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Keywords:

  • scid mouse;
  • colitis;
  • inflammatory bowel disease;
  • regulatory T cells;
  • enteric bacteria

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Adoptive transfer of CD4+ T cells into scid mice leads to a chronic colitis in the recipients. The transferred CD4+ T cells accumulate in the intestinal lamina propria (LP), express an activated Th1 phenotype and proliferate vigously when exposed ex vivo to enteric bacterial antigens. As LP CD4+ T cells from normal BALB/c mice do not respond to enteric bacterial antigens, we have investigated whether colonic LP-derived CD4+ T cells from normal mice suppress the antibacterial response of CD4+ T cells from scid mice with colitis. LP-derived CD4+ T cells cocultured with bone marrow-derived dendritic cells effectively suppress the antibacterial proliferative response of CD4+ T cells from scid mice with colitis. The majority of these LP T-reg cells display a nonactivated phenotype and suppression is independent of antigen exposure, is partly mediated by soluble factor(s) different from IL-10 and TGF-β, and is not prevented by the addition of high doses of IL-2 to the assay culture. Functionally and phenotypically the T-reg cells of the present study differ from previously described subsets of T-reg cells. The presence of T cells with a regulatory potential in the normal colonic mucosa suggests a role for these cells in the maintenance of local immune homeostasis of the gut.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The intestine harbours a complex microflora composed of a large variety of indigenous aerobic and anaerobic bacteria [1]. These bacteria are in close proximity to a vast number of intestinal lymphocytes, but the immunological mechanisms, which regulate the interaction between the mucosal immune system and the intestinal microflora, are poorly understood. Especially, it is unclear how the mucosal immune system is able to mount a rapid and effective response against pathogenic bacteria, viruses and parasites while remaining tolerant to the resident enteric microflora and dietary antigens.

Several lines of evidence suggest that inflammatory bowel disease (IBD) is the result of a breakdown of tolerance towards the normal enteric microflora [2–4]. It has been demonstrated that lamina propria (LP) mononuclear cells from IBD patients respond to autologous faecal extracts, which were not the case in normal controls [4]. Reactivity against enteric bacteria has also been demonstrated in several animal models, including the scid-transfer model of colitis [3,5,6]. In this model, scid mice transplanted with low numbers of purified CD4+ T cells from healthy congenic donor mice develop a chronic colitis 2–3 months post-transplantation [7–10]. The disease is characterized by degeneration of crypts, giant cell formation, epithelial ulcerations and the presence of elevated numbers of CD4+ T cells in the colonic LP. We have previously demonstrated that CD4+ T cells from scid mice with IBD, in contrast to cells from normal BALB/c mice, respond by vigours proliferation and cytokine secretion when exposed to enteric bacterial extracts [6].

Accumulated data suggest that regulatory T cells play an important role in the induction and maintenance of tolerance in the normal intestine [11,12]. Both CD4+ and CD8+ T cells have been demonstrated to act as regulatory cells [13–15], and their effect can be mediated by the release of regulatory cytokines such as TGF-β, IL-10 and IL-4 [13,14], as well as by cell-to-cell interactions [16–18]. In the scid-transfer model, Powrie et al. [10] reported that CD45RBlow T cells prevent the development of colitis induced by CD45RBhigh T cells due to the secretion of IL-10 and TGF-β by the regulatory CD45RBlow T cells [19,20]. Also so-called T regulatory-1 (Tr1) cells have been reported to inhibit the development of IBD in scid mice [21]. These cells were generated in vitro from TCR/OVA transgenic CD4+ T cells by repetitive antigen-specific stimulation in the presence of IL-10. The in vivo function of the Tr1 cells was dependent on antigen-specific activation, as only mice receiving OVA were protected from disease.

The studies mentioned above suggest that scid mice with colitis are lacking regulatory cell subsets capable of controlling inappropriate cellular responses such as extensive proliferation and dysbalanced cytokine secretion. In the present study we have investigated the capacity of isolated CD4+ T cells from the colonic LP of normal mice to suppress the extensive proliferation of enterobacteria-reactive Th1-cells from scid mice with colitis.

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Mice

Six week-old female BALB/c and severe combined immunodeficiency (scid) mice homozygous for the scid/scid mutation were purchased at Bomholtgaard (Ry, Denmark). The mice were kept in a controlled microbial environment at the animal facility at University of Copenhagen.

Induction and assessment of IBD

IBD was induced in scid mice by transplantation of CD4+ T cells from normal BALB/c mice. The induction was performed as described previously [7]. In brief, CD4+ T cells were positively selected from spleen single cell suspensions using anti-CD4 mAb coated Dynabeads and detach-a-bead from Dynal (Oslo, Norway). The CD4+ T cells (>98% pure assessed by flow cytometry) were then stimulated in vitro for 3 days with 4 µg/ml ConA (Sigma, St Louis, MO, USA). Scid mice were injected i.p. with 3 × 105 of the activated CD4+ T cells and monitored every week for weight loss, loose stools, bloody diarrhoea and rectal prolapse.

Isolation of CD4+ T cells from scid and BALB/c mice

CD4+ T cells were isolated from both normal BALB/c mice and scid mice with IBD (displaying at least two of the above mentioned clinical signs of IBD). LP CD4+ T cells were isolated by cutting the colon in small pieces and incubate these in PBS with 2 mM EDTA for 45 min at 37°C. After washing with PBS, the gut pieces were incubated for 1 h at 37°C in medium containing 1 mg/ml collagenase (Clostridiopeptidase A, Sigma) and 5 µg/ml DNAse (Boehringer Mannheim, Mannheim, Germany). A single cell suspension was made and the cells were separated from fat by resuspending in 40% (v/v) percoll (Pharmacia & Upjohn, Peapack, NJ, USA) and centrifuging at 625 × g for 25 min After an additional wash, CD4+ T cells were isolated with anti-CD4 Dynabeads and detach-a-beads. In addition, CD4+ T cells were isolated directly from lymph nodes and spleen by anti-CD4 Dynabeads and detach-a-beads from single cell suspensions.

In some experiments CD25+ were positively selected from normal LP CD4+ T cells: cells were reacted with biotinylated anti-CD25 mAb (Pharmingen,Ca,USA) for 10 min, washed and exposed to streptavidin microbead (Miltenyi Biotech, Belgisch Gladbach, Germany) and further processed according to the manufacture's description on a magnetic column system. CD25+ cells were washed and added to the assay culture system (see below).

Preparation of dendritic cells and regulatory cells

Regulatory cells were prepared by exposing LP CD4+ T cells from BALB/c mice to bone marrow-derived dendritic cells (DC). DC were prepared as described previously [6]. In brief, bone marrow cells were depleted for CD4+, CD8+, B220+ and MHC-II+ cells by MACS microbead system (Miltenyi Biotech). The depleted cells were cultured for 9 days in UltraCulture medium (BioWhittaker, Walkersville, MD, USA) supplemented with 0·5% FCS, 5 ng/ml Gm-CSF (Peprotech, Tocky Hill, NJ, USA) and 10 ng/ml Flt-3 ligand (R & D Systems, Minneapolis, MN, USA). The resulting DC, 2 × 104 cells, were added to each well of a 96-well round-bottom microtitre plate together with 105 LP CD4+ T cells from normal BALB/c mice and cocultured for 2–6 days, harvested, washed and added to the proliferation assay as described below.

Proliferatory and regulatory culture assays

Irradiated BALB/c splenocytes were used as antigen presenting cells (APC). They were pulsed overnight with faecal extract (400 µg protein/ml). Preparation of the extract and pulsing of splenocytes were performed exactly as described previously [6]. The antigen-pulsed cells were washed, irradiated (2000 rad), and 105 cells were added to each well of a 96-well flat-bottom microtitreplate. LP CD4+ T-cells (105 from scid mice with colitis) were then added to the irradiated spleen cells. The cells were cultured for 5 days and proliferation was measured by adding 0·5 µCi (Amersham,UK) to each well for the last 18 h of the culture period. Then the cells were harvested to count the incorporated thymidine.

The regulatory potentials of different cell populations (see below) were assessed by adding 12·5 × 103−5 × 104 of these cells to the proliferation assay described above. In some experiments murine IL-10 (20 ng/ml) (Peprotech, Rocky Hill, NJ, USA), 2 µg/ml anti-IL-10 antibody (Pharmingen), 10 ng/ml recombinant mouse TGF-bRII/Fc (R & D, cat.no. 532-R2, USA) or 1000 units IL-2/ml (Chiron, CA, USA) were added to the assay cultures.

When physical separation of regulatory and responder cells was needed, a transwell system was applied. The cells were separated using tissue culture inserts with Anopore membrane (Nunc, Roskilde, Denmark) in normal 96-well microtitre plates. Responder cells and APC were placed in the lower chamber whereas cells assayed for regulatory capacity and APC were placed in the upper chamber. All assay cultures were set up in 4–6 replicates.

Flow cytometry

Freshly isolated LP CD4+ T cells, or LP CD4+ T cells cocultured with DCs for 6 days, or LP CD4+ T cells cocultured with DCs for 6 days and in transwells for 5 days, were harvested and stained for 30 min on ice for expression of CD45RB (PE-conjugated rat anti-CD45RB, clone 16 A, Pharmingen), CD69 (PE-conjugated hamster anti-CD69, clone H1·2F3, Pharmingen) and CD3 (FITC-conjugated hamster anti-CD3, clone 145–2C11, Pharmingen). Subsequently, the cells were washed 3 times, resuspended in PBS and analysed using a FACScalibur flow cytometer (Becton Dickinson). Data were analysed using cellquest software (Becton Dickinson). Specificity of the antibodies were confirmed by isotype matched control mAbs (data not shown).

Anti-cytokine ELISA

Supernatants from the cell-cultures were analysed for the cytokines IL-2, IL-10, IFN-γ and TNF-α by ELISA as described previously [5]. In brief, maxisorp microtitre plates (Nunc, Roskilde, Denmark) were coated with the relevant anticytokine catching mAb (Pharmingen, San Diego, CA) and incubated overnight at 4°C. Unspecific binding sites were blocked with 10% (w/v) FCS and the plates were incubated with standards and samples. Then biotinylated anticytokine mAbs (Pharmingen) were added, followed by streptavidin-peroxidase (Sigma). As a substrate ABTS (2,2′-Azino-bis(3-ethylbenzenthiazoline-6-sulphonic acid) in 0·1 M citric acid) with hydrogen peroxide was used, and the plates were read at 405 nm. Anti-TGF-β levels were measured using a commercial anti-TGF-β kit (Pharmingen), following the manufacturer's instructions.

Statistics

Comparisons between cell cultures without and with regulatory cells added were performed using nonparametic Wilcoxon tests for paired differences.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Antigen-induced proliferation of LP T cells from CD4+ T cell transplanted scid mice

As demonstrated previously, LP CD4+ T cells from scid mice with colitis proliferate vigously when exposed to enterobacterial antigen extract [6]. Figure 1 shows the antigen-induced proliferation of or LP CD4+ T cells from scid mice prior to and during the course of colitis development. Already at day 10 following T cell transfer, prior to weight loss, diarrhoe and histolopathological signs of colitis [6]. CD4+ T cells from gut LP proliferated when exposed to antigen-pulsed APC. With time, as the disease and weight loss progressed, the antigen-induced proliferation of CD4+ T cells increased further. However, proliferation decreased at full blown colitis (weight loss of approx. 25%).

image

Figure 1. Enterobacterial antigen induces proliferation of LP CD4+ T cells from scid mice transplanted with 3 × 105 congenic CD4+ T blast cells. The average body weights of the group of mice tested at the different time periods are included. Each datapoint represents proliferation of a cell pool from 3 to 5 mice assayed in four replicate cultures in the presence of antigen pulsed APC (▪) or in the presence of unpulsed APC (□). Vertical bars represent 2 SD values. The experiment was terminated at day 65 when the transplanted scid mice showed a 25% loss of weight.

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Regulatory potential of freshly isolated and cultured LP CD4+ T cells

In contrast to scid mice with colitis, LP CD4+ T cells from normal BALB/c mice do not proliferate when exposed to APC pulsed with enterobacterial antigen [6]. The primary aim of the present study was therefore to investigate the possible existence of regulatory cells, within the population of colonic LP CD4+ T cells from normal mice, with the capacity to inhibit the antibacterial response of CD4+ T cells from scid mice with colitis.

The regulatory potential of freshly isolated colonic LP CD4+ Tcells from normal mice was compared with that of LP CD4+ T cells cocultured for varying periods with DC. As shown in Fig. 2a, addition of fresh, purified LP CD4+ T cells to the responder cell culture did not significantly influence the antibacterial response of CD4+ T cells from scid mice with colitis. However, LP CD4+ T cells cocultured with bone marrow-derived DC for 2, 4 and 6 days and subsequently added to the responder cell cultures exerted a strong inhibitory activity. These inhibitory or suppressive cells are in the following referred to as T-reg cells. As little as 12·500 T-reg cells significantly inhibited the proliferative activity of 100·000 responder cells (data not included). Figure 2b includes data showing that T-reg cells are also generated by DC coculture of CD4+ T cells from mesenteric lymph nodes and spleen although these regulatory cells are less efficient. In contrast, DC CD4+ T cell cocultures obtained from axillary lymph nodes (AxLN) do not display any regulatory activity. The suppression induced by T-reg cells is a very reproducable phenomenon. Table 1 depicts the results from eight separate experiments showing an average suppression of the responder LP CD4+ T cell proliferation of 91%. Table 1 also shows that T-reg cells do only proliferate marginally when exposed to antigen-pulsed APC.

image

Figure 2. (a) Colonic LP CD4+ T cells from normal mice cocultured for 2–6 days with bone marrow derived DC significantly (P < 0·05) inhibit the proliferation of enterobacterial reactive LP CD4+ T-cells from scid mice with colitis. I, 105 colitic LP CD4+ T cells exposed to antigen pulsed APC; II, as I, but with the addition of 5 × 104 freshly purified BALB/c LP CD4+ T cells; III-V as I, but with the addition of 5 × 104 BALB/c LP CD4+ T cells cocultured with DC for 2, 4 and 6 days, respectively. Vertical bars represent 2 SD values. (b) CD4+ T cells obtained from colonic LP, mesenteric lymph nodes and spleen and cocultured for 6 days with DC significantly inhibit (P < 0·05) the proliferation of enterobacterial reactive LP CD4+ T-cells from scid mice with colitis. Notice that CD4+ T cells from axillary lymph nodes do not possess a suppressive potential. I, 105 LP CD4+ T cells exposed to antigen pulsed APC; II-V, as I, but with the addition of of 5 × 104 BALB/c LP, mesenteric lymph node, spleen and axillary lymph node CD4+ T cells, respectively, exposed to antigen pulsed APC for 6 days. Vertical bars represent 2 SD values.

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Table 1.  Five separate experiments to demonstrate the level of suppression mediated by T-reg cells. Each entry represents the mean cpm values of four replicate cultures.
ExperimentResponder cells (cpm)Responder cells + T-reg cells (cpm)T-reg cells (cpm)Inhibition (%)
  1. SD were less than 10% of the mean cpm values. Responder cells are 105 LP CD4+ T cells from colitic scid mice and T-reg cells are 5 × 104 LP CD4+ T cells from normal BALB/c mice.

1 3242 310 66690
2 2030 190 46891
3293225543 74681
477673 554150999
5751253091564696
Mean ± SEM   91 ± 3

In separate experiments, purified LP CD4+ T cells from colitic scid mice were cocultured with DC for six days and assayed for inhibitory activity. However the recovered T cells from these cocultures proliferate strongly when exposed to APC pulsed with enterobacterial antigenty (data not included). Thus no regulatory activity was detected in LP CD4+ T cells recovered from diseased scid mice.

Phenotype of cells with regulatory activity

Table 2 summarizes the FACS data on the percentage of CD69, CD45 and CD25 of purified LP CD4+ T cells from normal mice prior to and after coculture with DC for 6 days and subsequent coculture with antigen pulsed APC in the assay culture for 5 days. The data show that>98% of the harvested cells from the cocultures are TCR/CD3 positive clearly illustrating the lack of ‘contaminating’ DC, which stick firmly to the plastic surface, during T-reg cell harvesting. More than one third of the native CD4+ T cells express the CD69 activation molecule whereas only 16–20% of the cultured CD4+ T cells retained this marker after DC coculture. The ratio between CD45RBhigh and CD45RBlow cells increases from 1:1 in native LP CD4+ T cells to 5:1–10:1 in CD4+ T cells after coculture. CD25+ cells are present in freshly prepared LP CD4+ T cells, but their frequency declines during the six days period of coculture period, a period where the number of CD4+ T cells decreases by 5–10 times (data not shown). Thus, it appears that nonproliferating, nonactivated (CD69low), nonmemory (CD45RBhigh) CD4+ T cells are enriched by the present DC coculture conditions.

Table 2.  Summary of the FACS data obtained from freshly prepared LP CD4+ T cells, six days of DC and LP CD4+ T cell cocultures (T-reg cells) and five days T-reg assay cultures
 Phenotype of naïve and cultured cells
CD3+CD45highCD45lowCD69+CD69CD25+CD25
  1. nd, not done.

LP CD4+ T cells 95–98%49%46%36%62%24%74%
LP CD4+ cells in DC coculture 99%82%16%16%84%15%84%
T-reg cells in assay culture100%90%10%20%80%ndnd

CD25+ LP CD4+ T cells from normal colonic mucosa possess regulatory capacity

The majority of regulary CD4+ T cell subsets so far described constitutively express the IL-2 α-chain receptor CD25 [16–18]. Since 25% of freshly prepared +LP CD4+ T cells express CD25 we purified these by magnetic bead separation procedures and assayed them for suppressor activity of colitic scid CD4+ T cells. Figure 3 shows that the CD4+ CD25+ T cells, in contrast to CD25 cells (data not included), indeed possess significant suppressive activity although not at the same high levels as T-reg cells generated from the DC cocultures (compare Fig. 3 and Table 1).

image

Figure 3. Regulatory activity of purified CD4+CD25+ cells from lamina propria of normal BALB/c colon. Proliferation of: I, colitic scid 105 LP CD4+ T cells exposed to antigen pulsed APC; II, 105 LP CD4+ of normal BALB/c CD4+ T cells; III, as I, but with the addition of positively selected LP CD4+CD25+ from normal BALB/c (significantly different from I p < 0·05); IV, LP CD4+CD25+ T cells from normal BALB/c exposed to antigen pulsed APC. Vertical bars represent 2 SD values.

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T-reg cells secrete soluble regulatory factor(s) in an antigen independent manner

To investigate if the T-reg cells mediate their effect via a soluble factor or through cell-to-cell contact, and whether T-reg cell function depends on antigen exposure, we applied a transwell system. In these experiments, T-reg cells are physically separated from the responder cells and exposed to unpulsed or antigen pulsed APC. Table 3 shows the results of five separate experiments. Although separation of regulatory and responder cells resulted in less inhibition of the proliferative response of enterobacteria reactive CD4+ T cells (compare Tables 1 and 3), these data indicate that suppression is mediated both by soluble factor(s) as well as by cell-to-cell contact mechanisms. In addition, the data in Table 2 show that suppression occurs both in the presence and absence of antigen exposure of the T-reg cells.

Table 3.  Suppression by T-reg cells in transwell cultures and T-reg cell dependence of antigen exposure. T-reg cells, 5 × 104, were cultured in the upper chamber in the presence of antigen-pulsed or nonpulsed APC. Responder cells, 105 LP CD4+ antigen-exposed T cells from colitic scid mice and T-reg cells, were cultured in the lower chamber.
ExperimentInhibition (%)
+ antigen– antigen
  1. Each single experiment was performed in 4–6 replicate assay cultures. *not significant 36.

17236
24051
36181
43320 ns*
54751
Mean ± SEM51 ± 752 ± 10

The role of cytokines for T-reg cell function

The presence of IL-2, IL-10, IFN-γ and TNF-α in day 6 DC LP CD4+ T cell cocultures was examined by ELISA in several separate coculture experiments. None of these cytokines were detected except for IL-10, which in two of the experiments reached levels of 5–8 ng/ml in the culture supernatants (data not included). No cytokines were detected in pure DC cultures. In accordance with these data, Fig. 4 shows that anti-IL-10 Ab as well as an antagonistic soluble TGFβ ligand added to the regulatory cell assay culture did not block the inhibitory effect of the T-reg cells. The efficiacy of anti-IL-10 Ab and TGFβ ligand was verified in separate experiments (data not shown). Finally, data in Fig. 4 shows that addition of 1000 units/ml IL-2 to the assay cultures increased the responder cell proliferation as well as proliferation of Treg cells themselves. However, addition of IL-2 did not block the suppressive effect T-reg cells.

image

Figure 4. (a) Anti IL-10 mAb and TGFβ do not block LP T-reg cell function. Columns represent: I, proliferation of 1 × 105 CD4+ T cells from scid mice with colitis exposed to antigen-pulsed APC; II, as I but with addition of 5 × 104 LP T-reg cells; III as I, but with addition of anti-IL-10 Ab; IV, as II, but with the addition of anti-IL-10 AB; V, as I, but with the addition of TGFβ ligand; VI, as II, but with the addition of TGFβ. Vertical bars represent 2 SD values. (b) IL-2 does not block the suppressor activity of LP T-reg cells. Columns represent: I, proliferation of 1 × 105 CD4+ T cells from scid mice with colitis exposed to antigen- pulsed APC; II, as I but with addition of 5 × 104 LP T-reg cells; III, as I, but with addition of 1000 units/ml of IL-2; IV, as II, but with the addition of IL-2; V, T-reg cells exposed to antigen-pulsed APC and IL-2.

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DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Development of the T-reg cell phenotype

It is now well established that regulatory cells play an important part in the maintenance of immune homeostasis including mucosal tolerance by preventing inappropriate responses to commensal bacteria [11,12]. These regulatory T cells are believed to be generated by exposure of the intestinal immune system to soluble, monomeric antigen [13,22,23]. Several subpopulations of regulatory T cells have been described in the past, including Th3, Tr1, CD45RBlow and CD4+CD25+ cells [11,12,16–20].

In scid mice with CD4+ T cell transfer colitis the tolerance towards the commensal bacteria flora is absent or severely down-regulated. In this disease the transferred CD4+ T cells respond vigorously ex vivo against enterobacterial extracts [6]. In the present study, we have for the first time, demonstrated the presence of T-reg cells generated in vitro from LP CD4+ T cells of the normal colonic lamina propria which can inhibit proliferation of enterobacteria reactive, disease-inducing CD4+ T cells from scid mice with colitis. In contrast, coculture of DC and scid colitis CD4+ T cells did not result in development of T-reg cells. Instead the CD4+ T cells retained their high proliferative responsiveness for enterobacterial antigens. Thus the abnormally high reactivity against enterobacterial antigens in scid mice with colitis is explainable from the absence of local regulatory cells in the diseased mice.

The generation of T-reg cells was antigen independent, but functional maturation required a period of 2–6 days in coculture with bone marrow derived DC. In contrast, freshly isolated unseparated LP CD4+ T cells were unable to mediate significant suppression, although freshly separated LP CD4+ CD25+ T cells, in contrast to CD25 cells, possessed moderate suppressive activity. Experiments are in progress to determine whether T-reg cells develop from CD25 positive or negative precursors.

The nature of the interactions between DC and LP CD4+ T cells, which leads to the regulatory effector capacity of the latter cell subset, is unknown. DC-derived cytokines such as TGF-β and IL-10 were not detected in the culture supernatants, but levels below the detection levels of our ELISA could have acted as differentiation factors for a regulatory T cell precursor subset present in the LP CD4+ T cell population, as has been demonstrated with other T cell populations [14,19,20,24]. However, our attempts to enhance quantitatively or qualitatively the generation of T-reg cells by the addition of TGF-β and/or IL-10 to the LP CD4+ T cells/DC coculture were unsuccessful (data not included), suggesting that other DC-derived factors are needed to obtain full regulatory capacity in the T-reg cell population. DC bound and/or secreted B7-molecules, which might bind to and stimulate through CTLA-4 molecules expressed by the cocultured LP CD4+ T cells, could be involved as such interactions have been described to promote the generation of some regulatory T cell subsets [25–27].

In contrast to CD4+ T cells from LP, mesenteric lymph nodes and spleen, CD4+ T cells purified from the axillary lymph nodes (AxLN), a lymphoid organ supposingly not exposed to gut enterobacterial antigens [28,29], did not develop into T-reg cells by DC coculture. The fact that T-reg cells can not be generated from the axillary lymph nodes, suggests that antigen experience in vivo is essential for the development of the present subset of T-reg cells and that CD4+ T cells from LP and their migrants are exposed to gut-derived antigens in the tolerogenic environment of the gut mucosa. Our observations are in line with the general view on the acquisition and maintenance of mucosal tolerance which assumes that a constant exposure to enteric bacterial antigens leads to generation of antigen specific regulatory T cell subsets [13,22,23].

One fourth of the freshly prepared LP CD4+ cells constitutively express CD25. These cells were shown to possess moderate regulatory activity, and are thus sharing the CD25 phenotype with the major subsets of T-reg cells [16–18]. However, it is not clear why T-reg cells require a period of several days in coculture with DC before being able to mediate their strong regulatory functions, because at this stage the fraction of CD25 expressing cells had decreased to 15%. In addition, the number of LP CD4+ T cells in the day 6 DC coculture declines by 80% and the surviving cells display a nonactivated (CD69low), nonmemory (CD45RBhigh), phenotype and they do not proliferate when exposed to antigen-pulsed APC (Table 1). T-reg cells might thus fit into the category of regulatory cells that during the intrathymic selection process, due to a high avidity for self, become relatively resistant to stimulatory signals instead of being deleted [16]. As suggested by our data, regulatory cells, as those described in the present work, might preferentially migrate to sites with a high loads of foreign antigen such as the GALT rather than to the relatively antigen free environment of the axillary lymph nodes.

Mechanisms of suppression

Several mechanisms have been proposed to explain how regulatory T cells mediate their suppression. In the scid mouse model of colitis it has been demonstrated that Tr1 and CD45RBlow cells mediate in vivo regulation via the production of suppressive cytokines such as TGF-β, IL-10 and IL-4 [19–21]. Socalled ‘professional suppressor’ CD4+CD25+ cells [16–18] have also been reported to mediate suppression by IL-10 secretion [30]. In vitro, however, most authors agree that the hyporesponsive, nonproliferative subset of ‘professional suppressor’ T cells as well as CD38+CD45RBlow regulatory cells, appear to mediate their activity independently of cytokines by direct cell-to-cell contact mechanisms and via activation through their TCR [16–18].

Our results in the transwell system show that the LP T-reg cells mature and function in vitro independently of antigen exposure, and that they can act in vitro through the release of soluble, diffusable factor(s), although not so efficient as in a single well system. A number of studies have shown that both Tr1, Th3 and CD45RBlow regulatory T cell subsets mediate their effect by secretion of IL-10 [14,19–21]. However, although we found that an exogenous source of IL-10 severely inhibited proliferation of enterobacterial reactive T cells from colitic scid mice (data not included), studies of the DC culture supernatants with ELISA and antibody-mediated blocking experiments indicated that LP T-reg cells do not function by IL-10 secretion. As mentioned above TGF-β has also been shown to play a role in suppression mediated by regulatory T cells [14,26,29]. However, we were unable to detect any TGF-β production by LP T-reg cells or block the function of these cells with a soluble TGF-β ligand. Addition of high amounts of IL-2 to the responder cell culture did not prevent suppression in our system, as observed for some [31,32], but not for other T-reg cell subsets [30]. This suggests that LP T-reg cells do not act by inhibition of the transcription of IL-2 in the responder population, as reported for CD4+CD25+ activation-dependent T-reg cells [33], or as an IL-2 sink draining endogeneously secreted IL-2. The naïve end nonactivated phenotype of the LP T-reg cells in the present study is not in direct opposition to other regulatory T-cell subsets described. Thus regulatory CD69 negative T-cells were generated in cocultures with immature DC [34]. In addition, although CD4+CD25+ regulatory T-cells display a modest increase in activation and memory markers, these cells may also contain significant proportions of naïve and resting cells [33,35].

In conclusion, the present results demonstrate that T-reg cells generated from the normal colonic LP can inhibit enteroantigen-induced proliferation of CD4+ T cells from scid mice with chronic colitis. Functionally and phenotypically the LP T-reg cells of the present study differ from the majority of previously described subsets of T-reg cells: by displaying an nonactivated, naive phenotype, and suppress without being exposed to antigen. In addition, LP T-reg cells can function independently of direct cell-to-cell contact. Our observations might lead to a more profound understanding of the basis for the maintenance of the normal gut mucosal immune homeostasis. In particular our data may be useful for new insight in the pathogenesis of human inflammatory bowel diseases. Thus the gut mucosal T lymphocytes in these patients display an abnormally high immune reactivity towards the enteric microflora [4] perhaps reflecting the absence of T-reg cells in these diseases.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Evelyn Kury is greatly acknowledged for her expert technical assistance. This work was supported by the Colitis Crohn Association, Physician SCE Friis and Wife's Fund, NovoNordic Fund and from The Danish Medical Research Agency.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  • 1
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