Phenotypic characterization of regulatory CD4+CD25+ T cells in rats
Leigh A. Stephens1,2,
A. Neil Barclay1 and
Don Mason1
1 Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK 2 Present address: Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK
Correspondence to: L. Stephens, Institute of Cell, Animal and Population Biology, University of Edinburgh, Ashworth Laboratories, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK. E-mail: leigh.stephens{at}ed.ac.uk
Transmitting editor: T. Hünig
 |
Abstract
|
---|
CD25 has become widely used as a marker for a subset of regulatory CD4+ T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4+CD25+ regulatory T cells in rats. In the thymus, CD4+CD8CD25+ cells were found to have a more mature phenotype than the corresponding CD4+CD8CD25 cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4+CD25+ cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4+CD25+ T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4+ T cells in rats and no further subdivision of the regulatory component of CD4+CD25CD45RClow T cells has yet been achieved.
Keywords: CD4+CD25+, regulatory T cell, tolerance
 |
Introduction
|
---|
Evidence has accumulated in recent years for the existence of regulatory CD4+ T cells that have immune suppressive activity and play a role in maintaining immunological tolerance. In particular, regulatory T cell activity has been shown to be enriched in a minority subset of CD4+ T cells that express CD25, and are found in both the thymus and periphery of mice, rats and humans (1,2). The range of disease states which cells of this phenotype have now been shown to regulate is wide and includes organ-specific autoimmune disease (including gastritis, thyroiditis and insulin-dependent diabetes) (37), inflammatory bowel disease (8), inflammatory lung disease (9), allograft rejection (1012), graft versus host disease (1315), allergy (16), sterilizing immunity to infectious organisms such as Leishmania major (17), and, less beneficially, CD4+CD25+ cells also hinder tumour immunity (18). CD4+CD25+ T cells also have immune suppressive activity in vitro, and can suppress the proliferation and cytokine production of other T cells, both CD4+ and CD8+, via an as yet undefined cell-contact dependent mechanism (19,20). However, CD4+CD25+ T cells act not only on T cells, but have also been shown to regulate cells of the innate immune system, as demonstrated by their ability to prevent Helicobacter hepaticus-triggered colitis in T cell-deficient mice (21).
Although there are now many studies that show an important role for CD4+CD25+ T cells in tolerance, many aspects of their biology remain unclear, including precise details regarding their development, specificity and mechanism of action. Another problem that hinders studies of regulatory T cells, particularly in humans, is the lack of a unique marker that defines all cells with regulatory activity, as CD25 has a number of limitations in this regard. As a molecule that is induced on all T cells after activation, CD25 cannot distinguish regulatory T cells from conventional activated T cells which also express CD25. Another limitation is that CD25 is not expressed on all CD4+ T cells with regulatory activity. Although regulatory T cells in both the thymus and periphery are enriched in the CD4+CD25+ T cell subset, in the periphery of rodents there is evidence also for regulatory CD4+CD25 T cells present within the memory CD45RClow subset in rats (CD45RBlow in mice) (6,7,12,16,22). The relationship between the CD25+ and CD25 regulatory cells in the periphery has not yet been established, and could either be the result of instability of this marker on a single regulatory T cell subset or due to the existence of different lineages of regulatory cells that perhaps even have separate mechanisms of action.
It is clear, therefore, that one important goal of research in the regulatory T cell field is the better definition of the phenotype of these cells and a search for markers that more specifically label only those cells with regulatory activity. With this aim in mind, we have phenotypically characterized CD4+CD25+ T cells from the thymus, blood, spleen and lymph nodes of rats using the currently available antibodies in this species, and have tested the subsets of CD25+ T cells so identified for their regulatory capacity in vitro and in vivo.
 |
Methods
|
---|
Antibodies
The following mouse anti-rat IgG1 antibodies used in these experiments were supernatants produced from hybridomas in the Sir William Dunn School of Pathology, Oxford, by Mike Puklavec: OX1 (anti-CD45), OX2 (anti-CD200), OX6 [anti-MHC class II (RT-1B)], OX7 [anti-Thy1.1 (CD90)], OX8 (anti-CD8
), OX18 [anti-MHC class I (RT-1A)], OX19 (anti-CD5), OX22 (anti-CD45RC), OX26 [anti-transferrin receptor (CD71)], OX39 (anti-CD25), OX40 [anti-OX40 (CD134)], OX44 (anti-CD53), OX45 (anti-CD48), OX47 (anti-CD147), OX48 (23), OX50 (anti-CD44), OX52 (anti-CD6) (24), OX54 (anti-CD2), OX56 (anti-CD43), OX61 (anti-CD26), OX62 (
M290 integrin), OX85 (anti-CD62L), IA-29 (anti-ICAM), JJ319 (anti-CD28), R73 (anti-TCR
ß), V65 (anti-
TCR), W3/25 (anti-CD4), W3/13 (anti-CD43), WT1 [anti-LFA-1 (CD11a)], 2G7 (IgG2a, anti-human TGFß, cross-reactive with rat) (25) and 10/78 [anti-NKR-P1A (CD161a)]. OX21 (mouse IgG1, reactive with human Factor I) was used as an isotype control antibody. Some of these antibodies were purified and biotinylated using standard methods. Cy5-conjugated W3/25 and R73 (conjugated by Ulf Yrlid, Sir William Dunn School of Pathology, Oxford) were also used in some experiments. 3H5 [anti-rat B7-1 (CD80)] and 24F [anti-rat-B7-2 (CD86)] were used as purified antibodies. References for all of these antibodies, unless given above, can be found in (26). Anti-rat OX40phycoerythrin (PE), anti-rat CD25FITC and fluorescein- conjugated isotype control mAb were obtained from Serotec (Oxford, UK). PE-conjugated rat-adsorbed donkey anti-mouse IgG F(ab')2 (DAMPE) (Jackson Laboratories, West Grove, PA) was used for the detection of unconjugated mouse mAb and streptavidinQuantum Red (Sigma, St Louis, MO) to detect staining of biotinylated antibodies.
Flow cytometry
For flow cytometric analysis, cells were stained with appropriately diluted antibody for 30 min on ice, washed twice with PBS/2% FCS and analysed on a FACSCalibur (Becton Dickinson, Oxford, UK) using CellQuest software (Becton Dickinson). For triple-colour analysis, cells were incubated first with the unconjugated mAb, washed, and detected with DAMPE. After 20 min blocking with 5% normal mouse serum in PBS, the cells were then co-incubated with the FITC- and biotin-conjugated antibodies, washed, and incubated for a further 30 min with streptavidinQuantum Red.
Purification of T cell subsets
Donor peripheral cells were obtained from pooled single-cell suspensions of cervical and mesenteric lymph nodes and spleen (after lysis of red blood cells) of 8- to 16-week-old female PVG.RT1u rats. Enrichment for CD4+ cells involved incubation for 4050 min on ice with the mouse mAb OX7, OX8, OX12 or OX33, OX42 and 10/78, followed by washing and two consecutive rounds of depletion with goat anti-mouse IgG-coated M450 magnetic beads (Dynal, Oslo, Norway). The efficiency of the depletion was confirmed by labelling cells pre- and post-depletion with DAMPE for 20 min for flow cytometric analysis and the percentage of contaminating cells was consistently <2%, with CD4+ T cell enrichment of 8491%. Enrichment of CD4+CD8 thymocytes was performed by negative selection of CD8+ thymocytes from 5- to 12-week-old female PVG.RT1u rats, by rosette depletion with OX8-coated sheep red blood cells (27). The resulting population of cells typically contained 80% single-positive CD4+CD8 thymocytes and 20% CD4CD8 thymocytes, and <2% contaminating CD8+ cells. For in vivo experiments, fractionation of the enriched CD4+ T cells on the basis of CD25 expression and a second marker was then performed by staining cells with FITC- and PE-labelled antibodies, followed by separation with FITCmultisort magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) used according to the manufacturers instructions and then further separation with antiPE magnetic beads (Miltenyi Biotec). For in vitro experiments requiring fewer cells, the enriched CD4+ T cells were stained as above and sorted on a MoFlo high-speed cell sorter (DakoCytomation; Dako, Ely, UK). In both cases, the purity of the fractionated cells after sorting was analysed on the flow cytometer and the percentage of CD4+ cells in each subset determined.
Induction and monitoring of insulin-dependent diabetes mellitus in PVG.RT1u rats
PVG.RT1u rats were bred and housed in specific pathogen-free conditions at the Sir William Dunn School of Pathology, Oxford. Insulin-dependent diabetes mellitus was induced in female PVG.RT1u rats by a protocol involving thymectomy at 6 weeks of age, followed by four doses of 250-rad
-irradiation at 2-week intervals, beginning 2 weeks after thymectomy, and described in more detail elsewhere (28,29). Cells to be tested for regulatory activity were injected i.v. shortly after the last irradiation. The cell dose was chosen on the basis of the known number of CD4+CD25+ or CD4+CD25CD45RClow cells required to give protection (6). Animals were subsequently weighed twice weekly for at least 12 weeks and those exhibiting weight loss were tested for the development of diabetes, as assessed by measurement of blood glucose levels using Glucostix (Bayer Diagnostics, Newburgh, UK) and confirmed by the presence of glycosuria with Clinistix (Bayer Diagnostics). Animals were considered diabetic if their blood glucose levels were >16 mM and they also tested positive for glycosuria. The onset of diabetes in control animals occurred 58 weeks after the last irradiation.
In vitro proliferation assays
Cells were cultured in triplicate in U-bottomed 96-well plates in RPMI supplemented with 10% heat-inactivated FCS, 50 IU/ml penicillin, 50 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate and 25 µM 2-mercaptoethanol. Responder T cells were added at 105 cells per well, with 105
-irradiated (3000 rad) mixture of spleen and lymph node cells as a source of antigen-presenting cells (APC). In co-culture experiments, increasing numbers of CD25+ cells were titrated into wells with the fixed dose of CD25 cells indicated above. Cells were stimulated with 2.5 µg/ml concanavalin A. Cells were pulsed at 72 h with 0.5 µCi [3H]thymidine and cultured a further 18 h before harvesting onto glass fibre filter mats and counting. Results shown are the mean ± SD of triplicate wells.
Immunoprecipitation and protein sequencing
OX48 antibody (purified on a Protein G column by FPLC) was covalently coupled to CNBr-4B Sepharose (Pharmacia) as recommended by the manufacturer. OX48-coupled Sepharose beads were then mixed with pre-cleared sodium deoxycholate-solubilized rat spleen lysates [prepared as described in (30)] and rotated overnight at 4°C. The Sepharose beads were then pelleted by centrifugation, washed in deoxycholate buffer and the protein eluted in 0.5% SDS at 55°C. The eluted material was concentrated using spin concentrators (Amicon) and loaded onto a NuPage 412% Bis-Tris minigel (Invitrogen) under non-reducing conditions. The major band of
70-kDa on SDSPAGE was digested with trypsin, and then peptides were separated and sequenced by automated Edman degradation in a Perkin-Elmer Procise 494A protein sequencer (Applied Biosystems).
RNA extraction and RT-PCR
Total RNA was isolated from 106 FACS-sorted CD25+ or CD25 CD8TCR
ß+ rat thymocytes using Tri Reagent (Sigma) according to the manufacturers instructions. Positive control RNA was purified from p815 cells transfected with rat CD80 or CD86 and negative control RNA was purified from untransfected p815 cells (all kindly provided by Hideo Yagita, Tokyo, Japan). mRNA was reverse transcribed using SuperScriptII RNase H reverse transcriptase (Gibco/BRL, Life Technologies, Paisley, UK) according to the manufacturers instructions in a 20 µl reaction volume. Reverse transcriptase-negative control reactions (RT) were prepared using equivalent amounts of RNA in water. PCR reactions were performed with 1 µl of cDNA or RT in a 20 µl reaction mix containing 2 µl 10 x PCR Buffer (Clontech), 0.6 µl dNTPs each at 10 mM (Sigma), 2 µl each of forward and reverse primers (10 µM; synthesized by Sigma-Genosys), and 0.5 µl Taq DNA polymerase (Clontech). The primer sequences used were: sense 5'-CTG CTG CTG GTT GGT CTA TTC-3' and antisense 5'-GTG GTC GTA CGT CGT GTT GA-3' to amplify a 573-bp fragment of rat CD80, and sense 5'-AAA CAT AAG CCC GAG TGA GC-3' and antisense 5'-TGC AAT CTG GAT CCA AGT CT-3' to amplify a 607-bp fragment of rat CD86. ß-Actin was used as an internal control for RT-PCR and to normalize cDNA concentration in the samples, and the primers used were: sense 5'-ATG CCA TCC TGC GTC TGG ACC TGG-3' and antisense 5'-AGC ATT TGC GGT GCA CGA TGG AGG G-3'. Reactions were performed in a PTC-0225 DNA Engine Tetrad (MJ Research). The PCR reaction was first subjected to a denaturation step at 94°C for 2 min, followed by cycles of 30 s annealing at 62°C, 45 s of elongation at 72°C and 30 s of denaturation at 94°C. In total, 34 cycles were performed for CD80 and CD86 reactions and 24 cycles for ß-actin. PCR fragments were visualized by electrophoresis on a 1% agarose gel containing ethidium bromide and images were recorded using GeneSnap Version 4.00.00 (Syngene, Cambridge, UK).
 |
Results
|
---|
Phenotypic analysis of rat CD4+CD25+ T cells in the thymus and periphery
CD4+ T cells from the thymus, lymph nodes, spleen and blood of rats were screened ex vivo with an extensive panel of antibodies reactive with rat lymphocyte cell-surface molecules to search for markers that are differentially expressed on CD25+ and CD25 T cells. Both CD25+ and CD25 subsets of CD4+CD8 thymocytes and peripheral CD4+ T cells were found to have equivalent uniformly positive expression of the following markers: TCR
ß, CD2, CD5, CD6, CD26, CD28, CD43 (as detected by OX56), CD45, CD147 and MHC class II (this last marker only being present on a minority subset of peripheral T cells, but weakly expressed on most CD4+CD8 thymocytes); and lacked detectable cell-surface expression of TCR
, NKR-P1A, CD71 (transferrin receptor), transforming growth factor-ß and OX62 (data not shown). Differences in the phenotype of the two subsets were found with respect to the markers shown in Fig. 1. There were no differences between spleen and lymph node cells with respect to any of the markers examined, and the phenotype of cells in blood was also identical to that of spleen and lymph nodes, with the only difference being that, as already described for thoracic duct lymphocytes (6), there was a slightly lower percentage of CD25+ cells in blood compared with spleen and lymph nodes.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 1. Phenotypic analysis of cell-surface proteins expressed by CD25+ and CD25 CD4+ T cell subsets in rat thymus, spleen, lymph nodes and blood. Cells are gated on CD4+CD25+ lymphocytes (bold line) or CD4+CD25 lymphocytes (thin line). The dotted line indicates the negative control gate for each tissue. Thymocytes were depleted of CD8+ cells before staining. Profiles shown are representative of at least three experiments using pooled samples for each tissue analysed.
|
|
The most striking phenotypic differences in CD25+ compared with CD25 CD4+ T cells in both the thymus and the periphery were found with respect to expression of OX40 (CD134), OX48, CD80 (B7-1) and CD86 (B7-2). CD25+ cells had a greatly increased proportion of cells staining positively for each of these markers compared to mostly negative expression on CD25 cells. These markers were therefore chosen to study further as potential candidates for further functional subdivision of regulatory T cells.
Some phenotypic differences were found between CD25+ and CD25 cells in the thymus, but not in the periphery, and were therefore considered to indicate different maturational states of the two subsets in the thymus. In the thymus, CD25+ cells were predominantly CD44hiCD53hiThy1lo and thus had a more mature phenotype than CD25 thymocytes, with closer resemblance to the phenotype of peripheral T cells (Fig. 1). In the periphery, however, the difference in the levels of expression of these molecules on CD25+ and CD25 cells was minimal, with most CD4+ peripheral cells appearing CD44hiCD53hiCD90(Thy1)/low, although consistently there was detection of slightly higher expression of CD44 on peripheral CD25+ cells compared with CD25 cells (Fig. 1).
There were other more subtle differences in the expression of certain markers on CD25+ compared with CD25 cells, both in the thymus and periphery. CD25+ cells had slightly higher levels of expression of MHC class I, CD48 and CD54 (ICAM) in comparison to CD25 cells. CD200 (OX2) was expressed on all CD4+CD8 thymocytes with marginally lower levels on CD25+ versus CD25 thymocytes, but was expressed on a higher percentage of CD25+ cells in the periphery. LFA-1 was expressed in higher levels by CD25+ cells in the periphery, although there was no differential expression of this marker on thymocytes. CD62L expression was similar for CD25+ and CD25 thymocytes, but the percentage of CD62Llow cells was slightly increased in the peripheral CD25+ subset. As described previously, CD25+ cells have mostly low to intermediate expression of CD45RC in the periphery (6). There was also a small proportion (
16%) of peripheral CD4+CD25+ cells expressing CD8. The regulatory potential of this subset is unknown, as previous experiments on regulatory T cells in rats have been done with CD4+ cells that were negatively selected using anti-CD8 antibodies (6). In addition, there was differential expression with respect to the isoform of CD43 recognized by the antibody W3/13, although both subsets had uniform high expression of CD43 when stained with another antibody OX56. CD25+ cells had an increased proportion of W3/13low cells compared with CD25 cells, both in the thymus and periphery. The W3/13 antibody is thought to be inhibited in its binding to highly glycosylated forms of CD43 (31).
OX40 expression does not functionally subdivide CD25+ or CD25 regulatory CD4+ T cells
It is known that the T cells from normal rats that can transfer protection from autoimmune diabetes to syngeneic lymphopenic recipients are enriched in the CD4+CD25+ subset in both the thymus and periphery, but that additionally there is less potent regulatory activity present in the peripheral antigen-experienced CD4+CD25Thy1CD45RClow T cell subset (6,32). OX40 was found to be expressed almost exclusively by CD45RClow cells in the periphery, and within this subset was expressed on
45% of CD25+ cells and
20% of CD25 cells (Fig. 2A). OX40 is induced on T cells after activation and is thought to play a role in the survival of memory cells (33). One hypothesis considered initially was that OX40 might be used as a marker to distinguish conventional activated T cells (co-expressing CD25 and OX40) from regulatory T cells (expressing CD25 and lacking OX40). Unexpectedly, however, OX40 was not found purely on peripheral T cells and there was great enrichment of OX40 expression on CD25+ thymocytes (Fig. 1). In addition, a previously unrecognized observation was that, unlike most CD4+ T cells in the periphery, CD25 CD4+CD8 thymocytes in rats express OX40, albeit at low levels (Fig. 1). An alternative hypothesis was that OX40 might be a better marker than CD25 for regulatory T cells.


View larger version (52K):
[in this window]
[in a new window]
|
Fig. 2. OX40 is not a useful marker for regulatory T cell activity in vivo. (A) OX40 expression is enriched in CD4+CD45RClow peripheral T cells. Peripheral T cells (spleen and lymph nodes) were negatively selected for CD4 enrichment (>90% CD4+) and stained with OX40PE, OX39FITC (CD25) and OX22Cy5 for analysis on the flow cytometer. Shown are density plots of CD25 and OX40 expression on gated cells as indicated. (B) The ability of sorted CD4+ T cell subsets to protect against autoimmune diabetes. CD4+ T cells were enriched from the spleen and lymph nodes of PVG.RT1u rats, and fractionated further with magnetic beads according to both CD25 and OX40 expression. In the experiments with CD25OX40CD45RClow cells, CD4+ cells were depleted of CD45RChigh cells before CD25/OX40 sorting. The purity of the sorted subsets is indicated in Table 1. Cell subsets were tested for their ability to transfer protection from autoimmune diabetes to syngeneic TxX recipients. The dose of cells injected (x106) was: 0.71 CD25+OX40+, 0.71 CD25+OX40, 4 CD25OX40+, 4 CD25OX40CD45RClow and 20 CD45RChigh. The results are pooled from a total of five experiments.
|
|
We tested these various possibilities by subdividing CD4+CD45RClowThy1 peripheral T cells according to expression of both CD25 and OX40. The hallmark of regulatory T cells is their ability to inhibit autoimmune and inflammatory disease in vivo, and the fractionated T cell subsets were tested for their ability to protect thymectomized and irradiated (TxX) PVG.RT1u rats from developing autoimmune diabetes. The results of a series of five experiments are shown in Fig. 2(B). Both the OX40+ and OX40 subsets of CD4+CD25+ cells were able to prevent diabetes in TxX rats at the cell dose tested. Similarly, both OX40+ and OX40 subsets of CD4+CD25CD45RClow cells were capable of preventing diabetes at a higher cell dose, so OX40 expression was not capable of uniquely identifying those regulatory T cells present in the peripheral CD4+CD25 T cell pool. The purities of cells used for in vivo experiments after MACS sorting are shown in Table 1. The protection is not explained by the presence of contaminating cells, as the dose of contaminating cells is below that required to give protection in each case. Therefore, although OX40 expression was greatly enriched on cells with regulatory activity, it was not useful for their further functional subdivision within either the CD25+ or CD25 subsets.
Another useful correlate of the in vivo suppressive activity of regulatory CD4+CD25+ T cells is their ability to suppress the proliferation of other T cells after co-culture in vitro. This has been reported previously for these cells in mice (19,20), humans (3440) and, recently, also for rats (41). As fewer cells were needed for the in vitro experiments, we were able to use FACS-sorted cells, yielding a higher degree of purity of the cell populations compared with those used for the in vivo assays (Fig. 3A). Both OX40+ and OX40 subsets of CD25+ cells were hypoproliferative in the absence of exogenous IL-2, and suppressed the proliferation of both naive CD4+CD25OX40CD45RChigh and memory CD4+CD25OX40CD45RClow/int cells during co-culture in vitro (Fig. 3B and C). Although both subsets were suppressive, the in vitro assays revealed a slight increase in potency of the CD25+OX40+ cells compared with the CD25+OX40 cells in their suppressive activity (Fig. 3C). With regard to CD25CD45RClow cells, the OX40+ subset proliferated poorly compared with the OX40 subset in the absence of exogenous IL-2. However, in contrast to the CD25+ cells, neither subset of CD25 cells displayed the ability to suppress CD45RChigh cells in vitro, although CD25OX40+ marginally suppressed the proliferation of CD4+CD25OX40CD45RClow/int cells (35% suppression at 1:1 ratio). CD25OX40+ cells were also unable to suppress proliferation of whole CD4+CD25OX40 cells not fractionated according to CD45RC expression, even at a 1:1 ratio (data not shown); therefore, their ability to suppress T cell proliferation in vitro is at best extremely weak, despite their anergic phenotype.


View larger version (74K):
[in this window]
[in a new window]
|
Fig. 3. OX40 does not functionally subdivide CD25+ or CD25 CD4+CD45RClow peripheral regulatory T cells in vitro. Shown is a representative experiment of seven performed. (A) The purity of FACS-sorted CD4+ T cell subsets fractionated according to expression of CD25, OX40 and CD45RC. Enriched CD4+ T cells purified from the spleen and lymph nodes of 12-week-old PVG.RTIu rats were stained with fluorescently labelled mAb to CD25, OX40 and CD45RC, and sorted on a MoFlo. (B) In vitro proliferation of peripheral CD4+ T cell subsets. FACS-sorted T cell subsets (4 x 105) shown in (A) were stimulated with 2.5 µg/ml concanavalin A in the presence of 105 APC (irradiated spleen and lymph node cells) ± recombinant rat IL-2. At 72 h of culture, the cells were pulsed with 0.5 µCi [3H]thymidine for a further 18 h. (C) In vitro suppressive activity of CD4+ T cells fractionated according to CD25, OX40 and CD45RC expression. Different doses of the sorted cells indicated in the legend were tested for their ability to suppress proliferative T cell responses by titration into wells containing either 4 x 105 CD4+CD45RChigh cells (left) or CD4+CD45RClow/int cells (right), using the same conditions described in (B).
|
|
Determination of the specificity of OX48 mAb for rat CD86
The specificity of the OX48 mAb, which had not been previously defined, was of particular interest due to its ability to subdivide CD4+CD25+ T cells and its very low expression on CD4+CD25 cells. Early studies using this antibody had found it to be reactive against a protein of
70 kDa found on a subset of activated T cells and dendritic cells (23). In this study, a
70-kDa molecule was immunoprecipitated from rat spleen lysates using OX48-coated Protein GSepharose beads (Fig. 4A), digested into peptides, sequenced and compared to the sequence database. One of these peptides (SFDRDNQALR) was found to exactly match the sequence of rat B7-2 (CD86).


View larger version (83K):
[in this window]
[in a new window]
|
Fig. 4. OX48 mAb is specific for rat CD86. (A) OX48 protein was immunoprecipitated from deoxycholate-solubilized rat spleen Tween 40 membrane preparations with OX48-conjugated Sepharose and resolved in two lanes under non-reducing conditions by SDSPAGE. Molecular weight markers in kDa are indicated on the left. (B) Confirmation that OX48 is specific for CD86. CD86-transfected p815 cells, and control CD80-transfected or untransfected p815 cells (kindly provided by Hideo Yagita) were stained with OX48 mAb and DAMPE and analysed on the flow cytometer. 24F mAb was a positive control for CD86 expression, 3H5 was a positive control for CD80 expression and OX21 (shown as the unfilled histogram) was used as an isotype control for all three antibodies.
|
|
To confirm the specificity of OX48, rat CD86-transfected P815 cells (kindly provided by Hideo Yagita, Tokyo) were stained and analysed by flow cytometry. OX48 gave identical staining of CD86-transfected, but not wild-type or control CD80-transfected P815 cells, as the positive control mAb 24F (Fig. 4B). OX48 did not appear to bind the same epitope of CD86 as 24F because pre-incubation of rat dendritic cells with OX48 antibody did not block binding of PE-conjugated 24F (data not shown). However, OX48 appears to be able to block binding of CD86 with its ligand CD28, as addition of this antibody to T cell cultures led to a similar inhibition of proliferation as 24F [(23) and data not shown].
CD86 expression does not functionally subdivide CD4+CD25+ thymocytes in vitro
As CD4+CD25+ cells in both the thymus and periphery were found to be heterogeneous with regard to CD86 expression, and this marker was virtually absent on CD25 cells, it was tested for its utility in functionally subdividing CD25+ cells further with regard to their suppressive activity in vitro. Both CD86+ and CD86 subsets of CD4+CD8CD25+ thymocytes were found to be equally potent in suppressing polyclonal T cell proliferation in vitro (Fig. 5). CD80 was not used to subdivide CD25+ cells as it was expressed on the majority of these cells.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5. CD86 expression does not functionally subdivide CD4+CD25+ regulatory T cells in vitro. CD8-depleted thymocytes were sorted with antiFITC multisort and antiPE beads after indirect staining for CD86 with OX48 and DAMPE, followed by direct staining with CD25FITC. Left: 105 sorted thymocytes were stimulated with 2.5 µg/ml concanavalin A in the presence of 105 APC (irradiated spleen and lymph node cells) ± recombinant rat IL-2. Right: sorted cells were titrated into wells containing 105 CD4+CD8CD25 thymocytes at the indicated ratios. At 72 h of culture, the cells were pulsed with 0.5 µCi [3H]thymidine for a further 18 h. Shown is a representative of three experiments.
|
|
CD80 and CD86 mRNA is expressed in CD4+CD25+ T cells
Recently, it has been shown that CD80 expression on T cells in mice and humans not only occurs due to endogenous T cell production of this protein, but also due to its acquisition from APC (42,43). To determine whether CD4+CD25+ T cells were producing endogenous CD80 and CD86, the expression of CD80 and CD86 mRNA was examined. CD25+ and CD25 subsets of CD4+CD8TCR
ß+ thymocytes were sorted to high purity on a MoFlo cell sorter, after initial depletion of CD8-expressing cells. The purity of both sorted populations was >99.9% with regard to TCR
ß expression, and with regard to CD25 expression the CD25 cells were 99.9% pure and the CD25+ cells were >94% pure. Total RNA was purified from these cells, and RT-PCR analysis performed for CD80 and CD86, with ß-actin to normalize levels of RNA in the samples. Results shown in Fig. 6 revealed that CD4+CD25+ thymocytes express mRNA for both CD80 and CD86, indicating that at least part of the observed expression of these molecules is due to endogenous production. As noted for protein expression, there was differential expression of CD80 and CD86 mRNA in CD25+ compared to CD25 cells, with barely detectable expression of these transcripts in the CD25 subset of mature CD4+ thymocytes.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 6. Expression of CD80 and CD86 mRNA in CD4+CD8-25+ thymocytes. RT-PCR analysis for CD80 and CD86 was performed on RNA purified from CD25+ and CD25 subsets of CD4+CD8TCR ß+ thymocytes, sorted to high purity on a MoFlo cell sorter as described in the text. ß-Actin was used to indicate levels of mRNA in the samples. Control RT reactions did not result in the amplification of any DNA, thereby excluding the presence of contaminating genomic DNA in the RNA samples (data not shown). Results are representative of more than three experiments.
|
|
 |
Discussion
|
---|
This study describes the further phenotypic characterization of CD4+CD25+ regulatory T cells in rats and a search for more specific markers to distinguish regulatory T cells from conventional T cells. Based on this analysis it was apparent that CD25+ cells in the CD4+CD8 compartment of the thymus have a more mature phenotype than the corresponding CD25 cells with respect to expression of Thy1, CD53 and CD44. Thy1 is highly expressed on immature thymocytes in rats, expressed in lower levels on recent thymic emigrants and is lost on peripheral T cells within a week of emigration from the thymus (44). CD25+ cells in the thymus had a mostly Thy1lo phenotype, distinct from the Thy1 phenotype of most mature T cells and the Thy1hi phenotype of the majority of CD25CD4+CD8 thymocytes. Similarly, CD44 expression and CD53 expression were higher on the majority of CD25+ thymocytes compared with CD25CD4+CD8 thymocytes, corresponding more closely to the levels found on peripheral T cells. Thus, CD25+ thymocytes have a predominantly, but not exclusively, mature phenotype, suggesting that CD25 expression, and perhaps also regulatory function, is acquired relatively late in thymocyte maturation.
In the periphery, CD4+CD25+ cells were found to have increased expression of CD44, CD48, ICAM, LFA-1, CD200, OX40, CD80 and CD86 compared with CD25 cells, consistent with them having a more activated phenotype and in general agreement with previous observations in mice (45). However, in our search for better markers of regulatory T cell function we focused our functional analyses on those markers that were heterogeneous within the CD25+ population, but were present on only a minority of the CD25 subset and did not simply reflect different maturational states in the thymus. OX40 and CD86 fell into this category. However, despite the fact that CD4+CD25+ cells were heterogeneous with respect to OX40 and CD86 in both the thymus and the periphery, this was not found to correlate with heterogeneity in terms of regulatory T cell activity in vitro or in vivo. Thus, CD25 remains for now the most suitable marker available to enrich for T cells with regulatory activity in rats.
Similarly, reports of other attempts to subdivide CD4+CD25+ T cells in mice have not yet revealed a marker which is more specific than CD25 for ex vivo isolated CD4+ T cells with in vitro suppressive activity. In this regard, it has been shown that subdivision of CD4+CD25+ cells in mice according to expression of CD45RB, CD38, CD62L, CD103 and LAP did not reveal any functional differences in the ability of cells to mediate suppression of T cell proliferation in vitro (4650), although slight differences in potency were reported for CD103 (49). In contrast, it has been reported that the ability of CD4+CD25+ T cells from young NOD mice to delay diabetes in vivo in an adoptive transfer model was found solely in the CD62Lhigh subset, relating probably to differences in the homing properties of the two subsets (51). Similarly, the ability of whole CD4+CD8 thymocytes to prevent autoimmune diabetes was previously shown to be a property of the CD62Lhigh cell subset in rats (52) and mice (53).
We noted OX40 expression on a subset of the CD4+CD45RClowThy1CD25 cells that can also protect against diabetes in our model, but were unable to determine substantial regulatory activity in this subset in vitro or to show that all the in vivo regulatory activity of CD4+CD25 T cells resided exclusively in the OX40+ subset. This contrasts with recent reports of other markers in mice that have been suggested to be useful for the identification of the CD4+CD25CD45RBlow cells with regulatory activity, such as CD103 (49), latency-associated peptide (LAP) (50) and glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) (54). There is evidence to suggest that LAP+ cells comprise the major regulatory fraction of CD4+CD25CD45RBlow cells in mice, although in contrast to CD4+CD25+ cells, CD25LAP+ cells lacked in vitro regulatory activity (50). However, although CD4+CD25CD103+ cells were found to be regulatory in vivo and to a lesser extent in vitro, the regulatory potential of CD4+CD103CD45RBlow cells was not determined, and therefore it remains unknown whether or not all CD4+CD25 regulatory cells are CD103+ in mice (49). The same problem applies to the use of GITR as a marker for regulatory CD4+CD25 cells (54).
Although OX40 and CD86 were not found to be useful as markers for regulatory T cells in rats, these molecules might still be functionally important on regulatory T cells. OX40 is a molecule expressed by T cells after activation, and plays a role in the survival of antigen-experienced T cells and augmentation of immune responses (33). Attention to date has mainly focused on the role of OX40 on immune effector cells, as injection of agonistic antibodies to OX40 in mice can break peripheral T cell tolerance (55), and, conversely, neutralizing antibodies against OX40L can be beneficial in reducing inflammatory conditions in mice including colitis (56), experimental autoimmune encephalomyelitis (57) and collagen-induced arthritis (58). However, this study has shown that OX40 is not only expressed on activated effector T cells, but also on many regulatory CD4+CD25+ T cells in both the thymus and periphery, and in low levels on mature CD4+CD25 thymocytes. Similarly, in humans it has been noted that OX40 expression can be found on scattered medullary thymocytes (59,60). This previously unappreciated expression pattern of OX40 should be considered in the interpretation of studies involving therapeutic intervention by targeting of OX40+ T cells and its ligand. Further studies on CD25+ T cells from OX40/ mice will reveal whether OX40 plays any role in regulatory T cell survival or function.
The enrichment of both CD80 and CD86 expression on CD4+CD25+ T cells in the thymus and periphery has not been reported previously, even in published studies of global mRNA gene expression by CD25+ and CD25 CD4+ T cell subsets in mice (12,48,61). The reason for this is not clear, as we have found enrichment for CD80 and CD86 on CD4+CD25+ cells at both the protein and mRNA level, arguing that their expression on these cells is, at least in part, due to endogenous production and not solely protein acquisition from APC. Further studies will be required to determine the functional significance of CD80/CD86 expression on regulatory CD4+CD25+ T cells. It remains possible that some of the CD80/CD86 expressed by CD25+ T cells has been acquired from APC and such acquisition could act to limit the availability of co-stimulatory molecules on APC to other T cells at early stages of activation. This notion is consistent with reports that CD80 and CD86 expression is lowered on dendritic cells after interaction with CD25+ cells in vitro (62), and that in vitro suppression by CD25+ cells can be abrogated if the level of co-stimulation is increased with agonistic CD28 mAb (i.e. suppression only occurs when co-stimulation is limiting). However, it seems implausible that the exclusive action of regulatory CD4+CD25+ T cells can be via reduction of the co-stimulatory capacity of APC through CD80/CD86 acquisition because the suppressive activity of CD4+CD25+ T cells has also been demonstrated in certain APC-free culture systems, e.g. using beads coated with anti-CD3 and anti-CD28 (63). Nevertheless, it remains possible that CD80/CD86 expression by CD4+CD25+ T cells could be functionally important in their suppressive activity by providing a ligand for CTLA-4 on other activated T cells or on the regulatory CD4+CD25+ T cells themselves, which are known to be enriched in the expression of CTLA-4 in mice (5,8,64), humans (34) and now also in rats (41). In support of this possibility, it was shown by Chai et al. that T cell unresponsiveness induced by presentation of antigen to T cells using co-stimulation-deficient APC could be abrogated by the addition of antibodies to CD80 or CTLA-4 (65). As T cells were the only cells expressing CD80 in these experiments, the conclusion was drawn that T cell-expressed CD80 could induce T cell unresponsiveness via ligation of CTLA-4 on neighbouring or even the same T cells. This effect was reported not to occur when co-stimulatory molecules on APC were not limiting, thus showing that increasing co-stimulation could abrogate the suppressive effect of CD80 expression on T cells, just as it abrogates the suppressive activity of CD4+CD25+ T cells. These results suggest that one action of regulatory T cells is to limit responses to those cells that are least heavily dependent on co-stimulation. Such an action would be compatible with their involvement in the prevention of autoimmunity, as autoreactive cells are likely to have low-affinity TCR for self-antigens having avoided negative selection by virtue of this fact. B7-1/B7-2 double-knockout mice were reported to have greatly reduced numbers of CD4+CD25+ T cells (5), but the suppressive activity of the remaining cells has not been reported and could provide insight into the possible functional role of B7 expression on CD4+CD25+ T cells.
In summary, CD4+CD25+ T cells in rats had enriched, but heterogeneous, expression of OX40 and CD86 in both the thymus and periphery, although these markers were not useful for further subdivision of regulatory activity. It is possible that the expression of these molecules is a reflection of the activation state of regulatory T cells rather than a true lineage difference. We noted also that the majority of CD4+CD25+ cells in rats were distinguished from CD4+CD25 cells by expression of CD80. CD80 therefore joins other molecules that are known to be enriched and relatively homogeneously expressed on CD25+ cells, including CTLA-4 (5,8,64), GITR (48,66) and, more recently, the transcription factor Foxp3 (67). In addition, CD4+CD25+ T cells have been reported to be enriched in certain chemokine receptors such as CCR8 (68) and CCR5 (69). This study also demonstrated 10 differences in the phenotype of rat CD4+CD25+ cells from peripheral blood compared with spleen and lymph node with regard to any of the antibodies used for screening.
 |
Acknowledgements
|
---|
We are very grateful to Dr Hideo Yagita, Juntendo University School of Medicine, Tokyo, for providing us with rat CD86-transfected and control P815 cells, A. Willis, MRC Immunochemistry Unit, Oxford, for digestion of the immunoprecipitated OX48 protein and sequencing of peptides, Steve Simmonds for thymectomy and irradiation of rats, Nigel Rust for expert cell sorting on the MoFlo, Christy Toms for invaluable advice on PCR, and staff of the animal facility of the Sir William Dunn School of Pathology for care of the rats. This work was funded by Diabetes UK (Project Grant BDA:RD01:0002217).
 |
Abbreviations
|
---|
APCantigen-presenting cell
DAMdonkey anti-mouse
GITRglucocorticoid-induced tumour necrosis factor receptor family-related gene
LAPlatency-associated peptide
PEphycoerythrin
TxXthymectomized and irradiated
 |
References
|
---|
- Maloy, K. J. and Powrie, F. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816.[CrossRef][ISI][Medline]
- Curotto de Lafaille, M. A. and Lafaille, J. J. 2002. CD4+ regulatory T cells in autoimmunity and allergy. Curr. Opin. Immunol. 14:771.[CrossRef][ISI][Medline]
- Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. 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. Immunol. 155:1151.[Abstract]
- Asano, M., Toda, M., Sakaguchi, N. and Sakaguchi, S. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract]
- Salomon, B., Lenschow, D. J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A. and Bluestone, J. A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431.[ISI][Medline]
- Stephens, L. A. and Mason, D. 2000. 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 CD25 subpopulations. J. Immunol. 165:3105.[Abstract/Free Full Text]
- Furtado, G. C., Olivares-Villagomez, D., Curotto de Lafaille, M. A., Wensky, A. K., Latkowski, J. A. and Lafaille, J. J. 2001. Regulatory T cells in spontaneous autoimmune encephalomyelitis. Immunol. Rev. 182:122.[CrossRef][ISI][Medline]
- Read, S., Malmstrom, V. and Powrie, F. 2000. Cytotoxic T lymphocyte-associated Antigen 4 plays an essential role in the function of CD25+CD4+ regulatory T cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
- Hori, S., Carvalho, T. L. and Demengeot, J. 2002. CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice. Eur. J. Immunol. 32:1282.[CrossRef][ISI][Medline]
- Taylor, P. A., Noelle, R. J. and Blazar, B. R. 2001. CD4+CD25+ immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J. Exp. Med. 193:1311.[Abstract/Free Full Text]
- Hara, M., Kingsley, C. I., Niimi, M., Read, S., Turvey, S. E., Bushell, A. R., Morris, P. J., Powrie, F. and Wood, K. J. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789.[Abstract/Free Full Text]
- Graca, L., Thompson, S., Lin, C. Y., Adams, E., Cobbold, S. P. and Waldmann, H. 2002. Both CD4+CD25+ and CD4+CD25 regulatory cells mediate dominant transplantation tolerance. J. Immunol. 168:5558.[Abstract/Free Full Text]
- Cohen, J. L., Trenado, A., Vasey, D., Klatzmann, D. and Salomon, B. L. 2002. CD4+CD25+ immunoregulatory T Cells: new therapeutics for graft-versus-host disease. J. Exp. Med. 196:401.[Abstract/Free Full Text]
- Hoffmann, P., Ermann, J., Edinger, M., Fathman, C. G. and Strober, S. 2002. Donor-type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med. 196:389.[Abstract/Free Full Text]
- Taylor, P. A., Lees, C. J. and Blazar, B. R. 2002. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99:3493.[Abstract/Free Full Text]
- Curotto de Lafaille, M. A., Muriglan, S., Sunshine, M. J., Lei, Y., Kutchukhidze, N., Furtado, G. C., Wensky, A. K., Olivares-Villagomez, D. and Lafaille, J. J. 2001. Hyper immunoglobulin E response in mice with monoclonal populations of B and T lymphocytes. J. Exp. Med. 194:1349.[Abstract/Free Full Text]
- Belkaid, Y., Piccirillo, C. A., Mendez, S., Shevach, E. M. and Sacks, D. L. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502.[CrossRef][ISI][Medline]
- Shimizu, J., Yamazaki, S. and Sakaguchi, S. 1999. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163:5211.[Abstract/Free Full Text]
- Thornton, A. M. and Shevach, E. M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
- Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. and Sakaguchi, S. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract]
- Maloy, K. J., Salaun, L., Cahill, R., Dougan, G., Saunders, N. J. and Powrie, F. 2003. CD4+CD25+ TR cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197:111.[Abstract/Free Full Text]
- Annacker, O., Pimenta-Araujo, R., Burlen-Defranoux, O., Barbosa, T. C., Cumano, A. and Bandeira, A. 2001. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.[Abstract/Free Full Text]
- Somoza, C., Fernandez-Ruiz, E., Rebollo, A., Sanz, E., Ramirez, F. and Silva, A. 1990. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor. Immunology 70:210.[ISI][Medline]
- Carmo, M. A., Nunes, R. J., Oliveira, M. I., Tavares, P. A., Simoes, C., Parnes, J. R., Moreira, A. and Carmo, A. M. 2003. OX52 is the rat homologue of CD6: evidence for an effector function in the regulation of CD5 phosphorylation. J. Leukoc. Biol. 73:183.[Abstract/Free Full Text]
- Lucas, C., Bald, L. N., Fendly, B. M., Mora-Worms, M., Figari, I. S., Patzer, E. J. and Palladino, M. A. 1990. The autocrine production of transforming growth factor-ß1 during lymphocyte activation. J. Immunol. 145:1415.[Abstract/Free Full Text]
- van den Berg, T. K., Puklavec, M. J., Barclay, A. N. and Dijkstra, C. D. 2001. Monoclonal antibodies against rat leukocyte surface antigens. Immunol. Rev. 184:109.[CrossRef][ISI][Medline]
- Mason, D. W., Penhale, W. J. and Sedgwick, J. D. 1987. Preparation of lymphocyte subpopulations. In Klaus, G. G. B., ed., Lymphocytes: A Practical Approach, p. 35. IRL Press, Oxford.
- Fowell, D. and Mason, D. 1993. 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. Med. 177:627.[Abstract]
- Simmonds, S. and Mason, D. 1999. Induction of autoimmune disease by depletion of regulatory T cells. In Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. and Strober, W., eds, Current Protocols in Immunology, p. 15.12.1. Wiley, New York.
- Williams, A. F. and Barclay, A. N. 1986. Glycoprotein antigens of the lymphocyte surface and their purification by antibody affinity chromatography. In Weir, D. M., ed., Handbook of Experimental Immunology, p. 22.1. Blackwell Scientific, Oxford.
- Cyster, J. G., Shotton, D. M. and Williams, A. F. 1991. The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. EMBO J. 10:893.[Abstract]
- Stephens, L. A. and Mason, D. 2001. Characterization of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease. Microb. Infect. 3:905.[CrossRef][ISI][Medline]
- Rogers, P. R., Song, J., Gramaglia, I., Killeen, N. and Croft M. 2001. OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 15:445.[ISI][Medline]
- Stephens, L. A., Mottet, C., Mason, D. and Powrie, F. 2001. Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur. J. Immunol. 31:1247.[CrossRef][ISI][Medline]
- Taams, L. S., Smith, J., Rustin, M. H., Salmon, M., Poulter, L. W. and Akbar, A. N. 2001. Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis-prone population. Eur. J. Immunol. 31:1122.[CrossRef][ISI][Medline]
- Levings, M. K., Sangregorio, R. and Roncarolo, M. G. 2001. Human CD25+CD4+T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295.[Abstract/Free Full Text]
- Jonuleit, H., Schmitt, E., Stassen, M., Tuettenberg, A., Knop, J. and Enk, A. H. 2001. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285.[Abstract/Free Full Text]
- Dieckmann, D., Plottner, H., Berchtold, S., Berger, T. and Schuler, G. 2001. Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 193:1303.[Abstract/Free Full Text]
- Baecher-Allan, C., Brown, J. A., Freeman, G. J. and Hafler, D. A. 2001. CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167:1245.[Abstract/Free Full Text]
- Ng, W. F., Duggan, P. J., Ponchel, F., Matarese, G., Lombardi, G., Edwards, A. D., Isaacs, J. D. and Lechler, R. I. 2001. Human CD4+CD25+ cells: a naturally occurring population of regulatory T cells. Blood 98:2736.[Abstract/Free Full Text]
- Lin, C. H. and Hunig, T. 2003. Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur. J. Immunol. 33:626.[CrossRef][ISI][Medline]
- Sabzevari, H., Kantor, J., Jaigirdar, A., Tagaya, Y., Naramura, M., Hodge, J., Bernon, J. and Schlom, J. 2001. Acquisition of CD80 (B7-1) by T cells. J. Immunol. 166:2505.[Abstract/Free Full Text]
- Tatari-Calderone, Z., Semnani, R. T., Nutman, T. B., Schlom, J. and Sabzevari, H. 2002. Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction. J. Immunol. 169:6162.[Abstract/Free Full Text]
- Hosseinzadeh, H. and Goldschneider, I. 1993. Recent thymic emigrants in the rat express a unique antigenic phenotype and undergo post-thymic maturation in peripheral lymphoid tissues. J. Immunol. 150:1670.[Abstract/Free Full Text]
- Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
- Thornton, A. M. and Shevach, E. M. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
- Kuniyasu, Y., Takahashi, T., Itoh, M., Shimizu, J., Toda, G. and Sakaguchi, S. 2000. Naturally anergic and suppressive CD25+CD4+ T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation. Int. Immunol. 12:1145.[Abstract/Free Full Text]
- McHugh, R. S., Whitters, M. J., Piccirillo, C. A., Young, D. A., Shevach, E. M., Collins, M. and Byrne, M. C. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[ISI][Medline]
- Lehmann, J., Huehn, J., de la Rosa, M., Maszyna, F., Kretschmer, U., Krenn, V, Brunner, M., Scheffold, A. and Hamann, A. 2002. Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells. Proc. Natl Acad. Sci. USA 99:13031.[Abstract/Free Full Text]
- Oida, T., Zhang, X., Goto, M., Hachimura, S., Totsuka, M., Kaminogawa, S. and Weiner, H. L. 2003. CD4+CD25 T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism. J. Immunol. 170:2516.[Abstract/Free Full Text]
- Szanya, V., Ermann, J., Taylor, C., Holness, C. and Fathman, C. G. 2002. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J. Immunol. 169:2461.[Abstract/Free Full Text]
- Seddon, B., Saoudi, A., Nicholson, M. and Mason, D. 1996. CD4+CD8 thymocytes that express L-selectin protect rats from diabetes upon adoptive transfer. Eur. J. Immunol. 26:2702.[ISI][Medline]
- Herbelin, A., Gombert, J. M., Lepault, F., Bach, J. F. and Chatenoud, L. 1998. Mature mainstream TCR alpha beta+CD4+ thymocytes expressing L-selectin mediate active tolerance in the nonobese diabetic mouse. J. Immunol. 161:2620.[Abstract/Free Full Text]
- Uraushihara, K., Kanai, T., Ko, K., Totsuka, T., Makita, S., Iiyama, R., Nakamura, T. and Watanabe, M. 2003. Regulation of murine inflammatory bowel disease by CD25+ and CD25 CD4+ Glucocorticoid-induced TNF receptor family-related gene+ regulatory T cells. J. Immunol. 171:708716.[Abstract/Free Full Text]
- Bansal-Pakala, P., Jember, A. G. and Croft, M. 2001. Signaling through OX40 (CD134) breaks peripheral T-cell tolerance. Nat. Med. 7:907.[CrossRef][ISI][Medline]
- Malmstrom, V., Shipton, D., Singh, B., Al-Shamkhani, A., Puklavec, M. J., Barclay, A. N. and Powrie, F. 2001. CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice. J. Immunol. 166:6972.[Abstract/Free Full Text]
- Nohara, C., Akiba, H., Nakajima, A., Inoue, A., Koh, C. S., Ohshima, H., Yagita, H., Mizuno, Y. and Okumura, K. 2001. Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells. J. Immunol. 166:2108.[Abstract/Free Full Text]
- Yoshioka, T., Nakajima, A., Akiba, H., Ishiwata, T., Asano, G., Yoshino, S., Yagita, H. and Okumura, K. 2000. Contribution of OX40/OX40 ligand interaction to the pathogenesis of rheumatoid arthritis. Eur. J. Immunol. 30:2815.[CrossRef][ISI][Medline]
- Durkop, H., Latza, U., Himmelreich, P. and Stein, H. 1995. Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues. Br. J. Haematol. 91:927.[ISI][Medline]
- Onodera, J., Nagata, T., Fujihara, K., Ohuchi, M., Ishii, N., Sugamura, K. and Itoyama, Y. 2000. Expression of OX40 and OX40 ligand (gp34) in the normal and myasthenic thymus. Acta Neurol. Scand. 102:236.[CrossRef][ISI][Medline]
- Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A. and Rudensky, A. 2002. Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol. 3:33.[CrossRef][ISI][Medline]
- Cederbom, L., Hall, H. and Ivars, F. 2000. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30:1538.[CrossRef][ISI][Medline]
- Ermann, J., Szanya, V., Ford, G. S., Paragas, V., Fathman, C. G. and Lejon, K. 2001. CD4+CD25+ T cells facilitate the induction of T cell anergy. J. Immunol. 167:4271.[Abstract/Free Full Text]
- Takahashi, T., Tagami, T., Yamazaki, S., Uede, T., Shimizu, J., Sakaguchi, N., Mak, T. W. and Sakaguchi, S. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated Antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
- Chai, J. G., Vendetti, S., Amofah, E., Dyson, J. and Lechler, R. 2000. CD152 ligation by CD80 on T cells is required for the induction of unresponsiveness by costimulation-deficient antigen presentation. J. Immunol. 165:3037.[Abstract/Free Full Text]
- Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. and Sakaguchi, S. 2002. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 22:22.
- Hori, S., Nomura, T. and Sakaguchi, S. 2003. Control of regulatory T cell development by the transcription factor FOXP3. Science 299:1057.[Abstract/Free Full Text]
- Iellem, A., Mariani, M., Lang, R., Recalde, H., Panina-Bordignon, P., Sinigaglia, F. and DAmbrosio, D. 2001. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+CD25+ regulatory T cells. J. Exp. Med. 194:847.[Abstract/Free Full Text]
- Bystry, R. S., Aluvihare, V., Welch, K. A., Kallikourdis, M. and Betz, A. G. 2001. B cells and professional APCs recruit regulatory T cells via CCL4. Nat. Immunol. 2:1126.[CrossRef][ISI][Medline]