Abstract: CD4⁺CD25⁺ T cells played a critical role in the establishment and maintenance of peripheral tolerance via adoptive transfer. However, whether one or more molecules in CD4⁺CD25⁺ T cells that could independently mediate peripheral tolerance was disputed by worldwide researchers. In the present study, one soluble antigen-specific factor was extracted from splenic lymphocytes lysates of OVA-tolerant mice (named OVA Immune Tolerance Factor, OVA-ITF) with molecular mass <3 ku which could establish OVA-specific immune tolerance in recipient mice via transfer treated and induce the same effect of peripheral tolerance as those of splenic lymphocytes from OVA-tolerant mice. Treated with OVA-ITF to naive BALB/c mice resulted in significant suppression of DTH reaction and T cell proliferation in an antigen-specific manner as well as a significant increase in the percentage of CD4⁺CD25⁺ T cells within the CD4⁺ T cell subset in peripheral blood. Present study showed that OVA-TIF was produced by CD4⁺CD25⁺ T cell subset and could induce OVA-specific peripheral tolerance independently in vivo with TGF-β₁ as its main suppressive cytokine in recipient mice. These results suggested that OVA-TIF is a novel, low MW factor and totally different from other suppressive components reported previously which have important implications for expanding new potential therapeutic routes of prevention and control of graft rejection, autoimmune and related diseases.

INTRODUCTION

Establishment and breakdown of peripheral immune tolerance has close relation with autoimmune diseases (Kukreja et al., 2002; Van Amelsfort et al., 2004; Viglietta et al., 2004; Lindley et al., 2005; Atfy et al., 2009; La Cava, 2008), graft-rejection (Hall et al., 2007; Fu et al., 2010), cancers (Onizuka et al., 1999; Shimizu et al., 1999; Mougiakakos et al., 2010; Nishikawa and Sakaguchi, 2010) and some of parasitic and virus diseases (Long et al., 2003; Vahlenkamp et al., 2005; Bourreau et al., 2009; Rodrigues et al., 2009; Wongyanin et al., 2010; Langhans et al., 2010; Weiss et al., 2010). At present, several mechanisms for the induction of tolerance to self-antigens have been reported in cluding clonal deletion of autoreactive T cells in duction of T cell anergy, active suppression by regulatory T cells and present of pathogenic auto-reactive T cells (Kukreja et al., 2002; Viglietta et al., 2004; Kappler et al., 1987; Ota et al., 1990; Sakaguchi, 2004; Shevach et al., 2001). There is growing evidence that CD4⁺CD25⁺ regulatory T (Treg) cells play a crucial role in the establishment and maintenance of peripheral tolerance (Cobbold et al., 2003; Kohm et al., 2002; Sakaguchi, 2004; Cohen et al., 2002) which was first demonstrated by the induction of body-wide autoimmune diseases in athymic nude mice after injection with CD4⁺ T cells depleted of CD25⁺ T cells (Sakaguchi et al., 1995; Takahashi et al., 1998). Therefore, Tregs cells would be the target to determine the clinical effectiveness of novel therapy to modulate Tregs in vivo besides the conventional treatments (Atfy et al., 2009).
Recently, many specific cell therapies based on Treg cells and other T cell subsets were developed (Grupp and June, 2010; Lee et al., 2010; Turtle and Riddell, 2010) which have been presented to be promising approaches for cancer immunotherapy. Adoptive Cell Therapy (ACT) has been proved to be an effective treatment for viral infections which also could induce regression of cancer in early stage clinical trials and would be a particularly important and efficacious modality in the period following Hematopoietic Stem Cell Transplantation (HSCT) (Grupp and June, 2010). However, the cell therapies have lots of shortcomings (Miyara et al., 2009; Schumacher and Restifo, 2009) and the major limitation is the rapid decline in viability and function of the transplanted cells. Consequently, new therapeutic routes of these diseases should be reconsidered.

Previous studies showed several cell-surface markers are associated with Treg-cell phenotype and function and the most prominent molecules are cytotoxic T lymphocyteassociated protein 4 (CTLA-4) and Glucocorticoid-Induced TNFR-Related protein (GITR) (McHugh et al., 2002; Shimizu et al., 2002). Additionally, IL-10 and TGF-β although, rarely expressed in vitro might have functional importance for Treg cells in vivo, particularly in the context of disease (Hara et al., 2001; Nakamura et al., 2001). Although, these molecules are related to the regulation of suppressor activity by CD4⁺CD25⁺ T cells, they are not candidate molecules for the independent establishment of peripheral tolerance. Transfer treated of spleen cell lysates from tolerant mice could help to establish peripheral tolerance to certain antigens (HGG or HIgG) in naive mice (Jones and Kaplan, 1977; Taniguchi and Miller, 1978). This lysate-induced tolerance was acted in an antigen-specific manner that the immune response to related antigens was significantly suppressed while to other antigens was not affected. However, most of these cell lysates are >10 ku in molecular weight which would induce immune reaction of receptor to these exotic antigens. The origin and chemical nature were also unknown.

In the present study, a novel and smaller (<3 ku) soluble antigen-specific factor named OVA Immune Tolerant Factor (OVA-ITF) was identified which was generated by CD4⁺CD25⁺ T cells in OVA tolerant mice and could mediate OVA specific tolerance in naive mice. The findings would provide direct molecular evidence on the mechanism of Treg-mediated peripheral tolerance and expand new routes for clinical therapy.

MATERIALS AND METHODS

Mice: Female BALB/c mice (4 weeks old) were originally purchased from the Animal Center at the Fourth Military Medical University, Xi’an, P.R. China and housed under specific pathogen-free conditions in the facility with free access to food and water.

Induction of OVA-tolerant mice, preparation of Tolerant Splenic Lymphocytes (TSLC) and different tolerant T cell subsets and OVA Immune Tolerance Factor (OVA-ITF) purification: BALB/c mice received i.v. 3 times (5 day intervals) with 5 μg of OVA (Sigma) in 0.1 mL of PBS. Control mice received an equivalent volume of PBS. Then each mouse were injected s.c with 250 μg OVA in 0.1 mL PBS at 3 and 12 days after the last injection.

Lymphocytes were prepared from OVA-tolerant mice at 6 days after the last injection. Spleens were removed and pooled in D-Hank’s solution with 5% heat-inactivated FCS (HyClone, USA). Then the tissue was dispersed by passing through a stainless steel mesh. Lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation, rinsed twice with D-Hank’s solution and then resuspended in RPMI 1640 medium (HyClone, USA). The final solutions were split into two equal portions. The lymphocyte concentration in one portion was adjusted to 1x10⁸ cells mL^-1 for cell adoptive transfer. The other one was adjusted to 1.5x10⁸ cells mL^-1 for sorting different tolerant T cell subsets.

Different tolerant T cell subsets were sorted using flow cytometry. Single lymphocyte suspension of OVA-tolerant mice was prepared by negative selection using MACS microbeads labeled with various mAbs (StemCell Biotec) according to the manufacturer’s suggested protocol. The CD4⁺CD25¯ and CD4⁺CD25⁺ T cells were obtained using anti-PE coated microbeads labeled with PE-conjugated anti-CD25 (StemCell Biotec). The purity was consistently >95% or 90% for CD4⁺ or CD4⁺CD25⁺T cell preparations, respectively. The purified CD4⁺CD25⁺ T cells were adjusted to a concentration of 6x10⁶ cells mL^-1 in PBS for cell adoptive transfer and preparation of OVA-ITF.

The final CD4⁺CD25⁺ T cells were frozen-thawed at -20 and 37°C for 3 times and then centrifuged at 12,000xg for 15 min to remove cell debris. The supernatant was filtered through ultrafiltration tubes by centrifugation at 7000xg for 35-40 min at room temperature according to suggested protocol (Vivaspin2, Vivascience, Sartorius Group, Germany) and separated into three fractions that contained molecules of <3 ku, 3∼5 ku and >5 ku in molecular weight.

Each fraction was transferred to BALB/c mice and the tolerated effect was evaluated using the lymphocyte proliferation assay. The results showed that the optima effect was molecules of <3 ku fraction (data requested from researchers) which was named as OVA-ITF. As a control, splenic lymphocyte suspensions of naive mice were also prepared and the fraction with the molecules of <3 ku was harvested (named as naive ITF) and transferred to naive mouse.

To address the origin of OVA-ITF, purified CD4¯, CD4⁺CD25¯ and CD4⁺CD25⁺ T cells were resuspended in RPMI 1640 medium, respectively and the final concentrations were adjusted to 9x10⁶ cells mL^-1. Fractions with the same molecular weight as OVA-ITF were harvested according to the protocols above. These fractions were referred to as ITF_CD4¯, ITF_CD4+CD25+ and ITF_CD4+CD25¯.

Transfer treated and immunization: BALB/c mice were randomly assigned to eight groups. The first seven groups received i.v. with TSLC, OVA-ITF, naive ITF, isolated CD4⁺CD25⁺ T cells, ITF_CD4+CD25+, ITF_CD4¯, ITF_CD4+CD25¯ in a volume of 0.4 mL (day 0) and the last group was injected with PBS as blank control. The blood were collected at 1, 3, 7 and 10 days later to determine the percentage of CD4⁺CD25⁺ T cells in peripheral blood after adoptive transfer and Peripheral Blood Mononuclear Cells (PBMCs) were also analyzed using flow cytometry.

The cell-transferred mice were immunized s.c. with 250 μg of OVA on day 1 and sacrificed on day 5 to test for lymphocyte proliferation and cytokine secretion. ITF-treated mice (transferred with OVA-ITF, naive ITF, ITF_CD4+CD25+, ITF_CD4¯, ITF_CD4+CD25¯) as well as PBS control mice were also immunized s.c. with the same dose of OVA on day 3 and sacrificed on day 9. Then spleens of these mice were harvested and to prepare lymphocytes cultured in vitro.

For DTH reactions response, cell-transferred mice were immunized s.c. on day 1 with 100 μg of OVA in 50 μL of PBS and an equivalent volume of complete Freund’s adjuvant (CFA, Sigma). ITF-treated mice and PBS control mice were also immunized with the same dose of OVA and 50 μL of CFA on day 3.

Flow cytometry analysis: The whole blood aliquots (100 μL) with heparin were incubated with a mixture of FITC-labeled rat anti-mouse CD4 and PE-labeled rat anti-mouse CD25 (Biolegend, USA; 2 μL/100 μL blood) or their isotype control mAbs for 30 min at room temperature. A 2 mL aliquot of FACS lysing solution (BD Pharmingen) was added to each mixture and incubated for 10 min at room temperature. Then the solution were centrifuged at 800xg for 5 min and the labeled PBMCs were resuspended in 500 μL PBS for analysis, analyzed on a FACScan flow cytometer (BD Biosciences) and quantified with WinMDI 2.9 (WinRAR) software.

Proliferation assay: Lymphocyte proliferation was determined by MTT assay as previous studies with some modification (Keller et al., 2005; Peng et al., 2009; Liu et al., 2010). Briefly, Lymphocytes (1x10⁶ cells well^-1) were cultured alone or with OVA (500 μg mL^-1, Sigma), BSA (750 μg mL^-1, Roche, USA) for 68 h at 37°C in 96 well flat-bottom plates (Costar, NY, USA) in RPMI 1640 cell culture medium supplemented with 10% heat-inactivated FCS, 5x10^-5 M 2 ME (Biotech) and antibiotics (100 U mL^-1 penicillin and 100 U mL^-1 streptomycin), 10 mM Hepes buffer (Sigma), 2 mM L-glutamine (Amresco) and 1 mM sodium pyruvate (Amresco). Cultures were added MTT (5 mg mL^-1, Amresco) with 10 μL per well at 64 h later. Before harvest, DMSO (Amresco) was added with 100 μL per well and the mixtures were oscillated for 10 min. The absorbance was measured at 630 nm using a Model 680 Microplate Reader (BIO-RAD). The proliferation values were presented as a stimulation index value (SI value).

Cytokine ELISA: For cytokine assays, lymphocytes (5x10⁶ cells well^-1) were cultured with OVA (500 μg mL^-1, Sigma) in 24 well flat-bottom plates (Costar, NY, USA) in RPMI 1640 cell culture medium at 37°C. Supernatants were harvested 48 and 72 h later for detection of IL-10 and TGF-β₁, respectively.

The levels of IL-10 and TGF-β₁ were measured using commercial ELISA kits (IL-10, Jingmei Biotech, China, detection limit, 7 pg mL^-1; TGF-β₁, Uscnlife, USA, detection limit, 7.8 pg mL^-1) according to the manufacturer’s instructions.

Delayed-Type Hypersensitivity (DTH) reaction: About 6 days after immunization, mice were challenged s.c. with 50 μg OVA in 20 μL PBS in the left ear. Ear thickness was measured 24 h after challenge with a micrometer caliper (Mitutoyo, Japan). Results are shown as the difference between left and right ear thickness.

Statistical analysis: Statistical analyses were performed with the Student’s t-test between experimental and control groups. Results are expressed as mean±Standard Deviation (SD) and p-values <0.05 or <0.01 represented significant or highly significant differences between groups, respectively.

RESULTS AND DISCUSSION

The OVA-specific peripheral tolerant model in BALB/c mice was induced using normal mice which were firstly received i.v. 3 times with OVA solution or PBS and then immunized twice with OVA. The OVA-specific lymphocyte proliferation and the percentage of splenic CD4⁺CD25⁺ T cells were analyzed. The proliferation of OVA-specific T cells was significant lower in OVA-induced mice compared with control mice (p<0.01) whereas no significant suppression of BSA-specific T cell proliferation was observed in OVA-induced mice (Fig. 1a). The CD4⁺CD25⁺ T cell subset was significantly increased in groups that received i.v. with antigens.

Fig. 1:	Establishment of OVA-specific tolerance in BALB/c mice. (a) Lymphocytes were cultured with or without OVA (500 μg mL-1) or BSA (750 μg mL-1) for 72 h. The results are shown using the mean±SD of two independent experiments with six mice. (b) Flow cytometry analyses of splenic lymphocytes stained with CD4 and CD25. One representive experiment of six mice from two independent experiments was shown

Compared with naive mice (9.97±1.38%), the percentage of splenic CD4⁺CD25⁺ T cells was significantly increased in OVA-induced mice (15.37±0.22%, p<0.01) while that was slightly increased in control mice (11.23±0.41%) (Fig. 1b). These results suggested that OVA-specific tolerance had been induced in BALB/c mice.

Previous studies indicated that the splenocytes, CD4⁺ T cells and CD4⁺CD25⁺ T cells could transfer the antigen-specific tolerance which would be used to the prevention and control of autoimmune diseases and transplantation rejection (Graca et al., 2002; Becker et al., 2003; Morgan et al., 2005). To determine whether OVA-ITF could transfer OVA-specific tolerance to normal BALB/c mice, the OVA-ITF, splenic lymphocytes, naive ITF or an equivalent volume of PBS were injected into recipient mice. Then mice were tested for DTH reaction and sacrificed to determine OVA-specific T cell proliferation. The DTH reactions to OVA of mice that received OVA-ITF and lymphocytes were significantly reduced to that of naive ITF and PBS control mice (p<0.01) (Fig. 2a) which prompted that OVA-ITF could transfer peripheral tolerance in an OVA-specific manner. The proliferation of OVA-specific T cells was significantly suppressed by OVA-ITF and TSLC from OVA tolerant mice (p<0.01). In contrast, the transfer of naive ITF from naive mice had little effect on the proliferation of OVA-specific T cells. BSA stimulation also had no significant effect on splenic T cell proliferation in any experimental group, though the response to BSA was partly suppressed by both OVA-ITF and TSLC as a result of antigen-driven bystander suppression (Fig. 2b). These results suggested that tolerance induced by OVA-ITF was antigen-specific.

Preliminary experiments indicated that maximum suppression of lymphocyte proliferation occurred at different time in mice that received OVA-ITF compared with that received TSLC (data not shown) which revealed that the time of OVA-specific tolerance establishment was present to be diverse.

Considering the importance of CD4⁺CD25⁺ T cells in the induction and maintenance of peripheral tolerance, kinetic analysis of CD4⁺CD25⁺ T cell subset in peripheral blood due to peripheral tolerance established by OVA-ITF were performed. The percentage of CD4⁺CD25⁺ T cells in CD4⁺ T cells from mice received OVA-ITF was significantly increased (p<0.01) after injection with peaks of 10.106±0.108% on day 7 in peripheral blood. While the percentage of CD4⁺CD25⁺ T cells in mice received TSLC (positive control) also significantly increased but no change was detected in mice received naive ITF.

Fig. 2:

The regulatory functions of OVA-ITF from splenic lymphocytes in DTH reaction and lymphocyte proliferation assays. a) OVA-ITF or lymphocytes from OVA-tolerant mice, naive ITF or PBS were transferred into naive BALB/c mice and then the mice were immunized s.c. with 100 μg OVA/CFA. Ear challenge was performed 6 days after the immunization with 50 μg OVA in 20 μL PBS. The DTH reaction was measured by the increase of ear thickness 24 h later. The results are shown using the mean±SD from two independent experiments with six mice per group. ▲, p<0.01 vs naive ITF or PBS control. b) OVA-ITF, lymphocytes, naive ITF or PBS solution were transferred to mice followed by immunization with 250 μg OVA. Splenic lymphocytes were isolated and cultured with or without OVA (500 μg mL-1) or BSA (750 μg mL-1) for 72 h. The results are shown using the mean±SD of six mice from two independent experiments. ▲, p<0.01

Fig. 3:	Kinetic analysis of CD4+CD25+ T cells in CD4+ T cells from peripheral blood before and after adoptive transfer. The results are shown using the mean±SD from two independent experiments with six mice per group

Interestingly, a peak of CD4⁺CD25⁺ T cells in peripheral blood could reach only 3 days after injection of TSLC (Fig. 3).

To address the origin of OVA-ITF, ITF_CD4+CD25+, ITF_CD4¯, ITF_CD4+CD25¯ as well as CD4⁺CD25⁺ T cells from OVA-tolerant mice were transferred into recipient mice. Then DTH reaction and OVA-specific T cell proliferation were determined. The DTH reaction and OVA-specific T cell proliferation were significantly (p<0.01) reduced in mice received ITF_CD4+CD25+ or CD4⁺CD25⁺ T cells (Fig. 4a, b) which hinted that OVA specific tolerance could be induced by ITF_CD4+CD25+ or CD4⁺CD25⁺ T cells. However, no significant effect was observed in the mice injected with ITF_CD4¯ and ITF_CD4+CD25¯. The proliferative responses of splenic lymphocytes from mice received ITF_CD4+CD25+, ITF_CD4¯, ITF_CD4+CD25¯ and CD4⁺CD25⁺ T cell exhibited no significantly different with those received BSA (control) (Fig. 4b). But the DTH reaction and OVA-specific T cell proliferation of splenic lymphocytes in the ITF_CD4+CD25+ injected mice were nearly the same as those of OVA-ITF treated. These results suggested that the OVA-ITF was only produced by CD4⁺CD25⁺ T cell.

Generally, high levels of TGF-β and/or IL-10 would be observed in peripheral tolerant mice (Strid et al., 2004; Ostroukhova et al., 2004). In the present study, the supernatants of OVA-stimulated splenic lymphocytes were harvested from each group to measure the level of IL-10 and TGF-β₁. Significant high level of TGF-β₁ was detected in lymphocyte supernatants from mice transferred with OVA-ITF, TSLC, ITF_CD4+CD25+ and CD4⁺CD25⁺ T cells compared with naive ITF or PBS control mice (p<0.01). However, IL-10 was not detectable in lymphocyte supernatants from mice transferred with OVA-ITF or TSLC (Fig. 5a, b).

Fig. 4:

The regulatory functions of OVA-ITF from CD4+CD25+ T cells are shown in DTH reaction and lymphocyte proliferation assays. a) Mice received ITFCD4+CD25+, ITFCD4¯, ITFCD4+CD25¯, CD4+CD25+ T cells or PBS and then immunization s.c. with OVA. Ear challenge was performed with 50 μg OVA in 20 μL of PBS 6 days after immunization. The DTH reaction was measured by the increase in ear thickness 24 h later. The results are shown using the mean±SD from two independent experiments with six mice per group. ▲, p<0.01 vs PBS control. b) Mice received ITFCD4+CD25+, ITFCD4¯, ITFCD4+CD25¯, CD4+CD25+ T cells or PBS and then followed immunized s.c. with OVA. Splenic lymphocytes were isolated and cultured with or without OVA (500 μg mL-1) or BSA (750 μg mL-1) for 72 h. The results are shown using the mean±SD from two independent experiments

The level of IL-10 in mice received ITF_CD4+CD25+ was nearly equal to normal mice and that in mice transferred with CD4⁺CD25⁺ T cells was increased with nearly 2 fold. But no significant difference was observed in the level of IL-10 and TGF-β₁ secreted by lymphocytes in mice injected ITF_CD4¯ or ITF_CD4+CD25¯ (Fig. 5).

Fig. 5:

Cytokine production after adoptive transfer. Naive mice were injected i.v. with ITFCD4+CD25+, ITFCD4¯, ITFCD4+CD25¯, CD4+CD25+ T cells or PBS, followed by immunization with 250 μg of OVA. Naive mice served as an additional control. Splenic lymphocytes were cultured with OVA (500 μg mL-1). The supernatants were harvested after 48 h for IL-10 determination (Fig. 5a) and 72 h for TGF-β1 determination (Fig. 5b) using ELISA. The results are shown using the mean±SD from two independent experiments with six mice in each group. ▲, p<0.01 vs PBS control

Thus, OVA-ITF from splenic lymphocytes and CD4⁺CD25⁺ T cells displayed a high level of TGF-β1 and low of IL-10 profile in recipients which indicated that TGF-β₁ was the main suppressive cytokine in recipient mice transferred with OVA-ITF. In the present study, we firstly demonstrated that adoptive transfer of splenic lymphocytes and CD4⁺CD25⁺ T cells from OVA-tolerant mice was able to establish OVA-specific tolerance in recipient mice. A smaller (<3 ku) soluble antigen-specific factor (OVA-TIF) was also extracted from splenic lymphocyte lysates of OVA-tolerant mice which could establish OVA-specific immune tolerance in recipient mice via transfer treated and induce the same effect of peripheral tolerance as those of splenic lymphocytes from OVA-tolerant mice.

The percentage of CD4⁺CD25⁺ T cells could reflect the maintenance of peripheral tolerance. Usually, there is a significant higher percentage of CD4⁺CD25⁺ T cells in tolerant mice than normal mice (Zhang et al., 2001). In this study, CD4⁺CD25⁺ T cells in peripheral blood reached a peak 7 days after the treated with OVA-ITF which was 4 days delayed to lymphocyte transfer mice b672327_jaut the percentage of CD4⁺CD25⁺ T cells was not significantly different between the two groups. These results suggested that OVA-ITF could induce peripheral immune tolerance and there must be additional means of suppression enacted by the CD4⁺CD25⁺ T cells other than through production of OVA-TIF. The suppressive function of CD4⁺CD25⁺ T cells depended on interactions with multiple molecules on the surface of or secreted by Tregs and their corresponding receptors of target cells. During interaction, OVA-ITF might play a key role in determining the immunoregulatory function of CD4⁺CD25⁺ T cells.

Cytokine levels in lymphocyte supernatant also could reflect difference between OVA-ITF and CD4⁺CD25⁺ T cells. The IL-10 level in lymphocyte supernatant was significant lower in mice received OVA-ITF than those received CD4⁺CD25⁺ T cells. But no difference was observed in the level of TGF-β₁. A recent report indicates that the main action of IL-10 produced by Treg cells was to inhibit DC maturation, leading to reduction of MHC class II and co-stimulatory ligand expression (Houot et al., 2006). The immature Dcs would secrete IL-10 to induce the proliferation of CD4⁺CD25⁺ T cells that down-regulate the response of Ag-primed CD4⁺ T cells to normal mature DCs (Wakkach et al., 2003; Mahnke et al., 2007). In the study, adoptive transfer of CD4⁺CD25⁺ T cells could increase IL-10 and TGF-β₁ levels dramatically whereas only TGF-β₁ level was increased with OVA-ITF treated. These results suggested that the OVA-ITF would mainly affect level of TGF-β₁ cytokine which agreed with previous studies that TGF-β was required for Treg cells to mediate their suppressor function in the periphery (Nakamura et al., 2004; Marie et al., 2005; Li et al., 2006).

CONCLUSION

The smaller soluble antigen-specific factor (OVA-TIF), extracted from CD4⁺CD25⁺ T Cells of OVA tolerant mice was a novel and low MW factor and totally different from other suppressive components reported previously. OVA-TIF was produced by CD4⁺CD25⁺ T cell subset and could induce OVA-specific peripheral tolerance independently in vivo with TGF-β₁ as its main suppressive cytokine in recipient mice. These results have important implications for finding potential therapeutic route of prevention and control of graft rejection, autoimmune and related diseases. However, considering the clinical application of these disease-related tolerated factors, purification and physical evidence of the OVA-TIF would be further recognized using some other biochemical studies in the future.

ACKNOWLEDGEMENT

This project was supported by the China Postdoctoral Science Foundation (Grant no. 20070420219 and 200902607). The experiments comply with the current laws of the country in which the experiments were performed.