In this study, CD4 and CD8 T cells from patients with ICL, compared with those from controls, showed decreased STAT-5 phosphorylation in response to IL-7 that did not correlate with CD127 expression on T cells but correlated inversely in CD4 T cells with serum IL-7 levels. This decreased responsiveness was observed in total CD4 T cells, as well as within both central and effector memory subsets. Decreased IL-7 responsiveness was further supported by evidence of diminished gene upregulation and cycling of T cells after IL-7 stimulation. These data suggest a possible role of defective IL-7 signaling in the perturbation of T-cell homeostasis during ICL.
In our analysis, patients with ICL showed decreased responsiveness to IL-7, compared with control subjects, in the presence of elevated serum IL-7 levels. This phenomenon may be partially related to decreased IL-7Rα expression in CD4 T cells during ICL, observed in this and prior studies [1
]. As previously observed [1
], the present study confirmed that patients with ICL have a lower percentage of naive CD4 T cells, which express CD127 constitutively and could account for these differences. However, receptor expression and STAT-5 phosphorylation did not correlate in either CD4 T cells or CD8 T cells, although diminished responsiveness was observed predominantly in central and effector memory CD4 T cells. Because of limited sample availability, serum IL-7 analysis was only conducted in a subset of patients with ICL. In each of the patients with ICL we analyzed, serum IL-7 levels were markedly higher than reported levels in healthy adults, indicating that, at least among these patients, the defect did not seem to be limited IL-7 production or availability. The possibilities of anti-IL-7 antibodies or increased levels of sCD127 that could interfere by IL-7 binding were also excluded in our cohort. Alternatively, decreased IL-7 responsiveness ex vivo may be due to tachyphylaxis from exposure to high levels of IL-7 in vivo,
which could also explain why both CD4 T cells and CD8 T cells are affected. Supporting this, serum levels of IL-7 correlated negatively with IL-7 responsiveness in CD4 T cells during ICL, although this correlation was not observed in CD8 T cells. This could be related to the overall lower levels of CD127 expression in CD8 T cells or to the small sample size. Previous studies have also found that, while IL-7 is essential for homeostatic proliferation of CD4 T cells, memory CD8 T cells also rely on IL-15 [27
], although this relationship could be altered in the presence of high IL-7 levels [29
]. In addition, cytokine-mediated suppression of IL-7Rα transcription involves different molecular mechanisms in CD4 T cells and CD8 T cells [14
], as does stimulation via T-cell receptor versus IL-7 [30
To evaluate whether decreased responsiveness to IL-7 was due to decreased IL-7R expression, cells were also stimulated with IL-2, a γc chain cytokine that, like IL-7, signals via STAT-5 phosphorylation. CD4 T cells from patients with ICL showed only minimally decreased responsiveness to IL-2, indicating that although an overall defect in γc cytokine signaling may be contributing to decreased IL-7 responsiveness of CD4 T cells in ICL, the primary defect appears to lie in the IL-7 pathway itself, as this was not observed in CD8 T cells and responses to IL-15 appeared intact in a subset of our cohort.
The presence of increased proportions but not total counts of γδ T cells during ICL may simply be a function of depletion of CD4 T cells (which are αβ). However, elevated serum IL-7 levels may underlie expansion of γδ T cells, as previous studies have shown that IL-7 prevents T-cell precursors from differentiating into αβ T cells [31
]. Indeed, IL-7 levels were found to correlate strongly with the frequency of γδ T cells. Moreover, mice deficient in αβ T cells show increased homeostatic proliferation of γδ T cells, a process that was enhanced by both IL-15 and IL-7 [33
], while IL-7 knockout mice do not make γδ T cells [34
]. As many γδ T cells normally reside in the lymphoid tissue of the gastrointestinal tract, the increased frequencies we observed may also be a reflection of differential trafficking of these cells from gut mucosal tissue through peripheral blood. Lymph node, gut, and other tissue biopsy specimens from patients with ICL could clarify the question of possible CD4 T-cell redistribution in individuals with ICL.
A recent study of IL-7 signaling during HIV infection found that decreased IL-7 signaling correlated with total T-cell count, indicating the degree of IL-7 responsiveness may serve as a marker of the ability of lymphopenic patients to reconstitute T-cell counts [35
]. Whether the lymphopenia in ICL is secondary to decreased production, increased death, or redistribution of CD4 T cells remains unclear. Our data support that the defective IL-7 responsiveness may be contributing to both increased death of T cells, which has been previously described [19
], and inadequate cycling. Gene expression in PBMCs from patients with ICL, compared with those from healthy controls, showed overall lower increases in BCL2
induction after stimulation with IL-7, indicating that IL-7 may have less of an antiapoptotic effect during ICL. More importantly, there was clear evidence of decreased downregulation of p27kip1
in PBMCs from patients with ICL, a cyclin-dependent kinase inhibitor whose destabilization is the main mechanism of cycle progression after IL-7 stimulation. It thus appears that the T cells in patients with ICL may be unable to optimally respond to IL-7, despite the observed high serum levels.
Previous studies have shown increased turnover of CD4 T cells during ICL [1
]. A recent primate study showed that IL-7 induced T-cell redistribution from peripheral blood to secondary lymphoid organs, resulting in a transient drop in peripheral CD4 T cell numbers [37
]. This initial drop in CD4 T-cell count was also observed when IL-7 was administered to patients with HIV infection [20
]. In all studies, however, exogenous IL-7 resulted in a significant increase in peripheral T-cell counts in humans with lymphopenic conditions (HIV infection and cancer) without significant toxicity [20
], even though IL-7 signaling during HIV infection is defective [35
]. Further, the resulting T-cell expansions occurred primarily in the naive subset [22
], which is disproportionately depleted in ICL, and included significant expansion of CD8 T cells [20
], which may be beneficial given the association of very low CD8 T-cell counts with poor prognosis in ICL [1
In conclusion, in this study we demonstrated that T cells from patients with ICL display blunted in vitro responses to IL-7, which may reflect in vivo phenomena. Defective IL-7 signaling could, therefore, be important in the pathogenesis and persistence of lymphopenia in this setting and may have implications in the potential use of IL-7 as immunotherapy during ICL, which is currently being investigated in a phase I study (ClinicalTrials.gov identifier: NCT00839436). On the basis of these findings, IL-7 remains a potentially promising agent to study in patients with ICL, although ascertaining the appropriate dosing needed to obtain the desired effects of improved cycling and survival of T cells may be challenging.