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Keywords:

  • colitis;
  • ulcerative;
  • FOXP3 protein;
  • T-lymphocytes;
  • regulatory;
  • cytokines

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Background: CD4+CD25+ regulatory T cells (TR) can prevent or treat experimental murine colitis but little is known about their potential role in human inflammatory bowel disease (IBD). FOXP3 is a transcription factor that plays a critical role in the development and function of CD4+CD25+ TR. The aim of this study was to examine the presence and functional characteristics of TR cells in colonic lymphoid tissues in patients with ulcerative colitis (UC).

Methods: FOXP3 expression was assessed by flow cytometry, immunohistochemistry, and reverse-transcriptase polymerase chain reaction (RT-PCR). Functional characterization of CD4+CD25+ cells was analyzed by suppression of proliferation and secretion of cytokines by cocultured effector CD4+CD25 T cells.

Results: FOXP3+CD4+ T cells are increased in the lamina propria (LP) of inflamed and noninflamed areas of UC colon compared to normal colon. CD4+CD25+ T cells in UC mesenteric lymph nodes (MLN) express FOXP3 mRNA and protein and suppress the proliferation of autologous MLN CD4+CD25 T cells. The suppressor activity of MLN CD4+CD25+ T cells is cell contact-dependent but cytokine-independent. In addition, CD4+CD25+ T cells potently suppress the production of both Th1 (IFN-γ, IL-2) and Th2 (IL-5, IL-13) cytokines by cocultured CD4+CD25 T cells. FOXP3+ cells localized in the T-cell-rich areas of MLN and occasionally present in the follicles.

Conclusions: There is an expansion of FOXP3+CD4+ T cells in mucosal lymphoid tissues in UC. CD4+CD25+ isolated from UC MLN express FOXP3 and display features of TR cells in spite of active mucosal inflammation. These data suggest that their suppressor activity may be abrogated in vivo or they are unable to counterbalance the chronic mucosal inflammation in UC.

(Inflamm Bowel Dis 2007)

Studies in rodents and humans have identified the existence of a naturally occurring population of CD4+CD25+ regulatory T cells (TR) that suppress the proliferation and cytokine production of effector T cells upon in vitro TCR-mediated stimulation.1–5 TR cells can suppress the responses of autoreactive T cells in vivo and prevent the development of autoimmune diseases.6–10 FOXP3 is a member of the forkhead-winged helix family of transcription factors, which plays a critical role in the development and function of CD4+CD25+ TR.11–13 A mutation in the gene encoding FOXP3 was identified as the genetic defect underlying autoimmune and inflammatory disease in scurfy mice and in humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) or XLAAD (X-linked autoimmunity allergic dysregulation syndrome),14–16 establishing the importance of CD4+CD25+ TR in the maintenance of normal immune homeostasis.17 CD4+CD25+ TR are produced mainly in the thymus but can also be induced in the periphery under various stimulatory conditions.18–22 A recent study has shown that ex vivo TGF-β-induced FOXP3+ TR are able to suppress the development of experimental colitis upon CD4+CD62L+ T cell transfer in vivo.23 Adoptive transfer of CD4+CD25+ TR cells can cure established experimental colitis, suggesting their potential utility of ex vivo-generated TR for the treatment of inflammatory bowel disease (IBD).24 CD4+ CD25+ TR cells may also control intestinal inflammation induced by both innate and adaptive immune responses via IL-10 and TGF-β-dependent mechanisms.24

Specific T-cell subsets in the intestinal mucosa are poised with regulatory activity, therefore limiting the development of T-cell responses to intestinal commensal bacteria and the development of pathologic intestinal inflammation.24–27 Of these T-cell subsets, T regulatory 1 (Tr1), Th3, CD8+ TrE, and CD4+CD25+ natural TR cells are the ones best characterized thus far.25, 28–32 A lack of activation and/or expansion of these regulatory cells could, therefore, play a role in the uncontrolled inflammation seen in IBD.25

Although CD4+CD25+ TR cells could play an important role in mucosal inflammation, there is still little information regarding their localization in mucosal lymphoid tissues, the mechanisms by which these cells are generated and the potential functional impairment of CD4+CD25+ TR cells as the underlying abnormality in human IBD. Several recent studies have indicated an alteration of the frequency of these cells in the peripheral blood (PB) and the mucosa of patients with IBD.33–35 Since large numbers of mucosally activated lymphocytes reach the blood circulation after expansion and maturation within the mesenteric lymph nodes (MLN), we reasoned that the presence of CD4+CD25+ TR in MLN may be important in the control of effector T-cell expansion and differentiation.36 In the current study we examined the frequency of FOXP3+CD4+ T cells in mucosal lymphoid tissues in UC and examined the regulatory function of these cells in colonic draining MLN in severe UC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients

We studied 10 patients with severe UC who underwent surgery for medically refractory disease to characterize the regulatory activity of MLN CD4+CD25+ T cells. All patients except one were receiving mesalamine (5-ASA) and corticosteroids either intravenously or orally before their colectomy. One patient had received treatment with intravenous cyclosporine, which was discontinued prior to surgery. We also studied nine healthy controls and 13 additional patients with UC (six active, seven inactive) for the enumeration of FOXP3+ T cells in intestinal tissues by flow cytometry. Three of UC patients received oral mesalamine only, two were on mercaptopurine, two were on no medications, and two were on corticosteroids (some of the inflamed and noninflamed tissue samples were obtained from paired specimens). The study was approved by the Cedars-Sinai Institutional Review Board (IRB) (protocol #4359)

Antibodies and Reagents

The anti-CD25 PE-conjugated antibody (Ab) was purchased from Beckman Coulter (Fullerton, CA) and the CD4-APC or -TC conjugated Ab was from Caltag (Burlingame, CA). The CD8-PE and CD3-PE or -TC conjugated Ab was from Caltag. The FITC-conjugated antihuman FOXP3 antibody (clone PCH101) was from eBioscience (San Diego, CA) and the anti-FOXP3 236A/E7 was raised in the Monoclonal Antibodies Unit of the CNIO (Centro Nacional de Investigaciones Oncológicas) (provided by A.H.B.). The blocking anti-TGFβ1, -β2, -β3 Ab and recombinant human LAP (TGF-β1) was from R&D Systems (Minneapolis, MN). The neutralizing anti-IL-10 Ab (JES3-9D7) was from BioSource (Sunnyvale, CA). Anti-CD3 Ab (OKT3; obtained from ATCC, Rockville, MD) was purified from hybridoma culture supernatants using protein G-Sepharose affinity chromatography. GM-CSF and IL-4 were purchased from Peprotech (Rocky Hill, NJ). RPMI 1640 supplemented with L-glutamine and antibiotics was used throughout the experiments (complete RPMI).

Isolation and Purification of T-Cell Subsets

Allogeneic PBMC (peripheral blood mononuclear cells) were isolated from heparinized blood using Ficoll-Hypaque, irradiated at 3300 rads, and used as APC (antigen presenting cells). In some experiments, allogeneic dendritic cells (DC) were used as APC. DC were generated by plastic adherence of PBMC and culture in GM-CSF and IL-4 for 6 days as previously described.37 Lymphocytes were isolated from MLN from patients undergoing surgical resection for treatment-refractory UC following mechanical disruption and release of cells in complete RPMI. MLN lymphocytes were subsequently stained with CD4-FITC and CD25-PE and sorted into CD4+CD25 and CD4+CD25+ cells by FACS (fluorescence activated cell sorting). In some experiments CD4+CD25 and CD4+CD25+ cells were separated by MACS using the CD4+CD25+ regulatory T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA). The purity of CD4+CD25 and CD4+CD25+ cells with this method were >99% and >85%, respectively. Lamina propria lymphocytes (LPL) were isolated as previously described.38

FACS Analysis

LP and MLN lymphocytes were first stained for surface CD4 and CD25 using CD4-APC and CD25-PE for 20 minutes at 4°C in phosphate-buffered saline (PBS) plus 2% fetal calf serum (FCS), washed twice, and fixed. The cells were subsequently stained for intracellular FOXP3 using the anti-FOXP3-FITC (clone PCH101) and staining buffers as recommended by the manufacturer (eBioscience) and analyzed by FACS.

Proliferation Assays

T-cell proliferation assays were performed in FACS -or MACS-sorted CD4+CD25 (5 × 104/well) and CD4+CD25+ cells, cocultured in 96-round-bottom well plates with 2 × 104 APC or 104 DC and soluble OKT3 (100 ng/mL) for 5 days in triplicate. Following a 16-hour pulse with [3H]thymidine at 1 μCi/well, proliferation was analyzed in a scintillation counter. For transwell experiments, cells were cultured in 24-well plates, with or without a 0.4-μm transwell separating CD4+CD25+ (5 × 104 cells per well) cells from CD4+CD25 (5 × 104 cells per well).

Analysis of FOXP3 Expression by Reverse-transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from FACS-sorted CD4+CD25 and CD4+CD25+ MLN cells using TRIZOL reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). FOXP3 mRNA expression was analyzed in each cell subset using 1 μg of isolated RNA and 0.6 μM primers in a one-step RT-PCR reaction as recommended by the manufacturer (Qiagen, Valencia, CA). The cycle parameters were an initial melt at 95°C for 15 minutes, then 30 cycles: 94°C, 30 seconds; 55°C, 30 seconds; and 72°C, 60 seconds, followed by a final extension of 72°C, 10 minutes. Primers for FOXP3 were: sense 5′-GAA ACA GCA CAT TCC CAG AGT TC-3′, antisense 5′-CAA GAC AGT GGA AAC CTC ACT TC-3′ (product 476 bp). Amplification with β-actin primers sense 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′, antisense 5′-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3′ (product 661 bp) (ClonTech, Palo Alto, CA) was examined under identical conditions as an internal control to demonstrate equivalence of template. The PCR products were visualized with ethidium bromide after 1.5% agarose gel electrophoresis. The intensity of the bands was assessed by densitometry and the ratio of FOXP3 to β-actin mRNA was calculated (Scanalytics, Rockville, MD).

Immunohistochemistry and Double Immunoenzymatic Labeling

Four-μm paraffin sections were cut from paraffin blocks of MLN from patients with UC and MLN from unaffected individuals. Sections were dewaxed in citroclear (HD Supplies, Aylesbury, UK) and antigen retrieval was performed by microwave pressure cooking for 3 minutes at full pressure in 50 mM Tris; 2 mM EDTA, pH 9. Sections were incubated with hybridoma supernatant from the previously described39 236A/E7 FOXP3 monoclonal antibody for 30 minutes and immunoperoxidase detection was performed using the EnVision system (DakoCytomation Glostrup, Denmark). Sections were then counterstained with hematoxylin (Gill No. 3, Sigma, UK) and mounted in Aquamount (BDH, UK). Double enzymatic immunostaining for FOXP3 (236A/E7) and CD3 (1:80, F7.2.38, DakoCytomation) was also performed on the MLN paraffin sections. Double immunoenzymatic labeling of paraffin sections following the described pretreatment was carried out by means of the EnVision peroxidase and alkaline phosphatase kits (DakoCytomation). Primary antibodies were incubated for 30 minutes at room temperature and a diaminobenzidine (DAB) substrate (DakoCytomation) was then used for detection of antibody binding. Sections were then incubated for 30 minutes with the second antibody, then for an additional 30 minutes with the alkaline phosphatase EnVision kit (DakoCytomation). The second reaction was detected by means of a Vector blue alkaline phosphatase substrate kit (Vector Laboratories, Peterborough, UK). The sections were washed in tap water and mounted in aquamount (Merck, Poole, UK).40

Analysis of Cytokine Production

The cytokines IL-2, IL-5, IL-13, and IFN-γ were analyzed in culture supernatants using the cytokine Beadlyte Multiplex assay as recommended by the manufacturer (Upstate, Lake Placid, NY).

Statistical Analysis

Differences between the mean values for groups were analyzed by Student's t-test. A P-value of < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Increased Frequency of FOXP3+CD4+ T Cells in Mucosal Lymphoid Tissues in UC

We first examined the frequency of FOXP3+CD4+ T cells in the LP from normal and UC intestinal specimens. As shown in Figure 1A, the frequency of FOXP3+CD4+ T cells was significantly higher in both inflamed (15.5 ± 9.6%) and noninflamed (7.5 ± 2.6%) UC LPL compared to normal LPL (3.1 ± 1%). A representative dot plot of FOXP3 and CD25 staining of normal LPL and of a paired sample from inflamed and noninflamed UC LPL is shown in Figure 1B. Since CD25 expression has been found to be the best cell surface marker associated with FOXP3+CD4+ T cells, we analyzed the expression of FOXP3 among CD25+ and CD25CD4+ T cells in LPL and compared it with normal peripheral blood lymphocytes (PBL) as a reference point. There was a better correlation between CD25 and FOXP3 expression in PBL compared to LPL (Fig. 1C). The percentage of FOXP3+ cells among CD4+CD25+ cells was 85 ± 6% in PBL compared to only 46 ± 14% in normal LPL (P < 0.001) (Fig. 1C). There were no differences in the percentage of FOXP3+ cells among CD4+CD25 cells between PBL and LPL (2.2% versus 1.8%, P = 0.65). Interestingly, the frequency of FOXP3+ cells among CD4+CD25+ cells was significantly higher in inflamed or noninflamed UC LPL compared to normal LPL (Fig. 1C), suggesting that the majority of CD25+ T cells in the UC LP may be regulatory T cells.

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Figure 1. Increased frequency of FOXP3+CD4+ T cells in mucosal lymphoid tissues in UC. Cells from normal or UC colon were isolated and stained with CD4-APC, CD25-PE, and FOXP3-FITC and analyzed by flow cytometry. (A) Average FOXP3+CD4+ T cells from normal colon (n = 9), inflamed (I) (n = 6), and uninflamed (U) UC colon (n = 7) *P = 0.002, **P < 0.001. (B) Representative dot plot of CD25 versus FOXP3 or isotype control staining among CD4+ T cells isolated form normal LPL and a paired sample of inflamed (I) and uninflamed (U) UC colon. (C) Frequency of FOXP3+ among CD25+ and CD25CD4+ T cells isolated from normal LPL and inflamed (I) and uninflamed (U) UC LPL and normal PBL. *P = 0.005, **P = 0.003, ***P < 0.001, +P = 0.01. All comparisons were made to normal LPL.

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We next analyzed the correlation of CD25 and FOXP3 expression in colonic draining MLN in UC. We found that MLN CD4+CD25+ cells also expressed FOXP3 mRNA and protein, indicating that the majority of CD4+CD25+ T cells in UC MLN may in fact be TR (Fig. 2). When analyzed by flow cytometry, the percentage of FOXP3+ cells was 66 ± 8.5% among CD4+CD25+ compared to only 4.4 ± 2.4% among CD4+CD25 T cells isolated from UC MLN (Fig. 2B) (n = 6, P < 0.0001). The average percentage of FOXP3+CD4+ T cells in UC MLN was 12.4% (range, 9–22%, n = 6).

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Figure 2. FOXP3 expression in CD4+ T cells correlate with CD25 expression in UC MLN. (A) Mononuclear cells from UC MLN were stained with CD4-TC and CD25-PE and sorted into CD25+ and CD25 populations as shown in (A) and RNA was extracted. RT-PCR for β-actin (661 bp) and FOXP3 (476 bp) was performed as described in Materials and Methods. A representative experiment is shown in the middle panel and the FOXP3/β-actin ratio from 3 independent experiments among CD25+ and CD25 T cells as determined by densitometry is shown in the lower panel. *P = 0.03. (B) FOXP3 protein expression in CD4+ T cells from UC MLN was analyzed by flow cytometry. The mean percentage of FOXP3+ cells among CD25+ or CD4+CD25 cells from six different donors is shown in the lower panel. **P < 0.0001.

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MLN CD4+CD25+ Cells in Human UC Are Regulatory T Cells

The high FOXP3 expression by CD4+CD25+ cells from MLN in UC raised the possibility that these cells may be TR. Therefore, we purified MLN CD4+CD25+ cells by FACS and performed coculture experiments with autologous MLN CD4+CD25 cells as effectors in the presence of irradiated PBMC as APC and soluble OKT3 at 100 ng/mL. In preliminary experiments we determined that the optimal conditions to reveal TR activity by MLN CD4+CD25+ T cells was to use suboptimal T-cell receptor activation such as soluble OKT3 at a concentration of 10–100 ng/mL (data not shown). As shown in Figure 3A, CD4+CD25+ T cells suppressed the proliferation of autologous MLN CD4+CD25 cells in a dose-dependent manner. Maximal inhibition of proliferation was achieved at a ratio of responder to TR of 1:0.5 (Fig. 3A). Similar inhibition was observed when CD4+CD25+ were cocultured with CD4+CD25 cells in the presence of allogeneic DC (Fig. 3B). Maximal suppression of effector T-cell proliferation was seen at a responder to Treg ratio of 1:0.2. The average proliferation of CD4+CD25 cells in the presence of CD4+CD25+ T cells at a ratio of 1:0.5 was 18 ± 11% (Fig. 3C) (n = 10, *P = 0.001).

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Figure 3. CD4+CD25+ MLN T cells suppress the proliferation of autologous CD4+CD25 T cells. Sorted CD4+CD25 MLN T cells (responder) isolated from UC surgical specimens were cocultured with an increasing number of CD4+CD25+ T cells and (A) 2 × 104 irradiated allogeneic PBMC plus anti-CD3 (100 ng/mL) or (B) 104 immature allogeneic DC for 5 days in triplicate 96-round-bottom well plates but without OKT3. On day 5 cells were pulsed for 16 hours with [3H]thymidine at 1 μCi/well and proliferation was analyzed in a scintillation counter. (C) The mean percent of proliferation ± SEM of the responder to TR (ratio 1:0.5) coculture of 10 experiments from different UC MLN donors (*P = 0.001).

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MLN TR Suppression Is Cell Contact-Dependent but Cytokine-Independent

There is agreement that the in vitro suppression mediated by TR cells is mediated through cell contact. In some experimental systems the in vivo suppressive effects of TR cells are mediated through TGF-β and/or IL-10.41 In addition, CTLA-4 appears to play a critical role in the suppressive activity of murine TR in experimental colitis in vivo42 and some experimental systems in vitro.5, 43 To begin to elucidate the potential mechanism of immune suppression by UC MLN CD4+CD25+ we performed suppression assays with transwells and in the presence or absence of neutralizing antibodies to TGF-β, IL-10, or recombinant LAP (rLAP) (which blocks transmembrane TGF-β signaling). As shown in Figure 4A, the CD25+ TR-mediated suppression of CD4+CD25 cells is cell contact-dependent since separation of anti-CD3/APC stimulated CD25+ from CD4+CD25 cells by a transwell abrogated suppression of these cells. As a control, coculture of CD4+CD25 with CD25 cells did not have any significant effect on proliferation whether or not separated by a transwell (Fig. 4A). The suppression mediated by CD25+ TR cells was not reversed by the addition of a neutralizing anti-TGF-β1, -β2, -β3 Ab, anti-IL10 Ab, or rLAP (Fig. 4B). Addition of exogenous IL-2 restored the proliferation of effector CD25 T cells (Fig. 4B), indicating that the mechanism of suppression mediated by TR cells is through inhibition of IL-2 production or increased consumption.43 Our data show that TR-mediated suppression is cell contact-dependent but cytokine-independent.

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Figure 4. CD4+CD25+-induced suppression is cell contact-dependent but cytokine-independent. (A) Proliferation and suppression of UC MLN CD4+CD25 T cells by CD4+CD25+ or CD4+CD25 T cells. CD4+CD25+ and CD4+CD25 cells from the same donor were sorted by FACS and cultured alone, together with CD25 cells, or separated by a transwell. (B) UC MLN cells were sorted into CD4+CD25 and CD4+CD25+ and cocultured in the presence of 104 immature allogeneic DC alone or in the presence of blocking antibodies against TGF-β1, -2, -3, IL-10, or both, in the presence of recombinant LAP (10 μg/mL) or exogenous IL-2 (100 U/mL) for 5 days in triplicate 96-round-bottom well plates. On day 5 cells were pulsed for 16 hours with [3H]thymidine at 1 μCi/well and proliferation was analyzed in a scintillation counter. Representative of three experiments from different donors with similar results is shown.

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MLN TR Suppress the Production of Both Th1 and Th2 Cytokines by Effector CD4+ T Cells

Since we showed that CD4+CD25+ T cells from UC MLN can potently suppress the proliferation of effector CD4+CD25 T cells, it was important to determine whether they can also suppress the production of proinflammatory cytokines. In some recent studies, although the frequency and suppressor function of TR in some disease states, for example, rheumatoid arthritis, are preserved, their ability to inhibit the production of proinflammatory cytokines, which could account for the persistence of active inflammation, is lost.44 To determine if such a defect in cytokine suppression ability of UC MLN TR exists we cocultured CD4+CD25 T cells with allogeneic DC and CD4+CD25+ T cells at a 1:1 ratio for 5 days. Supernatants were collected and analyzed for the presence of IL-2, IFN-γ (Th1 cytokines), IL-5, and IL-13 (Th2 cytokines). As shown in Figure 5, CD4+CD25 T cells produced IL-2 and IFN-γ but also large amounts of the Th2 cytokines IL-5 and IL-13. Coculture of CD4+CD25 T cells with CD4+CD25+ TR cells at a 1:1 ratio resulted in almost complete inhibition of Th1 and Th2 cytokine secretion in the culture supernatants. Supernatants from cultures of CD4+CD25+ TR cells alone with dendritic cells contained few cytokines (Fig. 5). These data clearly indicate that CD4+CD25+ TR cells themselves produce no cytokines but that they potently suppress not only the proliferation but also the secretion of both Th1 and Th2 cytokines by effector CD4+CD25 T cells.

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Figure 5. CD4+CD25+ T cells potently suppress the production of both Th1 and Th2 cytokines. UC MLN cells were sorted into CD4+CD25 and CD4+CD25+ and cocultured in the presence of 104 immature allogeneic DC for 5 days. Supernatants were collected and analyzed for IL-2, IFN-γ, IL-5, and IL-13 content by the cytokine Beadlyte Multiplex assay. The amount of IL-13 detected in the culture supernatant of CD4+CD25 cells was 12,339 ± 215 pg/mL. Representative of two experiments with similar results is shown.

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Localization of FOXP3+ Cells in the T-Cell-Rich Areas of UC MLN

We next performed immunohistochemistry (IHC) to determine the localization of FOXP3+ cells in the different areas of MLN. As shown in Figure 6, intense staining of FOXP3+ cells was seen in both normal and UC MLN (Fig. 6A,B). FOXP3+ cells clustered in the T-cell-rich areas of MLN (Fig. 6C) and in some cases around the follicles (Fig. 6A). Double immunoenzymatic labeling revealed that FOXP3-expressing cells are CD3+ T cells (Fig. 6D). By flow cytometry all FOXP3+ cells are CD4+, but not CD8+ T cells (data not shown).

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Figure 6. Localization of FOXP3+ cells in colonic MLN. FOXP3 staining of normal (A) and UC (B) MLN (brown color). Numerous FOXP3+ cells are detected in the T-cell-rich areas of the MLN and occasional cells are present in the follicles including the mantle zone (A). (C,D) Double immunoenzymatic labeling for FOXP3 (brown) and CD3 (blue) in UC MLN. Magnification (A–C) ×100, (D) ×400.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The present study shows that FOXP3+ TR cells are expanded in inflamed and uninflamed colon in UC. Moreover, “functional” CD4+CD25+FOXP3+ TR cells are present in the colonic MLN of UC patients undergoing colectomy for medically refractory disease. These cells exhibit potent suppressor activity in vitro in terms of inhibiting the proliferation of effector CD4+CD25 T cells and the production of both Th1 and Th2 cytokines. The CD25+ TR function is cell contact-dependent but cytokine-independent.

Several reports have described the presence and functional characteristics of CD4+CD25hi T cells in PB and target tissues in patients with several autoimmune or immune-mediated diseases,44–47 including IBD,33–35, 48 but this is the first report, to our knowledge, of the detection of FOXP3+ TR in the organ draining MLN of any human autoimmune or immune-mediated disease. Since T-cell priming, expansion, and differentiation occurs in organ draining MLN, the presence of CD4+CD25+FOXP3+ TR in colonic MLN may inhibit the expansion and differentiation of antigen-primed effector CD4+ T cells by mucosally derived antigens. Recent experimental data support the notion that TR cell interactions in organ draining LN may be critical to suppress autoimmune disease.49, 50 Previous in vitro studies have indicated the ability of TR to inhibit the stimulatory capacity of dendritic cells and this may account for the main regulatory effect of TR in effector T-cell differentiation.43, 51 We found that FOXP3+ T cells are localized in the T-cell-rich areas in MLN and in some areas are present in the follicles, indicating that they could potentially be involved in the regulation of B-cell responses.52 In some cases, a dense population of FOXP3+ T cells were also found to localize in the T-/B-cell boundaries in UC MLN (Fig. 6B), indicating the potential interaction of theses cells with antigen-bearing DC.

How are CD4+CD25+FOXP3+ TR generated in UC MLN? One possibility is that FOXP3+ TR in UC MLN and LP may be generated and/or expanded locally in response to intestinal tissue or microbial antigens in an attempt to curtail the colonic inflammation. FOXP3+ TR may be induced by naïve CD4+CD25 T cells in the local MLN environment by tolerogenic/immature dendritic cells or in the presence of TGF-β.20, 21, 53 Another possibility is that CD4+CD25+FOXP3+ TR are thymus-derived and despite being anergic in vitro, are expanded in the regional lymph nodes by commensal bacterial or tissue antigens in the presence of IL-2.54, 55 A recent study also showed that TGF-β enhanced FOXP3 expression following T-cell activation and this resulted in the acquisition of TR activity.21, 23, 29 Can adoptive transfer of expanded human CD4+CD25+ TR improve or even cure established UC? Recent reports in experimental colitis have demonstrated that transfer of CD4+CD25+ T cells into mice with colitis led to resolution of the disease.56 CD4+CD25+ T cells were found to proliferate in the MLN and inflamed colon and to interact with clusters of CD11c+ cells and pathogenic T cells.56 These studies suggest that manipulation of CD4+CD25+ T cells may be beneficial in the treatment of established IBD. However, given the difficulty of TR cell expansion in vitro48 and our data of the presence and functional integrity of CD4+CD25+ TR in human UC MLN argue against such an approach in the management of human disease. Three additional studies and one anecdotal report have evaluated the CD25hi T cell subset in the peripheral blood and/or LP in patients with IBD,33, 34, 45, 48 and showed no functional defect of this cell subset in human IBD.

Why do CD4+CD25+FOXP3+ TR fail to control the development of colitis despite their increased frequency and potent suppressor activity in vitro? It is possible that the suppressive activity of MLN or LP CD4+CD25+FOXP3+ TR cells is abrogated in vivo through either costimulatory molecule or TLR signaling pathways.57–60 Using human TR cells it was recently shown that TLR8-signaling led to reversal of TR function through TLR8-MyD88-IRAK4 signaling.61 Another possibility is that strong TCR stimuli may abrogate the suppressive activity of MLN CD4+CD25+FOXP3+ TR cells or render the effector T cells resistant to suppression.62 The high number of functional TR cells in UC MLN may also to some extent limit the severity of inflammation in the affected colon despite their inability to reverse the disease process. Our data do not address the possibility that some aspects of their suppressive function may be impaired in vivo, such as, for example, the potential generation of bacteria-specific Tr1 cells or some other as-yet unidentified function.63

It is interesting to note that in UC MLN we noted a prominent Th2 response as evidenced by the high production of IL-5 and IL-13 (Fig. 5) when effector CD4+CD25 T cells were activated with allogeneic DC in the absence of CD4+CD25+ TR. CD4+CD25+ TR were able to potently suppress the secretion of both Th1 and Th2 cytokines by effector CD4+CD25 T cells. The Th2 cytokine profile observed in UC MLN is consistent with a recent study indicating the development of a Th2 response in UC, which is characterized by an increased IL-13 production by nonclassical CD1d-restricted NK T cells.64

In summary, we present evidence for the expansion and functional integrity of FOXP3+CD4+CD25+ TR in MLN in UC patients with active medically refractory disease. In addition, we demonstrate the expansion of FOXP3+CD4+ T cells in the LP of inflamed and noninflamed colon in UC. Our data argue against a functional impairment of CD4+CD25+ TR in human UC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. Stephan R. Targan (Cedars-Sinai Medical Center) for helpful comments and encouragement, Dr. Jacky Woo (PDL BioPharma) for performing the cytokine assays, and Mr. Alexander Smith (University of Oxford) for immunohistochemistry. Supported by grants from the Broad Medical Research Program in Inflammatory Bowel Diseases by the Eli and Edythe L. Broad Foundation (to K.A.P.), and the Leukaemia Research Fund (to A.H.B.).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES