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Effects of IL-7 on Early Human Thymocyte Progenitor Cells In Vitro and in SCID-hu Thy/Liv Mice¹

Laura A. Napolitano^2,*,, Cheryl A. Stoddart^*, Mary Beth Hanley^*, Eric Wieder³ and Joseph M. McCune^*,,

^* Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141; and Departments of Medicine and Microbiology and Immunology, University of California, San Francisco, CA 94143

	Abstract

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IL-7 is a critical component of thymopoiesis in animals and has recently been shown to play an important role in T cell homeostasis. Although there is increasing interest in the use of IL-7 for the treatment of lymphopenia caused by the HIV type 1, evidence that IL-7 may accelerate HIV replication has raised concerns regarding its use in this setting. We sought to identify the effects of IL-7 on human thymocyte survival and to determine the impact of IL-7 administration on in vivo HIV infection of the human thymus. Using in vitro analysis, we show that IL-7 provides potent anti-apoptotic and proliferative signals to early thymocyte progenitors. Analysis of CD34⁺ subpopulations demonstrates that surface IL-7 receptor is expressed on most CD34^highCD5⁺CD1a^- thymocytes and that this subpopulation appears to be one of the earliest maturation stages responsive to the effects of IL-7. Thus, IL-7 provides survival signals to human thymocytes before surface expression of CD1a. CD4⁺CD8⁺ thymocytes are relatively unresponsive to IL-7, although IL-7 protects these cells from dexamethasone-induced apoptosis. IL-7 has a predominantly proliferative effect on mature CD4⁺CD3⁺CD8^- and CD8⁺CD3⁺CD4^- thymocytes. In contrast to the in vitro findings, we observe that in vivo administration of IL-7 to SCID-hu Thy/Liv mice does not appear to enhance thymocyte survival nor does it appear to accelerate HIV infection. Given the growing interest in the use of IL-7 for the treatment of human immunodeficiency, these findings support additional investigation into its in vivo effects on thymopoiesis and HIV infection.

	Introduction

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Interleukin-7 is a cytokine that is essential for normal lymphocyte development (reviewed in Ref.1). Originally described as a B cell growth factor (2), IL-7 has subsequently been found to be essential for thymopoiesis in rodents. "Knockout" models deficient in either IL-7 or in the -chain of the IL-7R demonstrate a massive reduction of cells within both the thymus and peripheral lymphoid tissues (3, 4). Over-expression of Bcl-2 in IL-7R knockout mice overcomes deficits in T cell development, strongly suggesting that IL-7 provides an anti-apoptotic signal that is essential for thymocyte survival (5, 6).

Produced primarily by stromal cells within the bone marrow and lymphoid tissues (7, 8, 9, 10, 11), IL-7 binds to a receptor consisting of an -chain (shared with thymic stromal lymphopoietin) dimerized with a common -chain that is also a component of the IL-2, -4, -7, -9, and -15 receptors (12). Signals transmitted through the IL-7R appear to exert at least two major effects on developing lymphocytes: cell survival and differentiation (13, 14). Most data on the function of IL-7 have been derived from mouse models, and the specific effects of IL-7 on human thymopoiesis are still being elucidated. Mutation of the human IL-7R is associated with a specific defect in T lymphopoiesis (15), suggesting that at least one of the important survival signals provided by IL-7 is targeted to a T lineage progenitor cell. Abs that block the function of IL-7 inhibit the in vitro differentiation of human CD34⁺ progenitors derived from thymus or fetal liver to the CD4⁺CD3^-CD8^- (intrathymic T progenitor (ITTP)⁴ cell, also termed "CD4 intermediate single-positive (ISP)") stage, suggesting that IL-7 is important to the survival of the ITTP (13, 16). More recently, analysis of IL-7 effects on the survival of selected human thymocyte subpopulations showed that IL-7 enhances the survival of CD3^-/low thymocytes and increases TCR rearrangement (17). IL-7 also appears to facilitate the survival of positively selected CD4⁺CD8⁺ double-positive (DP) thymocytes in murine studies (18).

Several recent studies suggest that IL-7 also plays an important role in T cell homeostasis (11, 19, 20, 21), and there is growing interest in its therapeutic application as an immunomodulatory agent in the setting of human immunodeficiency. Nevertheless, the fact that IL-7 has been shown to enhance HIV infection, replication, and cytopathicity (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) has raised some concerns regarding its potential use in HIV disease. We sought to identify more specifically the effects of IL-7 on human thymocyte survival and to study the effects of IL-7 on the HIV-infected human thymus.

	Materials and Methods

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Thymus

Human fetal thymus tissue was acquired from Advanced Biosciences Resources (Alameda, CA) and processed immediately after receipt.

Cell culture

Human fetal thymocyte cultures were established using two independent systems: hanging drop culture and thymic organ culture (TOC). These systems were established under serum-free conditions so that the specific effects of IL-7 could be better characterized. Comparison of Yssel’s serum-free medium (Gemini Bio-Products, Woodland, CA) to RPMI supplemented with 10% FBS demonstrated no differences in thymocyte survival between these medium preparations (data not shown).

Hanging drop cultures. Single-cell suspensions of thymocytes and thymic stroma were cultured in a hanging drop system adapted from the technique of Merkenschlager et al. (33) as follows: thymi were washed, gently sheared with sterile knives in serum-free Yssel’s medium, and treated with 0.2 mg/ml collagenase B (Roche, Indianapolis, IN) and 100 U/ml DNase (Calbiochem, San Diego, CA) for 45 min at room temperature. Cell suspensions were vigorously pipetted and passed through a 70-µm cell strainer to obtain a single-cell suspension of thymocytes (comprising >95% of total cells) and thymic stroma (comprising <5% of total cells). Cells were centrifuged and resuspended at 50 x 10⁶ cells/ml in serum-free Yssel’s medium with or without recombinant human IL-7 (R&D Systems, Minneapolis, MN), and 40 µl of cells were plated into a 20-µl Terasaki-type well (Ge-Nunc Module; Nalge Nunc International, Rochester, NY). Plates were inverted and cells were cultured as hanging drops in a humidified chamber at 37°C with 5% CO₂. Cultures were terminated after 1–3 days, and cells were counted and subjected to multiparameter flow cytometry. Viable cell counts were performed using a standard hemocytometer with trypan blue exclusion. The number of thymocytes within each thymocyte subpopulation was determined by multiplying the total number of viable thymocytes (determined by cell count) by the percentage of each thymocyte subpopulation within the live thymocyte gate, as determined by flow cytometry.

TOCs. Thymi were dissected into small pieces (1–3 mm³) using sterile knives, transferred directly onto sterile filters (Millipore, Bedford, MA), and placed atop Gelfoam (Pharmacia-Upjohn, Kalamazoo, MI) rafts in 1 ml serum-free Yssel’s medium with or without IL-7 in 12-well plates. Cultures were incubated at 37°C in 5% CO₂ and terminated after 3–7 days. Individual thymus pieces were dispersed and washed before staining for FACS analysis.

Dexamethasone treatment. Thymocytes were cultured in 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO) in serum-free Yssel’s medium in the presence or absence of recombinant human IL-7.

FACS staining and analysis

Dispersed thymocytes from hanging drops and TOCs were washed, pelleted, and resuspended in 50 µl of Ab mixture as detailed below. Surface staining was performed in the dark at 4°C for 25 min.

Four-color phenotypic analysis. Phenotypic surface staining was performed in calcium/magnesium-free PBS supplemented with 1% BSA (PBS BSA), using fluoresceinated Ab against: CD3-FITC, CD34-PE, CD3-PerCP (all from BD Biosciences, San Jose, CA), and CD8-allophycocyanin (Caltag Laboratories, Burlingame, CA).

Apoptosis measurement. Three-color phenotypic surface analysis was performed in PBS BSA supplemented with 1.5 mM CaCl₂ by staining with the following fluoresceinated Ab against: CD4-PE (BD Biosciences), CD3-PerCP, and CD8-allophycocyanin (Caltag Laboratories) or with CD34-PE-Cy5 (Beckman Coulter, Brea, CA), CD1a-PE, and CD5-allophycocyanin (both from Caltag Laboratories). To label apoptotic cells, annexin V-green fluorescent protein (kindly provided by J. Ernst, University of California, San Francisco, CA) was added for the final 10 min.

Detection of cell proliferation. Thymocytes were incubated in 10 µM 5-bromo-2'-deoxyuridine (BrdU, Roche) for 14–20 h (hanging drops) or in 100 µM BrdU for 24 h TOC before harvest. Phenotypic surface staining was performed in PBS BSA using one of the three-color phenotypic panels described in Apoptosis measurement. When surface staining was completed, cells were rinsed, pelleted, and then fixed and permeablized in 1% paraformaldehyde/0.01% Tween 20 in PBS for 1 h in the dark at room temperature. After rinsing, cells were resuspended in anti-BrdU-FITC Ab (BD Biosciences) diluted in 10 mg/ml DNase I (Sigma-Aldrich) for 30 min in the dark at room temperature. Cells were rinsed and subjected to immediate FACS analysis. Alternatively, movement through the cell cycle was assessed by intracellular staining for Ki67 (see below).

Detection of intracellular proteins. Phenotypic surface staining, fixation, and permeabilization were performed as previously described for the detection of cell proliferation. Intracellular staining was performed as follows: HIV p24 using 1 mg/ml human gammaglobulin (Gemini Bio-Products) was added as a blocking reagent during fixation and permeabilization. After rinsing, cells were resuspended in anti-p24-FITC Ab (Beckman Coulter) in PBS BSA for 30 min in the dark at 4°C. Cells were rinsed and subjected to immediate FACS analysis. For Bcl-2 following fixation and permeabilization, cells were rinsed and resuspended in anti-Bcl-2-FITC Ab (DAKO, Carpinteria, CA) in PBS BSA and incubated for 30 min in the dark at 4°C. Cells were rinsed and subjected to immediate FACS analysis. For Ki67, after fixation and permeabilization, cells were rinsed and resuspended in PBS BSA containing 1 mg/ml human gammaglobulin and anti-Ki67-FITC (BD PharMingen, San Diego, CA). Cells were incubated for 30 min in the dark at 4°C, rinsed, and subjected to immediate FACS analysis.

IL-7R analysis. Surface staining for the IL-7R chain (CD127) was performed on thymocytes freshly dispersed from thymus tissue. Staining was performed in PBS BSA using fluoresceinated Ab against: CD127-PE (Beckman Coulter), CD8-FITC, CD3-PerCP (both from BD Biosciences), and CD4-allophycocyanin (Caltag Laboratories) or with CD1a FITC (BD PharMingen), CD127-PE, CD34-PE-Cy5, and CD5-allophycocyanin. Quantitative analysis of surface IL-7R was conducted using the Quantikine detection kit (BD Biosciences) according to the manufacturer’s protocol. Calculations were performed using a fluorochrome to protein ratio of 0.9, as specified by the manufacturer of the anti-CD127-PE Ab, and assumed a saturating Ab concentration with an average of 1.5 CD127 molecules bound to each Ab.

Data analysis. FACS data were acquired with a FACSCalibur or FACS Vantage flow cytometer. Analysis of FACS data was performed with CellQuest (BD Biosciences) or FlowJo (Tree Star, San Carlos, CA) software.

IL-7 treatment of SCID-hu Thy/Liv mice

All procedures involving SCID-hu Thy/Liv mice were approved by the Committee on Animal Research of the University of California (San Francisco, CA). SCID-hu Thy/Liv mice were generated as previously described (34, 35) and maintained under pathogen-free conditions. Animals in a given cohort were constructed from human fetal tissue from a single donor. IL-7-treated mice were injected i.p. with 0.5, 1.5, or 5 µg of carrier-free human IL-7 (R&D Systems) every 12 h for 7–21 days. The biologic activity of IL-7 was validated by a T cell proliferation assay performed by the manufacturer just before shipment. In experiments where IL-7 was administered to HIV-infected mice, the Thy/Liv implants were directly inoculated with 50 µl of virus (2000 tissue culture-50% infective dose) or with sterile tissue culture medium. Implants were harvested at the indicated time points, placed into sterile calcium/magnesium-free PBS supplemented with 2% FBS, and dispersed into a single-cell suspension. Thymocytes were then counted and aliquoted for p24 ELISA, branched DNA assay for HIV RNA, and FACS analysis, as previously described (36, 37, 38). In some cases, if adequate cells were available, analyses of thymocyte proliferation (Ki67 measurement) and apoptosis were also performed.

Plasma IL-7 determinations

EDTA plasma from SCID-hu Thy/Liv mice was prepared from blood obtained at the time of Thy/Liv organ harvest and stored at -70°C. Aliquots were thawed and analyzed in duplicate for human IL-7 using a commercially available, high sensitivity immunoassay (QuantikineHS IL-7 Immunoassay kit, R&D Systems).

Data analysis

Data were imported into StatView (version 5.0, Cary, NC) from Microsoft Excel or FlowJo (Tree Star) for graphing and statistical analysis. Unpaired t test analysis was used to calculate the probability values for all experiments unless otherwise indicated.

	Results

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IL-7 promotes the survival of selected thymocyte subpopulations

The effects of IL-7 on the viability of human thymocytes were examined. Human thymocytes were cultured in serum-free medium in the absence or presence of IL-7. Phenotypic subpopulation analysis was performed on the input cells using multiparameter flow cytometry (Fig. 1). After 2 days of culture, viable cells were counted and phenotypic analysis was repeated (Fig. 2A). Overall thymocyte viability was 50% on day 2 in the absence of IL-7 and 75% in the presence of IL-7 (p = 0.008, data not shown). The survival of ITTP cells was especially dependent upon the presence of IL-7. In the absence of IL-7, only 8% of ITTPs were recovered after 2 days of culture whereas the number of ITTP reached 115% of the input cell number when IL-7 was present (p < 0.0001). Thus, IL-7 alone was sufficient to preserve the survival of serum-starved ITTPs. CD34⁺ thymocyte survival was also significantly enhanced by IL-7, increasing from 10 to 48% of input cells in the presence of IL-7 (p = 0.0002). The effect of IL-7 on the viability of DP thymocytes was less pronounced (p = 0.01). Further analysis was performed on immature (CD3^-/low), partially mature (CD3^int), and mature (CD3^high) DP cells to examine the effects of IL-7 on the viability of these subpopulations. The most pronounced effect of IL-7 was seen in the CD3^high DP cells (p = 0.0001, data not shown). The recovery of CD8⁺CD4^-CD3⁺ (CD8 SP or SP8) medullary thymocytes was enhanced in the presence of IL-7 (p = 0.002), but no statistically significant effect was seen in CD4⁺CD8^-CD3⁺ (CD4 SP or SP4) cells.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Analysis of human thymocyte subpopulations. Representative characterization of thymocyte maturation stages using multiparameter flow cytometry with four-color phenotypic analysis. Thymocyte subpopulations are listed in order from the most immature to the most mature subpopulations in the lower panel on the left. Representative percentage distributions in fetal thymus are derived from at least three experiments and are listed on the right of the lower panel. Percentages are expressed for all thymocytes within the live gate.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. IL-7 promotes survival of selected human thymocyte subpopulations by inhibiting apoptosis and stimulating proliferation. Hanging drop cultures were established in serum-free medium in the presence or absence of IL-7. A, IL-7 promotes the viability of CD34+, ITTP, DP, and SP8 subpopulations. Cell count and multiparameter flow cytometry were performed on input cells and repeated after 2 days in culture. The most profound survival effect was seen in the CD34+ and ITTP subpopulations. Findings are representative of three experiments, each performed in triplicate. B, IL-7 exerts a potent anti-apoptotic effect on the ITTP and CD34+ subpopulations in a dose-dependent manner. The anti-apoptotic effects on more mature thymocytes are significant but less pronounced. C, IL-7 induces a strong dose-dependent proliferative effect on the CD34+, ITTP, and SP subpopulations but not on DP thymocytes. B and C, Hanging drop cultures were established with 0–1250 ng/ml of IL-7 and analyzed after 2 days in culture. Findings are representative of three experiments each performed in triplicate. Similar findings were obtained in three experiments using the TOC system. D, Surface expression of the IL-7R correlates with the biologic response to IL-7. Percentages of IL-7R+ cells and receptor density (IL-7R per IL-7R+ cell) are displayed. Analysis was performed on uncultured cells, freshly isolated from thymus. Percentages are representative of at least five experiments, and quantification of receptor density is representative of at least two experiments.

To more completely dissect the components of the survival signal provided by IL-7, we measured its effects on human thymocyte apoptosis and proliferation (Fig. 2, B and C). Thymocytes were cultured in serum-free medium containing 0–1250 ng/ml IL-7 and analyzed after 2 days of culture. Using multiparameter flow cytometry with phenotypic analysis, apoptosis was measured by surface binding of annexin V to cells within the live gate, thus identifying cells in early stages of apoptosis. There was a marked, dose-dependent anti-apoptotic effect of IL-7 on the ITTP subpopulation (Fig. 2B). This effect was significant at 10 ng/ml (p = 0.02) and maximum at 50 ng/ml (p = 0.002). A strong anti-apoptotic effect was also observed in CD34⁺ thymocytes (p = 0.004 at 50 ng/ml). No anti-apoptotic effect was detected on the total DP subpopulation, although there was a significant effect on the CD3^-/low and the CD3^high DP subpopulations (p = 0.006 and 0.004, respectively, data not shown). A small, but significant, reduction in apoptosis was detected in SP4 and SP8 cells (p = 0.004 and 0.003, respectively, at 50 ng/ml). Proliferation of thymocytes was measured by incorporation of BrdU using multiparameter flow cytometry (Fig. 2C). Similar to results seen in the apoptosis assay, IL-7 exerted potent proliferative effects on the earliest thymocyte progenitors, e.g., the CD34⁺ and ITTP subpopulations. The degree of IL-7-induced proliferation in these subpopulations was comparable, with a significant effect detectable at 10 ng/ml (p ≤ 0.05) and a maximum effect at 50 ng/ml (p < 0.005). To exclude the possibility that the observed effects of IL-7 on ITTP cells were not simply due to a primary effect on CD34⁺ cells followed by rapid maturation to the ITTP stage in culture, the effects of IL-7 were measured on sorted cell subpopulations placed into culture in the absence or presence of IL-7 for 2 days. The anti-apoptotic and proliferative effects of IL-7 were similar on sorted CD34⁺CD1a⁺CD3^-CD4^-CD8^- and CD34^-CD1a⁺CD3^-CD4⁺CD8^- subpopulations (data not shown). In contrast to its relatively minor anti-apoptotic effects on SP4 and SP8 thymocytes (Fig. 2B), IL-7 induced a strong proliferative response in these subpopulations (p < 0.0001). This effect was significant at a concentration as low as 2 ng/ml (p ≤ 0.05, data not shown) and maximum at 50 ng/ml. There was a minor but significant effect of IL-7 on DP proliferation that was seen predominantly in the more mature CD3^high DP cells and to a lesser degree in the more immature CD3^-/low DP cells (data not shown).

Surface expression of the IL-7R was measured on human thymocytes using multiparameter flow cytometry with an Ab directed against the IL-7R chain (Fig. 2D). Approximately 75% of CD34⁺ cells and 80% of ITTP cells expressed surface IL-7R. The fraction of IL-7R-positive cells decreased considerably after maturation to the DP stage, with fewer than 40% of DP cells expressing IL-7R, and then increased again to >65% on more mature SP4 and SP8 thymocytes. Analysis of DP subpopulations showed that a higher fraction of immature CD3^-/low DP cells and more mature CD3^high DP cells expressed IL-7R (49% and 56%, respectively, data not shown) compared with CD3^int DP cells (of which 31% were IL-7R-positive, data not shown). The surface receptor density of the IL-7R was calculated using standardized PE beads to calculate the number of PE-conjugated Abs bound to the surface of each cell. Analysis of IL-7R-positive cells revealed an approximate IL-7R density of 2000 IL-7 receptors on the surface of CD34⁺ and ITTP cells, 1200 receptors on DP cells, and 2500 receptors on SP4 and SP8 cells.

Effects of IL-7 on CD34⁺ thymocytes

To determine the effects of IL-7 on cells that have recently emigrated from the bone marrow into the thymus, further analysis was performed on CD34⁺ thymocytes (Fig. 3). As previously described (39), the surface expression of CD34 was highest on the least mature CD34^highCD5^-CD1a^- thymocytes and decreased progressively with further maturation to the CD34^highCD5⁺CD1a^-, the CD34^highCD5⁺CD1a⁺, and the CD34⁺/^-CD5⁺CD1a⁺CD4⁺ (ITTP) stages (data not shown). The number of rare CD34^highCD5^-CD1a^- thymocytes (≤0.2% of all thymocytes) was markedly reduced after 2 days in serum-free culture, and the addition of IL-7 had no significant effect on the survival of this subpopulation. In contrast, IL-7 significantly decreased apoptosis (p = 0.0007) and enhanced proliferation (p = 0.02) of the CD34^highCD5⁺CD1a^- subpopulation. IL-7 had similar, although somewhat less pronounced, effects on the CD34^highCD5⁺CD1a⁺ subpopulation (p = 0.02 and 0.04 for apoptosis and proliferation, respectively), which is regarded to be the earliest stage of thymocytes committed to the T cell lineage (39). Analysis of IL-7R expression on the CD34⁺ subpopulation (Fig. 3, lower panel) revealed surface receptor expression of 28% on CD34^highCD5^-CD1a^- thymocytes, 86% on CD34^highCD5⁺CD1a^- thymocytes, and 52% on CD34^highCD5⁺CD1a⁺ thymocytes. The expression of surface IL-7R thus correlated with the biologic response to IL-7 in each of these subpopulations.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. CD34highCD5+CD1a- thymocytes express surface IL-7R and demonstrate an anti-apoptotic and proliferative response to IL-7. Hanging drop cultures (upper panel) were established in serum-free medium in the presence or absence of IL-7 at 10 ng/ml. Cells were harvested and analyzed by multiparameter flow cytometry after 2 days in culture. Effects of IL-7 on apoptosis (upper left) and proliferation (upper right) of CD34+ thymocyte subpopulations are displayed. Findings are representative of six experiments each performed in triplicate. Results were similar at 50 ng/ml of IL-7. Surface expression of IL-7R on CD34high thymocyte subsets (lower panel). Analysis was performed on uncultured cells that had been freshly isolated from thymus. Receptor expression correlates with the observed biologic response to IL-7. Data are representative of three experiments.

IL-7-mediated up-regulation of Bcl-2 has been shown to promote the survival of murine and human T cells (5, 17, 18, 40). Because over-expression of Bcl-2 abrogates glucocorticoid-induced apoptosis of murine thymocytes (41), we examined the effects of IL-7 on glucocorticoid (dexamethasone) treatment of human thymocytes. Thymocytes were cultured with dexamethasone under serum-free conditions for 2 days in the presence or absence of IL-7 (Fig. 4, upper panel). Dexamethasone treatment was associated with a marked increase in apoptosis of the ITTP and DP subpopulations. In the presence of dexamethasone, the fraction of apoptotic ITTP cells increased from 20 to 57% (p = 0.003) and apoptotic DP cells increased from 7 to 47% (p < 0.0001). Less than 1% of input ITTP cells and 14% of input DP cells remained alive after 2 days of culture with dexamethasone (data not shown). Dexamethasone induced a more moderate increase in the fraction of apoptotic CD34⁺ thymocytes (from 7 to 25%, p = 0.03, data not shown). SP8 thymocytes were relatively unaffected by dexamethasone treatment, while SP4 cells showed a small but significant increase in apoptosis. The addition of IL-7 at the initiation of culture with dexamethasone resulted in a significant reduction in the number of apoptotic ITTP and DP thymocytes. IL-7 completely inhibited dexamethasone-induced apoptosis of ITTP thymocytes (p = 0.0008) and afforded near-complete protection from dexamethasone-induced apoptosis to CD34⁺ thymocytes (p = 0.05, data not shown) and DP thymocytes (p < 0.0001). Further analysis of CD3^-/low, CD3^int, and CD3^high DP subpopulations revealed that IL-7 conferred protection from dexamethasone-induced apoptosis at all stages of DP maturation (data not shown). SP thymocytes remained relatively resistant to dexamethasone-induced apoptosis in the presence or absence of IL-7.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Effects of IL-7 on dexamethasone-induced apoptosis and Bcl-2 expression. Hanging drop cultures were established in the absence or presence of 50 ng/ml of IL-7 and analyzed after 2 days in culture. IL-7 prevents dexamethasone (dex)-induced apoptosis in ITTP and DP thymocytes (upper panel). Findings are representative of three experiments each performed in triplicate. IL-7 increases intracellular Bcl-2 in ITTP and SP thymocytes, but not in DP thymocytes (lower panel). Findings are representative of three experiments each performed in triplicate. Experiments in the upper and lower panels were performed separately. MFI, mean fluorescence intensity.

Effects of IL-7 on HIV-infected and uninfected thymus in SCID-hu Thy/Live mice

Effects of IL-7 on HIV-infected SCID-hu Thy/Liv mice. HIV infection of the human thymus is associated with thymic destruction and accelerated disease progression (42, 43, 44, 45, 46). Given the profound effects of IL-7 on the survival of human thymocytes, it is possible that IL-7 might prevent or attenuate HIV-mediated destruction of the thymus. However, IL-7 has also been shown to enhance HIV replication and cytopathicity (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). Thus, it is unclear whether IL-7 might enhance thymocyte survival in the HIV-infected thymus or, alternatively, enhance HIV-mediated thymocyte destruction.

The relative contribution of these counterposing effects was assessed directly by administration of human IL-7 to HIV-infected SCID-hu Thy/Liv mice. Mice from a single SCID-hu cohort were inoculated with the HIV molecular clone NL4–3 and treated with IL-7 at a dose of 1 µg/day beginning at the time of viral inoculation. Thymus implants were collected and analyzed 7, 14, and 21 days after inoculation. HIV replication was assayed by three separate methods: cell-associated p24 and HIV RNA (measured on total implant thymocytes) and the percentage of infected cells within each thymocyte subpopulation (determined using multiparameter flow cytometry to detect intracellular p24). Infection with NL4–3 in the absence of IL-7 led to time-dependent increases in HIV RNA and cell-associated p24, with kinetics similar to those typically obtained after inoculation with this virus in this model (Fig. 5A) (36). IL-7 treatment was not associated with any apparent increase in HIV replication (Fig. 5, A and B). In both the IL-7-treated and untreated groups, HIV replication was barely detectable above background values 7 days after HIV inoculation (Fig. 5, A and B), whereas 14 and 21 days after inoculation evidence of HIV infection was detectable but not significantly different between the IL-7-treated and untreated groups.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Effects of IL-7 on HIV-infected and uninfected SCID-hu Thy/Liv human thymus implants. A–C, Implants were inoculated with NL4-3 and harvested weekly for 3 wk. IL-7 treatment does not increase HIV viral burden in implant thymocytes as determined by cell-associated p24 ELISA or HIV bDNA (A) or by flow cytometric intracellular p24 analysis of thymocyte subpopulations. SP8 cells expressing intracellular p24 most likely represent cells that were previously infected at the DP stage (B). IL-7 treatment also has no apparent effect on the extent of loss of various thymocyte subpopulations (C). Analysis was performed on two uninfected animals, five IL-7-treated animals inoculated with NL4-3, and six mock-treated animals inoculated with NL4-3 per time point, with the exception of day 14 when there were six animals in the IL-7-treated group. None of the observed changes were statistically significant. The dotted line in the right panel (A) represents the lower limit of detection for the bDNA assay. A–C, Data are from animals receiving IL-7 at a dose of 1 µg/day and are representative of three independent experiments including dosing regimens of 1, 3, or 10 µg per day. D, Uninfected SCID-hu Thy/Liv mice were treated with IL-7 for 12 days. IL-7 treatment was not associated with any significant change in the number of thymocytes or in the fraction of apoptotic or proliferating thymocytes within any of the analyzed subpopulations. Analysis was performed on 26 IL-7-treated animals and 29 untreated animals. Displayed data are from mice receiving IL-7 at a dose of 3 µg/day and are representative of analysis performed in three separate SCID-hu cohorts.

Effects of IL-7 on uninfected SCID-hu Thy/Liv mice. The cellular effects of IL-7 on the uninfected SCID-hu Thy/Liv thymus were studied in three separate cohorts of mice. In contrast to the effects of IL-7 measured on thymocytes in vitro, treatment of IL-7 in SCID-hu Thy/Liv mice in vivo was not associated with a significant reduction in apoptosis or increase in proliferation (as measured by Ki67) in any thymocyte subpopulation (Fig. 5D).

Bioavailability of IL-7 in SCID-hu Thy/Liv mice. Studies were performed to evaluate the bioavailability and biologic effects of IL-7 in SCID-hu Thy/Liv mice. IL-7 was administered at a dosage of 1, 3, or 10 µg per day. IL-7 therapy was well tolerated, although a 5–10% weight loss was noted in mice receiving IL-7 at 10 µg/day (data not shown). Circulating peak and trough levels of human IL-7 were measured in the plasma of animals receiving either 10 µg/day of human IL-7 (5 µg every 12 h) or saline injection. Peak IL-7 levels, measured 60 min after IL-7 administration, were generally above the upper limits of assay detection despite multiple dilutions (range from 40 pg/ml to >4266 pg/ml in five mice). Trough levels of IL-7, measured 12 h after dosing, ranged from 17 to 70 pg/ml. In contrast, peak and trough levels of IL-7 were undetectable (<0.1 pg/ml) in mice given saline injection. Thus, circulating levels of IL-7 were 2–4 logs higher in IL-7-treated mice. Evidence of IL-7 bioavailability in parenchymal spaces was manifested by the presence of splenomegaly in IL-7-treated animals (Fig. 6). Splenic weight increased in a dose-dependent manner with IL-7 treatment, resulting in a 40% increase in animals receiving 3 µg/day (p = 0.0006) and a 90% increase in animals receiving 10 µg/day (Mann Whitney U test, p < 0.0001).

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Exogenous IL-7 induces splenomegaly in SCID-hu Thy/Liv mice. Bioavailability of IL-7 is documented by the development of splenomegaly in IL-7-treated mice. Spleen weight is increased in a dose-dependent manner with IL-7 treatment. Data represent combined findings from four separate cohorts of uninfected and HIV-infected mice treated with IL-7 at 0, 3, or 10 µg/day for 11–18 days. Increases in spleen weight were present in both infected and uninfected mice, and there were no differences between these two groups. Data on splenomegaly are not available for mice treated with IL-7 at a dose of 1 µg/day.

	Discussion

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We sought to identify the effects of IL-7 on developing thymocytes that had recently emigrated from the bone marrow into the thymus. Subpopulations of thymocytes expressing high levels of CD34 have previously been aligned into a lineage relationship that ranges from those expressing CD34 but not expressing CD5 or CD1a (extremely rare multipotential progenitors with myeloid and lymphoid potential), to those co-expressing CD34 and CD5 (rare multipotential progenitors that retain the capacity to differentiate into NK cells, dendritic cells, and T cells), to those expressing CD34, CD5 and CD1a (believed to be the earliest stage committed to the T cell lineage) (39, 49). Whereas CD34^highCD5^-CD1a^- thymocytes express very low levels of surface IL-7R and do not appear to respond to IL-7-mediated survival and expansion signals, over 80% of CD34^highCD5⁺CD1a^- thymocytes express IL-7R and these cells demonstrate a clear anti-apoptotic and proliferative response to IL-7 treatment. This suggests that the multipotential CD34⁺CD5⁺CD1a^- thymocyte may represent the earliest stage at which IL-7 exerts a survival effect. This finding is notable in light of previous reports that IL-7 is capable of expanding cells within the NKT and dendritic cell lineages (50, 51). Nevertheless, it is difficult to exclude the possibility that IL-7 acts at an even earlier stage of T lineage maturation, because such progenitors are extremely rare and difficult to examine. Furthermore, multipotential CD34^highCD5^-CD1a^- thymocytes may require a synergistic combination of hematopoietic cytokines when cultured under serum-free conditions, and IL-7 alone may not be sufficient to promote survival or expansion of these cells.

Responsiveness to IL-7 is maintained in the T lineage-committed CD34⁺CD5⁺CD1a⁺ subpopulation, and thymocytes become especially responsive to the effects of IL-7 upon maturation to the ITTP stage. Because the survival, expansion, and maturation of ITTP cells is required for the generation of adequate numbers of DP thymocytes, the survival signal provided by IL-7 at this stage is likely to be a critical factor in human thymopoiesis. In contrast, DP thymocytes are less responsive to IL-7 survival signals. Because >95% of DP thymocytes are destined to undergo apoptosis during the process of thymocyte selection, it is not surprising that this subpopulation would have lower levels of IL-7R and a diminished anti-apoptotic and proliferative response to IL-7. Medullary SP thymocytes express high levels of IL-7R and demonstrate a strong proliferative response to IL-7. Although we observed that treatment of thymocytes with IL-7 results in enhanced recovery of SP8 in comparison to SP4 thymocytes (Fig. 2A), the survival signals conferred by IL-7 are identical in these two subpopulations (Fig. 2, B and C). This makes it unlikely that the increased ratio of SP8 to SP4 cells is attributable to a differential survival signal of IL-7 on SP4 and SP8 thymocytes. These data are consistent with previous studies demonstrating that IL-7 may influence the maturation of DP thymocytes and induce preferential differentiation to SP8 cells (14, 52). It is also possible that these findings are partly a result of in vitro artifact; Merkenschlager et al. (33) reported artifactual decreases in the CD4/CD8 ratio with hanging drop cultures.

As has been previously observed (5, 17, 18, 40), we found that the survival effects of IL-7 on human thymocyte subpopulations correlated with the induction of Bcl-2. The greatest induction of Bcl-2 expression by IL-7 was seen in ITTPs, which exhibited the most profound anti-apoptotic response to IL-7. Glucocorticoid-induced apoptosis of thymocytes has been shown to be mediated through a "mitochondrial" pathway and is reversible by over-expression of Bcl-2 (41, 53). Accordingly, we found that IL-7 reversed the anti-apoptotic effect of dexamethasone on CD34⁺ and ITTP thymocytes. Interestingly, IL-7 also significantly reduced dexamethasone-induced apoptosis of DP thymocytes. Given the absence of IL-7-mediated Bcl-2 up-regulation in DP thymocytes, it is unlikely that the reduction in dexamethasone-mediated-DP apoptosis was related to alterations in Bcl-2. Similarly, the absence of an anti-apoptotic or proliferative effect of IL-7 on DP thymocytes makes it unlikely that IL-7 reversal of dexamethasone-induced DP apoptosis involves previously defined survival signals provided by IL-7. The effects of IL-7 are known to be mediated through at least two distinct signaling pathways in human thymocytes, phosphatidylinositol-3-kinase and STAT-5 (13). Previous studies have demonstrated that protein complex formation between STAT-5 and cytosolic glucocorticoid receptors can result in inhibition of glucocorticoid effects (54). Thus, it is conceivable that IL-7-induced increases in intracellular STAT-5 may confer protection to glucocorticoid-mediated apoptosis or that IL-7 antagonizes glucocorticoid effects through an alternative pathway. The interposing effects of IL-7 on glucocorticoid-induced apoptosis of DP thymocytes is of particular interest given the independently proposed roles of IL-7 and glucocorticoid signals during thymocyte selection (53, 55, 56). A more complete understanding of the functional interactions between these two mediators may better elucidate the processes involved in thymocyte differentiation and selection.

We were surprised that IL-7 did not appear to enhance the pace or extent of HIV infection in the SCID-hu Thy/Liv model. A growing literature has identified a variety of mechanisms by which IL-7 can up-regulate HIV replication including induction of cell activation or proliferation, up-regulation of CXCR4 expression, and NF-B-mediated activation of the HIV long terminal repeat (22, 23, 24, 25, 27, 30, 31, 32). In several studies, pretreatment with IL-7 has been shown to enhance HIV replication by rendering quiescent or CXCR4-negative lymphocytes susceptible to HIV infection (23, 27, 30, 31, 32). In contrast, thymocytes are highly susceptible to HIV infection (36, 37, 42, 43, 44, 46), and IL-7-mediated enhancement of HIV replication is seen in the absence of IL-7 pretreatment (29, 57). Work performed in a thymic reaggregate system has shown that contact between thymic epithelial cells and thymocytes is required for optimal HIV replication in vitro (58). Such conditions are present in vivo in the SCID-hu Thy/Liv organ that contains a complex network of functional thymic epithelial cells that interact closely with thymocytes in a manner indistinguishable from that of normal human thymus. The spread of infection in the Thy/Liv organ is relatively slow (HIV RNA and p24 remain undetectable 7 days after inoculation and cellular depletion is minimal 14 days postinoculation), providing adequate opportunity to detect IL-7-mediated enhancement of infection in this system. Nevertheless, IL-7 did not appear to augment HIV infection in SCID-hu Thy/Liv mice. Although some of our findings were similar to those reported previously, including a trend toward increased intracellular p24 in SP4 cells (29) and a transient and minor increase in HIV RNA 14 days after viral inoculation (26), we did not find convincing effects of IL-7 on HIV replication or thymocyte loss in the SCID-hu Thy/Liv model. Additional experiments were conducted with administration of up to 10 µg of IL-7 per day to determine what dose of IL-7 might be necessary to accelerate HIV infection in the SCID-hu Thy/Liv organ. Even at these high doses, no enhancement of infection was detected despite evidence of IL-7 bioavailability manifested by a 2–4 log increase in circulating IL-7 and the development of splenomegaly in IL-7-treated animals.

Several nonexclusive explanations for these findings could be considered. First, it is possible that the use of a highly cytopathic HIV, such as NL4-3, obscured IL-7-mediated enhancement of HIV infection and that such enhancement might be more evident in the context of infection with a less cytopathic virus. Second, IL-7 may not have the same effect on HIV replication in vivo that it does in vitro. For instance, in the intact thymus, IL-7-mediated increases in viral replication and cytopathicity might be counterbalanced by IL-7-mediated enhancement of thymopoiesis, an effect that may not be readily apparent in TOC or other in vitro systems. A third possibility is that endogenous levels of IL-7 (and other cytokines that may influence thymopoiesis or HIV replication) are sufficiently high in the SCID-hu Thy/Liv organ to obscure any additional effects of exogenous IL-7. Finally, exogenously administered IL-7 may simply be unable to penetrate into the Thy/Liv implant of this model. The high plasma levels of IL-7 in treated mice and the presence of biologic effects of IL-7 in other organs (e.g., the spleen) speak against, but do not rule out, this possibility.

We favor the hypothesis that endogenous production of IL-7 makes it more difficult to demonstrate an impact of exogenous IL-7 on thymopoiesis and HIV infection in vivo. Indeed, the in vitro effects of IL-7 on thymopoiesis have not been so clearly reproduced in vivo and may reflect a limited ability of exogenous IL-7 to exert effects beyond those induced by the intact cytokine milieu of the in vivo thymic microenvironment. Morrissey et al. (59, 60) found that IL-7 treatment of normal and immunodeficient mice was associated with an increase in peripheral splenocytes and lymphocytes (both B and T cells were expanded) but found no increase in thymic cellularity. Similarly, emerging data from studies of non-human primates demonstrate that administration of exogenous IL-7 to immunocompetent and immunodeficient animals is associated with peripheral T cell expansion, lymphadenopathy, and splenomegaly, but there is no evidence of increased thymopoiesis (61, 62) or enhanced SIV replication (61). Thus, our findings in SCID-hu Thy/Liv mice are similar to findings reported in other in vivo models. Conversely, IL-7 has been shown to increase in vivo thymopoiesis as measured by the formation of TCR rearrangement excision circles in aging (63) and immunodeficient mice (17). Because peripheral human T cell analysis is not easily conducted in SCID-hu Thy/Liv mice (64), we did not measure TCR rearrangement and cannot exclude the possibility that IL-7 enhances TCR rearrangement excision circles formation in this model. Finally, our data are not consistent with evidence that exogenous IL-7 enhances thymopoiesis in mice after bone marrow transplantation (65, 66). It has been demonstrated that the effects of IL-7 in this setting are attributable to radiation-induced damage to IL-7-producing thymic stromal cells resulting in decreased endogenous IL-7 production in transplanted mice (67). Consequently, the use of IL-7 in these models represents IL-7 replacement in the setting of relative IL-7 deficiency, whereas our model involves the use of exogenous IL-7 in animals with presumably intact endogenous IL-7 production. When considering these differences in the context of our current data, it is possible that a relative deficiency of IL-7 in the in vitro setting may account for the disparate in vitro and in vivo effects of IL-7 on thymopoiesis and HIV replication.

In conclusion, although the absence of increased thymopoiesis in our in vivo analysis of HIV-infected and uninfected SCID-hu mice is discrepant with in vitro results in this study and those reported elsewhere, these findings are consistent with in vivo findings reported by others. In contrast, the observation that IL-7 does not appear to accelerate in vivo HIV infection of the thymus has not been previously reported. We hypothesize that endogenous production of IL-7 may diminish the impact of exogenous IL-7 on thymopoiesis and HIV infection in vivo. Clearly, factors unique to this animal model may limit the generalization of these observations, and these findings may not be readily extendable to the peripheral T cell compartment where accelerated in vivo infection could be driven independently by IL-7. Nevertheless, the apparent absence of enhanced in vivo HIV infection in the setting of a 2–4 log increase in circulating IL-7 levels is noteworthy. Given the possibility that IL-7 might facilitate T cell production and expansion in humans, additional in vivo studies are warranted to further investigate the potential benefits and risks of IL-7 therapy in HIV-infected individuals.

	Acknowledgments

 
We are grateful to Kaveh Bastani, Mary Beth Moreno, Jose Rivera, Jennifer Bare, and Sofiya Galkina for their expert assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI01597 (to L.A.N.), AI05418 (to C.A.S.), and AI43864 and AI40312 (to J.M.M.). J.M.M. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist and a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

² Address correspondence and reprint requests to Dr. Laura A. Napolitano, Gladstone Institute of Virology and Immunology, University of California, P.O. Box 419100, San Francisco, CA 94141-9100. E-mail address: lnapoli{at}itsa.ucsf.edu

³ Current address: Department of Blood and Marrow Transplantation, University of Texas MD Anderson Cancer Center, Houston, TX 77030.

⁴ Abbreviations used in this paper: ITTP, intrathymic T progenitor CD4⁺CD3^-CD8^- thymocyte; DP, CD4⁺CD8⁺ double-positive thymocyte; TOC, thymic organ culture; BrdU, 5-bromo-2'-deoxyuridine; SP, single-positive; SP8, CD8⁺CD4^-CD3⁺ thymocyte; SP4, CD4⁺CD8^-CD3⁺ thymocyte.

Received for publication November 11, 2002. Accepted for publication May 2, 2003.

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