Aberrant gene expression by CD25+CD4+ immunoregulatory T cells in autoimmune-prone rats carrying the human T cell leukemia virus type-I gene

Hiroko Hayase1, Akihiro Ishizu1, Hitoshi Ikeda1,3, Yukiko Miyatake1, Tomohisa Baba1, Masato Higuchi1, Asami Abe1, Utano Tomaru1 and Takashi Yoshiki1,2

1 Department of Pathology/Pathophysiology, Division of Pathophysiological Science, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan
2 Genetic Lab, Co., Ltd, Kita-9, Nishi-15, Chuo-ku, Sapporo 060-0009, Japan
3 Present address: Section of Pathology, Hakodate Central General Hospital, Hakodate 040-8585, Japan

Correspondence to: A. Ishizu; E-mail: aishizu{at}med.hokudai.ac.jp


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 
Transgenic rats expressing the env-pX gene of human T cell leukemia virus type-I under the control of the viral long terminal repeat promoter (env-pX rats) developed systemic autoimmune diseases. Prior to disease manifestation, the immunosuppressive function of CD25+CD4+ T (T-reg) cells was impaired in these rats. Since T cell differentiation appeared to be disordered in env-pX rats, we assumed that the impairment of T-reg cells might be caused by an abortive differentiation in the thymus. However, reciprocal bone marrow transfers between env-pX and wild-type rats revealed that direct effects of the transgene unrelated to the thymus framework induced the abnormality of T-reg cells. To identify molecular changes, comparative analyses were done between env-pX and wild-type T-reg cells. Expression of the Foxp3 gene and cell-surface markers supported a naive phenotype for env-pX T-reg cells. Array analyses of gene expression showed some interesting profiles, e.g. up-regulation of genes associated with the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in env-pX T-reg cells. Additionally, expression of the suppressor of cytokine signaling (SOCS) family genes, which inhibit the JAK/STAT signals, was extremely low in env-pX T-reg cells. These findings suggest that the transgene may mediate the down-regulation of the SOCS family genes and that subsequent excess signals through the JAK/STAT pathways may result in the loss of function of env-pX T-reg cells. We suggest that investigation of the pathology of T-reg cells in our autoimmune-prone rat model may aid in understanding the roles of T-reg cells in human autoimmune diseases.

Keywords: animal model, Foxp3, JAK/STAT, SOCS, T-reg


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 
Human T cell leukemia virus type-I (HTLV-I) is pathogenically associated with not only adult T cell leukemia (1, 2) but also a number of inflammatory diseases such as myelopathy (3, 4), uveitis (5) and probably arthropathy (6), Sjögren's syndrome (7), T cell alveolitis (7, 8) and infective dermatitis (9). We reported earlier that transgenic rats expressing the env-pX gene of HTLV-I under the control of the viral long terminal repeat promoter developed a wide spectrum of collagen vascular diseases, including destructive polyarthritis resembling rheumatoid arthritis, necrotizing arteritis mimicking polyarteritis nodosa, sialoadenitis similar to Sjögren's syndrome, myocarditis, myositis and dermatitis (10). The transgene was expressed constitutively in all the organs of these rats (env-pX rats) without tissue or cell specificity. Since rheumatoid factors and anti-nuclear and anti-DNA autoantibodies were present in sera, env-pX rats seemed to be a prototype model for autoimmune diseases. Prior to development of diseases, progenitors for B cells and osteoclasts were shown to increase in the bone marrow (BM) (11). Peripheral T cells were pre-activated to express CD54 (ICAM-1) and CD80/86 before these rats developed diseases and showed a high response against several mitogenic stimuli in vitro (12). Thymus framework carrying the transgene was responsible for the development of autoreactive T cell-mediated necrotizing arteritis, thus suggesting that T cell differentiation in the thymus might be disordered in these rats (13). Recently, we found that immunoregulatory functions of peripheral CD25+CD4+ T (T-reg) cells were impaired in young env-pX rats without disease manifestation, though the number of the cells was equivalent to that in age-matched wild-type WKAH rats (14). On the other hand, it is known that T-reg cells are generated in the normal thymus (15, 16). Therefore, in the present study, we aimed to determine if functional alterations of T-reg cells in env-pX rats would be caused by an abortive differentiation in the thymus or by direct effects of the transgene on these cells. In addition, to examine aberrant molecular expression in env-pX T-reg cells, comparative analyses were done between env-pX and control WKAH rats, using flow cytometry, cDNA arrays and real time quantitative reverse transcriptase (RT)–PCR.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 
Rats
Six-week-old male env-pX rats [HTLV-I env-pX transgenic rats established in WKAH strains (10)] and non-transgenic WKAH rats were used. These rats were maintained at the Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine. Experiments using animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals in Hokkaido University Graduate School of Medicine.

Antibodies
Anti-rat CD3, CD4, CD25 (IL-2R{alpha} chain), CD28, CD45RC, CD54 (ICAM-1), CD122 (IL-2Rß chain),and TCR mAbs were purchased from Pharmingen (San Diego, CA, USA).

BM transfer
Mononuclear cells were prepared from the BM of env-pX and WKAH rats using Lympholyte Rat (Cedarlane, Ontario, Canada) and were then used as BM cells. Microscopic examinations revealed that all env-pX rats used as BM donors were disease-free. The BM cells were injected via the tail vein of recipient rats that had been lethally irradiated using 12 Gy from a 60Co source. BM cells from one donor (1 x 107 per rat) were transplanted into one recipient. In each group of donor/recipient combination, at least three pairs of transplantation were done. Two months after the transplantation, all rats were killed and CD25+CD4+ T cells were isolated, as described below.

Cell sorting
Mononuclear cells were prepared from the spleen of each rat, using Lympholyte Rat, then stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 mAbs. CD25+CD4+ and CD25CD4+ T cells were isolated using FACSVantage (Becton Dickinson, Franklin Lakes, NJ, USA). Purity of the sorted cells exceeded 95%.

Cell proliferation assay
Mononuclear cells were prepared from the cervical lymph nodes of WKAH rats, using Lympholyte Rat. After 1 h of incubation in plastic dishes, adherent and non-adherent cells were collected. The adherent cells were treated with mitomycin C (25 µg ml–1) for 1 h and served as antigen-presenting cells (APCs). CD25CD4+ T cells were separated from the non-adherent cells, using a magnetic cell sorting system (Miltenyi Biotec, Bergisch Gladbach, Germany), and served as responders. The responder cells (1 x 105) and mitomycin C-treated APCs (2 x 104) were mixed in tissue culture wells (96-well round-bottom plates) coated with anti-CD3 antibody, as described (12). Splenic CD25+CD4+ T cells (2 x 104) isolated from rats with BM transplantation using FACSVantage were added to the wells, and these cells were incubated for 96 h. [3H]Thymidine ([3H]TdR) (18.5 kBq) was pulsed 16 h prior to harvest of the cells. Proliferation of cells was quantified by [3H]TdR uptake.

Extraction of total RNA
Total RNA was extracted from FACS-sorted CD25+CD4+ and CD25CD4+ T cells using RNAeasy columns (Qiagen, Valencia, CA, USA).

The cDNA array analysis
For cDNA array analysis, we prepared original filters equipped with 271 rat genes. Details on the filter preparation are described elsewhere (17). Total RNA of each sample was treated with DNase I (TAKARA Shuzo, Kyoto, Japan), and poly (A)+ mRNA was purified using the mRNA purification kit (MagExtractor, TOYOBO, Osaka, Japan). Biotin-labeled cDNA probes were generated using Gene Navigator cDNA Amplification System ver.2 (TOYOBO), and then hybridized to the cDNA array filters using PerfectHyb Hybridization Solution (TOYOBO), according to the manufacturer's instructions. Hybridized signals were developed using Phototope Star kits (New England Biolab, Beverly, MA, USA), detected using FluorS and Quantity One v4.2.1 software (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using ImaGene 4.0 software (BioDiscovery, Segundo, CA, USA). Intensities less than the mean value of signals on spots of the negative control DNA fragment were excluded as false signals. Mean value of the intensity at spots of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for standardization of each filter.

Real time quantitative RT–PCR
The purified total RNA was reverse transcribed using M-MLV RT (Invitrogen, Carlsbad, CA, USA), and the random-primed cDNA served as a template. Real time quantitative PCR was done, using SYBR Green I dye (Applied Biosystems, Foster City, CA, USA). Primer sets for the Foxp3, JAK1, STAT1, suppressor of cytokine signaling (SOCS) family and GAPDH genes are listed in Table 1. Amplification was carried out at 45 cycles of two-step PCR (95°C for 30 s, 60°C for 30 s) after the initial denaturalization (95°C, 15 min), using an ABI PRISM 7700 Sequence Detector System (Applied Biosystems). The amount of specific mRNA was quantified at the point where the system detected the uptake in exponential phase of PCR accumulation, and the ratio to that of the GAPDH gene was calculated for each sample.


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Table 1. Primers used for the real time quantitative RT–PCR

 
Statistics
For cell proliferation assay and real time quantitative RT–PCR, the Mann–Whitney U test was applied for statistical analysis. A P-value of <0.05 was regarded as significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 
The transgene in BM cells but not in the thymus framework was responsible for functional alterations of T-reg cells in env-pX rats
To determine which was mainly implicated in the impairment of immunoregulatory function of env-pX T-reg cells, the transgene in the thymus framework or in the T-reg cells, reciprocal BM transfers were done between disease-free env-pX rats and wild-type WKAH rats. Splenic CD25+CD4+ T cells were isolated 2 months post-transplantation, after which the immunosuppressive function of the cells was assayed (Fig. 1). When CD25+CD4+ T cells from lethally irradiated env-pX rats reconstituted by WKAH BM cells were added to the mixed culture of WKAH CD25CD4+ T cells and mitomycin C-treated APCs, anti-CD3 antibody-induced cell proliferation was significantly suppressed as in the control experiments using CD25+CD4+ T cells from WKAH to WKAH BM transfers. By contrast, the immunosuppressive function of CD25+CD4+ T cells from lethally irradiated WKAH rats reconstituted by env-pX BM cells was completely absent as in the control experiments with BM transfers from env-pX to env-pX rats. These findings clearly indicated that the transgene in BM cells rather than in the thymus framework was critically involved in the pathology of T-reg cells in env-pX rats.



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Fig. 1. Analysis of the immunosuppressive function of CD25+CD4+ T cells from rats that had undergone BM replacement. Six-week-old disease-free env-pX rats and wild-type WKAH rats were lethally irradiated and then reconstituted by reciprocal transplantation of BM cells. Two months later, splenic CD25+CD4+ T cells were isolated from recipients, using FACSVantage. These cells (2 x 104) were added to mixed cultures of WKAH CD25CD4+ responder T cells (1 x 105) and mitomycin C-treated APCs (2 x 104) in tissue culture wells coated with anti-CD3 antibody (hatched columns) or uncoated (open columns). After 96 h of incubation, cell proliferation was measured based on [3H]TdR uptake. The uptake when responder cells were stimulated by anti-CD3 antibody in the absence of T-reg cells was set as 100. Data are represented as mean ± SD of percentage in experiments done in triplicate. (**P < 0.01.)

 
Expression level of the Foxp3 gene was equivalent in env-pX and WKAH T-reg cells
The Foxp3 is a master gene and the best marker for T-reg cells (18, 19). The real time quantitative RT–PCR revealed that the Foxp3 gene was expressed at a significantly higher level in env-pX CD25+CD4+ T cells than in CD25CD4+ T cells (Fig. 2). The relative expression (when the expression level in CD25CD4+ T cells was set as 1) reached 26.2 ± 3.9 in env-pX rats, which was equivalent to the value (31.0 ± 14.0) in wild-type WKAH rats.



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Fig. 2. Comparative analyses of mRNA expression of the Foxp3 genes in CD25+CD4+ (hatched columns) and CD25CD4+ T cells (open columns) of env-pX and WKAH rats, using real time quantitative RT–PCR. Splenic CD25+CD4+ and CD25CD4+ T cells were isolated from env-pX and WKAH rats, respectively (6-week-old, disease-free), using FACSVantage. Total RNA extracted from the cells was reverse transcribed, and then the random-primed cDNA served as a template. The real time quantitative PCR monitored by the SYBR Green I dye was carried out, using the ABI PRISM 7700 Sequence Detector System. Amounts of the specific mRNA were quantified at the point where the system detected the uptake in exponential phase of PCR accumulation, and the ratio to the housekeeping GAPDH gene was calculated for each sample. The expression in CD25CD4+ T cells from each group of rats was set as 1 and data are represented as mean ± SD of relative expression in experiments done in triplicate. (n.s.: not significant)

 
Difference in surface molecules on T-reg cells was nil between env-pX and WKAH rats
We previously reported that there was no significant difference in the surface expression of CD25 (IL-2R{alpha} chain), CD80, CD86 and membrane-bound transforming growth factor (TGF)-ß1 on T-reg cells of env-pX and WKAH rats (14). In the present study, we found that surface expression of TCR, CD28, CD45RC and CD122 (IL-2Rß chain) on env-pX T-reg cells was equivalent to that on WKAH T-reg cells (Fig. 3). The expression of CD54 (ICAM-1) on env-pX T-reg cells was also similar to that on WKAH T-reg cells (data not shown). The combined evidence suggests that env-pX T-reg cells may not exhibit an activation phenotype because cell-surface expression of molecules including CD25, CD45RC, CD54, CD80, CD86 and CD122 are normally altered when T cells are activated (12, 2023).



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Fig. 3. Comparative analyses of cell-surface molecules on T-reg cells of env-pX and WKAH rats. CD4+ T cells were separated from the spleen of each rat (6-week-old, disease-free), using the magnetic cell sorting system, and then stained with PE-conjugated anti-CD25 and FITC-conjugated antibodies for TCR, CD28, CD45RC or CD122 (IL-2Rß chain). Flow cytometry was done using FACSCalibur (Becton Dickinson), and then CD25+ cells were gated to obtain histograms using Cell Quest software (Becton Dickinson). Lines and dots represent env-pX and WKAH T-reg cells, respectively. Experiments were conducted at least twice, and representative results are shown.

 
Comparison of gene expression profiles in T-reg cells of env-pX and WKAH rats
For comparative analyses of gene expression profiles in T-reg cells of env-pX and WKAH rats, cDNA array analysis was done using original filters equipped with 271 probes for rat genes associated with apoptosis, signal transduction, cell cycle regulation and so on (17). About one-third of the genes tested could be evaluated. Expression levels of many genes were higher in env-pX T-reg cells than those in WKAH T-reg cells (Table 2), while a smaller number of genes had decreased in env-pX T-reg cells (Table 3). The expression levels of CD25 (IL-2R{alpha} chain) and CD122 (IL-2Rß chain) genes were increased in env-pX T-reg cells (3.7- and 4.8-fold, respectively). However, in our experiments, no significant difference in the surface expression of CD25 (14) or CD122 (Fig. 3) on T-reg cells was evident between env-pX and WKAH rats, suggesting that the transgene in env-pX T-reg cells did not induce cell-surface protein expression, but increased mRNA expression of CD25 and CD122 in the cells. A similar observation was noted for CD54 (ICAM-1, data not shown). Further investigations for the putative post-transcriptional events in env-pX T-reg cells are needed to understand the discrepancy.


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Table 2. Genes expressed at a higher level in env-pX T-reg cells than in WKAH T-reg cellsa

 

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Table 3. Genes expressed at a lower level in env-pX T-reg cells than in WKAH T-reg cellsa

 
Some characteristic gene expression profiles were recognized in env-pX T-reg cells, e.g. lack of regulation of cell cycle [expression of both activators, including cyclins and cyclin-dependent kinases (CDKs), and CDK inhibitors were altered], dysregulation of apoptosis (expression of apoptosis-inducible genes such as caspases was increased, while other apoptosis inducers FADD and TRADD, and anti-apoptotic molecules such as Bcl-2, were decreased) and up-regulation of genes associated with the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways (expression of JAK1, JAK2, STAT2 and STAT6 was increased).

High expression of the JAK1 and STAT1 genes and low expression of the SOCS family genes in env-pX T-reg cells
Using real time quantitative RT–PCR, we examined the expression of the JAK1 and STAT1 genes. The expression level of JAK1 gene was significantly higher in env-pX T-reg cells than in WKAH T-reg cells (Fig. 4A), corresponding to the cDNA array results. A similar tendency was observed in the STAT1 gene that could not be evaluated by the cDNA array for unknown reasons (data not shown). The SOCS family molecules have been shown to inhibit the JAK/STAT pathways activated by several cytokines (24, 25). When we examined the family, SOCS1, SOCS2, SOCS3 and cytokine-inducible Src homology 2 protein (CIS) genes, all were at extremely low expression levels in env-pX T-reg cells compared with findings in WKAH T-reg cells (Fig. 4B).



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Fig. 4. Comparative analyses of mRNA expression of the JAK1 and SOCS family genes in T-reg cells of env-pX (hatched columns) and WKAH rats (open columns), using real time quantitative RT–PCR. Splenic CD25+CD4+ T cells were isolated from env-pX and WKAH rats (6-week-old, disease-free), using FACSVantage. Total RNA extracted from the cells was reverse transcribed, and then the random-primed cDNA served as a template. The real time quantitative PCR monitored by the SYBR Green I dye was carried out, using the ABI PRISM 7700 Sequence Detector System. Amounts of the specific mRNA were quantified at the point where the system detected the uptake in exponential phase of PCR accumulation, and the ratio to the housekeeping GAPDH gene was calculated for each sample. The expression of each gene in WKAH T-reg cells was set as 1, and data are represented as mean ± SD of relative expression in experiments done in triplicate. (*P < 0.05, **P < 0.01.)

 

    Discusssion
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 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 
Peripheral CD25+CD4+ T (T-reg) cells are engaged in inhibiting proliferation of autoreactive T cells and in the maintenance of immunologic self-tolerance (26). Athymic nude mice which had been given peripheral lymphocytes-depleted T-reg cells from histocompatible BALB/c mice developed T cell-mediated autoimmune diseases, including gastritis, thyroiditis and insulin-dependent diabetes mellitus, and adoptive transfer of BALB/c T-reg cells to these mice suppressed development of the diseases (2729). Recent studies revealed that the transcription factor Foxp3 is a critical mediator of the development of T-reg cells (18, 19). Scurfy mice, in which the Foxp3 gene is deficient so that T-reg cells are not generated, develop fatal lymphoproliferative and autoimmune disorders (30). In addition, it has been shown that the number of T-reg cells is reduced in autoimmune-prone strains such as non-obese diabetic, New Zealand Black (NZB), New Zealand White (NZW) and (NZB x NZW) F1 mice (31). The combined evidence suggests that lack of T-reg cells may be pathogenically associated with the development of autoimmune diseases in mice.

On the other hand, we have reported that functional but not quantitative alterations of T-reg cells are evident in HTLV-I env-pX transgenic rats before they develop autoimmune diseases (14). In these rats, autoreactive T cells against the vasculature may not be eliminated in the thymus and T cell-mediated necrotizing arteritis occurs (13). Since the commitment to T-reg cells by Foxp3 has been suggested to occur in the normal thymus (15, 16, 32), we asked if the functional alterations of env-pX transgenic T-reg cells are caused by an abortive differentiation of T cells in the env-pX thymus. Contrary to our expectation, reciprocal BM transfers between disease-free env-pX rats and wild-type WKAH rats suggest that the abnormality of T-reg cells in env-pX rats is caused by direct effects of the transgene rather than by an abortive differentiation in the thymus.

Expression level of the Foxp3 gene in CD25+CD4+ T cells was equivalent in env-pX and wild-type WKAH rats. Although peripheral T cells from env-pX rats are ready to be activated in vitro (12) and activated T cells express CD25 on the cell surface (22), our results of the real time quantitative RT–PCR for the Foxp3 gene suggest that CD25+CD4+ T cells isolated from env-pX rats before they developed diseases might not contain activated CD4+ T cells expressing CD25. In addition, a significant difference in expression of cell-surface molecules including TCR, CD25 (IL-2R{alpha} chain), CD28, CD45RC, CD54 (ICAM-1), CD80, CD86, CD122 (IL-2Rß chain) and membrane-bound TGF-ß1 on T-reg cells was not evident in env-pX and WKAH rats. It is shown that expression of CD122 was increased in human leukemia cells transformed by HTLV-I (22, 23). The lower expression level of p40Tax (a product of the env-pX gene) in our transgenic model than in HTLV-I-transformed T cells (10) may be associated with the difference between human and rat cells with regard to the effect of the HTLV-I gene on expression of CD122. These findings suggest that env-pX T-reg cells may be phenotypically naive.

However, the cDNA array analysis showed some characteristic features of gene expression of env-pX T-reg cells, suggesting lack of regulation of the cell cycle, dysregulation of apoptosis and increased signal transduction through the JAK/STAT pathways. The aberrant expressions of cell cycle-related genes may correspond to our finding that env-pX T-reg cells show autologous and anti-CD3 antibody-induced proliferation (14). As alterations in gene expression of apoptosis-related molecules in env-pX T-reg cells were contradictory, it remains to be clarified whether env-pX T-reg cells would be prone to apoptosis. Expression of genes that belong to the mitogen-activated protein (MAP) kinase cascade such as MAP kinase p42 and MAP kinase kinase-3, those in the downstream of JAKs (33), was also increased in env-pX T-reg cells (see Table 2). Moreover, the expression level of genes targeted by the JAK/STAT signals including cyclins and p21Waf1 (34) in T-reg cells was higher in env-pX rats than in wild-type rats. The collective evidence suggests that the JAK/STAT pathways are activated in env-pX T-reg cells. This may be also related to the loss of anergic features of T-reg cells in env-pX rats (14).

Many cytokines, if not all, that are related to immune responses utilize the JAK/STAT pathways (35, 36). The SOCS family molecules were shown to complete a negative feedback loop to attenuate signal transduction through the JAK/STAT pathways (24, 25). Recent studies have shown that the SOCS family genes are expressed at high levels in T-reg cells, suggesting that these molecules contribute to the anergic and immunosuppressive phenotypes of these cells (37). Real time quantitative RT–PCR showed increased expression levels of the JAK1 and STAT1 genes in env-pX T-reg cells. Although it remains to be clarified if phosphorylation of the JAK/STAT kinases is actually augmented in env-pX T-reg cells, our data do correspond to the report that the JAK/STAT pathways were activated in T cells transformed by HTLV-I (38, 39). Interestingly, we noted a marked and significant reduction of the SOCS family, SOCS1, SOCS2, SOCS3 and CIS genes in env-pX T-reg cells. Regulation of the expression of SOCS family molecules is poorly understood; however, IL-4, IL-12 and IFN-{gamma} have been shown to increase expression of these genes in T cells (40). In our cDNA array analysis, the expression of the IL-12 p35 gene in env-pX T-reg cells was one-tenth of that in wild-type T-reg cells (see Table 2). This may relate to the low-level expression of the SOCS family genes in env-pX T-reg cells. Since p40Tax encoded by the transgene impacts on the expression of several host genes and modulates molecular functions (41), it is possible that unidentified pathways associated with the transgene are implicated in the down-regulation of the SOCS family genes in env-pX T-reg cells.

HTLV-I p40Tax associates with nuclear factor (NF)-{kappa}B and activates the JAK/STAT kinases (39). In env-pX T-reg cells, excess signals through the JAK/STAT pathways may be transduced by the activation of NF-{kappa}B and removal of the negative regulation mediated by the SOCS family molecules. Immunoregulatory functions of T-reg cells are attenuated when exposed to a high dose of IL-2 (26). Since IL-2 transduces the JAK/STAT signals (35, 36), it is considered that the excess JAK/STAT signals mediated by p40Tax and NF-{kappa}B, mimicking signals by IL-2, may result in the loss of immunoregulatory function of env-pX T-reg cells. Studies are ongoing to clarify the relationship between HTLV-I p40Tax and the down-regulation of the SOCS family molecules.

Mutations in the Foxp3 gene cause immune dysregulation, polyendocrinopathy, enteropathy and X-linked (IPEX) syndrome in humans (42). However, it remains unclear whether lack and/or dysfunction of Foxp3 play pathogenic roles in patients with other common autoimmune diseases. Analyses using not only mouse models with quantitative alterations of T-reg cells but also our rat model exhibiting functional alterations of the cells may aid in understanding the pathogenic roles of T-reg cells in patients with autoimmune diseases.


    Acknowledgements
 
We thank Tsutomu Osanai and the entire staff of the Institute of Animal Experimentation, Hokkaido University Graduate School of Medicine for maintenance of transgenic rats, and Ken-ichi Nakase, Chisato Sudo and Masayo Tateyama for technical assistance. This work was supported by grants from the Ministries of Education, Culture, Sports, Science and Technology, and Health, Labour and Welfare, of Japan, and from the Akiyama and Takeda Science Foundations.


    Abbreviations
 
APC   antigen-presenting cell
BM   bone marrow
CDK   cyclin-dependent kinase
CIS   cytokine-inducible Src homology 2 protein
GAPDH   glyceraldehyde-3-phosphate dehydrogenase
[3H]TdR   [3H]thymidine
HTLV-I   human T cell leukemia virus type-I
JAK   Janus kinase
MAP   mitogen-activated protein
NF   nuclear factor
NZB   New Zealand Black
NZW   New Zealand White
SOCS   suppressor of cytokine signaling
STAT   signal transducer and activator of transcription
TGF   transforming growth factor
RT   reverse transcriptase

    Notes
 
Transmitting editor: K. Okumura

Received 9 December 2004, accepted 7 February 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discusssion
 References
 

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