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International Immunology Advance Access originally published online on October 22, 2007
International Immunology 2007 19(12):1329-1339; doi:10.1093/intimm/dxm102
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© The Author 2007. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved.
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IL-7 decreases IL-7 receptor {alpha} (CD127) expression and induces the shedding of CD127 by human CD8+ T cells

Agatha Vranjkovic1,2,*, Angela M. Crawley1,*, Katrina Gee1,3, Ashok Kumar2,3 and Jonathan B. Angel1,2,4

1 Ottawa Health Research Institute, Ottawa, Ontario, Canada
2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada
3 Division of Virology and Molecular Immunology, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada
4 Division of Infectious Diseases, Ottawa Hospital—General Campus, Ottawa, Ontario, Canada

Correspondence to: J. B. Angel; E-mail: jangel{at}ohri.ca


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
IL-7 receptor {alpha} (CD127) signaling is essential for T-cell development and regulation of naive and memory T-cell homeostasis. Fewer CD8+ T cells from HIV-infected patients express CD127 compared with healthy individuals, suggesting that specific host and/or viral factors regulate IL-7 receptor expression. Factors relevant to HIV infection that could potentially decrease CD127 expression on human CD8+ T cells and the mechanisms by which this occurs were therefore evaluated. IL-7, but not HIV gp120, IL-1-ß, IL-6, IL-10, IL-13, transforming growth factor-ß or tumor necrosis factor-{alpha}, reduced CD127-surface expression and did so without altering CD127 mRNA expression. Furthermore, IL-7 did not increase the amount of cytoplasmic CD127 in CD8+ T cells. Interestingly, IL-7 induced the shedding of CD127 from CD8+ T cells, suggesting a mechanism that may contribute to the increased concentration of CD127 in the plasma of HIV+ individuals, a novel finding reported here. Naive CD8+ T cells are more sensitive to IL-7 that mediated the down-regulation of CD127, suggesting that these effects may have particular significance early in T-cell life cycle. Since CD127 down-regulation may be an important contributor to HIV-associated T-cell dysfunction, determining the mechanism thereof may prove to be of considerable significance.

Keywords: cytokines, {gamma}-chain cytokine receptors, HIV, receptor internalization, receptor secretion


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
IL-7 plays an essential role in the development and maintenance of T lymphocytes (1, 2). The biological effects of IL-7 are mediated via the hematopoietic IL-7 receptor (IL-7R) complex, a heterodimer of an IL-7 receptor {alpha}(CD127) chain, which binds IL-7 and thymic stromal lymphopoietin and the IL-2 receptor {gamma}(CD132) chain, commonly shared by many cytokines including IL-2, 4, 9, 15 and 21 (3). IL-7 is also a key regulator of circulating naive and memory T-cell homeostasis (4, 5), maintaining T-cell survival, inducing Bcl-2 and BCL-xL expression (anti-apoptotic factors), repressing Bax expression (pro-apoptotic factor) and inducing T-cell proliferation. In addition to stimulating CD8+ T-cell proliferation (6), IL-7 augments anti-viral, alloreactive and anti-tumor CTL responses (7). Furthermore, IL-7 responsiveness of CD8+ T cells influences the maintenance of naive and memory cell populations, effector cell expansion and differentiation and effector cell contraction (8).

Effector CD8+ T cells are particularly important in the eradication and containment of many viral infections. Primary infection with HIV elicits a strong and initially effective virus-specific CTL response coincident with a reduction in plasma viremia (9, 10); however, this is insufficient for complete viral clearance, and CTL function gradually declines. Nevertheless, circulating HIV-specific CD8+ T cells are present in patients with advanced disease suggesting that virus-specific CD8+ T cells persist, but are unresponsive to their cognate antigens (11).

Increased plasma IL-7 concentrations have been observed in HIV-infected patients and strongly correlate with CD4+ T lymphopenia (5); perhaps as a homeostatic response to T-cell depletion. Despite increased IL-7, CTL activity is not restored. It was previously demonstrated in this laboratory, and confirmed by others, that significantly fewer CD8+ T cells express CD127 in HIV-infected patients with uncontrolled plasma viremia compared with healthy individuals (12–15). Suppression of viremia with effective anti-retroviral therapy was associated with a greater proportion of CD8+ T cells expressing CD127 compared with untreated individuals (13, 16, 17), suggesting that ongoing HIV replication either directly or indirectly down-regulates CD127. In a previous study, IL-7 enhanced CTL function only in those HIV+ patients with pre-existing CD8+ T cell-mediated lytic activity, while circulating CD8+ T cells from IL-7-unresponsive patients had markedly decreased expression of CD127 (18); again demonstrating an association between decreased CD127 expression and declining CTL activity. In addition, CD8+ T cells from HIV+ patients are significantly less responsive to IL-7 than those from healthy individuals and the degree of lymphopenia seems to contribute further to this effect (19–21).

Murine CD127 expression on CD8+ T cells is down-regulated by IL-7 and other common IL-2 receptor {gamma}-chain ({gamma}C) cytokines (22). In addition, in vivo IL-7 treatment of macaques down-regulated CD127 expression in CD4+ and CD8+ T cells (23, 24). We have recently shown that IL-7 down-regulates CD127 expression on human thymocytes ex vivo (25). Similarly, others have shown that incubation of human PBMCs with IL-7 decreases cell-surface expression of CD127 on CD4+ T cells (12) and on naive and memory CD8+ T cells within 24–48 h of culture (16). The mechanisms by which IL-7 down-regulates CD127 expression on human T cells remain to be established. In mice, IL-7 down-regulates CD127 mRNA expression in lymph node T cells (22); however, in humans a similar decrease in mRNA is not observed until 3–5 days after exposure to IL-7 and therefore does not explain the decrease in surface CD127 expression observed within 24–48 h of culture (16, 25, 26).

The activity of HIV proteins with immunoregulatory activity, such as gp120 (27), and cytokines that have been correlated with viral replication or impaired T-cell function, including IL-1-ß, IL-6, IL-10, IL-13, transforming growth factor (TGF)-ß or tumor necrosis factor (TNF)-{alpha} (28–30), represent a number of candidate viral and host factors that may contribute to the decrease in CD127 expression on CD8+ T cells. The current study addresses potential mechanisms that alter the expression of CD127 on CD8+ T cells in order to better understand the potential role of CD127 expression in HIV infection and in other settings in which regulation of CD127 may be of significance.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Preparation of cells
All research conducted using blood from human subjects was approved by the Ottawa Hospital Research Ethics Board. PBMCs from healthy HIV seronegative volunteers were isolated by Ficoll-PaqueTM PLUS (Pharmacia Fine Chemicals, Piscataway, NJ, USA) gradient separation, washed twice in PBS and re-suspended in RPMI medium 1640 supplemented with penicillin–streptomycin and 10% FCS at a concentration of 106 cells ml–1. The CD8+ T cells were isolated from PBMCs using a MACS CD8+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA) in accordance with the manufacturer's instructions achieving a purity of >98–99% as verified by flow cytometry. Purified CD8+ T cells were re-suspended at 106 cells ml–1 in RPMI supplemented with penicillin–streptomycin and 20% FCS.

Cell stimulation
The PBMCs and purified CD8+ T cells were cultured with increasing concentrations of HIVIIIB gp120 (0, 0.5, 1, 5 and 10 µg ml–1) (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from ImmunoDiagnostics, Woburn, MA, USA); recombinant human IL-7 (10, 100, 1000 and 10 000 pg ml–1) (R & D Systems, Minneapolis, MN, USA); IL-1-ß, IL-6, IL-10, IL-13 (1, 10 and 100 ng ml–1) (BD Biosciences PharMingen, San Diego, CA, USA); TGF-ß (0, 1, 10 and 100 ng ml–1) or TNF-{alpha} (0, 1, 10 and 100 ng ml–1) (Sigma–Aldrich, St Louis, MO, USA). Following incubations of 0, 24, 48, 72 and 96 h, CD127, CD45RA and CD45RO expression on CD8+ T cells was analyzed by flow cytometry.

Evaluation of surface CD127 expression by flow cytometry
Flow cytometry analyses were performed using a Beckman Coulter ALTRA flow cytometer and the EXPO version 2.0 software package. Samples of 2 x 105 cells were incubated with saturating antibody concentrations for 20 min at room temperature. mAbs used included CD8–PC5 and CD127–PE, for two-color analysis; CD8–PC5, CD127–PE and CD45RA–FITC or CD45RO–FITC, for three-color analysis and CD8–PC5, CD127–PE, CD45RA–ECD and CD27–FITC or CD62L–FITC, for four-color analysis. All mAbs were purchased from Immunotech (Beckman Coulter, Marseille, France). The impact of a given stimulus on the expression of CD127 on CD8+ T cells was calculated as follows: [(% CD3+CD8+ T cells expressing CD127 in the presence of stimulus)/(% CD3+CD8+ T cells expressing CD127 in media alone)] x 100.

To exclude potential cell mortality resulting from incubation with cytokines as an explanation for results observed, 7-aminoactinomycin D (7-AAD) (Molecular Probes, OR, USA) or propidium iodide staining was performed on purified CD8+ T cell cultures and samples were analyzed by flow cytometry.

Semi-quantitative PCR
Following 16, 24 and 72 h incubations with IL-7 (10 000 pg ml–1), total RNA was isolated from purified CD8+ T-cell cultures (3 x 106 cells) using the RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Total RNA concentration was determined spectrophotometrically (GeneQuant Pro) and complementary DNA (cDNA) synthesis was conducted using the Advantage RT-for-PCR Kit (Clontech) as follows: 0.5 µg of total RNA (in 10 µl distilled water) and 1 µl of oligo(dT)18 were heated to 70°C for 2 min and added to 4 µl of 5x reaction buffer, 1 µl of deoxynucleoside triphosphate (dNTP) mix (10 mM each), 0.5 µl of recombinant RNase inhibitor and 1 µl of Moloney murine leukemia virus reverse transcriptase. The mixture was incubated at 42°C for 1 h and 94°C for 5 min to stop the cDNA synthesis reaction.

cDNA was PCR amplified with gene-specific primers for the CD127 gene (forward 5'-GAAGGTTGGAGAAAAGAGTC-3' and reverse 5'-CAAAATGCTGATGGTTAGTAA-3') to amplify cDNA encoding membrane-bound CD127 and primers to detect the housekeeping gene beta-actin (forward 5'-GAAACTACCTTCAACTCCATC-3' and reverse 5'-CGAGGCCAGGATGGAGCCGCC-3'). All semi-quantitative PCRs included the following: 2 µl cDNA, 50 µl of Taq PCR Master Mix (Qiagen), 2 µl each of forward and reverse primers (0.3 µM) and 44 µl of distilled water, for a final volume of 100 µl. Cycling conditions were 30 s at 95°C, 30 s at 52°C and 1 min at 72°C, for 30 cycles. PCR products were resolved by electrophoresis on 1% agarose gel, stained with ethidium bromide and visualized using a UV transluminator.

Quantitative real-time PCR for CD127
Following 16 and 24 h incubations with IL-7 (10 000 pg ml–1), total RNA was isolated from purified CD8+ T-cell cultures (3 x 106 cells) using RNeasy® Mini Kit (Qiagen) according to the manufacturer's instructions and treated with DNase (Qiagen). First-strand cDNA synthesis was conducted using the SuperScript II Reverse Transcriptase Kit (Invitrogen) as follows: 0.5 µg of total RNA (in 10 µl sterile water), 1 µl of oligo(dT)12–18 (500 µg ml–1) and 1 µl of dNTP mix (10 mM each) were heated to 65°C for 5 min and added to 4 µl 5x First-Strand Buffer, 2 µl of 0.1 M dithiothreitol and 1 µl (200 U) SuperScript II Reverse Transcriptase. The mixture was incubated at 42°C for 50 min and 70°C for 15 min. In addition to the use of DNase in sample preparation, the possibility of genomic DNA contamination was addressed by routinely conducting PCR analysis of some RNA samples incubated with reverse transcription reaction mixtures lacking the reverse transcriptase.

cDNA was PCR amplified with gene-specific primers for the CD127 gene (see previous section). All quantitative PCRs included the following: 25 µl of QuantiTect SYBR Green PCR Master Mix (Qiagen, Mississauga, Ontario, Canada), 3 µl each of forward and reverse primers (0.3 µM), 10 µl of a 1:100 dilution of cDNA, 1 µl MgCl2 (50 mM) and 8 µl of Rnase-free H2O for a final 50 µl reaction. Reactions were performed in a BIO-RAD iCycler iQ (Bio-Rad) with a 15-min activation of DNA polymerase at 95°C, followed by 50 cycles of denaturation at 95°C for 30 s, annealing at 60.3°C for 20 s, extension at 72°C for 30 s and measurement of sample fluorescence at 72°C for 5 s for quantification. Melting curve analysis confirmed the amplification of a single product with minimal primer–dimer formation. The housekeeping gene ribosomal protein S18 was amplified as above without adding MgCl2. S18 primers (forward 5'-CTGCCATTAAGGGTGTGG-3' and reverse 3'-TCCATCCTTTACATCCTTCTG-5') were designed using Beacon software 3.0. The CD127-specific PCR products were sequenced and homology to the known CD127 sequence was confirmed (data not shown), thereby confirming both primer specificity and suitability of the PCR product for use in generating standard curves for real-time PCR. To quantify CD127 or S18 transcript copy numbers, CD127 and S18 PCR products were isolated using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences). The number of mRNA copies per microliter was calculated using the following formula: [(6.022 x 1023) x (concentration of DNA sample in micrograms per microliter)]/[(weight of one copy of mRNA) x (106 µg)], where the weight (g) of one copy of mRNA = (number of base pairs in PCR product) x (325 g mol–1). Serial dilutions ranging from 1 x 1010 to 1 x 102 mRNA copies per microliter of purified CD127 or S18 PCR products were included in PCR experiments to generate standard curves from which sample mRNA copy numbers were extrapolated using the iCycler software. The CD127 mRNA copy numbers for each sample were normalized to the mRNA copy numbers of the S18 gene using a two-step formula: (i) normalization factor for individual(n), treatment(a) = [average S18 copy number for all individuals(1, 2, 3, ..., n) for treatment(a)]/[observed S18 copy number for individual(n)] and (ii) actual CD127 mRNA copy number for individual(n), treatment(a) = [observed CD127 copy number for individual(n), treatment(a)] x [normalization factor for individual(n), treatment(a)].

Evaluation of CD127 expression by immunofluorescence and confocal microscopy
Purified CD8+ T cells were treated with medium or IL-7 (10 000 pg ml–1) for 24 h. Cells (1 x 106) were fixed in 2% PFA, re-suspended in cold PBS + 1% antibody serum, permeabilized with 10% Triton X-100 for 15 min. Staining for CD127 was conducted as follows: (i) IgG1 mouse anti-human CD127 mAb (R & D Systems) for 20 min at 4°C and (ii) AlexaFluor 488-conjugated goat anti-mouse IgG (H + L) (Invitrogen) for 20 min at 4°C. Staining for CD132 was conducted as follows: (i) rat anti-human CD132 (BD PharMingen) for 20 min at 4°C and (ii) AlexaFluor 488-conjugated goat anti-rat IgG (H + L) (Invitrogen). Following each antibody incubation step, cells were washed with PBS + 1% antibody serum. After staining, cells were centrifuged to a pellet, re-suspended in 10 µl PBS, layered onto a microscope slid and visualized using a Zeiss laser sectioning microscope using a magnification of x110 and a x2 zoom. Images were subsequently processed using Adobe Photoshop 7.0 (Adobe, San Jose, CA, USA) to adjust brightness and contrast and to generate single-layered and overlaid images.

Detecting soluble CD127 by western blot
To determine if IL-7 induced the release of CD127, purified CD8+ T cells were cultured in medium or stimulated with IL-7 (10 000 pg ml–1). After 24 h, culture supernatants were collected and 30 µg of total protein was resolved by SDS–PAGE, blotted onto polyvinylidene fluoride membrane and probed with a goat polyclonal anti-human CD127 antibody (R & D Systems), followed by a donkey anti-goat-HP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins were detected by ECL chemiluminescence (Pierce Biotechnologies, Rockford, IL, USA). A soluble IL-7R-secreting cell line, WI-26VA4 (American Type Culture Collection, Manassas, VA, USA), was cultured in the presence of IL-7 and supernatants were collected and included in the western blot analyses as a positive control. Densitometry analysis of protein bands was performed using GeneTools Software (Syngene, Frederick, MD, USA).

To determine if soluble CD127 was present in human plasma in health and HIV disease, blood was collected from HIV– and HIV+ individuals not receiving anti-retroviral therapy and who provided informed consent. Blood was collected in heparin-containing tubes and plasma was collected following the first step of Ficoll-Paque gradient separation as described above. All samples were diluted 1:5 in 0.1% TritonX-100/PBS for 60 min at 37°C to kill any viral particles and then stored at –80°C. Samples (2 µl per well) were electrophoresed in a 6% SDS–PAGE gel under reducing conditions. A constant volume of plasma was loaded into each well since total plasma protein concentrations did not vary significantly among individuals. Serial dilutions of recombinant human CD127–Fc chimera protein (R & D Systems) were included as positive controls, also diluted in 0.1% TritonX-100/PBS. Detection of CD127 protein by western blot was conducted as described above.

Statistical analysis
The Student's t-test or paired t-test was used for data analysis of as appropriate and analysis of variance was used for multiple group comparisons using Sigma Stat 3.0 software (SPSS, Leesburg, VA, USA), where values of P ≤ 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
CD127 expression on CD8+ T cells in PBMC cell culture
A recent publication from this laboratory demonstrated that significantly fewer CD8+ T cells express CD127 in HIV-infected patients with uncontrolled viremia compared with healthy controls (13). This report prompted an investigation of the mechanisms that decrease CD127 expression on CD8+ T cells. As previously described (22), expression of CD127 on CD8+ T cells in PBMCs recovers after 24 h in culture and remains stable over time (Fig. 1B). No significant change in CD127 expression on CD8+ T cells was observed following incubation of PBMCs with HIV gp120, IL-1-ß, IL-6, IL-10, IL-13, TGF-ß or TNF-{alpha} (data not shown). Addition of IL-7 significantly decreased CD127 expression on CD8+ T cells in PBMC cultures (Fig. 1A). Treatment of PBMCs with IL-7 decreased CD127 expression on CD8+, CD8+CD45RA+ and CD8+CD45RO+ T cells (Fig. 1A). With concentrations of IL-7 up to 1000 pg ml–1, this occurred in a transient nature but was sustained with higher concentrations (10 000 pg ml–1) (Fig. 1A). Based on four-color flow cytometry analysis, CD127 expression on CD45RA+ cells occurs almost exclusively on CD45RA+CD27+ (Fig. 2) or CD45RA+CD62L+ cells (truly naive cells) (data not shown). Gating on CD45RA+ alone therefore reflects an accurate evaluation of the expression of CD127 on naive cells without requiring the use of additional cell-surface markers. Of note, incubation of IL-7 did not affect the proportion of CD8+ T cells that expressed CD45RA or CD45R0 (data not shown).


Figure 1
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  		Fig. 1. Effect of IL-7 on CD127 expression on CD8+ T cells within PBMC cultures. (A) Incubation of PBMCs with increasing concentrations of IL-7 (100–1000 pg ml–1) resulted in statistically significant yet transient decreases of CD127 expression on CD8+ T cells in PBMC cultures (n = 3, *P = 0.001; {dagger}P ≤ 0.001 by analysis of variance and P < 0.05 by Dunnett’s simultaneous test versus time 0). A sustained decrease in CD127 expression was observed following incubation with 10 000 pg ml–1 of IL-7. Representative dot plots of CD127 expression on CD8+ T cells in PBMCs cultured over time are also shown. (B) Expression of CD127 recovers after 24 h in medium alone and is sustained over time. (C) Incubation with IL-7 (1000 pg ml–1) resulted in a transient decrease of CD127 expression. (D) High concentrations of IL-7 (10 000 pg ml–1) resulted in a sustained down-regulation of CD127.

 

Figure 2
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  		Fig. 2. Gating on CD45RA+ cells reflects an accurate evaluation of the expression of CD127 on naive cells. Based on four-color flow cytometric analysis and gating on the CD3+CD8+CD45RA+ T-cell populations, CD127 expression on CD45RA+ cells occurs almost exclusively [78.4%/(78.4% + 0.4%) > 99%] on the CD45RA+CD27+ population (truly naive cells).

 
To exclude IL-7-induced selective mortality as a potential contributor to the observed results, 7-AAD staining was performed on PBMCs incubated with IL-7 (10 000 pg ml–1) for 96 h and no significant cell mortality was detected (data not shown).

IL-7 decreases CD127 expression on purified CD8+ T cells, which occurs mainly on naive CD8+ T cells
To determine if IL-7 acts directly on CD8+ T cells, purified CD8+ T cells were cultured with increasing concentrations of the cytokine. The expression of CD127 on CD8+ T cells was transiently decreased by IL-7 (100–1000 pg ml–1) and returned to baseline levels after 96 h of incubation (Fig. 3A). This occurred specifically on CD8+CD45RA+ T cells but not on CD8+CD45RO+ T cells (Fig. 3B and C, respectively). This effect was detected within the first 24 h and was sustained for 96 h on both CD45RA+ and CD45RO+ sub-populations with 10 000 pg ml–1 of IL-7 (Fig. 3B and C). As observed with PBMCs, HIV gp120, IL-1-ß, IL-6, IL-10, IL-13, TGF-ß or TNF-{alpha} did not alter CD127 expression on isolated CD8+ T cells (data not shown). Analysis of mean channel fluorescence (MCF) was also performed in parallel and IL-7 stimulation resulted in significant decreases in MCF (data not shown).


Figure 3
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  		Fig. 3. IL-7 decreases surface CD127 expression in purified CD8+ T-cell cultures. (A) The expression of CD127 in purified CD8+ T cells was transiently decreased by IL-7 (100–1000 pg ml–1) which returned to baseline levels by 96 h of incubation. The highest concentration of IL-7 (10 000 pg ml–1) resulted in a sustained decrease in CD127 expression (n = 3, *P < 0.001 and {dagger}P = 0.013 by analysis of variance (ANOVA) and P < 0.05 by Dunnett's simultaneous test versus time 0). To determine if this effect occurred on a specific sub-population of CD8+ T cells, the expression of CD127 on CD45RA+ (B) and CD45RO+ (C) CD8+ T cells was assessed. Treatment with IL-7 (100–1000 pg ml–1) transiently decreased CD127 expression almost exclusively on CD8+CD45RA+ T cells (B), which returned to baseline after 96 h. The highest concentration of IL-7 (10 000 pg ml–1) resulted in a sustained decrease of CD127 on both CD45RA+ and CD45RO+ sub-populations (C) (n = 3, *P = 0.020; {dagger}P = 0.017 and {ddagger}P < 0.001 by ANOVA or ANOVA on ranks as appropriate and P < 0.05 by Dunnett's simultaneous test versus time 0).

 
CD127 RNA expression is unchanged by IL-7
A semi-quantitative PCR method was developed to detect expression of mRNA that encoded membrane-bound CD127 and results were confirmed by quantitative real-time PCR. Treating purified CD8+ T cells with IL-7 (10 000 pg ml–1) for 24 (Fig. 4A), 48 and 72 h (data not shown) did not alter the expression of mRNA encoding the membrane form of CD127. These observations were confirmed by quantitative real-time PCR, in which IL-7 did not decrease the expression of CD127 mRNA from CD8+ T cells (Fig. 4B).


Figure 4
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  		Fig. 4. IL-7 has no effect on the expression of mRNA-encoding membrane-bound CD127 mRNA in CD8+ T cells. The effect of IL-7 on CD127 gene expression was studied to further evaluate the mechanism of CD127 down-regulation on CD8+ T cells. (A) Addition of IL-7 (10 000 pg ml–1) for 24 h did not alter the amount of mRNA encoding membrane-bound CD127 expressed by CD8+ T cells as determined by semi-quantitative PCR (n = 3). The upper bands represent the expression of CD127 and the lower bands represent the expression of the beta-actin housekeeping gene. The molecular weight marker is shown on the left. (B) The lack of change in the expression of mRNA encoding membrane-bound CD127 mRNA in IL-7-stimulated CD8+ T cells was confirmed by real-time PCR (n = 4). The expression of CD127 mRNA was normalized to the expression of the ribosomal subunit-18.

 
Cytokine receptor internalization
Since IL-7 transiently decreases CD127 expression on CD8+ T cells without affecting CD127 mRNA expression, other mechanisms may be involved in surface receptor down-regulation such as receptor internalization. After culture with media or IL-7 (10 000 pg ml–1) for 30 min to 24 h, cells were fixed and permeabilized and cell surface and cytoplasmic CD127 were stained with anti-CD127 mAbs. The effect of IL-2 on CD132 expression was used as a positive control for receptor internalization (31, 32). As expected, IL-2 increased cytoplasmic CD132 (Fig. 5A). In contrast to IL-2, we did not observe an increase in CD127 within the cytoplasm of IL-7-stimulated cells (Fig. 5B–D). IL-7 consistently decreased the expression of surface CD127 on CD8+ T cells compared with media controls in fixed-only (data not shown) or fixed and permeabilized cells (Fig. 5D), confirming the results obtained by flow cytometry (Fig. 3). Preliminary results of IL-7-stimulated CD8+ T cells pre-incubated with lactacystin, a known proteosome inhibitor, did not reveal an increase in cytosolic CD127 suggesting that proteosomal degradation does not account for the observed lack of internalized CD127.


Figure 5
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  		Fig. 5. IL-7 does not significantly increase cytoplasmic CD127 in CD8+ T cells. (A) As a positive control for the assessment of receptor internalization by confocal microscopy, the presence of cell and cytoplasmic CD132 was detected in fixed and permeabilized cells CD8+ T cells that were incubated with IL-2 for 30 min. (B) Untreated CD8+ T cells express abundant CD127 molecules on their cell surface after 30 min of culture and this remained unchanged in the presence of IL-7 (10 000 pg ml–1). (C) Similarly, IL-7 did not increase the presence of cytoplasmic CD127 after 2 h of culture. (D) Following 24 h of culture, the expression of surface CD127 on CD8+ T cells decreased significantly in response to IL-7, consistent with flow cytometry observations (Fig. 3), while the amount of cytoplasmic CD127 remained unchanged. Therefore, CD127 does not accumulate within the cytoplasm, suggesting that IL-7 does not induce receptor internalization. (E) A representative figure of CD8+ T cells cultured for 24 h with media or IL-7 and then fixed (but not permeabilized) and stained for surface CD127 expression is included as a control. Samples were visualized using a Zeiss laser-sectioning microscope using a magnification of x110 and a x2 zoom. Images were subsequently processed using Adobe Photoshop 7.0 (Adobe) to adjust sharpness, brightness and contrast and to generate single-layered and overlaid images. The image scale shown here is {approx}10 µm.

 
IL-7 induces CD127 shedding
An alternative mechanism by which IL-7 may down-regulate CD127 expression is via receptor shedding. Western blot analysis of supernatants collected from purified CD8+ T cells after 24 h of culture detected the presence of CD127 (Fig. 6). IL-7 increased the level of secreted CD127 in five of seven healthy donors (Fig. 6, lanes 1 and 4–7). In three of these samples, IL-7 (10 000 pg ml–1) increased the amount of CD127 detected compared with medium only (Fig. 6, lanes 4, 6 and 7). In two of seven samples, although undetected in medium controls, CD127 was detected when the cells were cultured in the presence of IL-7 (10 000 pg ml–1) (Fig. 6, lanes 1 and 5). In the remaining samples, no CD127 was detected in either control or IL-7-treated cells (Fig. 6, lanes 2 and 3). These results indicate that incubation of CD8+ T cells with IL-7 results in an increase of CD127 in culture supernatants.


Figure 6
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  		Fig. 6. Analysis of soluble CD127 in CD8+ T-cell culture supernatants. Western blot analysis of CD8+ T-cell culture supernatants was performed to determine if IL-7 induced the release of CD127. Increases in CD127 protein were detected in culture supernatants of CD8+ T cells incubated with IL-7 (1000 pg ml–1) for 24 h (n = 7). Equivalent total protein concentrations of culture supernatants collected from untreated WI-26VA4 cells were used as a negative control, while IL-7 (10 000 pg ml–1)-treated WI-26VA4 cells were included as a positive control.

 
Increased CD127 in the plasma of HIV+ individuals compared with HIV– individuals
Since increased plasma IL-7 is associated with HIV-induced lymphopenia (5) and IL-7 induces the shedding of CD127 in vitro (Fig. 6), the plasma of untreated HIV-infected patients was analyzed for the presence of CD127 protein. The CD127-specific western blot can detect differences in protein concentrations, as demonstrated in a dilution series of a recombinant human CD127–Fc chimera protein with a molecular mass of 80–90 kDa (Fig. 7A). Analysis of human plasma samples identified three protein bands of ~50, 90 and 150 kDa (Fig. 7B). This finding is similar to previous descriptions in mice in which multiple bands were detected by SDS–PAGE analysis of culture supernatants from cell lines transfected with murine CD127. In the mouse, these protein bands are thought to represent unglycosylated ({approx}49.6 kDa), glycosylated ({approx}68 kDa) and dimeric ({approx}153 kDa) forms of the receptor (33). Therefore, the multiple protein bands detected here likely represent similar forms of the receptor. Densitometry analysis of the glycosylated CD127 protein bands ({approx}90 kDa) indicated that plasma of untreated HIV+ individuals (mean CD4 counts 371 cells ml–1, mean viral load 59 436 copies µl–1) contained significantly more CD127 than HIV– individuals (Fig. 7B) (P = 0.0008, data not shown). Regression analysis of CD127 band density (90 kDa) suggests that there is an inverse correlation between plasma CD127 and CD4 counts in HIV+ individuals, although this did not reach statistical significance. There was no observable difference in the densities of the larger protein bands and the smallest protein bands were consistently diffuse and hence densitometry analysis was not performed.


Figure 7
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  		Fig. 7. Detection of soluble CD127 in human plasma in health and HIV disease. (A) Detection of different concentrations of human CD127–Fc chimera protein ({approx}80–90 kDa) by western Blot (lane 1 = 5 ng; lane 2 = 1 ng and lane 3 = 0.1 ng). (B) Analysis of plasma samples from HIV– (lanes 4–8) and HIV+ (lanes 9–14) individuals was conducted to determine whether soluble CD127 is present in human plasma and to determine if soluble CD127 is increased in HIV infection in vivo Densitometry analysis of the {approx}90 kDa protein bands, representing the glycosylated CD127 receptor, indicated that HIV+ individuals have a greater amount of plasma CD127 than HIV– individuals (P < 0.001).

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Reduced CD127 expression on CD8+ T cells of HIV-infected patients with uncontrolled viral replication may contribute to the observed decline in CTL activity and provides a rationale for investigating potential host and viral factors that may alter CD127 expression and the mechanisms thereof. Although HIV-specific CTLs persist in the circulation during HIV infection, they are unable to control viral replication (34) and may contribute to ensuing susceptibility to opportunistic infections and malignancies. Down-regulation of T cell-associated CD3-zeta chain (35), reduced perforin (36) and IFN-{gamma} expression (37), maturation arrest of CD8 effector T cells (34) and increased PD-1 expression (38–40) have been identified as potential causes for decreased CTL activity during HIV infection; however, the extent to which each of these contributes to impaired CTL activity and the mechanisms by which each of these occurs remain unclear.

The survival and proliferation of lymphoid progenitors is dependent on IL-7R signaling, however, the control of receptor expression remains poorly understood. Regulation of the murine CD127 gene expression in hematopoietic progenitors is dependent on the transcription factor PU.1 binding to a GGAA motif, affecting primarily pro-B cell development (41). In contrast, murine T cells do not express PU.1 but rather depend on a GA-binding protein binding to the same GGAA motif to regulate CD127 expression (42). Therefore, regulation of CD127 differs in distinct lymphoid lineages in part by the differential recruitment of factors to the same GGAA motif. Murine CD8+ T cells require the transcriptional repressor growth factor independence 1 (GFI1) for cytokine down-regulation of CD127 gene expression (22). The human CD127 promoter region includes multiple putative transcription factor binding sites, including GFI1 sites; however, similar gene regulation has not yet been described. In addition to reports on factors down-regulating CD127 in mice, glucocorticoids such as dexamethasone, have been shown to up-regulate human and mouse CD127 gene and protein expression (43, 44), suggesting that CD127 transcription is inducible.

Patients with HIV infection have increased circulating IL-7 concentrations, and this may be a compensatory response to the observed T-cell depletion (5, 45). Paradoxically, IL-7 may down-regulate the expression of its own receptor, as it occurs with other cytokines including IL-15 (46) whose receptor shares the CD132 chain with CD127. Cytokine-mediated down-regulation of CD127 expression has been reported in IL-2-treated PBMCs from HIV– individuals (47) and in HIV+ individuals receiving IL-2 therapy (19, 28).

In the present study, IL-7 decreased CD127-surface expression on CD8+ T cells in both PBMCs and isolated CD8+ T cells in vitro and are consistent with recent observations by Colle et al. (16). Concentrations of IL-7 in the plasma of HIV-infected individuals have been shown to be elevated up to 70 pg ml–1 (5, 45, 48–50) in the range of those which caused a transient decrease in the expression of CD127 in the experiments here (Figs 1 and 3) and suggest that these observed changes may be physiologically relevant. Moreover, these changes were most evident within the naive CD8+ T-cell population (CD45RA+). The regulation of CD127 early in the T-cell life cycle is thought to be associated with the competition for sometimes limiting sources of IL-7 in relation to the numbers of circulating T cells, particularly naive T cells which compete with one another for the receipt of IL-7 survival signals (22).

A possible mechanism for surface receptor down-regulation is receptor internalization, a common feature of many cytokine receptors. In the case of CD127, IL-7 (10–1000 pg ml–1) transiently decreases surface receptor expression on CD8+ T cells without affecting RNA expression. Therefore, we investigated the possibility of receptor internalization. Recent evidence suggests that IL-7-induced phosphorylation of tyrosine residues in the intracellular domain of CD127 may lead to an association with clathrin, a protein known to be required for receptor internalization (51), hence CD127 may be internalized by clathrin vesicles, but this has not yet been shown. Moreover, the amino acid sequence of CD127 contains two tyrosine signal sequences (401–404 YQDL and 449–452 YVTM) in the intracellular domain that are thought to be part of receptor internalization motifs targeting transmembrane proteins to endocytic or secretory pathways, as reported in the IL-2-{alpha}, -ß and -{gamma} receptors (31, 52). Although cytoplasmic CD127 was detected (Fig. 5B–D) on CD8+ T cells, IL-7 does seem to enhance receptor internalization. It was also noted that the amount of cytoplasmic CD127 in CD8+ T cells was significantly less than IL-2R {gamma} (Fig. 5A), suggesting that spontaneous receptor recycling or cytokine-mediated receptor internalization may not be a primary mechanism for down-regulating cell-surface expression. In support of these findings, it has been suggested by others that CD127 is not significantly internalized in HIV infection, despite the correlation between increased IL-7 production and decreased surface CD127 expression (12).

Since IL-7 decreases cell-surface CD127 expression without altering expression of mRNA encoding membrane-bound CD127, it is possible that CD127 expression may also be regulated post-transcriptionally. The transient nature of CD127 down-regulation on CD8+ T cells treated with up to 1000 pg ml–1 of IL-7 is consistent with the effects of IL-7 on CD127-surface expression on CD4+ T cells from healthy human donors (12) in the absence of changes in membrane-bound CD127 mRNA production (S. Sasson and A. Kelleher, personal communication). In contrast, a recent publication reports that human CD4+ T cells treated with IL-7 down-regulate both CD127 surface expression and production of total CD127 mRNA (i.e. transcripts that would either encode membrane bound or secreted receptor) (26), albeit only after 3–5 days of culture. In mice, IL-7 down-regulates the transcription mRNA-encoding CD127. There is no evidence to date to suggest the existence of an mRNA transcript encoding a secreted form of CD127 in mice (33, 53). These differences in CD127 gene regulation by IL-7 may be due to different experimental systems or inter-species variation. An example where such a difference exists is in in vivo treatment of HIV-infected individuals where IL-2 does not decrease surface CD127 expression on CD4+ or CD8+ T cells (28), while similar treatment of mice significantly decreases the expression of CD127 protein and mRNA in lymph node or splenic T cells (22, 47). Human–murine species differences in CD127 and transcription factor mRNA expression have become increasingly apparent. Decreased human CD127 mRNA expression was associated with decreased GA-binding protein expression (54), while increased CD127 mRNA expression was associated with increased expression of GFI1 which is known to suppress murine CD127. Despite that a spliced variant of CD127 encodes a secreted form of the receptor is known to exist (33), the effects of IL-7 and other IL-2R {gamma} cytokines on the expression of mRNA-encoding secreted CD127 have not been studied and is the focus of future research.

Since IL-7 did not induce an appreciable increase in cytoplasmic CD127, suggesting that the down-regulation of CD127 was not a result of receptor internalization, we investigated whether CD127 is shed from T cells. We were able to detect the release of CD127 from IL-7-stimulated CD8+ T cells in five of seven individuals as determined by western blot. Since western blot analysis is typically capable of detecting down to a minimum of ~0.1 ng ml–1 of protein, the fact that soluble CD127 was not observed in two individuals likely reflects the limited sensitivity of the assay used. The biological significance of soluble/circulating CD127 in vivo, its relevance in HIV infection and contribution to impaired IL-7 bioactivity remains to be investigated. Given IL-7 resulted in the release of CD127 in vitro and that increased plasma IL-7 is associated with HIV-induced lymphopenia (5) and decreased CD127 expression in HIV infection, we investigated whether CD127 could be found in human plasma in health and HIV disease. This study reports for the first time that CD127 can be found in human plasma. Moreover, we have documented the novel observation of an increase in plasma CD127 in HIV-infected individuals compared with uninfected individuals (Fig. 7B). It is possible that IL-7 induces secretion of CD127, that would bind circulating IL-7, and result in decreased IL-7 bioavailability, similar to the activity of other secreted cytokine receptors, such as the receptors for TNF-{alpha} and IL-6 which may have immunoregulatory functions (55). Other possible sources of CD127 (e.g. CD4+ T cells) or other factors inducing CD127 secretion (host and viral) may explain the increase of soluble CD127 in HIV infection compared with the relatively high concentrations of IL-7 required to induce CD127 secretion in vitro (Fig. 6).

Expression of CD127 on both naive and memory CD8+ and CD4+ T cells has recently been inversely correlated with CD8+ T-cell exhaustion in the context of persistent exposure to antigen resulting in the deletion of antigen-specific naive or memory cells (14, 56). Both latent [EBV and cytomegalovirus (CMV)] and chronic (HIV and hepatitis C virus) viral infections are associated CD127 down-regulation, the implications of which has yet to be fully described (14, 15, 57). The cellular mechanism linking antigen persistence to decreased CD127 expression is not known; however, the expression of CD127 on CMV-specific CD8+ T-cell subsets appears to confer antigen-independent proliferation in response to IL-7 in contrast to the proliferation of CD127– cells which required antigen and CD4 T-cell help or other cytokines (58). These data directly demonstrates that IL-7 down-regulates CD127 and suggests a biological consequence that may explain reported correlations of increased plasma IL-7 and decreased in CD127 in HIV patients (12, 45). Furthermore, the loss of CD127 expression has been correlated with the expansion of effector CD8+ T cell with cytolytic activity during chronic HIV infection (15, 17). Therefore, it appears that antigen persistence either via or in addition to cytokine control are mechanisms for the down-regulation of CD127 in vivo. The recent observation that HIV tat, to the exclusion of other HIV and non-HIV proteins, decreases CD127 on CD8+ T cells (59) suggests a degree of pathogen specificity and that CD127 down-regulation is not simply a non-specific response to immune activation.

These results support a role for IL-7 in the down-regulation of CD127 expression and impairment of CTL function observed in HIV infection. A consequence of down-regulating CD127 expression in states of chronic immune activation or persistent infection may be impaired CTL activity and memory cell development. As an increase of IL-7 occurs in the face of declining CTL activity, providing IL-7 therapeutically is unlikely to enhance HIV-specific cellular immunity, as hypothesized by others (50). This is supported by the observation that although therapeutic use of IL-7 in simian immunodeficiency virus-infected rhesus macaques resulted in an augmentation of CD4 counts following anti-retroviral treatment, it had no effect on the control of viral replication (23). Alternatively, enhancing IL-7 signaling by increasing the expression of or enhancing the function of CD127 may result in improved CD8+ T-cell function and enhanced control of viral replication. To this end, identifying the alteration of CD127 expression and function during HIV infection and determining the regulatory mechanisms may provide insights into the development of novel immune-based therapies for patients infected with HIV. Moreover, improved understanding of CD127 regulation may potentially provide valuable insights into normal T-cell physiology and the role of this receptor in other diseases in which cellular immune function and IL-7 activity may be altered.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Ontario HIV Treatment Network (OHTN) (ROBG131); OHTN Studentship to A.V.; and Canadian Institutes of Health Research (HOP84649) and OHTN fellowships to K.G. J.B.A. and A.K. are OHTN Career Scientists.


   Acknowledgements
 
The authors thank Paul MacPherson and Karen Copeland for critical review of the manuscript and Rejean Munger for assistance with the confocal microscope. A.V. is a Ph.D. candidate at the University of Ottawa and this work is submitted in partial requirement for the Ph.D. A.V. performed most of the research, analyzed data and assisted in the initial draft of the manuscript; A.M.C. assisted in the design of the research, performed research, analyzed and interpreted data and completed the draft of the manuscript; K.G. performed research; A.K. revised the manuscript and J.B.A. conceived the project, designed research, interpreted data and assisted in the completion of the manuscript. A.K. and J.B.A. are Ontario HIV Treatment Network (OHTN) Career Scientists. The authors declare no competing financial interests.


   Abbreviations
 
7-AAD, 7-aminoactinomycin D
CD127, IL-7 receptor {alpha}
CD132, IL-2 receptor {gamma}
cDNA, complementary DNA
CMV, cytomegalovirus
dNTP, deoxynucleoside triphosphate
GFI1, growth factor independence 1
MCF, mean channel fluorescence
TNF, tumor necrosis factor
TGF, transforming growth factor

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: A. Singer

Received 20 March 2007, accepted 20 September 2007.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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