, and T cell activation (data not shown). The results, combined, suggest an important role for IGF-1 on both central and peripheral compartments, a complementary role of thymic density, and no evident role of T cell activation. More specifically, the models estimate that GH-associated increases in NCD4 cells would be 52% lower in the absence of GH-associated increases in circulating IGF-1 and 71% lower in the absence of GH-associated changes in IGF-1 and thymic density. A similar role for IGF-1 and thymic density was found for GH-associated gains in total CD4+
T cells. Gains in PBMC TREC frequency also appeared to be highly dependent on IGF-1, suggesting that IGF-1 is a major indicator (and probable mediator) of most GH effects on the immune system. Additional statistical models suggest that altered T cell activation does not appear to influence GH-associated gains in T cells or PBMC TREC frequency. Another possible explanation for the unexpectedly low frequency of naive T cell TRECs is that GH treatment led to altered trafficking of RTEs. Thus, murine studies have demonstrated that GH therapy promotes the accumulation of RTEs in peripheral lymphoid tissues by increasing RTE expression of adhesion molecules (49
). IL-7 and thyroid hormone also have been found to promote accumulation of TREC-bearing naive T cells and RTEs in lymphoid tissues (51
Finally, the discrepancy between PBMC and naive TREC findings raises yet another important mechanistic question: are GH-associated T cell gains attributable to increased thymic function, peripheral expansion, or both? In this respect, we postulate that GH treatment enhances both thymopoiesis and peripheral naive T cell expansion, possibly through the effects of IGF-1. Evidence for central (thymopoietic) effects of GH and IGF-1 in this study include: (a) prominent increases in thymic density, PBMC TREC frequency, and naive T cells with GH treatment; (b) gains in thymic density in the GH arm that were positively correlated with gains in the percentage and absolute count of NCD4 cells (Spearman rank correlation coefficients were ≥0.70 and reached [P < 0.05] or trended toward [P < 0.12] statistical significance [data not shown]); and (c) a 235% increase in PBMC TREC frequency (P = 0.0001) in those GH recipients with higher gains in IGF-1. Similarly, repeated-measures analysis (described above) estimates that the level of circulating IGF-1 measured 1 month after GH initiation appears to explain 80%–100% of PBMC TREC frequency increases. Data from this study also suggest that GH treatment increases naive T cell expansion in the periphery. Thus, higher GH-associated gains in IGF-1 were associated with an increased frequency of PBMC TRECs but a decreased frequency of naive T cell TRECs. Higher gains in IGF-1 were also associated with a markedly higher increase in NCD4 cells. These findings suggest that IGF-1 may enhance naive T cell expansion, a hypothesis that is supported by ex vivo data from our laboratory demonstrating that human naive T cells express high levels of IGF-1 receptor and that IGF-1 treatment directly enhances human naive T cell proliferation (L.A. Napolitano, unpublished observations).
Given the above consideration, we propose that GH-associated increases in thymic density, PBMC TREC frequency, and NCD4 cells represent de novo T cell production, with possible increases in peripheral T cell expansion. GH treatment was associated with a 30% increase in the CD4+ T cell count, whereas the 1-year increase in the absence of GH was 13%, representing a 2.4-fold increase in CD4+ T cell recovery over 1 year (absolute increases of 73 versus 30 cells/μl per year, respectively). Of note, CD4+ T cell gains continued at least 3 months beyond GH discontinuation, and analysis of 7 patients suggested that, despite reinvolution of the thymus, T cell gains persisted for at least 1 year after GH discontinuation. Five GH recipients with a baseline CD4+ T cell count of less than 200 cells/μl experienced a sustained increase above the clinically significant threshold of 200 cells/μl, with mean CD4+ T cell counts increasing from a baseline of 149 to 261 cells/μl at the final study visit. While these findings hold great promise, additional research is necessary to determine whether thymic recovery is associated with correlates of immune protection. It is feasible that enhanced thymopoiesis may generate increased breadth of the TCR repertoire. Such changes could improve immunity against pathogens, including HIV-1. It is also important to determine whether a recovered thymus retains the ability to successfully execute positive and negative selection so that newly made T cells provide effective immune protection without increased autoreactivity. Additional studies are warranted to further explore the effects of GH on the quality of immune function and to explore specific mechanisms of GH effects on the central and peripheral immune compartments.
The encouraging results of this study invite further consideration of how enhancement of thymopoiesis might be applied to the care of patients with HIV-1 infection. Individuals with advanced lymphopenia despite effective ARV and extended virologic suppression (such as the participants in this study) are likely to have thymic involution (2
) and would be most likely to benefit from enhanced thymopoiesis. It is also possible that therapies designed to enhance thymopoiesis may offer benefit if coadministered at initiation of effective ARV. Nevertheless, we do not believe that the findings of this study support the general use of GH treatment in the setting of HIV-1 disease at this time, as it is not possible to determine whether these GH-associated changes in immunologic correlates confer true clinical benefit. Also, the predominance of white males in our study, and the selection of individuals most likely to benefit from increased thymopoiesis, may limit the generalizability of our findings. Finally, this study included only ARV-treated patients with well-suppressed HIV-1 viremia. The use of GH in the setting of uncontrolled HIV-1 replication has not been studied, and therefore, its use should be avoided in the absence of virologic control.
GH has been safely administered to HIV-1–infected patients for wasting and in research studies of HIV-1 lipodystrophy (30
). Most AEs in this study were mild and resolved with time or adjustment of GH dosing. Nonetheless, the potential benefit of GH must be weighed against the potential toxicity of treatment. Although we excluded enrollment of those at greatest risk for GH toxicity, we documented several adverse effects of GH treatment. Dose adjustment or discontinuation was required for diabetes or hyperglycemia in 22% of GH recipients, despite normal baseline glucose tolerance. All glucose abnormalities resolved with GH discontinuation. Of greater concern were 3 cases of lymphoma — 2 in patients who did not receive GH — detected incidentally by radiographic studies during screening or study visits. Since lymphoma is relatively common in the setting of advanced immunodeficiency, subjects at highest risk for lymphoma would likely be included in any study of immune reconstitution. This possibility should be considered in the design of future studies with GH, or other potential immune-based therapies such as IL-7, and emphasizes the need to identify the specific mechanisms that mediate thymic recovery so that more narrowly targeted therapies can be developed.
In summary, we have shown that GH treatment is associated with enhanced thymopoiesis and peripheral T cell recovery. Many immune effects appear to be mediated by IGF-1, and it is possible that GH or IGF-1 also enhances T cell gains by promoting expansion of peripheral T cells. To our knowledge, this is the first intervention to successfully increase thymic function in humans. These promising findings have significant applications for the development of immune-based therapies in HIV-1 disease and other immunodeficient states and present substantial opportunity to explore mechanisms that mediate thymic recovery.