|
|
|
|
|
|
|
||
ARTICLE |
CORRESPONDENCE Guy Gorochov: guy.gorochov{at}psl.aphp.fr
 Top Abstract RESULTSDISCUSSIONMATERIALS AND METHODSReferences |
|---|
production. Sarcoidosis is therefore associated with a global T reg cell subset amplification whose activity would be insufficient to control local inflammation. At the same time, peripheral T reg cells exert powerful antiproliferative activity that may account for the state of anergy. Altogether, these findings advance our conceptual understanding of immune regulation in a way that resolves the immune paradox of sarcoidosis and permit us to envisage a profound clinical impact of T reg cell manipulation on immunity.
C. Parizot and C. Badoual contributed equally to this work.
Sarcoidosis is characterized by the presence of noncaseating granulomas in a variety of organs, most commonly the lung. CD4+ and CD8+ T lymphocytes (1), as well as a few B lymphocytes, form a characteristic ring at granuloma periphery. Most granuloma-associated lymphocytes have a Th1 phenotype (2). Th1 cells secrete cytokines such as IL-2 and IFN-
and play a central role in the control of the cellular immune response. Mechanisms underlying formation, maintenance, and spontaneous resolution of sarcoid granulomas are poorly understood (3, 4). From a general standpoint, one could say that sarcoidosis represents an unresolved immunological paradox: affected organs are the staging ground for an intense immune response, yet at the same time, a state of anergy is established as indicated by suppression of immune response to tuberculin in sarcoidosis patients (5).
Regulatory cells, of which at least two types can be distinguished, are capable of inhibiting diverse immunopathologic phenomena by controlling the proliferation of CD4+ and CD8+ T lymphocytes in vivo (6, 7). The first type regulates immune responses via secretion of cytokines and corresponds to IL-10–producing Tr1 cells (8, 9) and TGF-ß–producing Th3 cells (10). The second type of regulatory T cell is the naturally occuring, or innate, regulatory T cell (T reg cell). The latter mediates suppression through mechanisms dependent on cell contact and is characterized by constitutive expression of CD25 (6). Naturally occurring T reg cells have also been described in humans (11–15), where they are mainly confined to the CD25bright subset of CD4+ cells (16). The potential role of T reg cells in human disease is currently the focus of intensive research efforts (17). It is proposed that modulation of either the number or function of T reg cells could prove beneficial in the treatment of autoimmunity and cancer (18–20).
We report here that sarcoidosis is characterized by expansion of the innate T reg cell subset and that purified T reg cells efficiently suppress naive T cell proliferation, but are unable to totally suppress TNF- secretion.
 |
RESULTS |
|---|
|
TopAbstractRESULTSDISCUSSIONMATERIALS AND METHODSReferences |
|---|
|
|
|
CD4+CD25bright T cells from a sarcoidosis patient express a polyclonal TCR repertoire
To address the question of whether CD4+CD25bright T cell numbers are increased during active sarcoidosis as a result of oligoclonal expansion, we investigated the T cell repertoire usage of sorted CD4+CD25bright T cells. As described previously (27, 28), we chose to study nine BV families that span >50% of the TCRBV repertoire expressed in healthy individuals (29, 30). We used the Immunoscope technique (31), which is based on the analysis of TCR CDR3 length polymorphism to assess the clonal composition of the CD4+CD25bright T cell subset. 5 x 105 of these cells, as well as a corresponding number of CD4+CD25– T cells, were purified from a lymph node obtained from one patient with active sarcoidosis. It was verified that the sorted CD4+CD25bright T cells used for repertoire analysis were indeed immunosuppressive (Fig. 2 a). Immunoscope analysis of cells isolated from one patient shows (Fig. 4) that both subsets are similarly diverse and also display similar TCRB CDR3 size distributions.
|
CD4+ FoxP3+ cells accumulate in the periphery of noncaseating granulomas
We subsequently sought to visualize where T reg cells were localized in affected organs using bicolor microscopic analysis. As shown in Fig. 5, T reg cells identified as CD4+FoxP3+ cells accumulate in the granuloma periphery. These cells are also clearly more abundant in sarcoidosis-involved lymph nodes than in normal lymphoid tissue from control subjects (6.48 ± 2.12% of CD4+ cells, n = 10 vs. 1.67 ± 0.67%, n = 2).
|
|
or TNF- secretion by autologous and allogeneic cells could play a central role in sarcoidosis physiopathology (3, 4). The effect of patients isolated T reg cells on cytokine production was therefore studied. Cell cultures of each of nine patients and nine controls were analyzed three times over a period of 5 d (Fig. 6 b). In the control branch of the study, T reg cells completely inhibited TNF- and IFN- secretion as measured on day 5 after stimulation (partial inhibition on day 3), whereas in the experimental arm, a clear residual cytokine secretion was apparent on day 5. These data are summarized in Fig. 6 c. Intriguingly, patients' T reg cells completely inhibit IL-2 production (mean residual secretion 4.21 ± 2.57%, n = 18 vs. 4.23 ± 2.90%, n=19, in controls), but not that of IFN- (19.02 ± 12.37% vs. 4.07 ± 2.91%) or TNF- (27.23 ± 15.29% vs. 2.45 ± 2.7%). In comparison, control T reg cells inhibit the secretion of all three cytokines.
We then sought to determine if the expanded CD4+CD25bright subset could itself represent a notable source of TNF- in patients suffering from sarcoidosis. In five patients and four controls, we obtained enough CD4+CD25bright cells to study cytokine secretion in an isolated manner. As shown, CD4+CD25bright cells contribute only slightly to the residual level of TNF- secretion observed in the patient group after 5 d of stimulation (Fig. 6 b). Further control experiments confirmed that early production (day 1) of TNF- is of accessory cell (AC) origin, whereas the bulk of TNF- released during the course of activation is produced by CD25– T cells (Fig. 6 d). As shown, only the latter source of TNF- is sensitive to T reg–mediated suppression.
We then examined whether this confirmed partial loss of regulatory function on cytokine secretion was the result of a decrease in T reg cell activity or to a resistance of the patients' CD4+CD25– cells to inhibition. To address this issue, "criss-cross" experiments were performed. T reg cells from patients were tested on healthy donors' responder cells, and vice versa (Fig. 6 c). T reg cells from patients do not completely block cytokine secretion from either patients or healthy controls. However, residual amounts of TNF- are also detected in the supernatants of sarcoidosis CD4+CD25– cells stimulated in the presence of control T reg cells.
The aforementioned results were obtained using low-dose anti-CD3 (16), and allogeneic ACs as stimulus according to previous defined protocols (12–14, 32, 33). To verify that the persistence of TNF- and IFN- would not in fact be secondary to strong allogeneic responses, we repeated the functional analyses of T reg cells using only autologous ACs and anti-CD3 (0.5 µg/ml). Under these conditions, TNF- and IFN- production are not totally suppressed by sarcoidosis T reg cells (n = 4) compared with controls (n = 5, P < 0.02, Fig. 6 d). Therefore, the incomplete control of TNF- and IFN- production is not only observed in the context of strong allogeneic responses.
Most IFN-/TNF-–secreting cells do not produce IL-2
It follows from our results that (a) TNF- and IFN- production and (b) IL-2 production would be differentially affected by the presence of T reg cells. We postulated that this could be partially explained by the fact that TNF- and IFN- are usually produced by cells that no longer (or never) made IL-2. To address that issue, we used four-color cytometry to monitor IL-2, TNF-, and IFN- at a single cell level in three patients and five controls after a short-term in vitro stimulation (Fig. 7). We show that IFN- and IL-2 production are almost mutually exclusive. Although some TNF-–producing CD4+ cells can secrete IL-2, a subset of CD4+IL2–TNF-+IFN-+ cells can be clearly defined in patients' and controls' blood (Fig. 7). Altogether, we conclude on the existence of a defect in regulatory function exerted by sarcoidosis T reg cells, as well as a concurrent relative resistance on the part of highly differentiated (TNF-+, IFN-+) autologous effector T cells.
|
|
DISCUSSION |
|---|
|
TopAbstractRESULTSDISCUSSIONMATERIALS AND METHODSReferences |
|---|
We show here that the T reg population is indeed globally amplified in circulating blood and BALF of patients presenting with active sarcoidosis. In vitro, the capacity of these cells to strongly inhibit the proliferation and IL-2 secretion of CD4+CD25– T lymphocytes contrasts with their inability to completely inhibit the secretion of TNF- (and to a lesser extent IFN-). The latter observation could be particularly relevant as TNF- plays a key role in granuloma formation (35) and because anti-TNF- drugs have proved useful in the treatment of refractory sarcoidosis (36). Because T reg cells do not completely block TNF- secretion, they might only partially control granuloma evolution in vivo. A limited effect of T reg cells on TNF- secretion has previously been reported in patients suffering from rheumatoid arthritis (37). T reg function was also found impaired in multiple sclerosis (38). Given our data, we cannot conclude that T reg cells found in sarcoidosis patients have an intrinsic defect. It is equally probable that these cells are simply incapable of controlling an abnormally abundant secretion of TNF-. Our analysis of cytokine production at a single cell level (Fig. 7) delineates nonoverlapping subsets of CD4+ cells as defined according to their cytokine secretion profile. In particular, a CD4+IL2–IFN-+TNF-+ subset can be clearly defined. More work will be necessary to determine whether the later subset would be less sensitive to T reg suppression functionality in sarcoidosis patients. In any case, it is possible that phenotypically and functionally distinct subsets of CD4+ cells would be differentially regulated by T reg cells.
Although unable to totally suppress TNF- and IFN- production in sarcoidosis patients, T reg cells exert a profound antiproliferative activity and are able to abolish IL-2 secretion as expected (mean suppression 95.8 ± 2.6%, Fig. 6 c). T cell expansion represents a crucial aspect of memory responses. Memory T lymphocytes specific for a given antigen are scarce. The latter could have their proliferation effectively suppressed when they encounter their recall antigen in lymphoid organs harboring an abnormal proportion of T reg cells. We show that T reg cells are indeed abundant in affected lymph nodes, but are infrequently found in the lymph nodes of healthy controls, lending support to a T reg cell/T memory disequilibrium as a possible mechanism of anergy.
We therefore postulate that the aforementioned paradox can be solved if one considers that T reg cells tightly control T cell homeostasis mainly through the control of T cell expansion (39), and that this mechanism is overly effective in sarcoidosis patients. Alternatively, the same mechanism would not efficiently control the inflammation of injured tissues.
In active sarcoidosis patients, the chemokine receptor CCR4 is preferentially expressed on CD4+CD25bright T reg cells irrespective of their tissue of origin. In contrast, CXCR3 is overexpressed on BALF CD4+CD25bright cells. It was recently demonstrated that the specific recruitment of T reg cells in human ovarian tumors is mediated by the secretion of the CCR4 ligand CCL22 (macrophage-derived chemokine) by tumor cells and microenvironmental macrophages (19). However, lung macrophages from sarcoidosis patients do not secrete CCL22 (40), whereas they are known to produce high levels of the CXCR3 ligand CXCL10 (IFN-–inducible 10-kd protein [IP-10]) (41). It is therefore possible that it is in response to IP-10 secretion by activated tissue macrophages that CXCR3+ T reg cells are recruited to affected organs. Only cells that down-modulate CXCR3 would recirculate to the periphery. To explore another potential mechanism that would induce T reg migration and expansion, we also studied the clonal diversity of this subset (Fig. 4). Using CDR3-length polymorphism analysis (27), we found that CD4+CD25bright T reg cells are polyclonal in one sarcoidosis patient. It is therefore unlikely that in this case CD4+CD25bright expansion would result from direct antigenic stimulation. Unfortunately, it was not possible until now to purify enough CD4+CD25bright cells from sarcoidosis BALF to perform additional reliable repertoire studies. It is known that oligoclonal population of ß+ CD4+ T cells collect at granulomatous sites (42–44) and at Kveim reaction sites (45). Mycobacterial catalase-peroxydase was recently identified as one target of the adaptive immune response in sarcoidosis (46), but it remains unknown whether granuloma-associated T cells actually include mycobacterial catalase-peroxydase–specific T cells. In light of the data presented here, it could be interesting to study the reactivity of sarcoidosis T reg cells against mycobacterial antigens. In a first step, it will be necessary to solve the issue of the T reg repertoire diversity at granulomatous sites of inflammation.
To our knowledge, there is no clinical situation in which a T reg amplification of such magnitude (up to 21% of CD4+ T cells) has been found in the blood of patients. It is possible that the observed spontaneous release of IL-2 by lung T lymphocytes in active pulmonary sarcoidosis could be one of the factors that participate to their amplification (47). Circulating CD4+CD25bright cells were reported elevated during chronic graft-versus-host disease (48), but never exceeded 11% of CD4+ T cells. Accumulation of T reg cells has also been observed in the joints of rheumatoid arthritis patients (49) and in Hodgkin lymphoma–infiltrated lymph nodes (50). More studies are underway to confirm whether the expansion of circulating T reg cells is specific enough to distinguish sarcoidosis from any other granulomatosis. There currently exists no gold standard to confirm the diagnosis of sarcoidosis. We will therefore work to determine the peripheral blood or BALF T reg cell concentration that supports this diagnosis.
Finally, it is proposed that modulation of either the number or function of T reg cells could represent a means of controlling autoimmunity or other immunopathologic conditions (7, 18). Our data clearly support this assertion, but also indicate that such potential therapies might be, in the long term, detrimental to immune surveillance. Indeed, sarcoidosis patients are predisposed to opportunistic infections (51) and cancer (52). It was previously proposed that the increased cancer risk would result from chronic inflammation and subsequent tissue damage in affected organs (52). In light of the data presented here, we propose that a T reg cell/Th1 imbalance could also be evoked to explain why sarcoidosis patients have such a predisposition.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractRESULTSDISCUSSIONMATERIALS AND METHODSReferences |
|---|
Cytometry.
Mononuclear cells isolated from peripheral blood, BALF, and LNs were stained with anti-CD4–PerCP, anti-CD25–PE, or anti-CD25-allophycocyanin, anti-CCR4–PE mAbs (BD Biosciences), anti-CCR2–PE, anti-CCR3–PE, anti-CCR5–PE, anti-CCR6–PE, anti-CCR7–PE, anti-CCR8–PE, anti-CCR9–PE, anti-CX3CR1–PE anti-CXCR4–PE, anti-CXCR6–PE and anti-CXCR3–FITC (R&D Systems). Intracellular detection of CTLA-4 with anti-CD152–PE was performed on fixed and permeabilized cells using Cytofix/Cytoperm (BD Biosciences). FACSCalibur (Becton Dickinson) acquired data were analyzed with WinMDI 2.8 software (http://facs.scripps.edu/software.html) on 500,000 events. Cells were sorted using a FacsVantage (Becton Dickinson).
Proliferation assay and cytokine detection.
Varying numbers of sorted CD4+CD25bright T cells were cocultured in supplemented medium (26) with 2.5 103 autologous CD4+CD25– responder T cells and 2.5 104 allogeneic or autologous T cell–depleted PBMCs (irradiated at 5,000 rad) in 96 U-bottom well plates coated with 0.5 µg/ml of OKT3 (Orthobiotech). On day 5, 1 µCi [3H]-thymidine (MP Biomedicals) was added for the final 16 h of culture and proliferation was determined on day 6 (Wallac; Perkin-Elmer). IL-2, IFN-, and TNF- levels were measured in days 1, 3, and 5 supernatants using a cytometric bead array kit (BD Biosciences). Intracellular detection of Th1 cytokines was performed on fresh PBMCs stimulated for 24 h with 5 ng/ml of PMA. 3 µg/ml of brefeldin A and 3 µg/ml of monensin were added after the first 2 h of culture in the presence of PMA (all obtained from Sigma-Aldrich). Stimulated cells were permeabilized and stained with anti-CD4–perCP, anti-IL-2-PE, anti–TNF-–allophycocyanin, and anti–IFN- –FITC (all obtained from BD Biosciences). None of the patients tested for proliferation and cytokine production were on steroids at the time of analysis.
TCRBV analysis.
Total RNA was extracted from FACS-sorted cells and reverse transcribed using a single-strand synthesis kit (Stratagene). Amplification reactions were performed using a BC1/BC2-specific primer (5'-CGGGCTGCTCCTTGAGGGGCTGCG-3') and a BV-specific primer (53). In brief, 2 µl RT product (corresponding to 2 x 104–105 CD4+CD25bright or CD4+CD25– cells) were brought to a final reaction volume of 50 µl containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3, 20 pmol of each oligonucleotide, 0.2 mM of each dNTP, and 2.5 U Taq DNA polymerase blocked by the addition of an anti-Taq mAb (Taq Start; CLONTECH Laboratories, Inc.). After an initial denaturation step of 3 min at 95°C, the reactions were subjected to 30 cycles of PCR (94°C for 30 s, 60°C for 1 min, 74°C for 1 min), followed by a final extension step of 5 min at 74°C. One nested BC oligonucleotide (5'-GTGCACCTCCTTCCCATTCA-3') was used dye-labeled (JOE Fluorophore; Applied Biosystems) in runoff reactions. 2 µL of PCR product was added to 8 µl of a mixture containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3, 0.2 mM each dNTP, 0.2 U Taq DNA polymerase, and 0.1 µM JOE Fluorophore–labeled oligonucleotide. The extension reaction consisted of a 3-min denaturation step at 95°C followed by 12 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min at 72°C. A final 10-min incubation at 72°C was performed. Runoff products were loaded on a 4% acrylamide-4 M urea sequencing gel and run on an ABI 377 DNA sequencer (Applied Biosystems). A mixture of dye-labeled size standards was also loaded on the sequencing gel to allow the precise determination of the sizes of the BC-BV runoff reaction products. The sizes and areas of the peaks corresponding to the DNA products were determined using the Immunoscope software (31). Observed peaks were usually separated by three bases and corresponded to in-frame transcripts of TCRs. Windows of analysis were centered on expected sizes corresponding to TCR transcripts encoding a 10 residue-long CDR3 region.
BV family-specific primers used were as follows: BV1, 5'-CCGCACAACAGTTCCCTGACTTGC-3'; BV2, 5'-GGCCACATACGAGCAAGGCGTCGA-3'; BV3, 5'-CGCTTCTTCCGGATTCTGGAGTCC-3'; BV4, 5'-TTCCCATCAGCCGCCCAAACCTAA-3'; BV5, 5'-AGCTCTGAGCTGAATGTGAACGCC-3'; BV7, 5'-CCTGAATGCCCCAACAGCTCTCTC-3'; BV8, 5'-CCATGATGCGGGGACTGGAGTTGC-3'; BV15, 5'-CAGGCACAGGCTAAATTCTCCCTG-3'; and BV16, 5'-GCCTGCAGAACTGGAGGATTCTG-3'.
Detection of FoxP3+ T cells.
Frozen lymph node tissue obtained from sarcoidosis patients (n = 10) and controls (n = 2) were stained with polyclonal goat anti–human FoxP3 (ab2481; Abcam) and mouse anti–human CD4 (MT310; DakoCytomation), followed by FITC-conjugated rat anti–mouse (145–095-166; Jackson ImmunoResearch Laboratories) and biotinylated rabbit anti–goat (E0466, DakoCytomation) followed by cyanine-3–conjugated streptavidin (PA43001; GE Healthcare). Fluorescent images of mounted sections were acquired with an epifluorescent microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) and analyzed with FluoUp image analysis software (Explora Nova).
Real-time FoxP3 PCR.
Real-time PCR was performed with a TaqMan assay on an ABI 7700 system (Applied Biosystems). Total RNA extracted from FACS sorted cells was immediately reverse transcribed in a 50 µL reaction volume (ProSTAR First Strand; Stratagene) according to the manufacturer's instructions. FoxP3 and HPRT-1 Assays-on-Demand gene expression probes (Hs 00203958 and 99999909, respectively; Applied Biosystems) were used. In each reaction, HPRT-1 was amplified as a housekeeping gene to calculate a standard curve and to correct for variations in target sample quantities. Relative copy numbers were calculated for each sample from the standard curve after normalization to HPRT-1 by the instrument software.
Single cell RT-PCR.
Peripheral blood lymphocytes were stained with anti–human CD4-FITC and anti–human CD25-PE (BD Biosciences). Single cells were sorted using a FACS Vantage (Becton Dickinson) into 96-well PCR plates (Abgene, Epsom). Each cell sample was dropped in 10 µl ice cold reaction buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.5 µg oligodT, 0.5 µg random primer, 10 mM DNTP, 10 mM DTT, 0.5% vol/vol NP-40, 12 U recombinant RNAsin (Promega), 25 U Stratascript RT (Stratagene), and 100 pmol of 3'FoxP3cDNA-(5'-AGGAGCCCTTGTCGGATGAT-3') and 3'CD3
cDNA-(5'-CTTGTTCCGAGCCCAG-3') specific primers. Plates were stored on ice until reverse transcription at 42°C for 1 h followed by a 10 min step at 94°C. Subsequently, 2.5 µl of cDNA or first PCR product were used to amplify transcripts by two steps of PCR in a final volume of 32.5 µl. In the first round, multiplex PCR reaction was performed using 0.4 µM of each following oligonucleotide: 5'FoxP3-ext (5'-TTCATGCACCAGCTCTCAACG-3'), 3'FoxP3-ext (5'-CTTCTCCAGCACCAGCTGCTG-3'), 5'CD3-ext (5'-GGGAACGGTGGGAACACTGC-3'), 3'CD3-ext (5'-AAAGCAAGGAGCAGAGTGGC-3'). Reactions were subjected after 5 min at 94°C to 8 cycles (94°C for 30 s, 60°C for 40 s, 72°C for 50 s), 32 cycles (94°C for 30 s, 55°C for 40 s, 72°C for 50 s), and a final elongation at 72°C for 5 min. In a second PCR round, each gene was amplified separately using nested primers 5'FoxP3-int (5'-GGCCTCCCACCTGGGATCAAC-3') and 3'FoxP3-int (5'-CGCCTGGCAGTGCTTGAGGAA-3') or 5'CD3-int (5'-GACTGGACCTGGGAAAACGC-3') and 3'CD3-int (5'-CAATGATGCCAGCCACGGTG-3'). PCR was performed as in the first step. Reaction products were visualized by electrophoresis on a 2% agarose gel.
Statistical analysis.
Comparisons between active and inactive sarcoidosis patients and control subjects were made using the nonparametric Mann-Whitney U test. Comparisons of the rate of circulating CD25bright CD4+ T cells variation during the evolution of the disease within the same individuals were made using the Wilcoxon Signed Rank test (54). p-values <0.05 were considered significant. Statistical analyses were performed with Statview 5.0 software (SAS Institute).
|
Acknowledgments |
|---|
M. Miyara was supported by the Fond d'Etude et de Recherche du Corps Médical des Hôpitaux de Paris and was the recipient of a Fondation Line Pomaret Delalande price hosted by Fondation pour la Recherche Médicale, Paris. This study was financially supported by the European Union (ATTACK project), by the Institut National de la Santé et de la Recherche Médicale, and by Assistance Publique-Hôpitaux de Paris (CIB Pitié-Salpétrière).
The authors have no conflicting financial interests.
Submitted: 30 March 2005
Accepted: 22 December 2005
|
References |
|---|
|
TopAbstractRESULTSDISCUSSIONMATERIALS AND METHODSReferences |
|---|
1 Hunninghake, G.W., and R.G. Crystal. 1981. Pulmonary sarcoidosis: a disorder mediated by excess helper T-lymphocyte activity at sites of disease activity. N. Engl. J. Med. 305:429–434.[Abstract]
2 Baumer, I., G. Zissel, M. Schlaak, and J. Muller-Quernheim. 1997. Th1/Th2 cell distribution in pulmonary sarcoidosis. Am. J. Respir. Cell Mol. Biol. 16:171–177.[Abstract]
3 Newman, L.S., C.S. Rose, and L.A. Maier. 1997. Sarcoidosis. N. Engl. J. Med. 336:1224–1234.
4 Baughman, R.P., E.E. Lower, and R.M. du Bois. 2003. Sarcoidosis. Lancet. 361:1111–1118.[CrossRef][Medline]
5 Lecossier, D., D. Valeyre, A. Loiseau, J. Cadranel, A. Tazi, J.P. Battesti, and A.J. Hance. 1991. Antigen-induced proliferative response of lavage and blood T lymphocytes. Comparison of cells from normal subjects and patients with sarcoidosis. Am. Rev. Respir. Dis. 144:861–868.[Medline]
6 Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor -chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151–1164.[Abstract]
7 Shevach, E.M. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389–400.[Medline]
8 Cottrez, F., S.D. Hurst, R.L. Coffman, and H. Groux. 2000. T regulatory cells 1 inhibit a Th2-specific response in vivo. J. Immunol. 165:4848–4853.
9 Groux, H., A. O'Garra, M. Bigler, M. Rouleau, S. Antonenko, J.E. de Vries, and M.G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 389:737–742.[CrossRef][Medline]
10 Weiner, H.L. 2001. Induction and mechanism of action of transforming growth factor-ß-secreting Th3 regulatory cells. Immunol. Rev. 182:207–214.[CrossRef][Medline]
11 Taams, L.S., J. Smith, M.H. Rustin, M. Salmon, L.W. Poulter, and A.N. Akbar. 2001. Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population. Eur. J. Immunol. 31:1122–1131.[CrossRef][Medline]
12 Dieckmann, D., H. Plottner, S. Berchtold, T. Berger, and G. Schuler. 2001. Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 193:1303–1310.
13 Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, and A.H. Enk. 2001. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285–1294.
14 Annunziato, F., L. Cosmi, F. Liotta, E. Lazzeri, R. Manetti, V. Vanini, P. Romagnani, E. Maggi, and S. Romagnani. 2002. Phenotype, localization, and mechanism of suppression of CD4+CD25+ human thymocytes. J. Exp. Med. 196:379–387.
15 Stephens, L.A., C. Mottet, D. Mason, and F. Powrie. 2001. Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur. J. Immunol. 31:1247–1254.[CrossRef][Medline]
16 Baecher-Allan, C., J.A. Brown, G.J. Freeman, and D.A. Hafler. 2001. CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167:1245–1253.
17 Baecher-Allan, C., and D.A. Hafler. 2004. Suppressor T cells in human diseases. J. Exp. Med. 200:273–276.
18 O'Garra, A., and P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat. Med. 10:801–805.[CrossRef][Medline]
19 Curiel, T.J., G. Coukos, L. Zou, X. Alvarez, P. Cheng, P. Mottram, M. Evdemon-Hogan, J.R. Conejo-Garcia, L. Zhang, M. Burow, et al. 2004. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10:942–949.[CrossRef][Medline]
20 Tarbell, K.V., S. Yamazaki, K. Olson, P. Toy, and R.M. Steinman. 2004. CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 199:1467–1477.
21 Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp 3. Science. 299:1057–1061.
22 Fontenot, J.D., M.A. Gavin, and A.Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330–336.[CrossRef][Medline]
23 Khattri, R., T. Cox, S.A. Yasayko, and F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4:337–342.[CrossRef][Medline]
24 Read, S., V. Malmstrom, and F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295–302.
25 Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T.W. Mak, and S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303–310.
26 Amoura, Z., C. Combadiere, S. Faure, C. Parizot, M. Miyara, D. Raphael, P. Ghillani, P. Debre, J.C. Piette, and G. Gorochov. 2003. Roles of CCR2 and CXCR3 in the T cell-mediated response occurring during lupus flares. Arthritis Rheum. 48:3487–3496.[CrossRef][Medline]
27 Gorochov, G., A.U. Neumann, A. Kereveur, C. Parizot, T. Li, C. Katlama, M. Karmochkine, G. Raguin, B. Autran, and P. Debre. 1998. Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy. Nat. Med. 4:215–221.[CrossRef][Medline]
28 Gorochov, G., A.U. Neumann, C. Parizot, T. Li, C. Katlama, and P. Debre. 2001. Down-regulation of CD8+ T-cell expansions in patients with human immunodeficiency virus infection receiving highly active combination therapy. Blood. 97:1787–1795.
29 Roglic, M., R.D. Macphee, S.R. Duncan, F.R. Sattler, and A.N. Theofilopoulos. 1997. T cell receptor (TCR) BV gene repertoires and clonal expansions of CD4 cells in patients with HIV infections. Clin. Exp. Immunol. 107:21–30.[CrossRef][Medline]
30 van den Beemd, R., P.P. Boor, E.G. van Lochem, W.C. Hop, A.W. Langerak, I.L. Wolvers-Tettero, H. Hooijkaas, and J.J. van Dongen. 2000. Flow cytometric analysis of the Vß repertoire in healthy controls. Cytometry. 40:336–345.[CrossRef][Medline]
31 Pannetier, C., M. Cochet, S. Darche, A. Casrouge, M. Zoller, and P. Kourilsky. 1993. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor ß chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA. 90:4319–4323.
32 Viguier, M., F. Lemaitre, O. Verola, M.S. Cho, G. Gorochov, L. Dubertret, H. Bachelez, P. Kourilsky, and L. Ferradini. 2004. Foxp3 expressing CD4+CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J. Immunol. 173:1444–1453.
33 Sugiyama, H., R. Gyulai, E. Toichi, E. Garaczi, S. Shimada, S.R. Stevens, T.S. McCormick, and K.D. Cooper. 2005. Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J. Immunol. 174:164–173.
34 Planck, A., K. Katchar, A. Eklund, S. Gripenback, and J. Grunewald. 2003. T-lymphocyte activity in HLA-DR17 positive patients with active and clinically recovered sarcoidosis. Sarcoidosis Vasc. Diffuse Lung Dis. 20:110–117.[Medline]
35 Kindler, V., A.P. Sappino, G.E. Grau, P.F. Piguet, and P. Vassalli. 1989. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell. 56:731–740.[CrossRef][Medline]
36 Baughman, R.P., and E.E. Lower. 2001. Infliximab for refractory sarcoidosis. Sarcoidosis Vasc. Diffuse Lung Dis. 18:70–74.[Medline]
37 Ehrenstein, M.R., J.G. Evans, A. Singh, S. Moore, G. Warnes, D.A. Isenberg, and C. Mauri. 2004. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF therapy. J. Exp. Med. 200:277–285.
38 Viglietta, V., C. Baecher-Allan, H.L. Weiner, and D.A. Hafler. 2004. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. 199:971–979.
39 Annacker, O., O. Burlen-Defranoux, R. Pimenta-Araujo, A. Cumano, and A. Bandeira. 2000. Regulatory CD4 T cells control the size of the peripheral activated/memory CD4 T cell compartment. J. Immunol. 164:3573–3580.
40 Inoue, T., S. Fujishima, E. Ikeda, O. Yoshie, N. Tsukamoto, S. Aiso, N. Aikawa, A. Kubo, K. Matsushima, and K. Yamaguchi. 2004. CCL22 and CCL17 in rat radiation pneumonitis and in human idiopathic pulmonary fibrosis. Eur. Respir. J. 24:49–56.
41 Agostini, C., M. Cassatella, R. Zambello, L. Trentin, S. Gasperini, A. Perin, F. Piazza, M. Siviero, M. Facco, M. Dziejman, et al. 1998. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J. Immunol. 161:6413–6420.
42 Forman, J.D., J.T. Klein, R.F. Silver, M.C. Liu, B.M. Greenlee, and D.R. Moller. 1994. Selective activation and accumulation of oligoclonal Vß-specific T cells in active pulmonary sarcoidosis. J. Clin. Invest. 94:1533–1542.[Medline]
43 Grunewald, J., C.H. Janson, A. Eklund, M. Ohrn, O. Olerup, U. Persson, and H. Wigzell. 1992. Restricted V 2.3 gene usage by CD4+ T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients correlates with HLA-DR 3. Eur. J. Immunol. 22:129–135.[Medline]
44 Forrester, J.M., Y. Wang, N. Ricalton, J.E. Fitzgerald, J. Loveless, L.S. Newman, T.E. King, and B.L. Kotzin. 1994. TCR expression of activated T cell clones in the lungs of patients with pulmonary sarcoidosis. J. Immunol. 153:4291–4302.[Abstract]
45 Klein, J.T., T.D. Horn, J.D. Forman, R.F. Silver, A.S. Teirstein, and D.R. Moller. 1995. Selection of oligoclonal Vß-specific T cells in the intradermal response to Kveim-Siltzbach reagent in individuals with sarcoidosis. J. Immunol. 154:1450–1460.[Abstract]
46 Song, Z., L. Marzilli, B.M. Greenlee, E.S. Chen, R.F. Silver, F.B. Askin, A.S. Teirstein, Y. Zhang, R.J. Cotter, and D.R. Moller. 2005. Mycobacterial catalase-peroxidase is a tissue antigen and target of the adaptive immune response in systemic sarcoidosis. J. Exp. Med. 201:755–767.
47 Pinkston, P., P.B. Bitterman, and R.G. Crystal. 1983. Spontaneous release of interleukin-2 by lung T lymphocytes in active pulmonary sarcoidosis. N. Engl. J. Med. 308:793–800.[Abstract]
48 Clark, F.J., R. Gregg, K. Piper, D. Dunnion, L. Freeman, M. Griffiths, G. Begum, P. Mahendra, C. Craddock, P. Moss, and R. Chakraverty. 2004. Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4+CD25high regulatory T cells. Blood. 103:2410–2416.
49 Cao, D., V. Malmstrom, C. Baecher-Allan, D. Hafler, L. Klareskog, and C. Trollmo. 2003. Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis. Eur. J. Immunol. 33:215–223.[CrossRef][Medline]
50 Marshall, N.A., L.E. Christie, L.R. Munro, D.J. Culligan, P.W. Johnston, R.N. Barker, and M.A. Vickers. 2004. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma. Blood. 103:1755–1762.
51 Ross, J.J., and J.D. Katz. 2002. Cryptococcal meningitis and sarcoidosis. Scand. J. Infect. Dis. 34:937–939.[CrossRef][Medline]
52 Askling, J., J. Grunewald, A. Eklund, G. Hillerdal, and A. Ekbom. 1999. Increased risk for cancer following sarcoidosis. Am. J. Respir. Crit. Care Med. 160:1668–1672.
53 Gorochov, G., P. Debre, V. Leblond, B. Sadat-Sowti, F. Sigaux, and B. Autran. 1994. Oligoclonal expansion of CD8+ CD57+ T cells with restricted T-cell receptor ß chain variability after bone marrow transplantation. Blood. 83:587–595.
54 Kuzma, J., and S. Bohnenblust. 1998. Basic Statistics For Health Science. McGraw-Hill Humanities/Social Sciences/Languages, New York, NY. 400 pp.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
Related Article
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
