In Vivo Control of Diabetogenic T-Cells by Regulatory CD4+CD25+ T-Cells Expressing Foxp3
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糖尿病学杂志 2005年第4期
the Hagedorn Research Institute, Gentofte, Denmark
ABSTRACT
To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitroeCactivated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3eC4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4+ fraction, as purified CD4+ T-cells prevented the accelerated diabetes. When CD25+ cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4+CD25+ cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4+CD25+ T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4+CD25eC cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4+CD25+ cells exhibit powerful regulatory potential in rat diabetes.
Autoimmune destruction of pancreatic -cells arises as a result of a loss of tolerance. Regulatory T-cells, particularly subsets of CD4+ T-cells, are essential for maintaining self-tolerance. Several forms of regulatory CD4+ cells are known: in vitroeCgenerated Tr1 and Th3 and naturally occurring CD4+CD25+ cells. Regulatory CD4+CD25+ T-cells can control the expansion of other CD4+ subsets (CD25eC) and pathogenic subsets (CD4+CD45RBhigh) in vitro (1eC4). They also control naive CD8+ cells (5) and memory CD8+ cells (6,7). However, reports on their ability to control pathogenic cells in vivo are fewer and more recent. In vitroeCgenerated regulatory CD4+ T-cell clones prevent colitis (8) and gastritis (9). Natural CD4+CD25+ cells prevent Th1- and Th2-induced colitis as well as Leishmania major infection (10), control active experimental autoimmune encephalomyelitis (11), and ameliorate established colitis (12).
In the NOD mouse model of human type 1 diabetes, regulatory T-cells were first defined as a subpopulation of cells expressing CD4+CD45RBlow or CD4+CD62L+ (13eC15). Later, these regulatory T-cells were shown to coexpress CD62L and CD25 (16).
In the other spontaneous model of type 1 diabetes, the DP-BB, regulatory cells have not been characterized with regard to their expression of CD25. Previously, spontaneous disease was prevented by a single transfer of spleen cells (17) and the prevention was enhanced by enrichment for CD4+ cells (18). However, transfer of leukocytes from normal rats has so far been successful only if the transfer takes place before the initiation of insulitis (i.e., at about age 1 month) (17,19); transfers conducted at 2 months accelerate diabetes (19). Protection is dependent on the engraftment of the ART2+ (formerly RT6) cells (20); in the control strain, the DR-BB, depletion of these induces diabetes (21). We recently showed that accelerated diabetes induced by the transfer of in vitro phorbol myristate acetate (PMA) plus ionomycineCactivated unfractionated splenocytes from acutely diabetic donors is delayed by transfer of DR-BBeCderived leukocytes (22).
In this study, we proposed to prevent diabetes using a new rat model where diabetes onset was accelerated by transfer of pure, in vitro PMA plus ionomycineCactivated T-cells; that is, without activated antigen-presenting cells as were used in our previous study (22). Using preactivated diabetogenic T-cells in the current study allowed us to explore a situation that is relevant to understanding how such already activated T-cells can be controlled in patients with ongoing -cell destruction. Establishing the conditions required for preventing diabetes by cotransferring various leukocytes from healthy DR-BB rats allowed us to narrow a powerful regulatory potential to CD4+CD25+ T-cells. This new model in rats is well suited to testing preventive strategies for preclinical proof-of-concept, as such studies in mice alone have not been entirely predictive (23). Moreover, it allowed us to track diabetes-inducing and -protective subsets in vivo.
RESEARCH DESIGN AND METHODS
DP-BB and DR-BB rats were taken from our colony at the Hagedorn Research Institute; the housing conditions have been previously described (22). During the period of these experiments, the incidence of spontaneous diabetes before age 120 days was 93% (n = 491). The DR-BB rats have been without diabetes for decades. The animals were tested to ensure they were free of viruses, bacteria, Mycoplasma, and fungi according to recommendations of the Federation of European Laboratory Animal Science Associations (http://www.felasa.org). All experiments were conducted with approval from the Danish Animal Experimentation Inspectorate.
Induction of diabetes in DR-BB rats and screening for diabetes.
Diabetes was induced in young DR-BB rats by treating them with PolyI:C (Sigma-Aldrich, St. Louis, MO) and concentrated hybridoma culture supernatant (clone DS4.23; secretes depleting rat antibody against rat ART2; kindly donated by Dr. J. Rozing), as previously described (22). These rats as well as DP-BB rats receiving cell transfers were screened from day 1 after intervention, as previously described (22). Diabetes was always confirmed by a blood glucose test >10 mmol/l.
Purification of diabetogenic T-cells and reactivation.
Spleens were excised within 8 days after diabetes onset and single-cell suspensions were made as previously described (22). The T-cells were purified on T-cell columns (R&D, Abingdon, U.K., or Cedarlane, Ontario, Canada) or with the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). We observed no differences in the ability to transfer diabetes related to the purification method used. Positive purification with the MACS system was performed with direct rat Pan T-cell microbeads after dead cells were removed with the dead cell removal kit (Miltenyi Biotech). A twofold higher than recommended amount of Pan T-Cell microbeads was added. The cells were activated in vitro, harvested, and analyzed by flow cytometry, as previously described (22), using the lower concentration of ionomycin (0.3 eol/l). Cells were transferred as previously described (22) into 25- to 35-day-old DP-BB rats.
Purification and transfer of protective cells from healthy DR-BB rats.
The spleen and/or mesenteric lymph nodes (mLNs) from healthy DR-BB rats age >65 days were removed into cold Hanks’ balanced salt solution without calcium and magnesium supplemented with 7.5% NaHCO3 (both from Life Technologies). A single-cell suspension was either transferred directly or further purified. Before all transfers, an aliquot of cells was used to analyze the subset distribution and estimate the frequency of apoptotic cells by flow cytometric analysis, as described below.
Unfractionated pooled cells were transferred 2, 3, or 4 days before the transfer of reactivated autoreactive cells, as indicated below. Cell subset distribution was as previously described (22, data not shown).
CD4+ cells were purified from mLNs by first labeling B-, NK-, and NKT-cells and CD8+ T-cells with purified antibodies (all mouse IgG1 isotype; CD45RA [OX-33], NKR-P1 [10/78], and CD8 [OX8], respectively; Pharmingen), then incubating cells with dynabeads M-450 rat anti-mouse IgG1 (Dynal Biotech, Oslo, Norway), according to the manufacturer’s description, except that only one bead was added per target cell. The purity of CD4+ cells was 96 ± 2%; <0.2% were CD8+. The CD4+ subset displayed a similar distribution of ART2high and CD25+ cells before and after purification (65 and 8.5%, respectively).
CD25+ or CD25eC subsets of pure CD4+ T-cells were obtained with the MACS system. CD4+ T-cells were purified from mLNs, dead cells were removed, and live cells were labeled with mouse anti-CD25 phycoerythrin (PE; OX39; Pharmingen) and then labeled with anti-PE microbeads before being fractionated. Before fractionation, the frequency of CD25+ cells among CD4+ cells was 8 ± 2%; after fractionation, the CD25+ fraction was 78 ± 8% pure, with a mean FL2-fluorescence intensity (MFI) of 128 ± 32. The corresponding CD25eC fraction was 98 ± 0.4% pure, with an MFI of 1.4 ± 0.4 (all data n = 7).
Monoclonal antibodies and flow cytometry.
The antibodies used to characterize cells before they were transferred have been previously described (22). Flow cytometry was performed on a FACSCalibur with 4th color option equipped with CellQuest and Attractor software (BD Biosciences, San Jose, CA), as previously described (22); 10,000 events were acquired within the live T-cell gate. In transfer experiments using cells obtained without the dead cell removal kit, the viability of the cells was always tested with propidium iodide and annexin V antibody (Pharmingen). The frequency of the wanted cell subset was calculated to include only cells within a live FSC/SSC gate.
In vivo tracking of the cells.
Purified CD4+ protective cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR) immediately before being transferred, as previously described (22). In vitroeCactivated diabetogenic cells were labeled with CellTracker Orange (CTO; Molecular Probes). In brief, the cells were resuspended in PBS with BSA at a concentration of 5 x 107 cells/ml, CTO was added (final concentration 25 eol/l), cells were incubated for 40 min at 37°C in the dark, and then cells were washed at least twice. Successful labeling was assessed by flow cytometry.
In two experiments, 10 x 106 CD4+ CFDA-SEeClabeled protective cells were transferred and 4 days later 3 x 106 CTO-labeled diabetogenic T-cells were transferred; in some recipients, only one subset was transferred. We removed cervical lymph nodes (cLN), pancreatic lymph nodes (panLN), mLNs, and spleens in ice-cold cell wash buffer (BD Biosciences) 2 or 4 days after the last transfer. Single-cell suspensions were stained first with biotinylated antieCT-cell receptor or anti-CD25 antibody and then streptavidin-APC conjugate and processed for flow cytometry, as previously described.
Foxp3 expression in CD4+CD25+ and CD4+CD25eC T-cells.
RNA was extracted from an aliquot of the purified CD4+CD25+ and CD4+CD25eC T-cells (before in vivo testing) with an RNA Microprep Kit (Stratagene). Quantitative differences in the levels of Foxp3 were determined using semiquantitative RT-PCR on cDNA made with Superscript (Life Technologies). [32P]-labeled dCTP was added to the PCRs, which were then run on standard sequencing gels (Gel-Mix 6; Life Technologies). Primer-specific bands were quantitated using a Typhoon 8600 Variable Mode Imager (Amersham-Pharmacia Biotech, Little Chalfont, U.K.). Primers specific for Foxp3 were 5'-GCTTGTTTGCTGTGCGGAGAC-3' and 5'-GTTTCTGAAGTAGGCGAACAT-3' (from GenBank accession no. XM_228771), and primers specific for the housekeeping gene G6pd were 5'-GACCTGCAGAGCTCCAATCAAC-3' and 5'-CACGACCCTCAGTACCAAAGGG-3' (from GenBank accession no. NM_017006) were used as an internal control in all reactions.
Statistical analysis.
Frequency data were analyzed by a log-rank test and differences in T-cell ratios were evaluated by Student’s t test. Equal or nonequal variance was evaluated by the F test.
RESULTS
Characterization and transfer of purified diabetogenic T-cells to prediabetic DP-BB rats.
Before the transfer, the frequency of T-cells among reactivated, live cells from diabetic DR-BB donors was 96 ± 3%, with >85% CD62L+ (69 ± 4% being CD4+ and 31 ± 4% being CD8+) (n = 10). The percentage of CD4+ T-cells positive for CD25 was 55 ± 5%; 77 ± 7% expressed the CD4 blast marker CD134 and <2% expressed CD122. Among the CD8+ T-cells, 69 ± 9% were positive for CD25. The marker CD90, which discriminates recent thymic emigrants from more experienced T-cells, was expressed on 10 ± 7% of the CD4+ T-cells and 44 ± 16% of the CD8+ T-cells. A minor fraction of T-cells (6 ± 2%) expressed ART2 on the day of transfer.
We transferred these in vitroeCactivated, pure T-cells intravenously into DP-BB rats age 25eC35 days and screened the rats for diabetes. Doses of 3, 5, 10, and 23 x 106 T-cells all resulted in an accelerated onset of diabetes compared with no transferred T-cells (Table 1). Diabetes developed in nearly 100% of the rats. The time to onset of diabetes decreased significantly by increasing the dosage to between 10 and 23 x 106.
Control of diabetes-inducing cells by unpurified cells from healthy donors.
We then tested the ability of unfractionated leukocytes from healthy DR-BB rats to prevent accelerated diabetes induced by transfer of 10 x 106 purified T-cells (Fig. 1). We transferred cells containing 64 x 106 T-cells as a means to prevent spontaneous diabetes (22). Subset analysis of the protective cells before transfer was similar to that previously described (22). Transfer of protective cells 3 days before rats received diabetogenic cells resulted in 0 of 4 rats becoming diabetic, while transfer 4 days before resulted in 1 of 10 rats becoming diabetic by age 62 days; transfer of only diabetogenic cells resulted in 32 of 34 rats developing diabetes by age 42eC59 days (Fig. 1). Transfer of protective cells 2 days before diabetogenic cell transfer produced a partial but significant prevention of accelerated diabetes, as 4 of 8 rats developed diabetes by age 43eC51 days. In all experiments, a group of DP-BB rats receiving only protective cells was included; of these, 97% (n = 29) were protected from developing spontaneous diabetes (data not shown).
Protection resides in the CD4+ subset of T-cells.
To test whether the observed protection resides in the CD4+ compartment of T-cells, we purified CD4+ T-cells from mLNs by negative methods. The CD4+ subset displayed a similar distribution of ART2high and CD25+ cells before and after purification (65 and 8.5%, respectively). We transferred different numbers of these cells 4 days before transferring diabetogenic cells (Table 2). The 43 x 106 CD4+ T-cells corresponded to the number present in the previous experiment with unfractionated cells. Accelerated diabetes induced by 5 x 106 diabetogenic T-cells was effectively prevented by 43 x 106 CD4+ T-cells, whereas accelerated diabetes induced by 3 x 106 diabetogenic T-cells was prevented by both 21.5 x 106 and 10 x 106 CD4+ T-cells. Two rats developed diabetes late in life; thus, the rats were protected against diabetes for the first period after transfer, but this protection was not long lasting. The rest of the cotransfer recipients all remained free of diabetes up to at least age 120 days (Table 2).
Homing of diabetogenic T-cells and CD4+ protective T-cells.
To evaluate the migration pattern of the diabetogenic and protective T-cells, we transferred protective CFDA-SEeClabeled CD4+ T-cells and then 4 days later transferred diabetogenic CTO-labeled T-cells. We found that 2 or 4 days after the last transfer diabetogenic cells had homed to panLNs, mLNs, and cLNs as well as the spleen, but with a significant preference for the panLNs (Fig. 2A). Protective cells displayed distinct CFDA-SE dilution, indicating cell divisions in all lymph nodes (Fig. 2B) and were found at equal frequencies (11eC21% of total live T-cells), with no difference between panLNs and other lymph nodes irrespective of transfer of protective cells only or of both cell types (data not shown). The lymph node cells were harvested 7 days after transfer of protective cells alone and stained with an anti-CD25 antibody (Fig. 2C). Both nondividing and dividing fractions of the CD4+ T-cells contained cells that coexpressed CD25.
Protection of CD4+CD25+ and CD4+CD25eC subsets in the accelerated model.
We next attempted to elucidate whether protection resided within the CD25+ or CD25eC subset of the 21.5 x 106 CD4+ T-cells. We transferred protective T-cells 4 days before transferring 3 x 106 diabetogenic T-cells (Fig. 3A). Diabetes onset was significantly delayed in rats receiving 2 x 106 CD4+CD25+ (1 of 6 developed diabetes at age 93 days), compared with controls receiving diabetogenic T-cells only (8 of 8 developed diabetes at age 52eC66 days). We also observed that five of eight rats receiving 20 x 106 CD4+CD25eC T-cells became diabetic at age 59eC76 days, which was significantly different from the finding in controls (Fig. 3A).
Next, we performed the protective transfers simultaneously with diabetogenic transfer of 3 x 106 T-cells (Fig. 3B). Diabetes onset was significantly delayed in rats receiving both diabetogenic and 2 x 106 CD4+CD25+ T-cells (2 of 12 became diabetic at age 78 and 85 days, respectively) compared with rats receiving diabetogenic cells only (8 of 9 developed diabetes at age 57eC71 days). However, this was not the case in rats receiving both diabetogenic and 20 x 106 CD4+CD25eC cells (5 of 11 developed diabetes at age 63eC97 days).
Protection of CD4+CD25+ and CD4+CD25eC subsets in the spontaneous diabetes model.
Because 2 x 106 CD4+CD25+ T-cells appeared to harbor a powerful regulatory potential, we tested whether transfer of these cells to otherwise untreated DP-BB rats could prevent spontaneous diabetes (Fig. 4). When the cells were transferred to 14 rats age 27 ± 2 days (Fig. 4A), only 3 rats became diabetic (at age 91eC103 days) as opposed to 93% in rats not receiving the transferred cells (age 77 ± 10 days). Moreover, only 2 of 18 rats became diabetic (age 71 and 85 days, respectively) among littermates that received transfer of the reciprocal subset of 20 x 106 CD4+CD25eC T-cells.
When 2 x 106 CD4+CD25+ T-cells were transferred to 10 rats age 42 days, none became diabetic (Fig. 4B). By contrast, 6 of 10 littermates became diabetic at age 83eC91 days after the reciprocal subset of 20 x 106 CD4+CD25eC T-cells were transferred, which was significantly different from the former group as well as from the controls.
Foxp3 expression in CD4+CD25+ and CD4+CD25eC subsets.
Because the transcription factor, Foxp3 is specifically expressed in CD4+CD25+ regulatory T-cells in mice and humans (24eC27), we evaluated Foxp3 mRNA levels in our cell subsets by RT-PCR. We found that the CD4+CD25+ regulatory T-cell subset expressed >40-fold more message for Foxp3 than the CD4+CD25eC subset (Fig. 5). Moreover, Foxp3 levels were almost undetectable in cells used for diabetogenic transfers (data not shown).
DISCUSSION
We previously observed that unfractionated cells from healthy donors could not fully prevent diabetes induced by unpurified PMA plus ionomycineCactivated splenocytes, but only delayed onset (22). In this study, we found that such protective cells prevent diabetes induced by purified PMA plus ionomycineCactivated T-cells. Thus, there appears to be a large number of cells within diabetes-inducing unpurified splenocytes that cannot be controlled. What subset this is needs to be formally tested, but antigen-presenting cells that are activated by PMA plus ionomycin are obvious candidates. In this study, however, we focused on defining subsets within the unfractionated cells that protect against diabetes in the purified T-cell model and against spontaneous diabetes because of the clinical relevance of these situations.
Indeed, transfer models have been widely used for studying diabetes development in BB rats (22,28eC32). In the present study, we extended these models and showed that a wide range (3 to 23 x 106) of reactivated, pure diabetogenic T-cells is able to accelerate diabetes in young DP-BB rats. The finding that the high number (23 x 106) is associated with more rapid diabetes development contrasts with the inflammatory bowel disease transfer model studied by Barthlott et al. (33), where only 1 x 106 or fewer cells transferred colitis. In that model it appears that massive homeostatic expansion of colitis-inducing cells is required within the recipient for disease to develop. In our model this seems not to be the case.
We found that the transfer of protective leukocytes needs to precede the transfer of diabetogenic T-cells to be 100% effective. There were 43 x 106 CD4+ T-cells within the unfractionated pool, which contained considerable and normal fractions of both ART2high and CD25+ cells and, in an interesting finding, as many as 17 x 106 CD45RChigh cells (data not shown), which are known to transfer wasting disease and autoimmunity, including diabetes (34eC36).
We further found that pure CD4+ T-cells control accelerated diabetes. This finding is new, as previously it has been reported only that the transfer of cells enriched for this subset can control spontaneous diabetes (18). Moreover, cotransfer of an equal dose of lymph node T-cells from untreated DR-BB rats containing both CD4 and CD8 subsets, with 60% being ART2high cells, prevents adoptively transferred diabetes in WAG nude rats (28). However, in this study, pure CD4+ T-cells protected the DP-BB recipient fully when they were transferred before diabetogenic cells.
our data with 10 x 106 protective, pure CD4+ cells containing <1 x 106 CD4+CD25+ T-cells and 3 x 106 diabetogenic T-cells, we can deduce that a ratio of protective to diabetogenic cells of 1:3 seems to approach the lower limit for complete prevention. When we fractionated CD4+ T-cells according to their level of CD25 expression, we observed that CD4+CD25+ cells at a ratio of 2:3 protective to diabetogenic cells prevent diabetes. This protection was evident whether cells were transferred before or together with the diabetogenic subset. This finding correlates with a true regulatory phenotype (37) compared with other models of autoimmunity in which regulatory T-cells are able to prevent adoptively transferred disease at similar ratios (11,33,38).
The most important finding was that CD4+CD25+ T-cells were able to prevent spontaneous diabetes development during the interval where the activation of autoreactive cells is believed to occur (19); that is, as late as age 42 days. Previous studies have shown that reconstitution by transfer of purified and normal T-cells into DP-BB recipients age 2 months precipitates the onset of diabetes rather than confers protection (19). In contrast, we show that transfer of the reciprocal 20 x 106 CD25eC subset is far less protective, although the diabetes incidence is lowered.
Other studies (39,40) in rat models of induced diabetes also point to a role for the CD4+CD25+ regulatory T-cells. Thus, CD4+CD25+ T-cells protect in the virus-induced diabetic DR-BB model (39). In the adult thymectomy and split-dose gamma irradiation PVG rat model, CD4+ single positive thymocytes expressing CD25 control diabetes, whereas peripheral CD4+ T-cells both positive and negative for CD25 were found to have regulatory functions (40). The fact that we also observed some protective potential in the CD4+CD25eC fraction supports the hypothesis that regulatory CD4+CD25eC cells also exist in rats, as in mice (38).
In the NOD mouse, simultaneous cotransfer of CD4+CD25+ cells and autoreactive cells isolated from inflamed pancreatic islets only delay diabetes development (16). This apparent discrepancy may be explained by the source of regulatory T-cells in the NOD mouse, as these, and those in all reported experiments, are harvested from prediabetic mice that later in life develop diabetes.
The fact that CD25 is also expressed on conventionally activated T-cells hampers its use as a marker for regulatory cells. In contrast, the transcription factor Foxp3 appears to be a specific marker, as it has been found only in cells with regulatory function and is expressed in naturally occurring CD4+CD25+ cells in both mice (24eC26) and humans (27). Recently, Foxp3-expressing CD4+CD25+ T-cells were shown to prevent diabetes in a mouse model (41), and retroviral transduction of Foxp3 in nonregulatory T-cells rendered these cells capable of inhibiting colitis (24,25). We found Foxp3 to be upregulated in rat CD4+CD25+ T-cells compared with CD4+CD25eC T-cells. Ours is the first study to report that Foxp3 is also a marker of naturally occurring regulatory CD4+CD25+ T-cells in the rat.
The transfer of CD4+ T-cells into the lymphopenic recipients was associated with immediate and massive expansion of the protective cells (Fig. 2), as evidenced by the serial dilution of CFDA-SE. Expansion of regulatory T-cells has been associated with their ability to prevent unwanted transfer-induced immune reactions (33). Although CD4+CD25+ regulatory cells are hyporesponsive to antigenic stimuli in vitro, they exhibit a robust proliferative response in vivo and upregulate their regulatory function (42). This in vivo expansion was recently demonstrated to be a response to an encounter with their cognate antigen rather than to lymphopenia (43). In our study, 10% of the remaining undivided cells also expressed CD25.
Expansion per se does not entirely explain the observed, specific protection by CD4+ or CD4+CD25+ cells, as CD4+CD25eC cells also divide after being transferred into lymphopenic hosts (42). We therefore suggest that there are functional differences and perhaps different mechanisms behind the ability of CD4+CD25+ and CD4+CD25eC T-cells to regulate the autoreactive T-cells. A tantalizing suggestion would be that CD4+CD25+ T-cells are able to prevent the expansion of the spontaneously occurring diabetogenic T-cells, as the transfer of as few as 2 x 106 CD4+CD25+ T-cells to 42 day-old DP-BB rats completely prevented diabetes. Because we tracked the autoreactive cells with CTO, we could not detect the number of cell divisions. However, in the inflammatory bowel disease model, it was shown that CD4+CD25+CD45RBlow regulatory T-cells regulate cell division in the CD4+CD45RBhigh compartment early after cotransfer, and that they themselves display a limited potential for expansion (38). It was thus demonstrated that CD4+CD25+ T-cells can regulate other CD4+ T-cells in lymphopenic recipients. In contrast, it has not been previously shown that regulatory cells control autoreactive effector CD8+ T-cells in vivo, albeit CD4+CD25+ T-cells can prevent intra-islet differentiation of CD8+ T-cells into effector cytotoxic T-lymphocytes in the TeteCtumor necrosis factor-/CD80 B6 model for type 1 diabetes (44). In DP-BB rats, both CD4 and CD8 T-cells act synergistically in inducing -cell destruction (28); our observation that this can be prevented by CD4+CD25+ T-cells is new.
We further found that diabetogenic T-cells preferentially homed to the panLNs. The ability to enter the lymph nodes correlated well with the high levels of CD62L on the cells before transfer. In NOD mice, the panLNs have been shown to be important for diabetes development (45); however, the involvement of CD62L for homing in transfer models is not clear (46,47).
In human type 1 diabetes, loss of -cell mass is believed to have considerably slower kinetics than in the BB rat model. Thus, the finding that regulatory cells controlled highly activated diabetogenic cells in our models suggests that they could possibly do so in human type 1 diabetes also. Tolerogenic strategies (achieved by T-celleCdepleting antibodies) have been able to halt or prevent human type 1 diabetes, apparently by reducing the number of autoreactive T-cells and thus favoring the number of regulatory T-cells (48,49). Because the feeding of insulin -chain has been shown to slow disease progression in NOD mice via induction of bystander immune suppression (50), another possibility would be to increase the number of regulatory cells in humans.
ACKNOWLEDGMENTS
The Hagedorn Research Institute is an independent basic research component of Novo Nordisk A/S. D.L. is the recipient of a Ph.D. fellowship from the Danish Research Academy.
The authors wish to thank Camilla Knudsen and Stine Bisgaard for expert technical help. Furthermore, we wish to thank our animal unit and our laboratory technicians’ trainees for taking care of the animals. We are grateful to Claus Haase for critical reading of the manuscript.
FOOTNOTES
D.L., T.L.H., L.H., and H.M. are employed by Novo Nordisk A/S; D.L., L.H., and H.M. hold stock in Novo Nordisk A/S; and Novo Nordisk A/S has contributed funds supporting the work of H.M.’s laboratory.
CFDA-SE carboxyfluorescein diacetate, succinimidyl ester; cLN, cervical lymph node; CTO, CellTracker Orange; MFI, mean fluorescence intensity; mLN, mesenteric lymph node; panLN, pancreatic lymph node; PE, phycoerythrin; PMA, phorbol myristate acetate
REFERENCES
Thornton AM, Shevach EM: CD4+CD25+ immunoregulatory T-cells suppress polyclonal T-cell activation in-vitro by inhibiting interleukin-2 production. J Exp Med188 :287 eC296,1998
Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S: Immunologic self-tolerance maintained by CD25+CD4+ regulatory T-cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med192 :303 eC309,2000
Chai JG, Tsang JYS, Lechler R, Simpson E, Dyson J, Scott D: CD4+CD25+ T-cells as immunoregulatory T-cells in vitro. Eur J Immunol32 :2365 eC2375,2002
Read S, Mauze S, Asseman C, Bean A, Coffman R, Powrie F: CD38+CD45RBlowCD4+ T-cells: a population of T-cells with immune regulatory activities in vitro. Eur J Immunol28 :3435 eC3447,1998
Piccirillo CA, Shevach EM: Cutting edge: control of CD8+ T-cell activation by CD4+CD25+ immunoregulatory cells. J Immunol167 :1137 eC1140,2001
Murakami M, Sakamoto A, Bender J, Kappler J, Marrack P: CD25+CD4+T-cells contribute to the control of memory CD8+ T-cells. Proc Natl Acad Sci U S A99 :8832 eC8837,2002
Kursar M, Bonhagen K, Fensterle J, Kohler A, Hurwitz R, Kamradt T, Kaufmann SHE, Mittrucker HW: Regulatory CD4+CD25+ T-cells restrict memory CD8+ T-cell responses. J Exp Med196 :1585 eC1592,2002
Groux H, Ogarra A, Bigler M, Rouleau M, Antonenko S, Devries JE, Roncarolo MG: A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature389 :737 eC742,1997
Suripayer E, Amar AZ, Thornton AM, Shevach EM: CD4+CD25+ T-cells inhibit both the induction and effector function of autoreactive T-cells and represent a unique lineage of immunoregulatory cells. J Immunol160 :1212 eC1218,1998
Xu DM, Liu HY, Komai-Koma M, Campbell C, McSharry C, Alexander J, Liew FY: CD4+CD25+ regulatory T-cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice. J Immunol170 :394 eC399,2003
Kohm AP, Carpentier PA, Anger HA, Miller SD: Cutting edge: CD4+CD25+ regulatory T-cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol169 :4712 eC4716,2002
Mottet C, Uhlig HH, Powrie F: Cutting edge: cure of colitis by CD4+CD25+ regulatory T-cells. J Immunol170 :3939 eC3943,2003
Shimada A, Charlton B, Rohane P, Taylor-Edwards C, Fathman CG: Immune regulation in type-1 diabetes. J Autoimmun9 :263 eC269,1996
Shimada A, Rohane P, Fathman CG, Charlton B: Pathogenic and protective roles of CD45RBlowCD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse. Diabetes45 :71 eC78,1996
Lepault F, Gagnerault MC: Characterization of peripheral regulatory CD4+ T-cells that prevent diabetes onset in nonobese diabetic mice. J Immunol164 :240 eC247,2000
Szanya V, Ermann J, Taylor C, Holness C, Fathman CG: The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J Immunol169 :2461 eC2465,2002
Burstein D, Mordes JP, Greiner DL, Stein D, Nakamura N, Handler ES, Rossini AA: Prevention of diabetes in BB/Wor rat by single transfusion of spleen cells: parameters that affect degree of protection. Diabetes38 :24 eC30,1989
Mordes JP, Gallina DL, Handler ES, Greiner DL, Nakamura N, Pelletier A, Rossini AA: Transfusions enriched for W3/25+ helper/inducer T lymphocytes prevent spontaneous diabetes in the BB/W rat. Diabetologia30 :22 eC26,1987
Ramanathan S, Poussier P: T-cell reconstitution of BB/W rats after the initiation of insulitis precipitates the onset of diabetes. J Immunol162 :5134 eC5142,1999
Mordes JP, Bortell R, Doukas J, Rigby M, Whalen B, Zipris D, Greiner DL, Rossini AA: The BB/Wor rat and the balance hypothesis of autoimmunity. Diabetes Metab Rev12 :103 eC109,1996
Greiner DL, Mordes JP, Handler ES, Angelillo M, Nakamura N, Rossini AA: Depletion of RT6.1+ T lymphocytes induces diabetes in resistant biobreeding/Worcester (BB/W) rats. J Exp Med166 :461 eC475,1987
Lundsgaard D, Markholst H: A co-transfer system in young prediabetic BB rats: reactivated autoreactive T-cells can be partly controlled. J Autoimmun21 :325 eC338,2003
Greiner DL, Rossini AA, Mordes JP: Translating data from animal models into methods for preventing human autoimmune diabetes mellitus: caveat emptor and primum non nocere. Clin Immunol100 :134 eC143,2001
Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4+CD25+ regulatory T-cells. Nat Immunol4 :330 eC336,2003
Hori S, Nomura T, Sakaguchi S: Control of regulatory T-cell development by the transcription factor Foxp3. Science299 :1057 eC1061,2003
Khattri R, Cox T, Yasayko SA, Ramsdell F: An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol4 :337 eC342,2003
Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF: Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25eC T-cells. J Clin Invest112 :1437 eC1443,2003
Whalen BJ, Greiner DL, Mordes JP, Rossini AA: Adoptive transfer of autoimmune diabetes mellitus to athymic rats: synergy of CD4+ and CD8+ T-cells and prevention by RT6+ T-cells. J Autoimmun7 :819 eC831,1994
Whalen BJ, Love LC, Mordes JP, Rossini AA, Greiner DL: Intravital dye-labeled diabetogenic rat T-cells retain dye, home to the pancreas, and induce diabetes. Transplant Proc31 :1611 eC1614,1999
Metroz-Dayer MD, Mouland A, Brideau C, Duhamel D, Poussier P: Adoptive transfer of diabetes in BB rats induced by CD4 T lymphocytes. Diabetes39 :928 eC932,1990
Edouard P, Hiserodt JC, Plamondon C, Poussier P: CD8+ T-cells are required for adoptive transfer of the BB rat diabetic syndrome. Diabetes42 :390 eC397,1993
MacKay P: Adoptive transfer of diabetes to and from old normoglycaemic BB rats. Diabetologia38 :145 eC152,1995
Barthlott T, Kassiotis G, Stockinger B: T-cell regulation as a side effect of homeostasis and competition. J Exp Med197 :451 eC460,2003
Powrie F, Mason D: OX-22high CD4+ T-cells induce wasting disease with multiple organ pathology: prevention by the OX-22low subset. J Exp Med172 :1701 eC1708,1990
Fowell D, Mason D: Evidence that the T-cell repertoire of normal rats contains cells with the potential to cause diabetes: characterization of the CD4+ T-cell subset that inhibits this autoimmune potential. J Exp Med177 :627 eC636,1993
Seddon B, Mason D: Regulatory T-cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4+CD45RCeC cells and CD4+CD8eC thymocytes. J Exp Med189 :279 eC288,1999
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T-cells expressing IL-2 receptor alpha-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol155 :1151 eC1164,1995
Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Barbosa TC, Cumano A, Bandeira A: CD25+ CD4+ T-cells regulate the expansion of peripheral CD4 T-cells through the production of IL-10. J Immunol166 :3008 eC3018,2001
Zipris D, Hillebrands JL, Welsh RM, Rozing J, Xie JX, Mordes JP, Greiner DL, Rossini AA: Infections that induce autoimmune diabetes in BBDR rats modulate CD4+CD25+ T-cell populations. J Immunol170 :3592 eC3602,2003
Stephens LA, Mason D: CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T-cells with this function are found in both CD25+ and CD25eC subpopulations. J Immunol165 :3105 eC3110,2000
Peng YF, Laouar Y, Li MO, Green EA, Flavell RA: TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T-cells responsible for protection against diabetes. Proc Natl Acad Sci U S A101 :4572 eC4577,2004
Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A: Homeostasis and anergy of CD4+CD25+ suppressor T-cells in vivo. Nat Immunol3 :33 eC41,2002
Cozzo C, Larkin J, Caton AJ: Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T-cells. J Immunol171 :5678 eC5682,2003
Green EA, Choi YW, Flavell RA: Pancreatic lymph node-derived CD4+CD25+ Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity16 :183 eC191,2002
Gagnerault MC, Luan JJ, Lotton C, Lepault F: Pancreatic lymph nodes are required for priming of beta cell reactive T-cells in NOD mice. J Exp Med196 :369 eC377,2002
Fabien N, Bergerot I, Orgiazzi J, Thivolet C: Lymphocyte function-associated antigen-1, integrin alpha-4, and L-selectin mediate T-cell homing to the pancreas in the model of adoptive transfer of diabetes in NOD mice. Diabetes45 :1181 eC1186,1996
Lepault F, Gagnerault MC, Faveeuw C, Bazin H, Boitard C: Lack of L-selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by mel-14 antibody treatment. Eur J Immunol25 :1502 eC1507,1995
Cooke A, Phillips JM, Parish NM: Tolerogenic strategies to halt or prevent type I diabetes. Nat Immunol2 :810 eC815,2001
Masteller EL, Bluestone JA: Immunotherapy of insulin-dependent diabetes mellitus. Curr Opin Immunol14 :652 eC659,2002
Polanski M, Melican NS, Zhang J, Weiner HL: Oral-administration of the immunodominant B-chain of insulin reduces diabetes in a co-transfer model of diabetes in the NOD mouse and is associated with a switch from Th1 to Th2 cytokines. J Autoimmun10 :339 eC346,1997(Dorthe Lundsgaard, Thomas)
ABSTRACT
To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitroeCactivated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3eC4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4+ fraction, as purified CD4+ T-cells prevented the accelerated diabetes. When CD25+ cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4+CD25+ cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4+CD25+ T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4+CD25eC cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4+CD25+ cells exhibit powerful regulatory potential in rat diabetes.
Autoimmune destruction of pancreatic -cells arises as a result of a loss of tolerance. Regulatory T-cells, particularly subsets of CD4+ T-cells, are essential for maintaining self-tolerance. Several forms of regulatory CD4+ cells are known: in vitroeCgenerated Tr1 and Th3 and naturally occurring CD4+CD25+ cells. Regulatory CD4+CD25+ T-cells can control the expansion of other CD4+ subsets (CD25eC) and pathogenic subsets (CD4+CD45RBhigh) in vitro (1eC4). They also control naive CD8+ cells (5) and memory CD8+ cells (6,7). However, reports on their ability to control pathogenic cells in vivo are fewer and more recent. In vitroeCgenerated regulatory CD4+ T-cell clones prevent colitis (8) and gastritis (9). Natural CD4+CD25+ cells prevent Th1- and Th2-induced colitis as well as Leishmania major infection (10), control active experimental autoimmune encephalomyelitis (11), and ameliorate established colitis (12).
In the NOD mouse model of human type 1 diabetes, regulatory T-cells were first defined as a subpopulation of cells expressing CD4+CD45RBlow or CD4+CD62L+ (13eC15). Later, these regulatory T-cells were shown to coexpress CD62L and CD25 (16).
In the other spontaneous model of type 1 diabetes, the DP-BB, regulatory cells have not been characterized with regard to their expression of CD25. Previously, spontaneous disease was prevented by a single transfer of spleen cells (17) and the prevention was enhanced by enrichment for CD4+ cells (18). However, transfer of leukocytes from normal rats has so far been successful only if the transfer takes place before the initiation of insulitis (i.e., at about age 1 month) (17,19); transfers conducted at 2 months accelerate diabetes (19). Protection is dependent on the engraftment of the ART2+ (formerly RT6) cells (20); in the control strain, the DR-BB, depletion of these induces diabetes (21). We recently showed that accelerated diabetes induced by the transfer of in vitro phorbol myristate acetate (PMA) plus ionomycineCactivated unfractionated splenocytes from acutely diabetic donors is delayed by transfer of DR-BBeCderived leukocytes (22).
In this study, we proposed to prevent diabetes using a new rat model where diabetes onset was accelerated by transfer of pure, in vitro PMA plus ionomycineCactivated T-cells; that is, without activated antigen-presenting cells as were used in our previous study (22). Using preactivated diabetogenic T-cells in the current study allowed us to explore a situation that is relevant to understanding how such already activated T-cells can be controlled in patients with ongoing -cell destruction. Establishing the conditions required for preventing diabetes by cotransferring various leukocytes from healthy DR-BB rats allowed us to narrow a powerful regulatory potential to CD4+CD25+ T-cells. This new model in rats is well suited to testing preventive strategies for preclinical proof-of-concept, as such studies in mice alone have not been entirely predictive (23). Moreover, it allowed us to track diabetes-inducing and -protective subsets in vivo.
RESEARCH DESIGN AND METHODS
DP-BB and DR-BB rats were taken from our colony at the Hagedorn Research Institute; the housing conditions have been previously described (22). During the period of these experiments, the incidence of spontaneous diabetes before age 120 days was 93% (n = 491). The DR-BB rats have been without diabetes for decades. The animals were tested to ensure they were free of viruses, bacteria, Mycoplasma, and fungi according to recommendations of the Federation of European Laboratory Animal Science Associations (http://www.felasa.org). All experiments were conducted with approval from the Danish Animal Experimentation Inspectorate.
Induction of diabetes in DR-BB rats and screening for diabetes.
Diabetes was induced in young DR-BB rats by treating them with PolyI:C (Sigma-Aldrich, St. Louis, MO) and concentrated hybridoma culture supernatant (clone DS4.23; secretes depleting rat antibody against rat ART2; kindly donated by Dr. J. Rozing), as previously described (22). These rats as well as DP-BB rats receiving cell transfers were screened from day 1 after intervention, as previously described (22). Diabetes was always confirmed by a blood glucose test >10 mmol/l.
Purification of diabetogenic T-cells and reactivation.
Spleens were excised within 8 days after diabetes onset and single-cell suspensions were made as previously described (22). The T-cells were purified on T-cell columns (R&D, Abingdon, U.K., or Cedarlane, Ontario, Canada) or with the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). We observed no differences in the ability to transfer diabetes related to the purification method used. Positive purification with the MACS system was performed with direct rat Pan T-cell microbeads after dead cells were removed with the dead cell removal kit (Miltenyi Biotech). A twofold higher than recommended amount of Pan T-Cell microbeads was added. The cells were activated in vitro, harvested, and analyzed by flow cytometry, as previously described (22), using the lower concentration of ionomycin (0.3 eol/l). Cells were transferred as previously described (22) into 25- to 35-day-old DP-BB rats.
Purification and transfer of protective cells from healthy DR-BB rats.
The spleen and/or mesenteric lymph nodes (mLNs) from healthy DR-BB rats age >65 days were removed into cold Hanks’ balanced salt solution without calcium and magnesium supplemented with 7.5% NaHCO3 (both from Life Technologies). A single-cell suspension was either transferred directly or further purified. Before all transfers, an aliquot of cells was used to analyze the subset distribution and estimate the frequency of apoptotic cells by flow cytometric analysis, as described below.
Unfractionated pooled cells were transferred 2, 3, or 4 days before the transfer of reactivated autoreactive cells, as indicated below. Cell subset distribution was as previously described (22, data not shown).
CD4+ cells were purified from mLNs by first labeling B-, NK-, and NKT-cells and CD8+ T-cells with purified antibodies (all mouse IgG1 isotype; CD45RA [OX-33], NKR-P1 [10/78], and CD8 [OX8], respectively; Pharmingen), then incubating cells with dynabeads M-450 rat anti-mouse IgG1 (Dynal Biotech, Oslo, Norway), according to the manufacturer’s description, except that only one bead was added per target cell. The purity of CD4+ cells was 96 ± 2%; <0.2% were CD8+. The CD4+ subset displayed a similar distribution of ART2high and CD25+ cells before and after purification (65 and 8.5%, respectively).
CD25+ or CD25eC subsets of pure CD4+ T-cells were obtained with the MACS system. CD4+ T-cells were purified from mLNs, dead cells were removed, and live cells were labeled with mouse anti-CD25 phycoerythrin (PE; OX39; Pharmingen) and then labeled with anti-PE microbeads before being fractionated. Before fractionation, the frequency of CD25+ cells among CD4+ cells was 8 ± 2%; after fractionation, the CD25+ fraction was 78 ± 8% pure, with a mean FL2-fluorescence intensity (MFI) of 128 ± 32. The corresponding CD25eC fraction was 98 ± 0.4% pure, with an MFI of 1.4 ± 0.4 (all data n = 7).
Monoclonal antibodies and flow cytometry.
The antibodies used to characterize cells before they were transferred have been previously described (22). Flow cytometry was performed on a FACSCalibur with 4th color option equipped with CellQuest and Attractor software (BD Biosciences, San Jose, CA), as previously described (22); 10,000 events were acquired within the live T-cell gate. In transfer experiments using cells obtained without the dead cell removal kit, the viability of the cells was always tested with propidium iodide and annexin V antibody (Pharmingen). The frequency of the wanted cell subset was calculated to include only cells within a live FSC/SSC gate.
In vivo tracking of the cells.
Purified CD4+ protective cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR) immediately before being transferred, as previously described (22). In vitroeCactivated diabetogenic cells were labeled with CellTracker Orange (CTO; Molecular Probes). In brief, the cells were resuspended in PBS with BSA at a concentration of 5 x 107 cells/ml, CTO was added (final concentration 25 eol/l), cells were incubated for 40 min at 37°C in the dark, and then cells were washed at least twice. Successful labeling was assessed by flow cytometry.
In two experiments, 10 x 106 CD4+ CFDA-SEeClabeled protective cells were transferred and 4 days later 3 x 106 CTO-labeled diabetogenic T-cells were transferred; in some recipients, only one subset was transferred. We removed cervical lymph nodes (cLN), pancreatic lymph nodes (panLN), mLNs, and spleens in ice-cold cell wash buffer (BD Biosciences) 2 or 4 days after the last transfer. Single-cell suspensions were stained first with biotinylated antieCT-cell receptor or anti-CD25 antibody and then streptavidin-APC conjugate and processed for flow cytometry, as previously described.
Foxp3 expression in CD4+CD25+ and CD4+CD25eC T-cells.
RNA was extracted from an aliquot of the purified CD4+CD25+ and CD4+CD25eC T-cells (before in vivo testing) with an RNA Microprep Kit (Stratagene). Quantitative differences in the levels of Foxp3 were determined using semiquantitative RT-PCR on cDNA made with Superscript (Life Technologies). [32P]-labeled dCTP was added to the PCRs, which were then run on standard sequencing gels (Gel-Mix 6; Life Technologies). Primer-specific bands were quantitated using a Typhoon 8600 Variable Mode Imager (Amersham-Pharmacia Biotech, Little Chalfont, U.K.). Primers specific for Foxp3 were 5'-GCTTGTTTGCTGTGCGGAGAC-3' and 5'-GTTTCTGAAGTAGGCGAACAT-3' (from GenBank accession no. XM_228771), and primers specific for the housekeeping gene G6pd were 5'-GACCTGCAGAGCTCCAATCAAC-3' and 5'-CACGACCCTCAGTACCAAAGGG-3' (from GenBank accession no. NM_017006) were used as an internal control in all reactions.
Statistical analysis.
Frequency data were analyzed by a log-rank test and differences in T-cell ratios were evaluated by Student’s t test. Equal or nonequal variance was evaluated by the F test.
RESULTS
Characterization and transfer of purified diabetogenic T-cells to prediabetic DP-BB rats.
Before the transfer, the frequency of T-cells among reactivated, live cells from diabetic DR-BB donors was 96 ± 3%, with >85% CD62L+ (69 ± 4% being CD4+ and 31 ± 4% being CD8+) (n = 10). The percentage of CD4+ T-cells positive for CD25 was 55 ± 5%; 77 ± 7% expressed the CD4 blast marker CD134 and <2% expressed CD122. Among the CD8+ T-cells, 69 ± 9% were positive for CD25. The marker CD90, which discriminates recent thymic emigrants from more experienced T-cells, was expressed on 10 ± 7% of the CD4+ T-cells and 44 ± 16% of the CD8+ T-cells. A minor fraction of T-cells (6 ± 2%) expressed ART2 on the day of transfer.
We transferred these in vitroeCactivated, pure T-cells intravenously into DP-BB rats age 25eC35 days and screened the rats for diabetes. Doses of 3, 5, 10, and 23 x 106 T-cells all resulted in an accelerated onset of diabetes compared with no transferred T-cells (Table 1). Diabetes developed in nearly 100% of the rats. The time to onset of diabetes decreased significantly by increasing the dosage to between 10 and 23 x 106.
Control of diabetes-inducing cells by unpurified cells from healthy donors.
We then tested the ability of unfractionated leukocytes from healthy DR-BB rats to prevent accelerated diabetes induced by transfer of 10 x 106 purified T-cells (Fig. 1). We transferred cells containing 64 x 106 T-cells as a means to prevent spontaneous diabetes (22). Subset analysis of the protective cells before transfer was similar to that previously described (22). Transfer of protective cells 3 days before rats received diabetogenic cells resulted in 0 of 4 rats becoming diabetic, while transfer 4 days before resulted in 1 of 10 rats becoming diabetic by age 62 days; transfer of only diabetogenic cells resulted in 32 of 34 rats developing diabetes by age 42eC59 days (Fig. 1). Transfer of protective cells 2 days before diabetogenic cell transfer produced a partial but significant prevention of accelerated diabetes, as 4 of 8 rats developed diabetes by age 43eC51 days. In all experiments, a group of DP-BB rats receiving only protective cells was included; of these, 97% (n = 29) were protected from developing spontaneous diabetes (data not shown).
Protection resides in the CD4+ subset of T-cells.
To test whether the observed protection resides in the CD4+ compartment of T-cells, we purified CD4+ T-cells from mLNs by negative methods. The CD4+ subset displayed a similar distribution of ART2high and CD25+ cells before and after purification (65 and 8.5%, respectively). We transferred different numbers of these cells 4 days before transferring diabetogenic cells (Table 2). The 43 x 106 CD4+ T-cells corresponded to the number present in the previous experiment with unfractionated cells. Accelerated diabetes induced by 5 x 106 diabetogenic T-cells was effectively prevented by 43 x 106 CD4+ T-cells, whereas accelerated diabetes induced by 3 x 106 diabetogenic T-cells was prevented by both 21.5 x 106 and 10 x 106 CD4+ T-cells. Two rats developed diabetes late in life; thus, the rats were protected against diabetes for the first period after transfer, but this protection was not long lasting. The rest of the cotransfer recipients all remained free of diabetes up to at least age 120 days (Table 2).
Homing of diabetogenic T-cells and CD4+ protective T-cells.
To evaluate the migration pattern of the diabetogenic and protective T-cells, we transferred protective CFDA-SEeClabeled CD4+ T-cells and then 4 days later transferred diabetogenic CTO-labeled T-cells. We found that 2 or 4 days after the last transfer diabetogenic cells had homed to panLNs, mLNs, and cLNs as well as the spleen, but with a significant preference for the panLNs (Fig. 2A). Protective cells displayed distinct CFDA-SE dilution, indicating cell divisions in all lymph nodes (Fig. 2B) and were found at equal frequencies (11eC21% of total live T-cells), with no difference between panLNs and other lymph nodes irrespective of transfer of protective cells only or of both cell types (data not shown). The lymph node cells were harvested 7 days after transfer of protective cells alone and stained with an anti-CD25 antibody (Fig. 2C). Both nondividing and dividing fractions of the CD4+ T-cells contained cells that coexpressed CD25.
Protection of CD4+CD25+ and CD4+CD25eC subsets in the accelerated model.
We next attempted to elucidate whether protection resided within the CD25+ or CD25eC subset of the 21.5 x 106 CD4+ T-cells. We transferred protective T-cells 4 days before transferring 3 x 106 diabetogenic T-cells (Fig. 3A). Diabetes onset was significantly delayed in rats receiving 2 x 106 CD4+CD25+ (1 of 6 developed diabetes at age 93 days), compared with controls receiving diabetogenic T-cells only (8 of 8 developed diabetes at age 52eC66 days). We also observed that five of eight rats receiving 20 x 106 CD4+CD25eC T-cells became diabetic at age 59eC76 days, which was significantly different from the finding in controls (Fig. 3A).
Next, we performed the protective transfers simultaneously with diabetogenic transfer of 3 x 106 T-cells (Fig. 3B). Diabetes onset was significantly delayed in rats receiving both diabetogenic and 2 x 106 CD4+CD25+ T-cells (2 of 12 became diabetic at age 78 and 85 days, respectively) compared with rats receiving diabetogenic cells only (8 of 9 developed diabetes at age 57eC71 days). However, this was not the case in rats receiving both diabetogenic and 20 x 106 CD4+CD25eC cells (5 of 11 developed diabetes at age 63eC97 days).
Protection of CD4+CD25+ and CD4+CD25eC subsets in the spontaneous diabetes model.
Because 2 x 106 CD4+CD25+ T-cells appeared to harbor a powerful regulatory potential, we tested whether transfer of these cells to otherwise untreated DP-BB rats could prevent spontaneous diabetes (Fig. 4). When the cells were transferred to 14 rats age 27 ± 2 days (Fig. 4A), only 3 rats became diabetic (at age 91eC103 days) as opposed to 93% in rats not receiving the transferred cells (age 77 ± 10 days). Moreover, only 2 of 18 rats became diabetic (age 71 and 85 days, respectively) among littermates that received transfer of the reciprocal subset of 20 x 106 CD4+CD25eC T-cells.
When 2 x 106 CD4+CD25+ T-cells were transferred to 10 rats age 42 days, none became diabetic (Fig. 4B). By contrast, 6 of 10 littermates became diabetic at age 83eC91 days after the reciprocal subset of 20 x 106 CD4+CD25eC T-cells were transferred, which was significantly different from the former group as well as from the controls.
Foxp3 expression in CD4+CD25+ and CD4+CD25eC subsets.
Because the transcription factor, Foxp3 is specifically expressed in CD4+CD25+ regulatory T-cells in mice and humans (24eC27), we evaluated Foxp3 mRNA levels in our cell subsets by RT-PCR. We found that the CD4+CD25+ regulatory T-cell subset expressed >40-fold more message for Foxp3 than the CD4+CD25eC subset (Fig. 5). Moreover, Foxp3 levels were almost undetectable in cells used for diabetogenic transfers (data not shown).
DISCUSSION
We previously observed that unfractionated cells from healthy donors could not fully prevent diabetes induced by unpurified PMA plus ionomycineCactivated splenocytes, but only delayed onset (22). In this study, we found that such protective cells prevent diabetes induced by purified PMA plus ionomycineCactivated T-cells. Thus, there appears to be a large number of cells within diabetes-inducing unpurified splenocytes that cannot be controlled. What subset this is needs to be formally tested, but antigen-presenting cells that are activated by PMA plus ionomycin are obvious candidates. In this study, however, we focused on defining subsets within the unfractionated cells that protect against diabetes in the purified T-cell model and against spontaneous diabetes because of the clinical relevance of these situations.
Indeed, transfer models have been widely used for studying diabetes development in BB rats (22,28eC32). In the present study, we extended these models and showed that a wide range (3 to 23 x 106) of reactivated, pure diabetogenic T-cells is able to accelerate diabetes in young DP-BB rats. The finding that the high number (23 x 106) is associated with more rapid diabetes development contrasts with the inflammatory bowel disease transfer model studied by Barthlott et al. (33), where only 1 x 106 or fewer cells transferred colitis. In that model it appears that massive homeostatic expansion of colitis-inducing cells is required within the recipient for disease to develop. In our model this seems not to be the case.
We found that the transfer of protective leukocytes needs to precede the transfer of diabetogenic T-cells to be 100% effective. There were 43 x 106 CD4+ T-cells within the unfractionated pool, which contained considerable and normal fractions of both ART2high and CD25+ cells and, in an interesting finding, as many as 17 x 106 CD45RChigh cells (data not shown), which are known to transfer wasting disease and autoimmunity, including diabetes (34eC36).
We further found that pure CD4+ T-cells control accelerated diabetes. This finding is new, as previously it has been reported only that the transfer of cells enriched for this subset can control spontaneous diabetes (18). Moreover, cotransfer of an equal dose of lymph node T-cells from untreated DR-BB rats containing both CD4 and CD8 subsets, with 60% being ART2high cells, prevents adoptively transferred diabetes in WAG nude rats (28). However, in this study, pure CD4+ T-cells protected the DP-BB recipient fully when they were transferred before diabetogenic cells.
our data with 10 x 106 protective, pure CD4+ cells containing <1 x 106 CD4+CD25+ T-cells and 3 x 106 diabetogenic T-cells, we can deduce that a ratio of protective to diabetogenic cells of 1:3 seems to approach the lower limit for complete prevention. When we fractionated CD4+ T-cells according to their level of CD25 expression, we observed that CD4+CD25+ cells at a ratio of 2:3 protective to diabetogenic cells prevent diabetes. This protection was evident whether cells were transferred before or together with the diabetogenic subset. This finding correlates with a true regulatory phenotype (37) compared with other models of autoimmunity in which regulatory T-cells are able to prevent adoptively transferred disease at similar ratios (11,33,38).
The most important finding was that CD4+CD25+ T-cells were able to prevent spontaneous diabetes development during the interval where the activation of autoreactive cells is believed to occur (19); that is, as late as age 42 days. Previous studies have shown that reconstitution by transfer of purified and normal T-cells into DP-BB recipients age 2 months precipitates the onset of diabetes rather than confers protection (19). In contrast, we show that transfer of the reciprocal 20 x 106 CD25eC subset is far less protective, although the diabetes incidence is lowered.
Other studies (39,40) in rat models of induced diabetes also point to a role for the CD4+CD25+ regulatory T-cells. Thus, CD4+CD25+ T-cells protect in the virus-induced diabetic DR-BB model (39). In the adult thymectomy and split-dose gamma irradiation PVG rat model, CD4+ single positive thymocytes expressing CD25 control diabetes, whereas peripheral CD4+ T-cells both positive and negative for CD25 were found to have regulatory functions (40). The fact that we also observed some protective potential in the CD4+CD25eC fraction supports the hypothesis that regulatory CD4+CD25eC cells also exist in rats, as in mice (38).
In the NOD mouse, simultaneous cotransfer of CD4+CD25+ cells and autoreactive cells isolated from inflamed pancreatic islets only delay diabetes development (16). This apparent discrepancy may be explained by the source of regulatory T-cells in the NOD mouse, as these, and those in all reported experiments, are harvested from prediabetic mice that later in life develop diabetes.
The fact that CD25 is also expressed on conventionally activated T-cells hampers its use as a marker for regulatory cells. In contrast, the transcription factor Foxp3 appears to be a specific marker, as it has been found only in cells with regulatory function and is expressed in naturally occurring CD4+CD25+ cells in both mice (24eC26) and humans (27). Recently, Foxp3-expressing CD4+CD25+ T-cells were shown to prevent diabetes in a mouse model (41), and retroviral transduction of Foxp3 in nonregulatory T-cells rendered these cells capable of inhibiting colitis (24,25). We found Foxp3 to be upregulated in rat CD4+CD25+ T-cells compared with CD4+CD25eC T-cells. Ours is the first study to report that Foxp3 is also a marker of naturally occurring regulatory CD4+CD25+ T-cells in the rat.
The transfer of CD4+ T-cells into the lymphopenic recipients was associated with immediate and massive expansion of the protective cells (Fig. 2), as evidenced by the serial dilution of CFDA-SE. Expansion of regulatory T-cells has been associated with their ability to prevent unwanted transfer-induced immune reactions (33). Although CD4+CD25+ regulatory cells are hyporesponsive to antigenic stimuli in vitro, they exhibit a robust proliferative response in vivo and upregulate their regulatory function (42). This in vivo expansion was recently demonstrated to be a response to an encounter with their cognate antigen rather than to lymphopenia (43). In our study, 10% of the remaining undivided cells also expressed CD25.
Expansion per se does not entirely explain the observed, specific protection by CD4+ or CD4+CD25+ cells, as CD4+CD25eC cells also divide after being transferred into lymphopenic hosts (42). We therefore suggest that there are functional differences and perhaps different mechanisms behind the ability of CD4+CD25+ and CD4+CD25eC T-cells to regulate the autoreactive T-cells. A tantalizing suggestion would be that CD4+CD25+ T-cells are able to prevent the expansion of the spontaneously occurring diabetogenic T-cells, as the transfer of as few as 2 x 106 CD4+CD25+ T-cells to 42 day-old DP-BB rats completely prevented diabetes. Because we tracked the autoreactive cells with CTO, we could not detect the number of cell divisions. However, in the inflammatory bowel disease model, it was shown that CD4+CD25+CD45RBlow regulatory T-cells regulate cell division in the CD4+CD45RBhigh compartment early after cotransfer, and that they themselves display a limited potential for expansion (38). It was thus demonstrated that CD4+CD25+ T-cells can regulate other CD4+ T-cells in lymphopenic recipients. In contrast, it has not been previously shown that regulatory cells control autoreactive effector CD8+ T-cells in vivo, albeit CD4+CD25+ T-cells can prevent intra-islet differentiation of CD8+ T-cells into effector cytotoxic T-lymphocytes in the TeteCtumor necrosis factor-/CD80 B6 model for type 1 diabetes (44). In DP-BB rats, both CD4 and CD8 T-cells act synergistically in inducing -cell destruction (28); our observation that this can be prevented by CD4+CD25+ T-cells is new.
We further found that diabetogenic T-cells preferentially homed to the panLNs. The ability to enter the lymph nodes correlated well with the high levels of CD62L on the cells before transfer. In NOD mice, the panLNs have been shown to be important for diabetes development (45); however, the involvement of CD62L for homing in transfer models is not clear (46,47).
In human type 1 diabetes, loss of -cell mass is believed to have considerably slower kinetics than in the BB rat model. Thus, the finding that regulatory cells controlled highly activated diabetogenic cells in our models suggests that they could possibly do so in human type 1 diabetes also. Tolerogenic strategies (achieved by T-celleCdepleting antibodies) have been able to halt or prevent human type 1 diabetes, apparently by reducing the number of autoreactive T-cells and thus favoring the number of regulatory T-cells (48,49). Because the feeding of insulin -chain has been shown to slow disease progression in NOD mice via induction of bystander immune suppression (50), another possibility would be to increase the number of regulatory cells in humans.
ACKNOWLEDGMENTS
The Hagedorn Research Institute is an independent basic research component of Novo Nordisk A/S. D.L. is the recipient of a Ph.D. fellowship from the Danish Research Academy.
The authors wish to thank Camilla Knudsen and Stine Bisgaard for expert technical help. Furthermore, we wish to thank our animal unit and our laboratory technicians’ trainees for taking care of the animals. We are grateful to Claus Haase for critical reading of the manuscript.
FOOTNOTES
D.L., T.L.H., L.H., and H.M. are employed by Novo Nordisk A/S; D.L., L.H., and H.M. hold stock in Novo Nordisk A/S; and Novo Nordisk A/S has contributed funds supporting the work of H.M.’s laboratory.
CFDA-SE carboxyfluorescein diacetate, succinimidyl ester; cLN, cervical lymph node; CTO, CellTracker Orange; MFI, mean fluorescence intensity; mLN, mesenteric lymph node; panLN, pancreatic lymph node; PE, phycoerythrin; PMA, phorbol myristate acetate
REFERENCES
Thornton AM, Shevach EM: CD4+CD25+ immunoregulatory T-cells suppress polyclonal T-cell activation in-vitro by inhibiting interleukin-2 production. J Exp Med188 :287 eC296,1998
Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S: Immunologic self-tolerance maintained by CD25+CD4+ regulatory T-cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med192 :303 eC309,2000
Chai JG, Tsang JYS, Lechler R, Simpson E, Dyson J, Scott D: CD4+CD25+ T-cells as immunoregulatory T-cells in vitro. Eur J Immunol32 :2365 eC2375,2002
Read S, Mauze S, Asseman C, Bean A, Coffman R, Powrie F: CD38+CD45RBlowCD4+ T-cells: a population of T-cells with immune regulatory activities in vitro. Eur J Immunol28 :3435 eC3447,1998
Piccirillo CA, Shevach EM: Cutting edge: control of CD8+ T-cell activation by CD4+CD25+ immunoregulatory cells. J Immunol167 :1137 eC1140,2001
Murakami M, Sakamoto A, Bender J, Kappler J, Marrack P: CD25+CD4+T-cells contribute to the control of memory CD8+ T-cells. Proc Natl Acad Sci U S A99 :8832 eC8837,2002
Kursar M, Bonhagen K, Fensterle J, Kohler A, Hurwitz R, Kamradt T, Kaufmann SHE, Mittrucker HW: Regulatory CD4+CD25+ T-cells restrict memory CD8+ T-cell responses. J Exp Med196 :1585 eC1592,2002
Groux H, Ogarra A, Bigler M, Rouleau M, Antonenko S, Devries JE, Roncarolo MG: A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature389 :737 eC742,1997
Suripayer E, Amar AZ, Thornton AM, Shevach EM: CD4+CD25+ T-cells inhibit both the induction and effector function of autoreactive T-cells and represent a unique lineage of immunoregulatory cells. J Immunol160 :1212 eC1218,1998
Xu DM, Liu HY, Komai-Koma M, Campbell C, McSharry C, Alexander J, Liew FY: CD4+CD25+ regulatory T-cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice. J Immunol170 :394 eC399,2003
Kohm AP, Carpentier PA, Anger HA, Miller SD: Cutting edge: CD4+CD25+ regulatory T-cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol169 :4712 eC4716,2002
Mottet C, Uhlig HH, Powrie F: Cutting edge: cure of colitis by CD4+CD25+ regulatory T-cells. J Immunol170 :3939 eC3943,2003
Shimada A, Charlton B, Rohane P, Taylor-Edwards C, Fathman CG: Immune regulation in type-1 diabetes. J Autoimmun9 :263 eC269,1996
Shimada A, Rohane P, Fathman CG, Charlton B: Pathogenic and protective roles of CD45RBlowCD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse. Diabetes45 :71 eC78,1996
Lepault F, Gagnerault MC: Characterization of peripheral regulatory CD4+ T-cells that prevent diabetes onset in nonobese diabetic mice. J Immunol164 :240 eC247,2000
Szanya V, Ermann J, Taylor C, Holness C, Fathman CG: The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J Immunol169 :2461 eC2465,2002
Burstein D, Mordes JP, Greiner DL, Stein D, Nakamura N, Handler ES, Rossini AA: Prevention of diabetes in BB/Wor rat by single transfusion of spleen cells: parameters that affect degree of protection. Diabetes38 :24 eC30,1989
Mordes JP, Gallina DL, Handler ES, Greiner DL, Nakamura N, Pelletier A, Rossini AA: Transfusions enriched for W3/25+ helper/inducer T lymphocytes prevent spontaneous diabetes in the BB/W rat. Diabetologia30 :22 eC26,1987
Ramanathan S, Poussier P: T-cell reconstitution of BB/W rats after the initiation of insulitis precipitates the onset of diabetes. J Immunol162 :5134 eC5142,1999
Mordes JP, Bortell R, Doukas J, Rigby M, Whalen B, Zipris D, Greiner DL, Rossini AA: The BB/Wor rat and the balance hypothesis of autoimmunity. Diabetes Metab Rev12 :103 eC109,1996
Greiner DL, Mordes JP, Handler ES, Angelillo M, Nakamura N, Rossini AA: Depletion of RT6.1+ T lymphocytes induces diabetes in resistant biobreeding/Worcester (BB/W) rats. J Exp Med166 :461 eC475,1987
Lundsgaard D, Markholst H: A co-transfer system in young prediabetic BB rats: reactivated autoreactive T-cells can be partly controlled. J Autoimmun21 :325 eC338,2003
Greiner DL, Rossini AA, Mordes JP: Translating data from animal models into methods for preventing human autoimmune diabetes mellitus: caveat emptor and primum non nocere. Clin Immunol100 :134 eC143,2001
Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4+CD25+ regulatory T-cells. Nat Immunol4 :330 eC336,2003
Hori S, Nomura T, Sakaguchi S: Control of regulatory T-cell development by the transcription factor Foxp3. Science299 :1057 eC1061,2003
Khattri R, Cox T, Yasayko SA, Ramsdell F: An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol4 :337 eC342,2003
Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF: Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25eC T-cells. J Clin Invest112 :1437 eC1443,2003
Whalen BJ, Greiner DL, Mordes JP, Rossini AA: Adoptive transfer of autoimmune diabetes mellitus to athymic rats: synergy of CD4+ and CD8+ T-cells and prevention by RT6+ T-cells. J Autoimmun7 :819 eC831,1994
Whalen BJ, Love LC, Mordes JP, Rossini AA, Greiner DL: Intravital dye-labeled diabetogenic rat T-cells retain dye, home to the pancreas, and induce diabetes. Transplant Proc31 :1611 eC1614,1999
Metroz-Dayer MD, Mouland A, Brideau C, Duhamel D, Poussier P: Adoptive transfer of diabetes in BB rats induced by CD4 T lymphocytes. Diabetes39 :928 eC932,1990
Edouard P, Hiserodt JC, Plamondon C, Poussier P: CD8+ T-cells are required for adoptive transfer of the BB rat diabetic syndrome. Diabetes42 :390 eC397,1993
MacKay P: Adoptive transfer of diabetes to and from old normoglycaemic BB rats. Diabetologia38 :145 eC152,1995
Barthlott T, Kassiotis G, Stockinger B: T-cell regulation as a side effect of homeostasis and competition. J Exp Med197 :451 eC460,2003
Powrie F, Mason D: OX-22high CD4+ T-cells induce wasting disease with multiple organ pathology: prevention by the OX-22low subset. J Exp Med172 :1701 eC1708,1990
Fowell D, Mason D: Evidence that the T-cell repertoire of normal rats contains cells with the potential to cause diabetes: characterization of the CD4+ T-cell subset that inhibits this autoimmune potential. J Exp Med177 :627 eC636,1993
Seddon B, Mason D: Regulatory T-cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4+CD45RCeC cells and CD4+CD8eC thymocytes. J Exp Med189 :279 eC288,1999
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T-cells expressing IL-2 receptor alpha-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol155 :1151 eC1164,1995
Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Barbosa TC, Cumano A, Bandeira A: CD25+ CD4+ T-cells regulate the expansion of peripheral CD4 T-cells through the production of IL-10. J Immunol166 :3008 eC3018,2001
Zipris D, Hillebrands JL, Welsh RM, Rozing J, Xie JX, Mordes JP, Greiner DL, Rossini AA: Infections that induce autoimmune diabetes in BBDR rats modulate CD4+CD25+ T-cell populations. J Immunol170 :3592 eC3602,2003
Stephens LA, Mason D: CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T-cells with this function are found in both CD25+ and CD25eC subpopulations. J Immunol165 :3105 eC3110,2000
Peng YF, Laouar Y, Li MO, Green EA, Flavell RA: TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T-cells responsible for protection against diabetes. Proc Natl Acad Sci U S A101 :4572 eC4577,2004
Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A: Homeostasis and anergy of CD4+CD25+ suppressor T-cells in vivo. Nat Immunol3 :33 eC41,2002
Cozzo C, Larkin J, Caton AJ: Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T-cells. J Immunol171 :5678 eC5682,2003
Green EA, Choi YW, Flavell RA: Pancreatic lymph node-derived CD4+CD25+ Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity16 :183 eC191,2002
Gagnerault MC, Luan JJ, Lotton C, Lepault F: Pancreatic lymph nodes are required for priming of beta cell reactive T-cells in NOD mice. J Exp Med196 :369 eC377,2002
Fabien N, Bergerot I, Orgiazzi J, Thivolet C: Lymphocyte function-associated antigen-1, integrin alpha-4, and L-selectin mediate T-cell homing to the pancreas in the model of adoptive transfer of diabetes in NOD mice. Diabetes45 :1181 eC1186,1996
Lepault F, Gagnerault MC, Faveeuw C, Bazin H, Boitard C: Lack of L-selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by mel-14 antibody treatment. Eur J Immunol25 :1502 eC1507,1995
Cooke A, Phillips JM, Parish NM: Tolerogenic strategies to halt or prevent type I diabetes. Nat Immunol2 :810 eC815,2001
Masteller EL, Bluestone JA: Immunotherapy of insulin-dependent diabetes mellitus. Curr Opin Immunol14 :652 eC659,2002
Polanski M, Melican NS, Zhang J, Weiner HL: Oral-administration of the immunodominant B-chain of insulin reduces diabetes in a co-transfer model of diabetes in the NOD mouse and is associated with a switch from Th1 to Th2 cytokines. J Autoimmun10 :339 eC346,1997(Dorthe Lundsgaard, Thomas)