Introduction
The adaptive immune system uses various potent effector mechanisms for the elimination of foreign pathogens. Because these mechanisms are potentially damaging to the host, an essential feature of the immune system is its ability to distinguish self from non-self antigens and to develop tolerance to the former. With regard to T cell tolerance, the immune system has evolved several strategies. Most autoreactive T cells are eliminated during (primary) maturation in the thymus, a process described as negative selection, resulting in central T cell tolerance. Autoreactive T cells that escape negative selection will nevertheless be prevented from being activated as they are confronted with auto-antigen in the periphery. Several mechanisms have been proposed to account for this peripheral tolerance. One of those is suppression by a subset of T cells that express both CD4 and CD25. Evidence for the important role of these cells is overwhelming [
1]. For example, when CD4
+ T cells isolated from peripheral lymphoid tissues of normal mice are depleted of CD4
+CD25
+ T cells and injected into
nu/
nu mice, the recipients develop a high incidence of organ-specific autoimmune disease [
2]. Co-transfer of the CD4
+CD25
+ population prevents the induction of disease. CD4
+CD25
- and CD4
+CD25
+ T cells are therefore often designated as, respectively, T
eff and T
reg cells. CD4
+CD25
+ T
reg cells are generated in the thymus. Their development is directed by relatively high-avidity interactions between the TCR and self-peptide ligands [
3‐
5]. The CD4
+CD25
+ T
reg cell population constitutes 5 to 10% of the mature CD4
+ cell population in the adult thymus and the peripheral lymphoid tissue and blood.
In vitro, CD4
+CD25
+ T
reg cells inhibit polyclonal T cell activation [
6,
7]. The suppression is mediated by a cytokine-independent, cell contact-dependent mechanism that requires activation of the CD4
+CD25
+ cells via the TCR with specific antigen [
8]. However, once stimulated, they are competent to suppress in an antigen-independent manner. Although the exact mechanism by which T
reg cells exert their regulatory function is still unknown, there are indications that interaction of transforming growth factor-β (TGF-β) with its receptor [
9‐
11], inhibition of IL-2 production [
6] or downregulation of co-stimulatory molecules on antigen-presenting cells [
12] could be involved.
T
reg cells have proved to be important in various animal models of autoimmune diseases. Administration of anti-CD25 antibody
in vivo induces organ-localised autoimmune diseases [
13]. Inoculation of CD4
+ T cells depleted of CD25
+ cells in
nu/nu mice results in autoimmune diseases such as gastritis, thyroiditis and insulitis [
2]. Thus, transfer of T
reg cells prevents autoimmune gastritis after neonatal thymectomy, and inhibits gastritis induced by H/K ATPase-reactive effector T cells [
14]. MBP-specific CD25
+CD4
+ T cells prevent spontaneous autoimmune encephalomyelitis in TCR-transgenic mice deficient in the recombination activating gene RAG-1 [
15]. Similarly, CD4
+CD25
+ T
reg cells suppress central nervous system inflammation during active experimental autoimmune encephalomyelitis [
16].
Collagen-induced arthritis (CIA) is a well-described animal model for rheumatoid arthritis. The disease is induced in genetically susceptible DBA/1 mice by immunisation with collagen type II (CII), and both T cell and B cell autoimmune responses are required for its development [
17‐
19]. IFN-γ receptor knock-out (IFN-γR KO) mice have been found to suffer an accelerated and more severe form of CIA [
20‐
23]. Moreover, knocking-out of the IFN-γ gene makes genetically resistant strains of mice susceptible to CIA [
24,
25]. These data indicate that deletion of the IFN-γ response somehow disrupts an endogenous protective mechanism against CIA.
Morgan and colleagues [
26] have recently demonstrated that CD25
+ T
reg cells are important in the pathogenesis of CIA. In the present study we confirmed the importance of T
reg cells in the pathogenesis of CIA by rendering wild-type DBA/1 mice deficient in T
reg cells by depleting anti-CD25 antibodies. Anti-CD25-treated mice developed a significantly more severe arthritis, comparable to the disease course in IFN-γR KO mice. Thus, we proposed that the higher susceptibility of IFN-γR KO DBA/1 mice to CIA might be ascribed to defects in the production (differentiation and homeostasis) or function of these CD4
+CD25
+ T
reg cells. We therefore determined the numbers of T
reg cells in central and peripheral lymphoid organs of IFN-γR KO and wild-type mice. We further investigated whether T
reg cells of IFN-γR KO mice have defects in the ability to suppress TCR-induced
in vitro proliferation of CD4
+CD25
- T
eff cells.
Materials and methods
Mice and experimental conditions
The generation and the basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene coding for the α-chain of the IFN-γ receptor (IFN-γR KO) have been described [
27]. These IFN-γR KO mice were backcrossed with DBA/1 wild-type mice for 10 generations to obtain the DBA/1 IFN-γR KO mice used in the present study. The homozygous IFN-γR KO mice were identified by PCR as described [
23]. Wild-type and IFN-γR KO DBA/1 mice were bred in the Experimental Animal Centre of the University of Leuven. The experiments were performed in mice 6 to 10 weeks old, but in each experiment the mutant and wild-type mice were age-matched within 5-day limits. The male : female ratio was kept between 0.8 and 1.3 in each experiment group, unless otherwise mentioned. All animal experiments were approved by the local ethical committee (University of Leuven).
Induction and clinical assessment of arthritis
Native chicken CII (Sigma-Aldrich, St Louis, MO, USA) was dissolved at 2 mg/ml in PBS containing 0.1 M acetic acid by stirring overnight at 6°C and emulsified in an equal volume of complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) with added heat-killed Mycobacterium butyricum (0.5 mg/ml). IFN-γR KO and wild-type mice were sensitised with a single intradermal injection at the base of the tail with 100 μl of the emulsion on day 0. From day 0 after immunisation, mice were examined for signs of arthritis five times a week. The disease severity was recorded with the following scoring system for each limb: score 0, normal; score 1, redness and/or swelling in one joint; score 2, redness and/or swelling in more than one joint; score 3, redness and/or swelling in the entire paw; score 4, deformity and/or ankylosis.
All cells were grown in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium), supplemented with 10% heat-inactivated FCS (Gibco, Paisley, UK), penicillin (100 IU/ml; Continental Pharma, Brussel, Belgium), streptomycin (100 μg/ml; Continental Pharma), 2 mM L-glutamine, 10 mM Hepes (Gibco), 0.1 mM nonessential amino acids (ICN, Asse Relegem, Belgium), 1 mM sodium pyruvate (Gibco) and 50 μM 2-mercaptoethanol (Fluka, AG, Switzerland).
Anti-CD25 IL-2Rα monoclonal antibody was produced by hybridoma PC61 in an INTEGRA CELLine CL1000 (Elscolab, Kruibeke, Belgium) and is a rat IgG1 antibody. The hybridoma supernatant was purified by Protein G-Sepharose chromatography (Amersham Biosciences, Roosendaal, The Netherlands) for administration in vivo.
The hamster monoclonal antibody, directed against the mouse CD3 complex, was prepared from the culture supernatant of 145-2C11 hybridoma cells [
28]. The antibodies were purified by affinity chromatography with Protein A-Sepharose (Amersham Biosciences). Batches of anti-CD3 antibody were tested for endotoxin content with the
Limulus amebocyte lysate QCL-1000 kit (Bio Whittaker) and were found to contain less than 3 ng/ml endotoxin.
Cell purification
Lymph nodes (axillary, inguinal and mesenteric) and spleens were harvested from mice 6 to 8 weeks old. Lymph nodes and spleens were gently cut into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Red blood cells were lysed by two consecutive incubations (5 and 3 min at 37°C) of the suspension in NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Remaining cells were washed, resuspended in cold PBS and counted. Lymph node preparations were then enriched for CD4+ T cells with the Mouse T cell CD4 Subset Column Kit (R&D systems, Abingdon, UK). To purify CD4+CD25+ and CD4+CD25- cells, the enriched CD4+ T cells were incubated for 20 min at 4°C with FITC-conjugated anti-CD25 and phycoerythrin (PE)-conjugated anti-CD4 antibodies (10 μg per 108 cells) in PBS containing 2% FCS. They were sorted by flow cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA, USA). The resultant purity of the CD4+CD25- population was 99%, whereas the purity of the CD4+CD25+ population varied from 96% to 99%. Alternatively, CD4+ T cells were labelled with PE-conjugated anti-CD25 monoclonal antibody, followed by incubation with magnetic-activated cell sorting (MACS) anti-PE beads (CD25 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25+ T cells were selected on an LS column in a magnetic field and the flow-through was collected as CD4+CD25- T cells. After removal of the column from the magnetic field, CD4+CD25+ T cells were flushed out by a plunger. The purity of the CD4+CD25- population was 99% and the purity of the CD4+CD25+ population varied from 90% to 95%.
T cell-depleted spleen suspensions were prepared by MACS (Miltenyi Biotec) and used as accessory cells (ACs). For MACS separation, the cell suspension was magnetically labelled with CD90 (Thy1.2) microbeads and passed through a CS separation column, placed in a magnetic field. The unlabelled CD90- cells ran through.
Flow cytometry
Single-cell suspensions (5 × 105 cells) were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (CD16/CD32; BD Biosciences Pharmingen, San Diego, CA, USA). Cells were washed with PBS containing 2% FCS and stained with the indicated FITC-conjugated antibodies (0.5 μg) for 30 min, washed twice and incubated for 30 min with the indicated PE- or biotin-conjugated antibodies. For the biotin-conjugated antibodies, a third staining step with streptavidin conjugated with peridinin chlorophyll a protein (PerCP) was performed. After washing, propidium iodide (Sigma-Aldrich) was added at a final concentration of 4 μg/ml to distinguish dead cells from living cells. Biotin-conjugated anti-CD25 (7D4), FITC-conjugated anti-CD25 (7D4), FITC-conjugated CD69 (H1.2F3), PE-conjugated anti-CD4 (RM4-5) and PerCP-conjugated streptavidin were purchased from BD Biosciences Pharmingen. FITC-conjugated anti-CD62L (MEL-14) and anti-CD44-FITC (IM7.8.1) were from CALTAG Laboratories (Burlingame, CA, USA).
For intracellular staining with anti-CTLA-4-PE (UC10-4F10-11; BD Biosciences Pharmingen), 106 cells were first labelled with anti-CD25-FITC as described above. Then, cells were fixed, permeabilised and stained with anti-CTLA-4-PE using the Cytofix/Cytoperm™ Kit (BD Biosciences Pharmingen) according to the recommendations of the manufacturers.
Flow-cytometric analysis was performed on a FACScan flow cytometer with Cell Quest software (Becton Dickinson).
Proliferation assays
CD4+CD25- cells (5 × 104 per well) were cultured in U-bottomed 96-well plates (200 μl) with ACs (5 × 104 per well, 30 Gy γ-irradiated or treated with mitomycin-C (Sigma-Aldrich)), 3 μg/ml anti-CD3 and the indicated numbers of CD4+CD25+ cells for 48 hours at 37°C in 7% CO2. Cultures were pulsed for the last 16 hours with 1 μCi of [3H]TdR and harvested. The suppressive activity of the Treg cells can be presented by plotting the percentage of inhibition (100 × (Radioactivity in condition without Treg cells – Radioactivity in condition with Treg cells)/Radioactivity in condition without Treg cells) against the number of Treg cells.
Antibody administration
DBA/1 mice were immunised with CII in CFA; 13 days after immunisation, the mice were treated every second day with 0.25 mg of anti-CD25 (PC61) or control IgG antibodies, for 4 weeks (injected intraperitoneally).
Histological examination
Forelimbs and hindlimbs were fixed in 10% formalin and decalcified with formic acid (31.5% (v/v) formic acid and 13% (w/v) sodium citrate). The paraffin sections were stained with haematoxylin and eosin.
Measurement of serum anti-CII antibodies
Blood samples were taken from the orbital sinus and were allowed to clot at room temperature for about 1 hour, and at 4°C overnight. Individual sera were tested by ELISA for antibodies directed against chicken CII. In brief, ELISA plates (Maxisorb; Nunc, Wiesbaden, Germany) were coated overnight at 4°C with native CII (1 μg/ml; 100 μl per well) in coating buffer (50 mM Tris-HCl, pH 8.5, 0.154 mM NaCl), followed by incubation for 2 hours with blocking buffer (50 mM Tris-HCl, pH 7.4, 0.154 mM NaCl and 0.1% caseine) to saturate non-specific binding sites. Serial twofold dilutions of the sera in assay buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl and 0.05% Tween 20) were added and incubated for 2 hours at room temperature. The plates were then incubated for 2 hours with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) in reaction buffer (100 mM sodium acetate/citric acid, pH 4.9) was added for a 10 min incubation and absorbance was determined at 450 nm. Plates were washed five times between each step with PBS containing 0.05% Tween 20. A serial twofold dilution series of a purified standard was included to permit a calculation of the antibody content of each sample. The standard was purified by affinity chromatography from pooled sera obtained from various arthritic wild-type and IFN-γR KO mice.
Quantitative RT-PCR
Isolated CD4+CD25+ and CD4+CD25- cells were pelleted and directly used for total RNA isolation, using the Micro-to-Midi Total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA (1 μg) was used for random primed cDNA synthesis with RAV-2 reverse transcriptase (Amersham, Aylesbury, Bucks., UK). The reaction mixture was incubated for 80 min at 42°C and the reverse transcriptase was inactivated by incubating the cDNA samples for 5 min at 95°C.
The cDNA samples were then subjected to real-time quantitative PCR, performed in the ABI prism 7700 sequence detector (Applied Biosystems, Foster City, CA) as previously described [
29]. The sequences of the forward (-FW) and reverse (-RV) primers and probes (-TP) for β-actin and Foxp3 were as follows: β-actin-FW, AGA GGG AAA TCG TGC GTG AC; β-actin-RV, CAA TAG TGA TGA CCT GGC CG T; β-actin-TP, CAC TGC CGC ATC CTC TTC CTC CC; Foxp3-FW, CCC AGG AAA GAC AGC AAC CTT; Foxp3-RV, TTC TCA CAA CCA GGC CAC TTG; Foxp3-TP, ATC CTA CCC ACT GCT GGC AAA TGG AGT C; TGF-β-FW, TGA CGT CAC TGG AGT TGT ACG G; TGF-β-RV, GGT TCA TGT CAT GGA TGG TGC; TGF-β-TP, TTC AGC GCT CAC TGC TCT TGT GAC AG. Probes were dual-labelled with 5'-FAM and 3'-TAMRA.
All primers and probes were designed with the assistance of the computer program Primer Express (AB) and were purchased from Eurogentec (Seraing, Belgium). The 5'-nuclease activity of the
Taq polymerase was used to cleave a nonextendable dual-labelled fluorogenic probe. Fluorescent emission was measured continuously during the PCR reaction. PCR amplifications were performed in a total volume of 25 μl containing 5 μl of cDNA, 12.5 μl of Universal PCR Master Mix, no AmpErase UNG (AB), each primer at 100 to 300 nM, and the corresponding detection probe at 200 nM. Each PCR amplification was performed in triplicate wells under the following conditions: 94°C for 10 min, followed by 40 or 45 cycles at 94°C for 15 s and 60°C for 1 min. cDNA plasmid standards, consisting of purified plasmid DNA specific for each individual target, were used to quantify the target gene in the unknown samples, as described [
29]. All results were normalised to β-actin and/or hypoxanthine–guanine phosphoribosyltransferase (HPRT) to compensate for differences in the amount of cDNA in all samples. Results were similar whether β-actin or HPRT was used as the housekeeping gene.
Discussion
We and others have previously demonstrated that IFN-γ(R) KO mice show an accelerated and more severe from of arthritis than their wild-type counterparts, indicating that endogenous IFN-γ acts as a protective factor in CIA [
20,
21,
24,
25]. Because CIA has been defined as a Th1-driven disease (reviewed in [
17]), the protective effect of IFN-γ in CIA constitutes an enigma that compromises the Th1/Th2 paradigm as a basis for explaining the regulation of autoimmune diseases. A clue to the enigma seemed to be the use of CFA in the induction procedure of CIA. In the absence of IFN-γ, CFA induces an extensive extramedullary myelopoiesis that goes together with an even more pronounced Th1 cytokine profile than in wild-type counterparts [
22,
36]. The data suggest that IFN-γ can, under certain circumstances, be a strong Th2 inducer, a finding that has recently been confirmed by others [
37]. Here, we tested the hypothesis that this protective action of IFN-γ is due to a stimulatory effect on T
reg cells. Specifically, we addressed the following two questions. Are T
reg cells important in modulating CIA? And, because we found that depletion of T
reg cells in wild-type mice increased the severity of CIA, can the higher susceptibility of IFN-γR KO mice to CIA be explained by defects in the number or function of their T
reg cells?
As to the first question, we found that administration of a T
reg cell-depleting anti-CD25 antibody to wild-type DBA/1 mice after CFA-assisted immunisation with CII resulted in accelerated and more severe arthritis. In fact, the disease course in these mice was comparable to that in IFN-γR KO mice [
20‐
23]. The actual depletion of T
reg cells was monitored by flow cytometry, and the authenticity of arthritis was verified histopathologically. These results are in line with those of Morgan and colleagues [
26], who showed that the administration of depleting anti-CD25 antibody before immunisation (days –28, –24, –21 and –14) hastened the onset of severe CIA. Because in our experiments antibodies were administered starting from day 11 or day 13 after immunisation, we can conclude that T
reg cells are important in the pathogenesis of CIA, not only in the immunisation phase but also in the effector phase. In contrast to the findings of Morgan and colleagues [
26], the accelerated and more severe course of arthritis was, in our experiments, not accompanied by a higher concentration of anti-collagen II antibodies, possibly due to the different regimen of anti-CD25 treatment. Indirect evidence for the involvement of T
reg cells in the pathogenesis of CIA comes from data of Min and colleagues [
38]. They found that the immune tolerance induced by oral feeding of CII before induction of CIA was mediated by IL-10-producing CD4
+CD25
+ T cells. Notably, in proteoglycan-induced arthritis, another model of autoimmune arthritis, it has been shown that CD4
+CD25
+ T
reg cells might not have a critical role [
39]. This might result from the use of a different auto-antigen.
To address the second question, we compared CD4
+CD25
+ cell numbers and T
reg cell function in IFN-γR KO DBA/1 mice with those in wild-type mice. According to our hypothesis we expected numbers of T
reg cells in IFN-γR KO mice to be lower. Counter to this expectation, in each of the six experiments done, we found a trend for a higher proportion of CD4
+CD25
+ T cells in the total CD4
+ cell population. This was true for thymic, splenic and lymph node CD4
+ cells, in both naive and immunised mice. Analysis of all data as one set revealed a significant difference of about 30% and 20% in naive and immunised mice, respectively. CD25 is not an exclusive marker of T
reg cells: especially in immunised mice, part of the CD4
+CD25
+ population might be effector rather than regulatory T cells [
40,
41]. Therefore, to exclude the possibility that we were comparing two completely different populations, we performed additional flow-cytometric characterisation studies on pre-sorted CD4
+CD25
+ cells.
Expression of CD44, CD69, CTLA-4 and CD62L in CD4
+CD25
+ cells from IFN-γR KO mice did not differ from expression in cells from corresponding wild-type mice, whether naive or immunised. However, because T
reg cells display an activated phenotype, activation markers might not be adequate to distinguish T
reg cells from activated T
eff cells. According to Fontenot and colleagues [
42] a specific marker for T
reg cells is Foxp3, because it is highly expressed in CD4
+CD25
+ T
reg cells and is virtually undetectable in both resting and activated T
eff cells. We examined Foxp3 expression by determining mRNA levels with PCR. After immunisation, CD4
+CD25
+ cells contained lower levels of Foxp3 mRNA than those of their naive counterparts. Moreover, mRNA levels in immunised IFN-γR KO mice were less than one-third of those in their wild-type counterparts, indicating that IFN-γR KO mice have a smaller number of T
reg cells or that expression of Foxp3 in each T
reg cell is lower.
Recently, Bruder and colleagues [
43] have shown linked expression of neuropilin-1 and Foxp3, thereby identifying neuropilin-1 as a specific surface marker for CD4
+CD25
+ T
reg cells able to distinguish them from both naive and recently activated CD4
+CD25
+ non-regulatory T cells.
Nishibori and colleagues [
44] demonstrated impaired development of T
reg cells in naive signal transduction and activators of transcription (STAT)-1-deficient mice, associated with an increased susceptibility to autoimmune disease. Because IFN-γ is among the strongest activators of STAT-1, these observations seem to conflict with ours. However, several cytokines, other than IFN-γ, can also activate STAT-1, including IFN-α, IFN-β, IL-6, IL-9, IL-11, oncostatin M, leukaemia inhibitory factor and the chemokines RANTES and macrophage inflammatory protein 1α [
45,
46].
To determine whether overall Treg cell activity would be lower in IFN-γR KO mice, we co-cultured increasing numbers of CD4+CD25+ T cells with fixed numbers of CD4+CD25- Teff cells and ACs in the presence of anti-CD3 antibody. We observed a dose-dependent inhibition of the proliferative responses by CD4+CD25+ Treg cells. By estimating numbers of CD4+CD25+ cells required to attain a selected level of suppression, we could compare suppressive activity in the different groups of mice. In naive mice, the inhibition curves were almost identical, whether the Treg cells were derived from wild-type or IFN-γR KO mice, indicating that endogenous IFN-γ is not an important regulator of the function of constitutive CD4+CD25+ Treg cells. In co-cultures of cells from immunised wild-type mice, the Treg suppressive capacity was about one-third of that in those from corresponding naive mice, and a further halving was noted in co-cultures of cells from immunised IFN-γR KO mice.
The observation that immunisation renders T
reg cells less suppressive is in line with results of Pasare and Medzhitov [
47], who found that microbial triggering of the Toll-like receptor (TLR) pathway by lipopolysaccharide or CpG, which are ligands for TLR4 and TLR9, respectively, blocked the suppressive effect of CD4
+CD25
+ T
reg cells. Because mycobacteria also contain TLR ligands, immunisation with CFA can be expected to affect T
reg cell activity similarly. The decrease in suppressive activity that takes place after TLR4 or TLR9 triggering was found to be dependent on IL-6 production [
47]. It might therefore be of interest to note that in our experiments, IL-6 production was enhanced after exposure to CFA-assisted immunisation, and this effect was even more pronounced in IFN-γR KO mice (P Matthys, unpublished data). This could provide an explanation for the fact that CD25
+ T
reg cells are totally functional before immunisation but lose (part of) their function after immunisation. However, the most important observation is the lower T
reg suppressive capacity in IFN-γR KO than in wild-type mice after CFA-assisted immunisation, because this supports our hypothesis that the protective effect of endogenous IFN-γ against CIA could be mediated in part by its stimulatory effect on T
reg cells.
Because the disease is barely detectable in wild-type mice on day 21 after immunisation, we investigated whether the decreased suppressive activity in immunised wild-type mice was further downregulated at a later time point (namely, day 35 after immunisation, when most of the animals show symptoms of arthritis). However, suppressive activity was not further downregulated to the level seen in homogeneous IFN-γR KO co-cultures, but was comparable to that seen in co-cultures from immunised wild-type mice on day 21 after immunisation (maximal inhibition 60%; data not shown). This indicates that the low suppressive activity as evident in immunised IFN-γR KO mice is restricted to conditions under which IFN-γ is abrogated.
The implication is that the CIA immunisation schedule induces a decrease in Treg activity and that endogenous IFN-γ largely counteracts this decrease. It therefore becomes important to know by what mechanism, direct or indirect, IFN-γ influences Treg cell function. Addition of anti-IFN-γ antibody to the co-cultures failed to affect suppressive activity (data not shown), indicating that the relevant IFN-γ effect takes place in vivo before sampling of the T cells. To examine the role of the different cell components, we tested suppressive activity in mixed co-cultures. CD4+CD25+ cells from immunised IFN-γR KO mice, confronted with Teff cells and ACs from immunised wild-type mice, were not less suppressive than wild-type CD4+CD25+ confronted with wild-type or IFN-γR KO Teff cells and ACs. This suggests that lower levels of suppression in homogeneous IFN-γR KO cultures result in part from the presence of IFN-γR KO-derived ACs. And, indeed, when CD4+CD25+ and Teff cells from immunised IFN-γR KO mice were co-cultured with ACs from immunised wild-type mice, suppressive activity was not inhibited. Finally, ACs from IFN-γR KO mice by themselves were unable to downregulate the activity of wild-type Treg cells acting on wild-type Teff cells. We therefore conclude that the in vivo effect of endogenous IFN-γ that accounts for the greater suppressive activity in wild-type mice than in IFN-γR KO mice concerns reprogramming of both ACs and Treg cells.
Because CD4+CD25+ T cells from immunised IFN-γR KO mice were not less suppressive than those of immunised wild-type mice in co-cultures with Teff cells and ACs from immunised wild-type mice, we can refute the proposition that the lower expression of Foxp3 in the CD4+CD25+ population from immunised IFN-γR KO mice is due to a smaller proportion of Treg cells and a larger number of activated Teff cells. Indeed, if the CD4+CD25+ population from immunised IFN-γR KO mice contained a higher proportion of activated Teff cells, suppression by these CD4+CD25+ cells should be lower, irrespective of the origin of the Teff cells and ACs. Another argument is that the addition of more CD4+CD25+ cells failed to improve suppression in co-cultures of cells from immunised IFN-γR KO mice. Our data are therefore more in line with the proposition of a lower Foxp3 expression level per cell.
Expression of Foxp3 could be downregulated by the interaction of T
reg cells with ACs. ACs might be source of TGF-β, which has been described to convert naive T cells into CD25
+ suppressor cells by inducing Foxp3 expression [
48]. Because IFN-γ and TGF-β act antagonistically with each other, the low levels of Foxp3 in arthritic IFN-γR KO mice might be due to inadequate amounts of TGF-β produced by ACs or other cells. However, quantitative PCR performed on isolated T
reg cells, on cells obtained from co-cultures (T
reg plus T
eff plus ACs) and on splenocytes from immunised IFN-γR KO and wild-type mice does not support the concept that the defective activity of T
reg cells
in vitro or
in vivo is due to defects in the production of TGF-β. ACs have also been shown to be able to reverse suppression by CD4
+CD25
+ cells through the GITR/GITR-ligand system [
49]. GITR (glucocorticoid-induced tumour necrosis factor receptor) is expressed on CD4
+CD25
+ T cells; GITR-ligand is initially upregulated on activated APCs. It remains to be determined whether this process involves a downregulation of Foxp3 expression. This or a similar mechanism might take place during the interaction of T
reg cells and ACs from immunised IFN-γR KO mice. Co-cultures with ACs of immunised wild-type mice might possibly normalise Foxp3 expression in the T
reg cells of immunised IFN-γR KO mice, together with their T
reg suppressive activity.
Authors' contributions
BDK, HK and TM performed the CIA induction and evaluation. HK, MVB and GL performed the cell purification. DB performed the quantitative PCR. TM and HK performed the flow cytometry. BDK and HK did the in vitro experiments. HK, GL and PM designed the study. All authors participated in the interpretation of the data. HK, AB, GL and PM prepared the manuscript. All authors read and approved the final manuscript.