The role of IL-7 during thymopoiesis has led to it being the focus of a number of therapeutic interventions. However, its small size and pleiotropic nature present problems for thymus-directed therapies. We have created a fusion molecule between the extracellular N-terminal domain of CCR9 and IL-7, which has the potential to overcome these difficulties. This novel fusion protein retains the thymopoietic activity of IL-7 and the ligand-binding ability of CCR9. As a thymopoietic agent, compared with IL-7, it shows an enhanced retention in the thymus, increased de novo T cell production, and increased thymic output. Old mice receiving the fusion protein show improved CD8 T cell responses and reduced viral load after infection with influenza virus compared with those receiving IL-7. This chimeric molecule offers a novel therapeutic strategy that may result in the production of an effective immunorestorative agent.

The use of therapeutic agents, such as cytokines, to improve immunity has been approached often in a haphazard manner, introducing the drug into the host at inappropriately high dosages at a distal site in the hope of reaching the target organ in sufficient quantity to have an effect. To help overcome the problem of dosage, chimeric variants of cytokines have been created. These molecules include cytokines conjugated with polyethylene glycol (1) and fusion to albumin (2) or to the Fc portion of IgG (3). Although production of such chimeras prolongs the t1/2 of cytokines and reduces dosing schedules, it does not completely address the problems associated with improving the cytokine’s targeting ability. One way of targeting a cytokine is to create a chimera between the cytokine and a single chain Ab (4), but such Abs, made in other species, often provoke immune responses. We report in this study a superior method for improving the targeting ability of cytokines, by using a chemokine receptor whose ligand is organ specific as the targeting moiety. This method has the advantage over the use of single chain Abs in that the receptor of interest can be cloned from the same species as the recipient of the therapy, thus causing no unwanted immune reaction.

IL-7 is a cytokine produced predominantly by class II+ thymic epithelial cells (5), which plays a central role in the survival and proliferation of thymocytes (6). In addition, IL-7 also has an effect on cells in the peripheral T cell pool, permitting the survival of naive and memory T cells (7). The role played by IL-7 in T cell development and homeostasis has made it the focus of numerous therapeutic interventions aimed at boosting thymopoiesis in the elderly (8), after bone marrow transplant (9), or following viral infection, for example with HIV (10). However, treatment with IL-7 is not without its problems: the use of high doses of this cytokine can lead to numerous side effects, such as osteoporosis (11), lymphoma (12), hyperproliferation (12), thrombocytopenia (13), neutropenia (13), and hemolytic anemia (13). In addition, high levels of IL-7 expression have also been shown to inhibit T cell development by inducing a dramatic block in the production of double-positive (DP)3 cells (14). Phillips et al. (15) have attempted to overcome these problems by incorporating an IL-7-secreting stromal cell line into the thymus with some success. Their method is not without its problems, including alterations to the thymic architecture, problems regulating the expression of IL-7, and the difficulty in translating the therapy to the clinic, which need to be addressed. We have taken an alternate approach to counter the adverse effects of IL-7 by creating a fusion protein between IL-7 and the extracellular region of the chemokine receptor CCR9. The chemokine CCL25, which is produced by the thymic stroma, is thought to play a major role in T cell development by acting as a chemoattractant for thymocytes, which express the CCL25 receptor, CCR9 (16). Unlike other CC chemokine receptors, CCR9 shows a strict specificity for its ligand CCL25 (17). When considered together with the high levels of CCL25 expressed in thymic tissue, CCR9 was deemed the ideal choice as a targeting component for IL-7. We show in this study the creation of a fusion protein between the N-terminal of CCR9 and IL-7 that shows enhanced thymopoietic ability, overcoming the problem of dosage previously associated with IL-7 therapy.

RNA was extracted from a male C57BL/6 mouse thymus using STAT-60 (AMS Biotechnology) and reverse transcribed, as previously described (18). The resulting cDNA was used for PCR with following primers (Oswel): CCR9, forward 5′-atg atg ccc aca gaa ctc aca agc-3′, reverse 5′-gct tgc aaa ctg cct gac aca tta-3′; IL-7, forward 5′-atg ttc cat gtt tct ttt aga tat-3′, reverse 5′-tgt tta tat act gcc ctt caa aat-3′.

The CCR9 and IL-7 PCR products together with the N-terminal portion of CCR9 to serve as a control were ligated into the pGEM-T vector (Promega) and transformed into JM109 competent cells.

CCR9 and IL-7 DNA were amplified by PCR using primers designed to create a novel restriction site NotI at the 3′ end of CCR9 and at the 5′ end of IL-7: CCR9, forward 5′-atg atg ccc aca gaa ctc aca agc-3′, reverse 5′-gcg gcc gcg tgc aaa ctg cct gac att at-3′; IL-7, forward 5′-ctc cgc ggc cgc atg ttc cat gtt tct-3′, reverse 5′-tgt tta tat act gcc ctt caa aat-3′ (Oswel). The resulting PCR products were subjected to restriction digest with NotI (Promega) and then used in a PCR with the forward CCR9 primer and the reverse IL-7 primer.

The CCR9/IL-7 fusion PCR product was ligated into the pcDNA4/HisMax TOPO TA expression vector (Invitrogen Life Technologies) and transformed into TOP10 competent cells.

Chinese hamster ovary cells were plated at a density of 5 × 104/ml in 60-mm culture dishes and transfected after 18 h in culture with 5 μg of EndoFree CCR9/IL-7 plasmid (Qiagen) in complete medium using poly(l-ornithine) (19) (Sigma-Aldrich). Stable transfectants were selected with Zeocin (Invitrogen Life Technologies).

In vivo experiments were conducted using young (6-wk) or old (20-mo) C57BL/6 male mice (Harlan Olac), as previously described (20). The anterior tibial muscle was first pretreated with 0.4 U/μl bovine hyaluronidase (Sigma-Aldrich). Two hours posthyaluronidase injection, 25 μl of circular control, IL-7, or fusion plasmid DNA (at 1 μg/μl) in normal saline was injected percutaneously in the anterior tibial muscle. An electrical field was applied to the muscle immediately following plasmid injection. The injected leg was held steady, and the 7-mm circular electrodes (BTX Tweezertrodes, VWR) were applied to the medial and lateral sides of the shaved lower hind limb with reasonable pressure to maintain contact with the skin surface. A voltage of 175 V/cm was applied in ten 20-ms square wave pulses at 1 Hz using a BTX ECM 830 electroporator. Both procedures were conducted under general anesthesia using the protocol described in Gollins et al. (21).

ELISA studies were performed on homogenized tissue and serum samples using reagents from R&D Systems.

Thymic output was assessed using the δEC assay (22). Quantification of the number of δEC genes was conducted by PCR using the Lightcycler (Roche) with primers specific for the Cδ exon 3 region: forward 5′-gct tcc aac ttc tca gtg c-3′, reverse 5′-ggg ggc aaa ata aaa tgg at-3′, the LightCycler FastStart DNA Master SYBR green I kit (Roche), and the cloned 205-bp product to generate the standard curve.

FACSCalibur (BD Biosciences) analysis of T cell phenotype was performed, as previously described (23). A total of 1 × 106 thymocytes was incubated with the following Abs: anti-CD8 FITC (clone 53-6.7), anti-CD4 CyChrome (clone H129.19), anti-CD4 R-PE (clone H129.19), anti-CD8 R-PE (clone 53-6.7), anti-CD3 R-PE (clone 17A2), anti-CD19 R-PE (clone ID3), anti-CD25 FITC (clone 7D4), and anti-CD44 CyChrome (clone IM7), or isotype-matched controls (all BD Biosciences). Thymocytes were fixed with 1% paraformaldehyde in PBS, and data analysis was performed using WinMDI software.

Following electroporation (as described above) with 1 μg/μl control, IL-7, or fusion constructs, 2 mo prior, 20-mo mice were anesthetized and intranasally infected with 50 hemagglutinin units of influenza A strain X31. After 6 days, single cell suspensions from lung tissue were stained for surface markers and intracellular cytokines and analyzed by flow cytometry, as previously described (24). Lung cells were incubated with anti-CD45RB FITC (clone R2a01; Caltag Laboratories), anti-CD4 PerCP (clone RM4-5), anti-CD8 allophycocyanin (clone 53-6.7), and anti-TNF PE (clone MP6-XT22) from BD Biosciences. Data were acquired on a FACSCalibur, and 10,000 lymphocyte events were analyzed with WinMDI software.

Viral load was assessed in the lung 6 days after virus challenge by PCR using the Lightcycler, using primers specific for the matrix protein of influenza A: forward 5′-gga tgt ttt tgc agg gaa ga-3′, reverse 5′-caa gcg cac cag ttg agt aa-3′, and the LightCycler FastStart DNA Master SYBR green I kit (Roche). We isolated the 287-bp product to provide the control sample required to generate the standard curve.

Statistical significance was evaluated using ANOVA and applying the Bonferroni correction using StatView software. Differences were considered significant if p < 0.0167.

The fusion plasmid was generated by first cloning the extracellular portion of murine CCR9 and murine IL-7, followed by the introduction of a NotI restriction enzyme site at the 3′ end of IL-7 and at the 5′ end of CCR9. The resulting products were digested with NotI and annealed, and the product was expanded by PCR to create the IL-7/CCR9 fusion gene. This fusion product was cloned into the mammalian expression vector, pcDNA4/HisMax TOPO TA, and was stably transfected into Chinese hamster ovary cells.

To assess whether the fusion protein exerted an effect in vivo, 1 μg/μl IL-7, fusion, or control plasmid (containing the N-terminal portion of CCR9) was electroporated into the anterior tibial muscle of mice. Muscle damage associated with the electrotransfer procedure arises from the intracellular presence and expression of plasmid DNA (25). The three plasmid constructs inserted all carried DNA capable of supporting gene expression and showed similar levels of muscle damage between each group (data not shown). Analysis of thymic tissue from these animals after 1 wk (Fig. 1,a) revealed significantly more IL-7 in the thymi of animals injected with the fusion and IL-7 plasmids compared with animals receiving the control plasmid control (p < 0.001). In addition, the results also showed significantly more IL-7 in animals after fusion treatment compared with treatment with IL-7 alone (p < 0.001). However, IL-7 levels were found to be below the level of detection in the plasma (data not shown). Urinary excretion is one of the major routes of elimination of IL-7 (26); therefore, we conducted analysis of IL-7 levels in the kidney (Fig. 1 b). This revealed that animals receiving the IL-7 and fusion plasmids had significantly more IL-7 than animals receiving the control plasmid (p < 0.001), whereas there was no difference in the IL-7 levels between the IL-7- and fusion-treated groups. These similar rates of excretion would suggest similar levels of production; this was confirmed by immunohistology of the anterior tibial muscles from treated animals. Again, a similar level of staining was found in animals receiving the IL-7 plasmid compared with those receiving the fusion plasmid (data not shown).

FIGURE 1.

IL-7 levels are higher in mice receiving fusion plasmid. a, Concentration of IL-7 in the thymi of mice, 6 wk of age, after treatment with 1 μg/μl control plasmid, IL-7, or the fusion plasmid. b, IL-7 levels in the kidneys of 6-wk mice after electroporation with 1 μg/μl sham control, IL-7, or the fusion plasmid. ∗, Denotes significant difference when compared with the control animals, p < 0.001, §, Denotes significant difference between IL-7- and fusion-treated animals, p < 0.001. Plots show medians with 5, 25, 75, and 95 percentiles of six mice.

FIGURE 1.

IL-7 levels are higher in mice receiving fusion plasmid. a, Concentration of IL-7 in the thymi of mice, 6 wk of age, after treatment with 1 μg/μl control plasmid, IL-7, or the fusion plasmid. b, IL-7 levels in the kidneys of 6-wk mice after electroporation with 1 μg/μl sham control, IL-7, or the fusion plasmid. ∗, Denotes significant difference when compared with the control animals, p < 0.001, §, Denotes significant difference between IL-7- and fusion-treated animals, p < 0.001. Plots show medians with 5, 25, 75, and 95 percentiles of six mice.

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To test whether increased intrathymic levels of IL-7 lead to increased thymic output, we conducted the δEC assay on animals 6 wk after treatment (Fig. 2). The δEC assay quantitates the excised TCR δ region from within the TCR α-chain (22), and the results revealed that splenic αβ T cells from animals treated with the fusion protein yielded significantly higher δEC numbers (p < 0.01) than animals receiving IL-7 alone (Fig. 2). However, because IL-7 increases the rate of peripheral cell turnover, the control group, receiving only CCR9, showed higher δEC levels, 0.29 ± 0.06 δEC/T cell, than either the IL-7 group, 0.075 ± 0.01 δEC/T cell, or the fusion group, 0.152 ± 0.02 δEC/T cell.

FIGURE 2.

Fusion-treated animals exhibit higher δEC numbers than treatment with IL-7. Number of δECs per 5 × 105 αβ T cells isolated from the spleen of mice 6 wk of age after treatment with 1 μg/μl IL-7 or the fusion plasmid. §, Denotes significant difference between IL-7- and fusion-treated animals, p < 0.01. Plots show medians with 5, 25, 75, and 95 percentiles of six mice.

FIGURE 2.

Fusion-treated animals exhibit higher δEC numbers than treatment with IL-7. Number of δECs per 5 × 105 αβ T cells isolated from the spleen of mice 6 wk of age after treatment with 1 μg/μl IL-7 or the fusion plasmid. §, Denotes significant difference between IL-7- and fusion-treated animals, p < 0.01. Plots show medians with 5, 25, 75, and 95 percentiles of six mice.

Close modal

We investigated whether the increased levels of IL-7 seen in the thymi of animals treated with either the IL-7 or the fusion plasmid had a restorative effect and allowed a renewal in thymopoiesis in aged animals. The morphology of thymi taken from old animals, 20 mo of age, after treatment with either IL-7, fusion, or the control plasmid, was analyzed by H&E staining (Fig. 3). A representative example of a control section revealed a loss in thymic structure (Fig. 3,a). The cellularity of the stroma was greatly reduced, with a loss of distinction between the cortical and medullary regions and an infiltration of white adipose tissue into the thymic stroma. After 8-wk treatment with IL-7 (Fig. 3,b), the cellularity of the thymus started to return, as well as a definable distinction between the cortex and medullar; however, some white adipose tissue remained. Fusion treatment (Fig. 3 c) revealed a complete restoration of the thymic architecture, with the stroma showing a high degree of cellularity, a clearly definable cortex and medullar, and the reformation of fine lobules, characteristic of young animals.

FIGURE 3.

Thymic architecture is restored after fusion treatment. Representative examples of thymic sections stained with H&E taken from 20-mo mice receiving the control (a), IL-7 (b), or fusion plasmid (c). Original magnification ×10. WAT indicates white adipose tissue; C, cortex; M, medullar.

FIGURE 3.

Thymic architecture is restored after fusion treatment. Representative examples of thymic sections stained with H&E taken from 20-mo mice receiving the control (a), IL-7 (b), or fusion plasmid (c). Original magnification ×10. WAT indicates white adipose tissue; C, cortex; M, medullar.

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Fusion treatment visibly demonstrated its restorative effect, but we also wanted to investigate its effect on thymopoiesis. Analysis of old mice, 1 wk after treatment, revealed the double-negative (DN) subset of fusion-treated animals (Fig. 4) and showed the greatest difference compared with the IL-7 and sham plasmid control animals, with the largest difference found in the CD44+CD25 subset. When comparing the data from all animals (Table I), a significant increase was found in the number of DN1 (CD44+CD25) cells of animals treated with IL-7 (p < 0.01) and fusion (p < 0.001) plasmids compared with control animals. Furthermore, there were significantly more DN1 cells in the fusion group (p < 0.001) compared with IL-7-treated animals. In addition, cell numbers were significantly enhanced in fusion-treated animals for the remaining subsets, DN2 (CD44+CD25+, p < 0.001), DN3 (CD44CD25+, p < 0.01), and the DN4 (CD44CD25, p < 0.01) subset, compared with control animals. There was no significant increase in the numbers of DP cells after treatment with IL-7 or fusion plasmid, but a significant increase was seen in the numbers of CD4-positive cells after IL-7 (p < 0.01) and fusion (p < 0.001) treatment when compared with the control group. Comparison between the fusion and IL-7 group also showed there to be a significant difference, with more CD4 cells in the fusion group (p < 0.05). A trend for an increase in the numbers of CD8 single-positive cells was also seen; however, these data neared significance. Taken together, these results suggest that the fusion plasmid is better able to restore thymopoiesis than IL-7 alone.

FIGURE 4.

Thymocyte numbers increase after treatment with fusion plasmid. A representative example of the DN subset obtained from 20-mo mice after 1-wk treatment with 1 μg/μl plasmid control, IL-7, or fusion plasmid.

FIGURE 4.

Thymocyte numbers increase after treatment with fusion plasmid. A representative example of the DN subset obtained from 20-mo mice after 1-wk treatment with 1 μg/μl plasmid control, IL-7, or fusion plasmid.

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Table I.

Thymocyte subset numbers following 1-wk treatment with 1 μg/μl plasmid control, IL-7, or fusion plasmid; showing averages and SD (n = 6)a

PopulationControl (±SD)IL-7 (±SD)Fusion (±SD)
CD44+CD25 (DN1) 0.81 ± 0.2 × 105 3.3 ± 0.1 × 105b 8.7 ± 0.3 × 105ce 
CD44+CD25+ (DN2) 0.82 ± 0.4 × 105 1.4 ± 0.5 × 105 2.2 ± 0.6 × 105b 
CD44CD25+ (DN3) 2.6 ± 0.9 × 105 3.5 ± 0.7 × 105 4.2 ± 1.2 × 105b 
CD44CD25 (DN4) 4.8 ± 5.5 × 105 6.8 ± 1.3 × 105 7.5 ± 1.8 × 105b 
    
CD4+CD8+ 18 ± 9.1 × 106 20 ± 2.9 × 106 25 ± 4 × 106 
CD4+CD8 4.5 ± 1.3 × 106 6.7 ± 1.1 × 106b 8.3 ± 0.8 × 106bd 
CD4CD8+ 2.3 ± 0.6 × 106 2.6 ± 0.5 × 106 3.1 ± 0.1 × 106 
PopulationControl (±SD)IL-7 (±SD)Fusion (±SD)
CD44+CD25 (DN1) 0.81 ± 0.2 × 105 3.3 ± 0.1 × 105b 8.7 ± 0.3 × 105ce 
CD44+CD25+ (DN2) 0.82 ± 0.4 × 105 1.4 ± 0.5 × 105 2.2 ± 0.6 × 105b 
CD44CD25+ (DN3) 2.6 ± 0.9 × 105 3.5 ± 0.7 × 105 4.2 ± 1.2 × 105b 
CD44CD25 (DN4) 4.8 ± 5.5 × 105 6.8 ± 1.3 × 105 7.5 ± 1.8 × 105b 
    
CD4+CD8+ 18 ± 9.1 × 106 20 ± 2.9 × 106 25 ± 4 × 106 
CD4+CD8 4.5 ± 1.3 × 106 6.7 ± 1.1 × 106b 8.3 ± 0.8 × 106bd 
CD4CD8+ 2.3 ± 0.6 × 106 2.6 ± 0.5 × 106 3.1 ± 0.1 × 106 
a

Denotes significant difference when compared with the control animals:

b

, p < 0.01;

c

, p < 0.001. Denotes significant difference between IL-7- and fusion-treated animals:

d

, p < 0.05;

e

, p < 0.001.

We then investigated whether the improved thymic output in fusion-treated animals translated into an improved in vivo response to pathogen. We infected 20-mo-old mice intranasally with influenza A strain X31 virus 2 mo after treatment with IL-7, fusion, or a sham plasmid control. Analysis of these animals 6 days postinfection indicated that there was significantly less viral load in the lungs of animals treated with IL-7 (p = 0.001) or fusion (p < 0.0001) plasmids when compared with those receiving the sham plasmid (Fig. 5,a). Substantial lung damage accompanies influenza infection, which is mediated by TNF (27), and is associated with cachexia. The percentage change in weight loss in response to viral infection (Fig. 5,b) appears to be reduced in fusion-treated animals compared with animals receiving IL-7 or the sham plasmid control; however, no significance was found. In addition, the number of CD8+ T cells producing TNF was significantly reduced (Fig. 5,c) in influenza-infected animals previously treated with IL-7 (p = 0.001) or fusion (p < 0.0001) plasmids compared with the sham group. The difference between IL-7 and fusion treatment approached significance (p = 0.0372). These results suggest that the fusion treatment may enable better control of the infection with a more efficient viral clearance and a reduction in the development of influenza virus-associated immunopathology. However, we found no change in the amount of TNF produced by CD4+ T cells (Fig. 5 d), nor did we see any difference in IFN-γ production by either CD4+ or CD8+ T cells (data not shown) following treatment with IL-7 or fusion plasmids.

FIGURE 5.

Fusion treatment causes a reduction in lung immunopathology following infection with influenza A strain X31 virus. a, Viral load in 20-mo mice treated with control plasmid, IL-7, or fusion plasmid. b, Percentage change in weight lost by animals receiving IL-7, fusion, or control plasmid. Results are the mean weight ± 1 SD of five to six mice. c, Number of CD8+TNF+ T cells in the lung after receiving IL-7, fusion, or control plasmid. d, Number of CD4+TNF+ T cells in the lung after the three different treatments. ∗, Denotes significant difference when compared with the control group, p < 0.001. Plots show medians with 5, 25, 75, and 95 percentiles of five to six mice.

FIGURE 5.

Fusion treatment causes a reduction in lung immunopathology following infection with influenza A strain X31 virus. a, Viral load in 20-mo mice treated with control plasmid, IL-7, or fusion plasmid. b, Percentage change in weight lost by animals receiving IL-7, fusion, or control plasmid. Results are the mean weight ± 1 SD of five to six mice. c, Number of CD8+TNF+ T cells in the lung after receiving IL-7, fusion, or control plasmid. d, Number of CD4+TNF+ T cells in the lung after the three different treatments. ∗, Denotes significant difference when compared with the control group, p < 0.001. Plots show medians with 5, 25, 75, and 95 percentiles of five to six mice.

Close modal

CD8+ T cell function is important in the clearance of influenza infection (28), and we wished to evaluate whether the improved viral clearance was associated with an increase in CD8+ T cells, given that we had hypothesized that there was renewed thymopoiesis in IL-7- and fusion-treated animals. CD45RB expression distinguishes naive T cells (CD45RBhigh) from activated cells (CD45RBlow). CD8+ T cells from fusion-treated animals were more numerous and showed lower levels of CD45RB expression than IL-7 and sham animals (Fig. 6, a and b), with a mean fluorescence intensity for fusion-treated animals of 17 compared with 28 for IL-7 treatment and 64 for the sham plasmid control animals. We were unable to detect any change in CD4+CD45RB expression or any difference in the numbers of CD4+CD45RBlow T cells (Fig. 6, c and d) after receiving the fusion or IL-7 plasmid. We did, however, find significantly more activated CD8+CD45RBlow cells in fusion (p < 0.0001)- and IL-7 (p = 0.0026)-treated animals compared with the sham control group (Fig. 6 b); in addition, there was a significant difference between fusion and IL-7 treatment (p = 0.0092).

FIGURE 6.

CD45RBlow expression in CD8+ cells is greater in fusion-treated animals after receiving influenza. a, Histogram showing CD8+CD45RB expression in IL-7, fusion, or control plasmid animals following intranasal infection with influenza virus. b, Total number of lung CD8+CD45RBlow cells following treatment with control, IL-7, or fusion plasmid. c, Histogram showing CD4+CD45RB expression in IL-7, fusion, or control plasmid animals. d, Total number of CD4+CD45RBlow cells isolated from the lung in animals that had received IL-7, fusion, or control plasmid. ∗, Denotes significant difference when compared with the control animals, p < 0.01. §, Denotes significant difference between IL-7- and fusion-treated animals, p = 0.0092. Plots show medians with 5, 25, 75, and 95 percentiles of five to six mice.

FIGURE 6.

CD45RBlow expression in CD8+ cells is greater in fusion-treated animals after receiving influenza. a, Histogram showing CD8+CD45RB expression in IL-7, fusion, or control plasmid animals following intranasal infection with influenza virus. b, Total number of lung CD8+CD45RBlow cells following treatment with control, IL-7, or fusion plasmid. c, Histogram showing CD4+CD45RB expression in IL-7, fusion, or control plasmid animals. d, Total number of CD4+CD45RBlow cells isolated from the lung in animals that had received IL-7, fusion, or control plasmid. ∗, Denotes significant difference when compared with the control animals, p < 0.01. §, Denotes significant difference between IL-7- and fusion-treated animals, p = 0.0092. Plots show medians with 5, 25, 75, and 95 percentiles of five to six mice.

Close modal

We report in this study the generation of a novel chimeric variant of IL-7 that has the ability to overcome the high doses currently necessary for a thymus-directed therapy. In addition, we show a superior method for improving the targeting ability of cytokines. We created the fusion molecule by fusing the extracellular domain of CCR9 to IL-7 with the aim of enhancing the retention of the fused molecule in the thymus. We chose to fuse CCR9 onto IL-7 because of the strict specificity for its ligand CCL25, and the high levels of CCL25 expressed by thymic tissue (17).

The fusion protein also showed an improved effect in vivo compared with IL-7 following expression of these plasmids. We found there to be significantly higher levels of IL-7 retained in the thymi of animals receiving fusion treatment, even though the similar amounts of IL-7 were produced by both treatment arms, suggesting that the fusion protein is preferentially retained in the thymus. This enhancement in IL-7 levels in the thymus after receiving the fusion protein was not considered an effect of gene dosage, as IL-7 levels in the kidney were not found to differ between the IL-7 and fusion group, indicating comparable clearance rates for both groups. Moreover, similar levels of expression were noted following immunohistology of the anterior tibial muscles of both groups. This improvement in thymopoiesis is due to an increased retention of the fusion protein, as fusion-treated animals showed significantly higher δEC levels than those treated with IL-7. This holds true even though the δEC levels were higher in control animals (CCR9 alone) compared with those receiving IL-7 or the fusion protein. This is because IL-7 is a potent regulator of T cell homeostasis and is able to enhance peripheral T cell expansion in a thymic independent manner (7). We chose to deliver the three therapies via plasmids, the downside of which being that vectors containing IL-7 continually produced the cytokine leading to complexity in interpreting the δEC data, as δEC values can be influenced by changes in peripheral T cell turnover. Despite this, the results showed the fusion protein to have a higher δEC value than animals receiving IL-7 alone. However, a direct assessment of whether the fusion protein shows better retention in the thymus would be to inject soluble fusion protein and IL-7. In addition, we cannot discount the possibility that the fusion protein binds other sites in the body, such as the gut, and this is currently being investigated.

The high levels of IL-7 detected after fusion treatment translated into a restoration of thymic architecture and a renewal of thymopoiesis. We demonstrated that fusion treatment caused regeneration of the thymus, defined by an increase in cellularity and the identification of distinct cortical and medullary regions. We also found there to be increased numbers of DN thymocytes after both IL-7 and fusion treatment, with the fusion protein showing a significantly greater number of DN1 cells compared with IL-7 treatment. In addition, the proportion of DN2, 3, and 4 cells also significantly increased after fusion treatment, whereas IL-7 alone failed to have such an effect, further indicating that the fusion protein is preferentially retained in the thymus. We also found fusion treatment to increase the number of CD4+ T cells, with no change in the number of DP cells. This is in contrast to a number of reports that show a decreased number of DP cells with overexpression of IL-7 (14) and an inhibition in the development of mature T cells following treatment with IL-7-secreting stromal cells (15). This may reflect the differing IL-7Rα expression profiles of these cells, the different concentrations of IL-7 and mode of delivery, as well as the timing of analysis. Our data were obtained only 1 wk after IL-7 supplementation, which might not have provided enough time for significant increases in the DP population from the DN progenitors to become apparent. However, we chose this time point to determine whether increases in these early T cell subsets translated into increased thymic output. We decided to use i.m. injection of plasmid DNA to deliver the IL-7, as it negated the difficulty of producing large quantities of highly pure cytokine. Intramuscular electrotransfer of DNA has the advantage that plasmids are retained for prolonged periods; in the absence of an immune response, the expression can last for over 6 mo and as long as 18 mo, in contrast to mitotic tissues in which plasmids can be rapidly lost. Additionally, electrotransfer reduces the variation seen with simple injection of naked DNA.

An increase in thymic output was evident from the δEC assay, with fusion animals showing significantly higher δEC levels in their splenic αβ T cell populations when compared with animals receiving IL-7 alone. This improvement in thymic output could be translated into an enhanced in vivo response to influenza virus, a virus that causes considerable morbidity and mortality in the elderly (29). This was demonstrated by a lower viral load in fusion- and IL-7-treated animals, which agrees with previous data reporting a lower viral burden during infection with influenza (30) and HSV type-1 (31) following treatment with rIL-7.

Substantial lung damage accompanies influenza infection, which is mediated by TNF (27). When TNF is released in large quantities, it is associated with cachexia, enhanced cell recruitment, and proliferation leading to airway occlusion (24). We show reduced numbers of CD8+ T cells expressing TNF in mice treated with IL-7, an effect that was more prominent by the addition of the fusion protein. This result is surprising because the number of activated (CD45RBlow) CD8+ T cells increased compared with control-treated mice, and viral titers were reduced. It is possible that TNF plays only a minor role in the clearance of influenza virus. In support of this hypothesis, our studies show that TNF neutralization does not affect clearance of pulmonary respiratory syncytial virus or influenza (24). The fusion protein may cause viral replication to be controlled more effectively by restoring the age-associated defects in effector memory cells more effectively than IL-7. Such age-related defects include the inability to mount a primary immune response against Ag (32). Indeed, it has been shown that when old and young mice were administered 75 hemagglutination units of influenza A virus, there were marked differences between the groups, with aged animals having fewer influenza-specific CD8+ T cells and a lower percentage of IFN-γ-producing cells, together with a decreased viral specific CTL activity (33). Such changes in effector cells in the elderly have been correlated to a reduced production of IL-7, suggesting a relationship between IL-7 and effector cells (34). It is also possible that these age-related defects in CD8+ effector function are caused in part by an alteration in CD4+ T cell-dependent help. Indeed, a recent study by Haynes et al. (35) has elegantly shown that the removal of aged CD4+ T cells from aged animals induced the production of new T cells, which exhibit robust responses to Ag ex vivo and in vivo. These data would suggest that the fusion protein, which is preferentially retained in the thymus, reduces the immunopathology following influenza infection by causing greater de novo T cell production, thus allowing fusion-treated animals to generate a more robust response to Ag.

We have described the creation of a novel fusion protein that shows enhanced thymopoiesis; however, if this fusion protein is to be developed further, its immunogenicity needs to be determined. The possibility that the fusion protein forms Abs that either block or neutralize the biological activity of IL-7 cannot be discounted, as a number of recombinant proteins are known to elicit Ab responses to varying degrees. Abs have been shown to develop in 20–75% of patients being treated with recombinant proteins, including erythropoietin (36), coagulation factor VIII (37), human growth hormone (hGH) (38), human GM-CSF (39), and IFNs α and β (IFN-α, IFN-β) (40, 41). Initial preparations of hGH contained an additional methionine residue at the N-terminal (methionine-human growth hormone; met-hGH), with the occurrence of Abs to met-hGH being measured in up to 75% of patients (38). Whereas more recent preparations of hGH that lack the extra methionine have been found to be less immunogenic, the incidence of Abs being found is 1.1% of patients (42). Although the mechanisms that underlie the generation of Abs to recombinant protein are not well understood, it is thought to involve peptide epitopes of the recombinant protein binding to HLA class II molecules (43). As with met-hGH, the fusion protein described in this work has the potential of being immunogenic, with responses being directed to the junctional sequence between the extracellular CCR9 domain and IL-7. Only by analyzing the binding ability of this novel peptide epitope contained in the fusion protein can we gauge the level of immunogenicity.

In summary, we have devised a novel therapeutic strategy by fusing the N-terminal domain of the chemokine receptor CCR9 to IL-7, causing an enhanced retention in the thymus and improved thymopoietic ability that we feel is a more effective immunorestorative agent than IL-7 alone.

We thank Professor Frances Gotch and Dr. Nesrina Imami for their critical review of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Biotechnology and Biological Sciences Research Council Experimental Research on Ageing Initiative, Grant 16279.

3

Abbreviations used in this paper: DP, double positive; δEC, δ excision circle; DN, double negative; hGH, human growth hormone; met-hGH, methionine-human growth hormone.

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