Chinese Medical Journal 2010;123(7):942-948
CD4+Foxp3+ regulatory T cells converted by rapamycin from peripheral CD4+CD25− naive T cells display more potent regulatory ability in vitro
CHEN Jian-fei, GAO Jie, ZHANG Dong, WANG Zi-han, ZHU Ji-ye
CHEN Jian-fei (Department of Hepatobiliary Surgery, Peking University People’s Hospital, Peking University Center for Transplantation, Beijing 100044, China)
GAO Jie (Department of Hepatobiliary Surgery, Peking University People’s Hospital, Peking University Center for Transplantation, Beijing 100044, China)
ZHANG Dong (Division of Plastic and Reconstructive Surgery, Department of Surgery, UPMC, Starzl Transplantation Institute, BST W 1500, Lothrop Street 200, Pittsburgh, PA 15261, USA)
WANG Zi-han (Department of Hepatobiliary Surgery, Peking University People’s Hospital, Peking University Center for Transplantation, Beijing 100044, China)
ZHU Ji-ye (Department of Hepatobiliary Surgery, Peking University People’s Hospital, Peking University Center for Transplantation, Beijing 100044, China)Correspondence to:ZHU Ji-ye,Department of Hepatobiliary Surgery, Peking University People’s Hospital, Peking University Center for Transplantation, Beijing 100044, China (Tel: 86-10- 88324176. Fax:86-10-68310585. E-mail:firstname.lastname@example.org)
Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells.
Methods Peripheral CD4+CD25− naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4+CD25− T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4+CD25+CD127− subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4+CD25+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×105 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4+CD25− T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×105/well) in 96-well round-bottom plates for 6 days. Then 1×105 or 0.25×105 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4+CD25− T cells using flow cytometry. Data were analyzed using CellQuest software.
Results We found that RAPA can convert peripheral CD4+CD25− naive T Cells to CD4+Foxp3+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity.
Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.
Naturally occurring CD4+CD25+ T-regulator (Treg) cells, which express the forkhead box protein 3 (Foxp3) transcription factor, are generated in the thymus and are characterized by the ability to suppress proliferation of T-effector (Teff) cells in vitro.1,2 Although the immunoregulatory ability of Treg is no longer contested, where and how this population of CD4 T cells is generated and developed remains largely unknown. The major debate centers on whether Treg are generated only in the thymus. Recent evidence suggests that Treg may also be induced in the periphery.3,4 One crucial issue is whether Treg can be induced or converted from normal peripheral CD4 T cells and if this occurs, whether this converted Tregs have suppressive ability.5,6
Rapamycin (RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, is an immunosuppressive agent widely used for preventing acute graft rejection in patients, and has been used to induce operational tolerance in mouse models.7 RAPA binds to FK506- binding protein-12, a highly conserved cytoplasmic receptor. The FK506-binding protein-12-RAPA complex then binds to and inhibits the activities of the serine/ threonine protein kinase mammalian target of RAPA, the activation of which is essential for protein translation and cell cycle initiation in T cells.8 Unlike other commonly used immunosuppressants, RAPA does not appear to interfere with tolerance induction.9,10 Some reports demonstrated that stimulation of CD4+ T cells from spleens of ovalbumin (OVA)-T cell receptor (TCR) transgenic mice in the presence of RAPA resulted in the selective expansion of naturally occurring CD4+CD25+ Treg cells, which can suppress allograft rejection in a model of allogeneic pancreatic islet transplantation.11
There is little information on how RAPA induces Teff cells to become Treg cells. In this paper, we presented evidence that RAPA can convert naive CD4+CD25− T cells into CD4+Foxp3+ regulatory T cells in vitro. We also found that the converted regulatory T cells could inhibit immune responses and maintain antigenic specificity in vitro.
Male C57BL/6 (H-2b), DBA/2 (H-2d), and C3H (H-2k) mice were provided by Beijing Vital River Laboratory Animal Co., Ltd. (Beijing, China) and were maintained in the animal facilities of Peking University People’s Hospital. Animal experiments were approved by the Local Animal Ethics Committee.
Single-cell suspensions were prepared from mouse spleens and lymph nodes of C57BL/6 mice, and red blood cells (RBCs) were removed using RBC lysis buffer (eBiosource, USA). CD4+CD25+ Treg cells were isolated using CD4+CD25+ Treg isolation kits (R&D Systems Inc., USA). CD4+CD25− T cells were isolated using T-cell enrichment columns (R&D Systems Inc.) and by subsequent deletion of fluorochrome-conjugated antibodies to mouse CD25, Ter119, CD11b, CD8, B220 and NK1.1 (eBiosource) positive cells using microbeads (Miltenyi Biotec, DE) and magnetic separation columns (MSC) (Miltenyi Biotec). CD4+CD25+CD127− fractions were isolated from the mixed lymphocyte reactions (MLR) with CD127 (eBiosource) negative selection, followed by CD4 (eBiosource) and CD25 positive selection using microbeads and MSC.
Purification of mitomycin C (MMC)-treated B cells
Single-cell suspensions were prepared from the spleens of DBA/2 mice. RBCs were removed using RBC lysis buffer, then B220+ cells were selected using microbeads and MSC. Purified B220+ cells were treated with 25 μg/ml MMC (Sigma, USA) at 37°C for 30 minutes.
Purification of maturation of dendritic cells (mDC)
Bone marrow cells were aspirated and harvested from DBA/2 mice femurs and tibias. RBCs were removed using RBC lysis buffer, then GR1, Ter119 and B220 (eBiosource) positive cells were depleted using microbeads and MSC. Purified bone marrow cells were cultured with 20 ng/ml granulocyte-macrophage colony-stimulating factor and treated with lipopolysaccharide (Sigma) on day 5. mDCs were separated by positive selection of CD86 on day 6.
Mixed lymphocyte reaction (MLR)
In the converted experiment, CD4+CD25− T cells were incubated for 6 days in 96-well round-bottom plates with MMC-treated B cells at a 1:1 ratio (1×105 T cells: 1×105 MMC-treated B cells) with RAPA (LC laboratories, USA) added to the plate at the different concentrations. CD4+CD25− T Cells were harvested after 6 days and were stained using the Foxp3 detection kit (eBiosource). Cells were analyzed for expression of Foxp3 using flow cytometry (BD Biosciences, USA).
A total of 1×105 Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, USA) labeled naive CD4+CD25− T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H mDCs (0.25×105/well) in 96-well round-bottom plates for 6 days. 1×105 or 0.25×105 converted Treg cells were added in every well as regulatory cells. Cells were harvested after 6 days culture and analyzed for proliferation of CFSE-labeled naive CD4+CD25− T cells using flow cytometry. Data were analyzed using CellQuest software.
Real-time polymerase chain reaction (rt-PCR)
mRNA was extracted from cells using an RNeasy mini-kit (Qiagen, NED). Reverse transcription to cDNA was performed using TaqMan reverse transcription reagents (Applied Biosystems, EU). Specific message levels were quantified by rt-PCR using the ABI 7300 Sequence Detection System (Applied Biosystems). A comparative threshold cycle (CT) method was used to determine foxp3 mRNA levels (Applied Biosystems, assay ID: Mm00475156_m1). Relative expression was analyzed against endogenous murine glyceraldehyde-3- phosphate dehydrogenase (GAPDH, Applied Biosystems, assay ID: Mm99999915_g1). For each sample, the Foxp3 CT value was normalized using the formula ΔCT= CTFoxp3−CTGAPDH. The mean ΔCT was determined, and the relative foxp3 mRNA expression was calculated with 2−ΔCT.
All data analyses were performed using Statistical Package for Social Sciences software (SPSS for Windows, version 13.0). Comparisons of data were analyzed by student’s t test. A P value <0.05 was considered statistically significant.
We determined whether RAPA can affect de novo expression of Foxp3 by peripheral T cells that were stimulated with MMC-treated DBA/2 mouse B cells as APCs. Highly purified (99% pure) CD4+CD25− T cells served as the Teff cells and were cultured with MMC-treated B cells and RAPA. We found that RAPA resulted in the conversion of 7%–8% of naive CD4+CD25− T cells into the Foxp3+ Treg phenotype after 6 days of culture. RAPA induction of Foxp3 is dose-dependent from 1 nmol/L to 100 nmol/L, with maximum conversion at a concentration of 100 nmol/L at day 6 (Figure 1).
Figure 1. Microbeads and MSC-isolated CD4+CD25− cells from naive mice were stimulated with MMC-treated B cells alone or in the presence of increasing doses of RAPA (1 nmol/L, 10 nmol/L, 100 nmol/L, 150 nmol/L) for 6 days in vitro. Foxp3 expression was monitored by FACS. The percentages of Foxp3+ cells within the total CD4+ T cell population are indicated. One representative result from at least three is shown (A). Induction of Foxp3 by RAPA is dose-dependent from 1 nmol/L to 100 nmol/L, peaking in the concentration of 100 nmol/L at day 6 and shown as means of three independent experiments (B). Error bars represent standard deviation.
Our method of Foxp3+ staining effectively distinguishes the Teff and the Treg populations, but results in cell inactivation. CD127− cells comprise >75% of the Foxp3+ cells in the entire population of CD4+ T cells.12,13 Therefore, we obtained CD4+CD25+CD127− T cells using microbeads and MSC from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC-treated B cells plus 100 nmol/L RAPA. We found that there was no difference in the percentage of Foxp3 expression between naive C57BL/6 CD4+CD25+ T cells and converted Treg by flow cytometry. Foxp3 expressions were (72.84±2.38)% and (75.90±1.59)% in naive CD4+CD25+ T cells and converted Treg (CD4+CD25+CD127−) cells, respectively (P >0.05. Figure 2). To determine the mechanism of the suppression, mRNA was isolated from naive C57BL/6 CD4+CD25+ T cells and from the converted Treg cell preparations. We analyzed Foxp3 gene expression of the two groups using rt-PCR. The Foxp3 levels were not different between the two groups.
We further determined whether CD4+CD25+CD127− T cells from the MLR can suppress alloantigen-triggered proliferation of naive CD4+CD25− T cells. As shown in Figure 3, CFSE-labeled naive C57BL/6 CD4+CD25− T cells underwent proliferation during a 6-day co-cultivation with either allogeneic DBA/2- or C3H-derived mDCs. We compared the suppressive ability of converted Treg cells (derived from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC-treated B cells plus 100 nmol/L RAPA) with C57BL/6 naive CD4+CD25+ T cells when they were cocultured with the same DBA/2 mDCs. Figures 3A and 3B show that the ability of converted Treg cells to suppress alloantigen-triggered proliferation of naive CD4+CD25− T cells was significantly higher than that of C57BL/6 naive CD4+CD25+ T cells. The inhibition rates were, respectively, (87.4±4.6)% and (68.1±3.9)% when CD4+CD25− T cells were cocultured with converted Treg cells or naive CD4+CD25+ T cells at a 1:1 ratio and (76.8±4.6)% and (53.5±6.1)% when CD4+CD25− T cells were cocultured with converted Treg cells or naive CD4+CD25+ T cells at a 4:1 ratio. We found that CD4+CD25+CD127− T cells obtained in the MLR with MMC-treated B cells of DBA/2 mice plus 100 nmol/L RAPA exert powerful inhibition on naive CD4+CD25− T cells stimulated with the DBA/2-derived mDCs.
Figure 2. Foxp3+ expression was compared between the C57BL/6 converted Treg cells (CD4+CD25+CD127− T cells) and C57BL/6 naive CD4+CD25+ T cells. One representative result from at least three experiments is shown.
The suppression of converted Treg cells tended to be alloantigen specific, as shown in Figures 3B and 3C. CD4+CD25+CD127− T cells converted during DBA/2 alloantigen stimulation suppressed C3H alloantigen- triggered naive CD4+CD25− T cell proliferation in suppression assay with lower efficiency. The inhibition rate decreased from (87.4±4.6)% to (69.4±5.0)% when CD4+CD25− T cells or converted Treg cells were added to the coculture with allogeneic DBA/2-derived mDCs at a 1:1 ratio and from (76.8±4.6)% to (53.5±6.1)% when CD4+CD25− T cells or converted Treg cells were added to the coculture with allogeneic C3H-derived mDCs at a 4:1 ratio.
Figure 3. C57BL/6 naive CD4+CD25+ T cells suppress CFSE-labeled C57BL/6 CD4+ CD25− T-cell proliferation triggered by the mature DBA/2 DCs. The horizontal bars gate the generation of the dividing cells and the numbers refer to the percentages of these cells. The percentages of these M1 cells may reflect the inhibition rate (A). C57BL/6 converted Treg cells (derived from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC–treated B cells plus 100 nmol/L RAPA) suppress CFSE-labeled C57BL/6 CD4+CD25− T cell proliferation triggered by the mature DBA/2 DCs (B). C57BL/6 converted Treg cells suppress CFSE-labeled C57BL/6 CD4+CD25− T-cell proliferation triggered by third party alloantigens (mature C3H DCs) at lower efficacy (C). The experiment was shown as means of 3 independent experiments. Error bars represent standard deviation. The rates of inhibition were (68.1±3.93)%, (87.4±4.55)%, (69.4±4.97)% at a 1:1 ratio (Teff:Treg), and were (54.8±4.2)%, (76.8±4.6)%, (53.5±6.1)% at a 4:1 ratio (Teff:Treg), and were (40.07±0.96)%, (40.07±0.96)%, (44.92±7.65)% at a 1:0 ratio (Teff:Treg). Statistical analyses was significant difference between the A, C and B (P <0.05), however no significant difference between the A and C (P >0.05) (D).
RAPA is widely used to effectively prevent transplant rejection. This drug is a potent and reasonably well-tolerated immunosuppressive agent, but its effects on graft-destructive Teff and graft-protective Treg cells are drastically different. Some studies demonstrated that RAPA efficacy is not due to selective expansion of naturally-occurring Treg, but induce suppressor functions in conventional CD4+ T cells upon TCR-mediated stimulation.14 RAPA may reverse the effects of AKT as a strong repressor after converting into the Treg phenotype in vitro and in vivo.15 In several other models, the ability of maturation-resistant, RAPA-conditioned DCs are markedly impaired in Foxp3− T cell allostimulatory capacity which favors the stimulation of murine alloantigen-specific CD4+CD25+Foxp3+ Treg cells.16 In all of the above mentioned studies, the researchers focus mainly on the effects of RAPA on Treg cells. However, only a few studies have examined the effects of RAPA on Teff cells.
The current study focuses on the role of RAPA during the conversion of Teff cells to Treg cells. Our experiments show that 100 nmol/L RAPA promotes conversion of about 7% of CD4+CD25− cells stimulated with MMC-treated B cells, with conversion peaking after 6 days of culture and acquisition of alloantigen-specificity. But when the concentration of RAPA exceeds 100 nmol/L, the number of converted CD4+CD25− T cells decreased. RAPA markedly enhanced the percentages of CD4+CD25+ Treg cells at a low dose, but it significantly reduced the cell numbers of all thymocyte subsets at a high dose.17 These results suggest an additional mechanism by which RAPA induces tolerance.
T-cell costimulation by APC, anti-CD3 and anti-CD28 plays important roles in the activation, proliferation and conversion of T cells. In addition to TCR recognition and binding to antigen-MHC, these constimulatory interactions provide a second activation signal. In the absence of costimulatory signals, T-cell responsiveness is impaired.18 Recent studies showed that treatment with transforming growth factor (TGF)-β and RAPA resulted in the conversion of naive CD4+Foxp3− T cells into the Foxp3+ Treg phenotype when stimulated with plate-bound anti-CD3 and anti-CD28.19 However, the Foxp3+ Treg cells were not alloantigen-specific. A previously-published report suggests that dendritic cells (DCs) partially abrogate the regulatory activity of Treg cells from the peripheral blood.20 B cells are seldom considered as professional APCs, and the majority of previous work has focused primarily on their role as terminal effectors as antibody producers.21-23 However, several reports suggest that B cell-induced Treg cells are crucial for resolving experimental autoimmune encephalomyelitis; only after reconstitution of mice with wild type B cells, did Treg numbers normalize and experimental autoimmune encephalomyelitis (EAE) symptoms disappear.24 Other studies suggest that B cells are able to directly block the proliferation of Treg cells.25,26 Therefore, in our experiments during the suppression assay, we used mDCs instead of MMC-treated B cells as APC. B cells are very poor APCs compared with DC, and although B cells have antigen-presenting function in MLR, B cells can also inhibit Treg cell proliferation.27-29 Thus, we selected mDCs as APCs to provide costimulation for CD4+CD25+CD127− T cells.
Other reports11 have shown that the CD4 and CD25 phenotype is essential for Treg cell function. CD4+CD25+ regulatory T cells have emerged as a unique subgroup of suppressor T cells that maintain peripheral immune tolerance. The extrathymic de novo generation of Treg cells is of considerable interest. Identification of Foxp3+ cells suggests that there may be a larger population of Treg cells in human peripheral blood than previously anticipated, although this is controversial due to unanticipated expression in several activated CD25− T cell populations.30,31 Foxp3 expression cannot be used to purify cells, and no cell surface markers can be used to isolate these and other T cell subsets to examine Treg cell activity. Activated CD4+ T cells expressing CD25 under neutral TCR stimulation conditions show no suppressive ability.5,6 For this reason, isolation of Treg cells cannot depend solely on CD25 as a marker. Using a Foxp3−/GFP fusion protein knock-in mouse, Fontenot et al reported that induction of Foxp3 expression in CD4+GFP− T cells may be obtained through fluorescence activated cell sorting.9 It has been recently reported that CD127 expression is down-regulated on CD4+ Treg cells, and is inversely correlated with Foxp3 expression.12,13 It was shown that 94% of CD4+CD25+CD127− T cells express Foxp3, suggesting that better cell surface markers for the identification of CD4+Foxp3+ T cells are CD127 and CD25.12 Similar results were observed in mice and humans.32 CD4+ T cells isolated from Foxp3-GFP knock-in mice were stained for CD127, and the vast majority of CD4+Foxp3+ T cells were CD127−.31,33 Others found that natural Treg cells constitutively expressed high amounts of folate receptor 4 (FR4). This claim will be substantiated in next experiment.34
Among many unsolved questions regarding Treg cells, arguably of most important, is to find ways to promote their suppressive activity or to increase their numbers for their potential clinical application. Since successful application of adoptive cell therapy requires a large number of Treg, and the frequency of alloantigen specific Treg in naive hosts is extremely low, attention has been turned to clonal expansion or induction of Treg.35 It has been shown that expanded Tregs retain their regulatory activity.36 So, it is important to study the production and suppression of induced Treg cells.
In our experiments, CD4+CD25− Teff cells stimulated with MMC-treated B cells and RAPA for 6 days were isolated using CD4, CD25 and CD127 markers to obtain the converted Treg cells (CD4+CD25+CD127− T cells). We observed 70%–75% CD4+Foxp3+ T cells in the CD4+CD25+CD127− T cell subset. We examined their ability to suppress allogeneic naive CD4+CD25− and to respond to allogeneic APCs,37 and we showed that CD4+CD25+CD127− cells are Treg cells. Our results demonstrate that these converted Tregs cells express the Foxp3+ gene and suppress allogeneic naive CD4+CD25− T cells. The CD4+CD25+CD127+ population cannot do so, despite some expression of Foxp3+. In addition, these converted Treg cells retained antigen specificity because they were cultivated with allogeneic B cells as APCs. Antigen specificity of converted Treg cells may render them more potent regulators of secondary immune responses in vitro. Whether the induction of allogeneic- specific Treg can cause skewing of the TCR repertoire of the Treg population towards specific antigens, or if an enhancement of the suppressive capacity on a per cell basis might play a role, requires further elucidation.38
In summary, our findings provide evidence that RAPA induces T regulatory cell conversion from T effector cells and this type of cell displays more potent regulatory ability in vitro; meanwhile our results uncover an additional mechanism for tolerance induction by RAPA.
1. Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25+CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001; 182: 18-32.
2. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299: 1057-1061.
3. Apostolou I, Sarukhan A, Klein L, von Boehmer H. Origin of regulatory T cells with known specificity for antigen. Nat Immunol 2002; 3: 756-763.
4. Zhang X, Izikson L, Liu L, Weiner HL. Activation of CD25+CD4+ regulatory T cells by oral antigen administration. J Immunol 2001; 167: 4245-4253.
5. Sakaguchi S. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 2000; 101: 455-458.
6. Shevach EM. CD4+CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2002; 2: 389-400.
7. Kahan BD, Camardo JS. Rapamycin: clinical results and future opportunities. Transplantation 2001; 72: 1181-1193.
8. Sehgal SN. Rapamune: mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin Biochem 1998; 31: 335-340.
9. Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med 1999; 5: 1298-1302.
10. Blaha P, Bigenzahn S, Koporc Z, Schmid M, Langer F, Selzer E, et al. The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade. Blood 2003; 101: 2886-2893.
11. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+ CD25+ FoxP3+ regulatory T cells. Blood 2005; 105: 4743-4748.
12. Liu WH, Putnam AL, Zhou XY, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ Treg cells. J Exp Med 2006; 203: 1701-1711.
13. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006; 203: 1693-1700.
14. Valmori D, Tosello V, Souleimanian NE, Godefroy E, Scotto L, Wang Y, et al. Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells. J Immunol 2006; 177: 944-949.
15. Haxhinasto S, Mathis D, Benoist C. The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J Exp Med 2008; 205: 565-574.
16. Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang ZL, Thomson AW. Rapamycin-conditioned DCs are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol 2007; 178: 7018-7031.
17. Qu Y, Zhang B, Zhao L, Liu G, Wei G, Ma H, et al. The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+ Foxp3+ T cells in mice. Transpl Immunol 2007; 17: 153-161.
18. Shanahan JC, Moreland LW, Carter RH. Upcoming biologic agents for the treatment of rheumatic diseases. Curr Opin Rheumatol 2003; 15: 226-236.
19. Gao W, Lu Y, El Essawy B, Oukka M, Kuchroo VK, Strom TB. Contrasting effects of cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells. Am J Transplant 2007; 7: 1722-1732.
20. Ahn JS, Krishnadas DK, Agrawal B. Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood. Int Immunol 2007; 19: 227-237.
21. Kelsoe G. Life and death in germinal centers. Immunity 1996; 4: 107-111.
22. Gulbranson-Judge A, Casamayor-Palleja M, MacLennan IC. Mutually dependent T and B cell responses in germinal centers. Ann N Y Acad Sci 1997; 815: 199-210.
23. van Rooijen N. Direct intrafollicular differentiation of memory B cells into plasma cells. Immunol Today 1990; 11: 154-157.
24. Mann MK, Maresz K, Shriver LP, Tan YP, Dittel BN. B cell regulation of CD4+CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J Immunol 2007; 178: 3447-3456.
25. Chen X, Jensen PE. Cutting edge: primary B lymphocytes preferentially expand allogeneic Foxp3+ CD4 T cells. J Immunol 2007; 179: 2046-2050.
26. Olson TS, Bamias G, Naganuma M, Rivera-Nieves J, Burcin TL, Ross W, et al. Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease. J Clin Invest 2004; 114: 389-398.
27. Lim HW, Hillsamer P, Banham AH, Kim CH. Cutting edge: direct suppression of B cells by CIM CD25 regulatory T cells. J Immunol 2005; 175: 4180-4183.
28. Zhao DM, Thornton AM, DiPaolo RJ, Shevach EM. Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood 2006; 107: 3925-3932.
29. Bystry RS, Aluvihare V, Welch KA, Kallikourdis M, Betz AG. B cells and professional APCs recruit regulatory T cells via CCL4. Nat Immunol 2001; 2: 1126-1132.
30. Ziegler SF. Foxp3: of mice and men. Ann Rev Immun 2006; 24: 209-226.
31. Fontenot JD, Dooley JL, Farr AG, Rudensky AY. Developmental regulation of Foxp3 expression during ontogeny. J Exp Med 2005; 202: 901-906.
32. Banham AH. Cell-surface IL-7 receptor expression facilitates the purification of Foxp3+ regulatory T cells. Trends In Immunology 2006; 27: 541-544.
33. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005; 22: 329-341.
34. Yamaguchi T, Hirota K, Nagahama K, Ohkawa K, Takahashi T, Nomura T, et al. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 2007; 27: 145-159.
35. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, et al. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 2004; 199: 1455-1465.
36. Ermann J, Hoffmann P, Edinger M, Dutt S, Blankenberg FG, Higgins JP, et al. Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. Blood 2005; 105: 2220-2226.
37. Suri-Payer E, Amar AZ, Thornton AM, Shevach EM. CD25+CD4+ T cells inhibit both the induction and the effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol 1998; 160: 1212-1218.
38. Coenen JJ, Koenen HJ, Emmer PM, van Rijssen E, Hilbrands LB, Joosten I. Allogeneic stimulation of naturally occurring CD4+CD25+ T cells induces strong regulatory capacity with increased donor-reactivity. Transpl Immunol 2007; 17: 237-242.