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Published ahead of print on February 8, 2006
J Am Soc Nephrol 17: 697-706, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2005090978
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Basic Immunology and Pathology

Foxp3-Transduced Polyclonal Regulatory T Cells Protect against Chronic Renal Injury from Adriamycin

Yuan Min Wang*, Geoff Yu Zhang*, Yiping Wang{dagger}, Min Hu*, Huiling Wu*, Debbie Watson*, Shohei Hori{ddagger},§, Ian E. Alexander||, David C.H. Harris{dagger} and Stephen I. Alexander*

* Centre for Kidney Research and || Gene Therapy Research Unit, Children’s Hospital at Westmead, and {dagger} Centre for Transplantation and Renal Research, University of Sydney at Westmead Millennium Institute, Westmead, New South Wales, Sydney, Australia; {ddagger} RIKEN Research Center for Allergy and Immunology, Yokohama, and § Japan Science and Technology Agency, Kawaguchi, Japan

Address correspondence to: Dr. Stephen I. Alexander, Centre for Kidney Research, Children’s Hospital at Westmead, Westmead, New South Wales 2145, Sydney, Australia. Phone: +61-2-9845-3408; Fax: +61-2-9845-3038; E-mail: stephena{at}chw.edu.au

Received for publication September 21, 2005. Accepted for publication December 13, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic proteinuric renal injury is a major cause of ESRD. Adriamycin nephropathy is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. Foxp3 is a gene that induces a regulatory T cell (Treg) phenotype. It was hypothesized that Foxp3-transduced Treg could protect against renal injury in Adriamycin nephropathy. CD4+ T cells were transduced with either a Foxp3-containing retrovirus or a control retrovirus. Foxp3-transduced T cells had a regulatory phenotype by functional and phenotypic assays. Adoptive transfer of Foxp3-transduced T cells protected against renal injury. Urinary protein excretion and serum creatinine were reduced (P < 0.05), and there was significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates (P < 0.01). It is concluded that Foxp3-transduced Treg cells may have a therapeutic role in protecting against immune injury and disease progression in chronic proteinuric renal disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulatory T cells (Treg) have a role in many human disease states, including autoimmune disease and responses to infection and cancer (1,2). Recently, defective Treg numbers and function have been shown in human diseases, including multiple sclerosis, psoriasis, rheumatoid arthritis, autoimmune polyglandular syndrome, and myasthenia gravis (3–5). Recovery from the autoimmune renal disease Goodpasture’s is associated with the expansion of antigen-specific Treg (6). The best characterized subset of Treg is the CD4+CD25+ T lymphocyte. Recent studies have indicated that the transcription factor gene Foxp3 is specifically expressed by CD4+CD25+ cells and programs their development and function (7–10). Therefore, cellular therapy using Treg to replace missing regulatory function is an attractive option for many immune-mediated diseases.

Forced expression of Foxp3 in naïve CD4+ T cells induces a Treg phenotype (11). This has been applied in diabetes using Foxp3 transduction of TCR transgenic T cells in NOD mice (12). Only TCR transgenic cells corrected diabetes, whereas transduced polyclonal CD4+ T cells did not. Similarly, Foxp3 transduction in a TCR transgenic model of rejection has been successful (13). However, other than correction of Treg deficiency, the only study using polyclonal T cells transduced with Foxp3 demonstrated that these are broadly protective against severe systemic autoimmune disease and contact dermatitis (14). Alternative strategies to direct Treg to inhibit pathogenic T cells have used expression in polyclonal Treg of MHC class I in combination with the pathogenic myelin peptide MBP in experimental allergic encephalitis (15). The recent description of the green fluorescence protein (GFP)-Foxp3 mouse suggests that Foxp3 expression identifies the Treg population and is expressed in {alpha}beta T cells (16). Hence, the more significant disease is found in Foxp3-deficient mice ("scurfy mice") that die from autoimmune disease within 3 wk of age, compared with those depleted of CD4+CD25+ T cells that develop autoimmune disease at a much slower rate. This suggests that Foxp3-expressing T cells may be more effective Treg than CD4+CD25+ T cells alone (16,17).

Chronic proteinuric renal disease is a major cause of ESRD (18). A regulatory CD4+ T cell population that secretes TGF-beta has been shown in rat mercuric-chloride autoimmune glomerulonephritis, a toxin-induced nephritis with secondary immune injury (19). Similarly, CD4+CD25+ lymphocytes can protect against anti–glomerular basement membrane disease that is induced by injection of specific anti-sera in mice (11,20). We have used Adriamycin (ADR)-induced nephropathy (AN) in mice as a model of chronic proteinuric renal disease (21,22). AN is induced in either mice or rats by a single injection of ADR. After the initial toxic injury, an immune-mediated chronic proteinuric renal disease that resembles human focal segmental glomerulosclerosis develops (23). The pathologic features of AN are nephrotic syndrome, focal glomerular sclerosis, tubular injury, and interstitial compartment expansion with mononuclear cell infiltrates that are composed largely of macrophages and T cells (21–24). Initial studies showed that depletion of CD8 T cells ameliorated AN and that depletion of CD4 T cells worsened disease (22,25). However, in immune-deficient SCID mice, AN is more severe than in BALB/c mice with lower doses of ADR and is still accompanied with heavy macrophage infiltrate, suggesting that the cognate immune system contains a subset of protective cells (unpublished data). The CD4+CD25+ subset of T cells therefore is a potential candidate for this role. We therefore wished to examine the effect of Treg on AN.

Treg were created by retroviral gene transfer of Foxp3 into CD4+ T cells. We evaluated the transduced T cells for regulatory markers and tested their ability to inhibit T cell activation in vivo. We then tested the effect of the Foxp3-transduced T cells on AN. We further depleted Treg using anti-CD25 antibodies. The results show that Foxp3-transduced CD4+ Treg can protect against renal functional and structural injury in vivo in this murine model of chronic proteinuric renal disease, whereas depletion of CD4+CD25+ T cells exacerbates AN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Male BALB/c mice were obtained from the ARC (Perth, Australia). All mice were maintained free of pathogens in the Westmead Hospital animal house. Eight-week-old mice that weighed 20 to 22 g were used in all groups.

Cell Culture and Retroviral Transduction
The cell lines that were used include NIH/3T3 and packaging cell line EcoPack2–293 (BD Biosciences, North Ryde, Australia). The cell lines were cultured in DMEM (Invitrogen, Carlsbad, CA). Foxp3/MIGR1 and MIGR1 vectors were provided by Dr. Hori (11) and Dr. Pear (26). The retroviral vectors Foxp3/MIGR1 and MIGR1 were transfected into the packaging cell line EcoPack2–293 using calcium phosphate, and the viral supernatant was collected (27). The viral titer was 106 cfu/ml. Mouse CD4+ T cells were isolated from BALB/c mouse spleens by MACS CD4+ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and stimulated by anti-CD3 mAb (5 µg/ml; BD Biosciences) and rIL-2 (100 IU/ml) for 24 h and then were infected with either Foxp3/MIGR1 or MIGR1 viral supernatant using retronectin (Edward Keller, Hallam, Australia) and Vivaspin (Sartorins, East Oakleigh, Australia). GFP-positive cells then were sorted by flow cytometry at day 7 after transduction (BD FACS Vantage SE).

Adoptive Transfer of Foxp3-Transduced Cells and Induction of AN
After sorting, 1 x 106 MIGR1/Foxp3- or MIGR1-transduced cells were injected into the tail vein of each BALB/c mouse. Mice were divided into four groups (five mice in each group): (1) The ADR group (received ADR only), (2) the ADR+Foxp3 group, (3) ADR+MIGR1 group, and (4) the control group. Both transduced groups were injected 1 wk before injection with ADR. ADR (doxorubicin hydrochloride; Pharmacia & Upjohn Pty Ltd., Perth, Australia) was injected via the tail vein of each nonanesthetized mouse (9.6 mg/kg). Mice in the control group were treated with saline only. Body weights were measured daily until day 28 after ADR. Blood, spleen, and kidney samples were obtained from mice in each group.

Depletion of CD4+ CD25+ Cells In Vivo for AN Model
A purified rat anti-mouse CD25 monoclonal IgG1 antibody (PC61; Bio Express, West Lebanon, NH) was used to deplete mouse CD4+ CD25+ cells in vivo. Five mice received an intravenous injection of 0.25 mg of PC61 2 d before ADR injection. Another dose of 0.25 mg of PC61 was injected intravenously 2 d after ADR injection. The efficacy of CD25 depletion was confirmed by flow cytometry analysis, using PE-conjugated anti-mouse CD25 and FITC-conjugated anti-mouse CD4 (ADR +PC61).

Renal Function
Renal function was assessed by measurement of urine protein and creatinine and of blood albumin and creatinine. Urine was collected from each mouse for 16 h before being killed. Urine volume, protein, and creatinine were measured. Blood samples for serum albumin and creatinine were obtained by cardiac puncture at day 28. Creatinine in serum and urine was measured by the CREA method, serum albumin was analyzed by the ALB method, and total urine protein was measured by the UCFP method on the Dimension Clinical Chemistry System (Dade Behring Ltd., Deerfield, IL).

Proliferation Assays
Ninety-six-well flat-bottom plates were coated with anti-CD3 mAb (5 µg/ml; BD Biosciences). Sorted CD4+CD25– cells (2.5 x 104 per well) were cultured alone or in the presence of a 1:1 ratio of transduced GFP-expressing cells (sorted from cells that were transduced with Foxp3 or MIGR1), or sorted CD4+CD25+ cells were cultured in a final volume of 200 µl of complete medium at 37°C for 72 h. Titration to a ratio of target:suppressor of 1:0.5 was also performed. During the last 16 h of culture, cells were pulsed with 3H-thymidine (1 µCi/well; MP Biomedicals, Seven Hills, Australia). Cells were assessed for thymidine incorporation in a Microbeta counter (Wallac Oy 1450 MicroBeta; Wallac, Melbourne, Australia).

RNA Isolation and Real-Time PCR
RNA was isolated from mouse lymphocytes, kidney, spleen, and lymph node using TRIZOL Reagent (Invitrogen, Life Technologies, Mount Waverly, Australia). The total amount of RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Life Technologies) and random primer (Promega, Madison, WI). Foxp3 mRNA levels were quantified by real-time PCR using the ABI PRISM 7700 (PE Applied Biosystems, Foster City, CA), and cDNA samples were subjected to real-time quantitative PCR analyses using primers and an internal fluorescence probe specific for Foxp3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR mixture contained 0.3 µM primers and 0.05 µM TaqMan probe and was cycled for 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The primers and TaqMan probe sequences for real-time PCR were as follows: Foxp3 primers 5'-TTGGCCAGCGCCATCTT-3' and 5'-TGCCTCCTCCAGAGAGAAGTG-3', Foxp3 probe 6FAMCAGCTGCTGCTCCAGMGBNFQ, GAPDH primers 5'-TGCACCACCAACTGCTTAGC-3' and 5'-GGAAGGCCATGCCAGTGA-3', and GAPDH probe VICCCTGGCCAAGGTCATCCATGACAACTT-TAMRA. The normalized value for Foxp3 mRNA expression was calculated as the relative quantity of Foxp3 divided by the relative quantity of GAPDH. Triplicate samples were run.

Reverse Transcription–PCR Conditions and Primers
The expression of TGF-beta, CTLA-4, CD103, and GITR was measured by reverse transcription–PCR (RT-PCR). RNA isolation and cDNA amplification were performed as above. cDNA was subjected to nested PCR amplification using external and internal primer couples, and beta-actin was used as a control. The PCR conditions were 95°C for 5 min (1 cycle); 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min (35 cycles) with the final cycle at 72°C for 7 min. The following primers were used: CTLA-4, 5'-TGTGCCACGACATTCACAG-3' and 5'-TTGGGGGCATTTTCACATAG-3'; GITR, 5'-AACGGAAGTGGCAACAACAC-3' and 5'-CTTGGGGCACAGAGGAAG A-3'; TGF-beta, 5'-TGGACCGCAACAACGCCATCTATGAGAAAA CC-3' and 5'-TGGAGCTGAAGCAATAGTTGGTATCCAGGGCT-3'; CD103, 5'-CGTGGAGAAGAAGGCAGAGT-3' and 5'-TCGGGGGTAAAGGTCATAGAT-3'; and beta-actin, 5'-TGGGTCAGAAGGACTCCTATG-3' and 5'-CAGGCAGCTCATAGCTCTTCT-3'. The PCR products were run on 1.5% agarose gels and visualized under ultraviolet light using gel-doc 1000 (Bio-Rad, Hercules, CA).

Flow Cytometric Analysis
Antibodies that were used for flow cytometry included FITC-conjugated anti-mouse CD25, phycoerythrin (PE)-conjugated anti-mouse CD3, and PE-conjugated anti-mouse CD4 (BD Bioscience). Foxp3 intracellular staining was performed by using PE anti-mouse Foxp3 Staining Set (clone FJK-16s; eBioscience, San Diego, CA) following the manufacturer’s instructions. All samples were analyzed on a FACScan analyzer (Becton Dickinson, Mountain View, CA). CellQuest software was used for analysis (Becton Dickinson, North Ryde, Australia).

Histology and Morphometric Evaluation
The kidneys were removed rapidly on day 28. Sagittal slices of renal tissue were fixed in neutral buffered formalin at room temperature for 24 h and embedded in paraffin for evaluation of pathology. Five-micrometer slices were stained with periodic acid-Schiff reagent and assessed by light microscopy. The remaining cortex of the same kidney was snap-frozen in liquid nitrogen and used for RNA and immunohistochemistry. In each biopsy, a semiquantitative score from two blinded, trained observers was used to evaluate the degree of renal injury, and a minimum of 10 consecutive fields at a magnification of x400 were assessed and scored in each section. The degree of renal injury was estimated by evaluating the percentage of renal injury per field and was graded on a scale of 0 to 4 (28).

Immunohistochemistry
Immunohistochemical staining was performed for CD4+ T cells, CD8+ T cells,and macrophages. Primary antibodies that were used in immunohistochemistry were rat anti-mouse L3T4 (Sigma, Balcatta, Australia) for CD4+, rat-anti-mouse Ly-2 (PharMingen, Inc., Sydney, Australia) for CD8+, and rat anti-mouse Mac-3 (PharMingen, Inc.) for macrophages. The secondary antibody was a biotinylated rabbit anti-rat Ig (Dako Corp., Carpinteria, CA). A section from each kidney or spleen was placed in OCT (Sakura Fintek Inc., Torrance, CA). Six-micrometer sections were cut, dried, and fixed in cold acetone. Endogenous peroxidase activity was blocked by incubating sections for 15 min in 0.3% (vol/vol) H2O2 solution. Endogenous avidin binding activity was blocked by incubating the sections with Biotin Blocking System (Dako). For control sections, normal rat Ig was used. Sections were incubated with secondary antibodies, 3,3-diaminobenzidine substrate-chromogen solution (Dako) was applied, and then sections were washed. Slides were counterstained with hematoxylin (Sigma). For assessment of interstitial infiltration, positively stained cells that were located in interstitium only were counted from five random cortical fields (Magnification, x400). Glomerular macrophage infiltration was evaluated as the number of macrophages per 10 glomerular cross-sections.

Assessment of Number of Adoptively Transferred Cells In Vivo
CD4+ T cells were purified by MACS CD4+ MicroBeads (Miltenyi Biotec) from splenocytes of B6STL-ptprca (Ly5.1) mice, and 1 x 106 CD4+ T cells were transferred by tail-vein injection into congenic C57Bl/6 mice (Ly5.2). Twenty-four hours after transfer, peripheral blood was assessed for relative expression of Ly5.1 CD4+ T lymphocytes by flow cytometry using FITC-conjugated anti-CD4 and PE-conjugated anti-Ly5.1.

Semiquantitative RT-PCR for GFP Expression
Semiquantitative RT-PCR for GFP expression was performed on tissues from each experimental group. The primers that were used to amplify the GFP sequences (Invitrogen) were 5'-CCTGAAGTTCATCTGCACCACC-3' and 5'-CTGCTGGTAGTGGTCGGCGAGC-3'. Amplification conditions were 95°C for 5 min, followed by 40 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min. beta-Actin primers served as the internal control. The sequences used are described above, and PCR conditions were the same as for GFP, except that the reaction was run for 30 cycles. RT-PCR fragments of GFP and beta-actin were electrophoresed on a 2% agarose gel. Signals were quantified by Alphalmager analysis system (Alpha Innotech, Quantum Scientific, Lane Cove, Australia).

Statistical Analyses
Statistical analysis was performed by one-way ANOVA for multiple comparisons. Results are expressed as the group mean ± SD. Two group differences were analyzed by t test. Two-tailed P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retroviral Transduction of Foxp3
Retroviral vectors that expressed Foxp3 and GFP were transfected into the packaging cell line EcoPack2–293, and viral supernatants were transduced into mouse CD4+ T cells. Retroviral transduction led to expression of GFP/Foxp3 in NIH/3T3 cell line and also in primary murine CD4 T cells (Figure 1A). EcoPack2–293, NIH/3T3 cell lines, and T cells that were transduced by either retroviral vectors expressed GFP. One week after transduction, 30% of Foxp3- and 50% of the control MIGR1-transduced T cells showed GFP expression by flow cytometry (Figure 1B).


Figure 1
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  		Figure 1. Expression of green fluorescence protein (GFP) in the EcoPack2–293 cell line, NIH/3T3 cell line, and T cells under reverse fluorescence microscope after transduction of both retroviral vectors (Foxp3/GFP and MIGR1/GFP). (A) The packaging cell line EcoPack2–293, NIH/3T3 cells, and transduced CD4+ T cells show expression of GFP after transfection or transduction with the Foxp3 and control MIGR1 retroviruses. (B) Flow cytometry of virally transduced CD4+ T cells shows 30 to 58% expression of GFP (transduced cells, solid black; untransduced CD4 T cells, gray overlay).

 
Transduction of Foxp3 Induces a Treg Phenotype
We examined the expression of Foxp3 mRNA by RT-PCR using Foxp3-specific primers and beta-actin as control. Sorted CD4+CD25+ cells showed expression of Foxp3, whereas CD4+CD25– cells did not (Figure 2A). The Foxp3-transduced NIH/3T3 cell lines showed strong Foxp3 expression, whereas cells that were transduced with control retroviral vector showed no Foxp3 expression (Figure 2B). One week after Foxp3 transduction, GFP-positive cells were sorted further by flow cytometry. After sorting, Foxp3-transduced cells had high levels of expression of Foxp3, TGF-beta, and CTLA-4 and lower expression of CD103 by RT-PCR (Figure 2C). Control vector (MIGR1)-transduced cells showed no expression of these Treg markers. GITR was found in all T cells possibly secondary to the activation step above.


Figure 2
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  		Figure 2. Foxp3 is specifically expressed in CD4+CD25+ T cells and Foxp3-transduced T cells. (A) PCR from 3T3 cell line before and after transduction. (B) The expression of Foxp3 mRNA in different cell populations by reverse transcription–PCR (RT-PCR) using Foxp3-specific primers and beta-actin as a control. (C) GFP-positive cells were sorted further by flow cytometry. After sorting, Foxp3-transduced cells showed stronger expression of Foxp3, TGF-beta, and CTLA-4 by RT-PCR. In cells that were transduced with control vector (MIGR1), there was no expression for these regulatory T cell (Treg) markers. (D) Real-time quantitative PCR of Foxp3 from purified T cell populations (each representative of three independent experiments). P < 0.01, CD4+ cells versus CD4– cells; P < 0.001, CD4+CD25+ cells versus CD4+CD25– cells. P < 0.001, Foxp3-transduced cells versus CD4+ cells and MIGR1-transduced cells (**P < 0.01, ***P < 0.001).

 
Foxp3 mRNA expression was assessed in transduced and control CD4+ T cell subsets. cDNA samples were subjected to real-time quantitative PCR analyses using primers and internal fluorescence probe specific for Foxp3 or GAPDH. Our results showed that Foxp3 mRNA level in CD4+CD25+ cells was 200-fold more abundant than in CD4+CD25– cells, and transduced Foxp3 T cells showed >200-fold higher expression of Foxp3 than MIGR1-transduced T cells (Figure 2D).

Foxp3-Transduced T Cells Inhibit CD4+CD25– Cell Proliferation In Vitro
Foxp3-transduced cells specifically inhibited proliferation of freshly prepared CD4+CD25– responder T cells when stimulated with anti-CD3 mAb. The proliferation of CD4+CD25– responder T cells was inhibited by freshly sorted CD4+CD25+ T cells (P < 0.05) as expected, and the Foxp3-transduced cells were able to inhibit proliferation (P < 0.02). In contrast, MIGR1-transduced CD4 T cells did not show any suppressive activity (P > 0.05; Figure 3A). Titration of the suppressor CD4+CD25+ T cells or the Foxp3-transduced CD4+ T cells had a suppressive effect at a ratio of 1:0.5 (Figure 3B).


Figure 3
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  		Figure 3. (A) Foxp3-transduced T cells inhibit proliferation activity in vitro. Suppression of proliferation of freshly isolated CD4+CD25– cells by CD4+CD25+ T cells and by Foxp3-transduced CD4+ T cells after stimulation with anti-CD3 and culture for 3 d. Cell proliferation was measured after 72-h culture by incorporation of [H]thymidine. CD4+CD25– or CD4+CD25+T cells were cultured alone or together with transduced Foxp3 (F) or control vector MIGR1 (M). The results shown are the mean ± SD of triplicate wells. Co-culture with Foxp3-transduced CD4+ T cells or CD4+CD25+ T cells suppressed proliferation, whereas co-culture with MIGR1 control-transduced CD4+ T cells did not suppress proliferation in co-culture (each representative of five independent experiments; NS P > 0.05, *P < 0.05, **P < 0.02). (B) Titration of CD4+CD25+ cells and Foxp3-transduced CD4+ T cells relative to CD4+CD25– at a ratio of 1:1 and of 1:0.5 with other conditions as described above.

 
Foxp3-Transduced T Cells Protected against Renal Damage Induced by ADR
All ADR-treated mice developed renal injury characterized by proteinuria, tubular cell atrophy and tubular dilation, focal segmental glomerulosclerosis, and interstitial expansion with infiltration of mononuclear cells. In the ADR group, tubules showed a decrease in height of tubular epithelial cells, vacuolization, and a moderate interstitial monocyte infiltration. Glomeruli were reduced in size with severe vacuolization and the expansion of the mesangium under periodic acid-Schiff staining (Figure 4). Similar morphologic changes were observed in the ADR+MIGR1 group. Light microscopic examination revealed that glomerular surface area was significantly diminished in both groups of mice, where the glomeruli were shrunken because of glomerulosclerosis. However, there was only mild damage of the glomeruli and tubules in ADR+Foxp3 group, compared with the ADR and ADR+MIGR1 groups (Figure 4). Morphometric analysis showed significantly less damage in glomeruli and tubules in the ADR+Foxp3 group, compared with the ADR and the ADR+MIGR1 groups (P < 0.01, one-way ANOVA tests), as well as reduced interstitial monocyte infiltration in ADR+Foxp3 group, compared with other two groups (P < 0.05, one-way ANOVA tests; Figure 5A).


Figure 4
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  		Figure 4. Histology of renal sections with reduced injury and cellular infiltration in mice that received Foxp3-transduced CD4 T cells. Magnification, x200.

 

Figure 5
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  		Figure 5. Renal histopathology, function, and body weight changes. (A) Semiquantitative scores of morphologic changes showed significantly less damage in glomeruli and tubules in the Foxp3 group, compared with the ADR group and the ADR+MIGR1 group (P < 0.01); reduced interstitial monocyte infiltration was shown in the Foxp3 group, compared with other two groups (P < 0.05). (B) Mice from ADR, ADR+MIGR1, and ADR+PC61 groups had significantly higher levels of urine protein secretion over 16 h compared with normal and ADR+Foxp3 groups (**P < 0.01, ***P < 0.001). ADR+PC61 had significantly worse proteinuria than ADR alone (P < 0.05). (C) Mice from ADR, ADR+MIGR1, and ADR+PC61 groups had significantly higher levels of serum creatinine compared with normal and ADR+Foxp3 groups (*P < 0.05, **P < 0.01). (D) ADR+Foxp3 group body weight was the same as the normal group and did not show weight loss found in the other ADR-treated groups (***P < 0.001).

 
A dense mononuclear cell infiltrate, consisting of macrophages and CD8+ and CD4+ T cells, was found in the interstitium in the ADR and ADR+MIGR1 groups of mice (Figure 6). There was significantly less macrophage infiltration in the ADR+Foxp3 group than in the other ADR-treated groups of mice (P < 0.01).


Figure 6
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  		Figure 6. Photomicrographs of immunohistochemical sections from normal, Adriamycin (ADR), ADR+Foxp3, and ADR+ MIGR1 groups of mice showing CD4+ T cell, CD8+ T cell, and macrophage infiltration. Magnification, x400.

 
Renal function was measured in each animal at the end of experiment. Urinary protein excretion was significantly reduced in ADR mice that were treated with Foxp3-transduced cells (ADR+Foxp3), compared with those that received ADR and those that received ADR and were treated with control vector (ADR+MIGR1; P < 0.001; Figure 5B), whereas proteinuria was worse in mice that were depleted of Treg (ADR+PC61). Serum creatinine was also significantly lower in the ADR+Foxp3 group than in the ADR and ADR+MIGR1 groups of mice (P < 0.01) but was not different between ADR and ADR+PC61 (Figure 5C). Furthermore, the ADR-Foxp3 group did not experience the weight loss seen in the other ADR-treated groups (P < 0.001; Figure 5D) after PC61 depletion of CD25 T cells (Figure 5E).

Relative Number of Transferred CD4 T Cells
For assessment of the relative number of transferred CD4 T cells, 1 x 106 CD4 T cells that were purified from a congenic Ly5.1-expressing mouse were transferred by tail-vein injection into a C57Bl/6 mouse of similar size as the experimental mice. At 24 h, the adoptively transferred cells composed 5.7% of the total CD4+ lymphocyte population (Figure 7A).


Figure 7
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  		Figure 7. (A) Adult C57Bl/6 (Ly5.2) mice received an intravenous injection of 1 x 106 CD4+ cells, which were isolated from a syngeneic B6STL-ptprca (Ly5.1) mouse. One day after injection, peripheral blood cells were analyzed for the frequency of adoptively transferred CD4+ cells. Ly5.1 expression was found on 5.7% of CD4+ cells in the C57Bl/6 mouse. (B) Semiquantitative RT-PCR analysis of GFP expression in spleen, lymph node, and kidney at 5 wk after injection with transduced cells. beta-Actin served as an internal control. All results are expressed as mean ratio of GFP densitometry score to beta-actin ± SD; n = 5 per group (*P < 0.05, **P < 0.01). (C) Real-time quantitative PCR of Foxp3 mRNA level from kidney and lymph nodes from mice 5 wk after injection of ADR. ADR+Foxp3 group had a significantly high level of Foxp3 mRNA level compared with other groups (*P < 0.05).

 
Location of the Transferred Transduced T Cells
Both transduced groups of CD4 T cells contained GFP. We therefore used RT-PCR for GFP and identified both Foxp3 and control MIGR1-transduced T cells in the spleen, draining lymph nodes, and kidneys of the AN mice (Figure 7B). We further assessed Foxp3 levels in the kidney and lymph nodes by real-time PCR to demonstrate high levels of Foxp3 within both the lymph nodes and the affected kidneys of mice that received Foxp3-transduced cells (Figure 7C).

Assessment of Foxp3 Expression in AN
Mononuclear cells were obtained from spleen, lymph node, and kidney of wild-type (WT) and AN mice and stained with CD4, CD25, and intracellular Foxp3. Treg as defined by Foxp3 expression composed 12 to 19% of CD4 T cells in WT spleen and lymph node but not in CD8 T cells (Figure 8A). No CD4 T cells were detected in WT kidney. Similar proportions of Foxp3-containing CD4 cells (11 to 17%) were found in AN spleen, lymph node, and kidney (Figure 8B). CD25 was expressed on a majority of Foxp3-containing CD4 T cells but was lower on cells within the kidney (Figure 8C).


Figure 8
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  		Figure 8. Flow cytometric analysis of Foxp3 expression by intracellular staining. (A) Foxp3 expression is predominantly in CD4+ cells. Analysis of live gated splenocytes and cells from lymph nodes of naïve Balb/c mice for CD4+ and CD8+ cells (no CD4 T cells were found in normal kidney). (B) Flow cytometry analysis of CD4+ cells of splenocytes and cells from lymph nodes and kidney from mice with ADR-induced nephropathy (AN). (C) CD25+ expression in Foxp3 CD4+ cells from splenocytes, lymph nodes, and kidneys from AN mice. (D) CD25+ expression in Foxp3 CD4+ cells from splenocytes, lymph nodes, and kidneys from AN mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Treg that were generated from naïve CD4+ T cells by retroviral gene transfer of Foxp3 were highly effective in the treatment of AN. The Foxp3-transduced cells expressed regulatory molecules, including CTLA-4, GITR, CD103, and TGF-beta. They demonstrated suppressive function in in vitro assays. Foxp3 transduction alone was sufficient to induce CD4+CD25+ Treg function in naïve T cells. The reduction in both Foxp3 expression of MIGR1-transduced T cells relative to CD4+ T cells and TGF-beta expression of MIGR1-transduced T cells relative to CD4+CD25– T cells is likely a function of the activation and culture conditions that are required for retroviral transduction. TGF-beta is a requirement for Treg survival, and its absence in the culture may explain the results suggestive of a loss of a Treg subset in the MIGR1-transduced controls (29). In addition, the effect of CD25 depletion in exacerbating AN may be tempered by Treg that contain Foxp3 but do not express CD25. Tissue-based Foxp3-expressing Treg have a larger proportion of cells that do not express CD25. Therefore, the depletion of CD25, although showing some effect, may not completely deplete all Treg, particularly those in the tissues (16).

Array studies of Foxp3-expressing CD4+ T cells suggest that they express a broad range of chemokine receptors CCR2, CCR4, and CCR8, and functional studies show that Treg protection against graft-versus-host disease is not effective in the absence of CCR5 (16,30). We showed previously that a range of chemokines, including CCL2, are expressed in the AN kidney and may help attract Treg to the kidney (31).

In neither chronic proteinuric renal disease in humans nor murine AN has a specific antigen been defined. Therefore, the strategy of pathogenic TCR antigen–specific T cells with regulatory properties is not currently an option in chronic proteinuric renal disease. However, the general effects of Foxp3-transduced T cells may limit their general use because of nonspecific effects in areas such as cancer immunosurveillance and infection.

The ability of polyclonal Treg to protect against renal injury raises a number of questions. Models of disease using TCR specific for pathogenic antigens seem more potent in treating disease (12,15). The strength of regulation seems to be enhanced by Treg that are specific for pathogenic antigens (32). However, in addition to their antigen specificity, Treg seem to have a more general immunosuppressive effect. Thus, although the predominant mode of action of Treg studied has been their effects on the cognate immune system, the data on the effect of Treg on the innate immune system may explain the effect of a polyclonal rather than TCR-directed population of CD4 Treg in AN. In a Helicobacter disease model in RAG-1–/– mice that were deficient in T and B cells, Treg ameliorated disease (33). Similarly, in a murine model of burn injury, there was evidence of CD4+ Treg suppressing innate immune activation through TLR2 and TLR4 (34). Data suggesting this have also been shown in vitro, where Treg impaired the maturation and antigen presentation of dendritic cells (35). This concurs with our previous data that CD4 depletion significantly aggravated AN in BALB/c mice with increased accumulation of macrophages (25).

This idea of regulation’s occurring against T cells that bear specific TCR and regulation’s occurring through the recognition of other activating markers such as CD25 or CD69 was addressed recently (36). The effects in our model suggest either a non–TCR-restricted effect or a direct regulation of innate effectors such as dendritic cells or macrophages.

Adoptive cell therapies are beginning to enter clinical application. The use of cytotoxic T cells in a T cell–depleted environment has reached clinical application in the treatment of malignancy, whereas virus-specific T cells have been used to treat cytomegalovirus and other viral diseases (37–41). Therapeutic cell therapies with regulatory T cells are still in their infancy. Depletion and transfer strategies in mice have been used successfully to demonstrate the effect of this regulatory subset on disease progression (1,42). In addition, the TCR specificity that is required for regulation may further limit the effectiveness of directly isolated Treg (12,15). A number of strategies to overcome this have been proposed, including ex vivo expansion of Treg using cytokines such as TGF-beta, IL-2, and IL-15 (43–45).

Of particular interest is the induction of specific regulation by forced expression of the regulatory gene Foxp3. Our studies show that previous administration of Foxp3 Treg can limit renal injury either by inhibiting pathogenic T cells or possibly by limiting innate immune injury and raise the possibility of these as a therapeutic strategy to protect against renal injury.


   Acknowledgments
 
This work was supported by a grant from the National Health and Medical Research Council of Australia (NHMRC grant 49414).

We thank the animal house staff in the Children’s Hospital Research Institute for care of the animals. We thank Mary Sartor and Sanda Lum for help in cell sorting and Maolin Zheng and Sharon Cunningham for help with gene transfer. We thank Mr. and Mrs. Apte for support and advice. We also thank Prof. Warren Pear for kindly providing the MIGR1 vectors.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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