Angela M. Crawley

Soluble IL-7Rα (sCD127) Inhibits IL-7 Activity and Is Increased in HIV Infection

Soluble CD127 (sCD127) appears to play an important role in the immunopathogenesis of several chronic infections, multiple sclerosis, and various cancers. The function of sCD127 and whether it influences IL-7 bioavailability or activity is unknown. In this study, we demonstrated that recombinant and native sources of sCD127 significantly inhibited IL-7–mediated STAT5 and Akt phosphorylation in CD8+ T cells. IL-7–mediated proliferation and Bcl-2 expression were similarly reduced by sCD127. In each case, native sCD127 inhibited IL-7 activity to a greater degree than rsCD127. Anti–IL-7 activity was inherent to human plasma and could be reversed by depletion of CD127, revealing for the first time the biological activity of naturally occurring sCD127. Plasma sCD127 concentrations were increased in HIV+ individuals compared with HIV− controls, correlated with IL-7 levels, and remained unchanged in HIV+ individuals following 1 y of effective antiretroviral therapy. Determining the regulation and function of sCD127 may be critical for understanding both the pathogenesis of diseases in which IL-7 likely has a role (e.g., HIV infection, cancer) and its potential impact on IL-7 as a therapeutic approach.

IL-7-dependent STAT-5 activation and CD8+ T cell proliferation are impaired in HIV infection

This study tests the hypothesis that IL‐7 signaling and activity of CD8+ T cells are impaired in HIV infection. IL‐7 is necessary for optimal CTL activity and T cell survival and proliferation. Defects in IL‐7R signaling may contribute to impaired activity of IL‐7 observed in progressive HIV disease. A decreased proportion of CD8+ T cells expressing the IL‐7Rα chain (CD127) in progressive HIV disease would be expected to affect IL‐7 activity. Alternatively, disease‐associated defects of remaining CD8+CD127+ T cells may influence IL‐7 responsiveness. Therefore, the IL‐7 responsiveness of CD8+CD127+ T cells from HIV– and untreated or treated HIV+ individuals was investigated. Blood was collected from HIV– and untreated or effectively treated HIV+ (<50 viral copies/ml for >1 year) individuals, and CD8+CD127+ T cells were isolated and cultured with IL‐7. Indicators of IL‐7 signaling (P‐STAT5) and activity (Bcl‐2 and proliferation) were evaluated by flow cytometry. Isolated CD8+CD127+ T cells from untreated HIV+ individuals expressed significantly less P‐STAT5 in response to IL‐7 compared with CD8+CD127+ T cells from HIV– individuals. In effectively treated HIV+ individuals, CD8+CD127+ T cells also expressed significantly lower levels of P‐STAT5 compared with HIV– individuals. IL‐7‐dependent proliferation of CD8+CD127+ T cells from untreated HIV+ individuals was similarly impaired. In contrast, IL‐7‐induced Bcl‐2 expression was not impaired in CD8+CD127+ T cells from HIV+ individuals. These data demonstrate that IL‐7/IL‐7R dysfunction in HIV infection may contribute to IL‐7‐specific signaling defects. Decreased, IL‐7‐dependent activation of STAT5 and impaired proliferation may negatively impact the maintenance of CD8+ T cell responsiveness in HIV infection.

The influence of HIV on CD127 expression and its potential implications for IL-7 therapy

Interleukin-7 (IL-7) is critical for early T-cell development and plays an important role in T-cell homeostasis, differentiation and function. Signalling via the IL-7 receptor is dependent on the expression of its components, IL-7Rα (CD127) and IL-2Rγ (CD132) and is mediated in part by alterations in CD127 expression levels in different cell subsets. Naïve and memory T-cells express high levels of CD127, while effector cells are CD127(lo) and retention of the receptor is thought to influence the development of memory cells. Reduced expression of CD127 has been associated with markers of disease severity in HIV infection and other chronic viral infections as well as in various cancers. In HIV infection, decreased CD127 expression on T-cells is correlated with reduced CD4(+) T-cell counts, increased viral replication and immune activation. The loss of IL-7 activity, due to decreased CD127 expression, may contribute to the observed loss of CD8(+) cytotoxic T lymphocyte (CTL) activity in HIV infection. The downregulation of CD127 expression in HIV infection may be due to host (e.g. IL-7, IL-4, immune activation) and/or viral (e.g. HIV-tat) factors and mechanisms of receptor regulation may differ by cell type. In addition, the expression of a soluble form of CD127 (sCD127) has been shown to be increased in HIV infection. This protein may affect IL-7 activity in vivo and therefore may have implications for IL-7-based therapies which are currently being tested in clinical trials. Understanding how CD127 is regulated during HIV infection will provide insight for the development of novel therapeutics to improve immune function and anti-viral T-cell activity.

Interleukin-4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γC) cytokines decrease CD127 expression on CD4+ and CD8+ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8+ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA+) CD8+ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8+ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8+ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8+ T cells, IL‐4 is a potential contributor to impaired CD8+ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.

IL-2 receptor γ chain cytokines differentially regulate human CD8+CD127+ and CD8+CD127− T cell division and susceptibility to apoptosis

Expression of IL-7 receptor α (CD127) is associated with naive and memory (i.e. non-effector) CD8+ T cell phenotypes. Effector CD8+ T cells are predominantly CD127− and most die by apoptosis. Therefore, CD127 appears to be a marker for CD8+ T cell differentiation, yet its role in CD8+ T cell survival and memory development is unclear. To address this, we investigated the cell death and cell division of isolated CD8+CD127+ and CD8+CD127− T cells in response to common IL-2 receptor γ chain (γC) cytokines other than IL-7. We show here that (i) memory cells (CD127+CD45RA−) divide frequently in response to either IL-2, -4 or -15; (ii) IL-2 and -15 enhance cell division in effector–memory-like cells (CD127−CD45RA+) while IL-4 enhances the cell division of effector cells (CD127−CD45RA−); (iii) CD8+CD127+ T cells are more sensitive to the anti-apoptotic effects of IL-2 or IL-15 than CD8+CD127− T cells and (iv) CD8+CD127+ T cell produce more Bcl-2 in response to IL-2 or IL-15 compared with CD8+CD127− T cells. Therefore, CD8+CD127+ and CD8+CD127− T cells differ in their responsiveness to cell division and anti-apoptotic signals from IL-2, -4 and -15. This suggests a role for γC cytokines in the pathogenesis of diseases in which CD127 expression is altered on CD8+ T cells such as in progressive viral infections and cancer.

Interleukin-7 enhances memory CD8+ T-cell recall responses in health but its activity is impaired in human immunodeficiency virus infection

Memory CD8+ T cells regain function during a recall response, but the requirement of signals in addition to antigen during a secondary immune response is unknown. In this study, the ability of interleukin‐7 (IL‐7) to enhance memory CD8+ CD45RA− CD127+ T‐cell responses in health and in human immunodeficiency virus (HIV) infection was investigated. CD8+ T‐cell‐depleted peripheral blood mononuclear cells (PBMCs) from HIV− and untreated HIV+ donors were pulsed with a cytomegalovirus/Epstein–Barr virus/influenza (CEF) peptide pool, and co‐cultured with autologous memory CD8+ T cells in the presence of IL‐7. Cell survival and the function of memory CD8+ T‐cell subsets were then evaluated. Memory CD8+ T‐cell proliferation and interferon‐γ (IFN‐γ) production was enhanced by the presence of antigen, and the addition of IL‐7 further enhanced antigen‐induced proliferation. In HIV+ individuals, the presence of antigen enhanced IFN‐γ production to a small degree but did not enhance proliferation. Lastly, IL‐7 did not enhance antigen‐mediated proliferation of memory CD8+ T cells from HIV+ individuals. IL‐7 therefore appears to have a role in secondary immune responses and its activity is impaired in memory CD8+ T cells from HIV+ individuals. These results further our understanding of the signals involved in secondary immune responses, and provide new insight into the loss of CD8+ T‐cell function in HIV infection.

Development of a Quantitative Bead Capture Assay for Soluble IL-7 Receptor Alpha in Human Plasma

Background IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. Methodology/Principal Findings We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assays range was 3.2–1000 ng/mL. The concentration of plasma sCD127 was 164±104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. Conclusions/Significance This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

Jak/STAT and PI3K signaling pathways have both common and distinct roles in IL-7-mediated activities in human CD8+ T cells

IL‐7 plays an important role in T cell survival, function, and memory cell development, yet the role of cytokine signaling pathways in these processes has not been fully elucidated. Moreover, the underlying mechanisms for the observed impairment of IL‐7 activity in diseases, such as HIV infection, breast cancer, and autoimmunity, are not well understood. It was therefore hypothesized that IL‐7‐induced signaling molecules could be linked with distinct IL‐7‐associated activities. To address this, the activation and functional associations of IL‐7‐induced signaling pathways, specifically antigen‐independent activities that are relevant to T cell homeostasis, were examined. Low concentrations of IL‐7 (100 pg/ml) are capable of activating the Jak‐STAT and PI3K signaling pathways, whereas higher concentrations (500–1000 pg/ml) were required to induce Bcl‐2 production and glucose uptake. Even higher concentrations of IL‐7 (10,000 pg/ml) were needed to induce cell proliferation and intracellular accumulation of perforin. Inhibition of Jak activation reduced IL‐7‐induced Bcl‐2 and perforin production, whereas inhibition of Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. This study suggests a complex control of IL‐7‐associated activities in the absence of antigen stimulation. These data may provide insights into mechanisms of impaired IL‐7 signaling and function in disease and could be relevant for the study of IL‐7‐based immunotherapeutics. Specifically, this study has linked STAT5 and PI3K activation to shared and distinct IL‐7‐associated activities in human CD8+ T cells.

In Vitro HIV Type 1 Infection Indirectly Alters CD127 Expression on CD8+ T Cells

Decreased expression of interleukin (IL)-7 receptor α (CD127) on CD8(+) T cells in progressive HIV disease suggests a role for CD127 regulation in HIV immunopathogenesis. The direct effect of HIV on CD127 expression has not been explored to explain these in vivo findings. Peripheral blood mononuclear cells (PBMCs) or isolated CD8(+) T cells from healthy individuals were cultured with either X4 (HIV-1(IIIB)), R5 (HIV-1(BaL)), dual tropic (HIV-1(CS204)), or replication-incompetent (HIV(8E5)) strains of HIV. Both X4 and R5 strains transiently decreased CD127 expression on CD8(+) T cells in PBMC cultures but had no effect on isolated CD8(+) T cell cultures. Isolated CD8(+) T cells exposed to either (1) PBMCs incubated with HIV and cultured in a transwell or (2) supernatants from PBMCs incubated with HIV resulted in decreased CD127 expression. Under no conditions did the replication-incompetent HIV strain affect CD127 expression. As observed in vivo, infection of PBMCs with HIV in vitro results in the downregulation of CD127 surface expression on CD8(+) T cells. Collectively, these data indicate that soluble factor(s) released as a result of HIV infection regulate CD127 expression. Further elucidation of the mechanism(s) of CD127 downregulation will provide important insights into the immunopathogenesis of HIV disease.

Complexed soluble IL-7 receptor α and IL-7 increase IL-7-mediated proliferation and viability of CD8⁺ T-cells in vitro.

Many soluble cytokine receptors inhibit cytokine bioactivity, while others prolong ligand activity. The biological role of an endogenous soluble form of IL-7Rα, or its therapeutic effects on CD8(+) T-cells are unknown. We demonstrate that recombinant IL-7Rα-Fc, when pre-incubated with IL-7, enhances IL-7-induced CD8(+) T-cell proliferation and viability of human or murine CD8(+) T-cells. Receptor blocking experiments confirmed IL-7-specific activity. These data demonstrate that exogenous soluble IL-7Rα significantly enhances CD8(+) T-cell responses to IL-7 in vitro and paves the way for future research to determine its therapeutic potential to restore impaired CD8(+) T-cell function in disease.