Skip Navigation LinksHome > November 2010 - Volume 51 - Issue 5 > Peripheral and Intestinal CD4+ T Cells With a Regulatory Phe...
Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0b013e3181e4d323
Original Articles: Gastroenterology

Peripheral and Intestinal CD4+ T Cells With a Regulatory Phenotype in Pediatric Patients With Inflammatory Bowel Disease

La Scaleia, Raffaella*; Morrone, Stefania*; Stoppacciaro, Antonella; Scarpino, Stefania; Antonelli, Manila*; Bianchi, Elettra*; Di Nardo, Giovanni; Oliva, Salvatore; Viola, Franca; Cucchiara, Salvatore; Santoni, Angela*; Palmieri, Gabriella*; Uccini, Stefania

Free Access
Article Outline
Collapse Box

Author Information

*Department of Experimental Medicine, Italy

Department of Clinical and Molecular Medicine, Italy

Department of Pediatrics, Pediatric Gastroenterology and Liver Unit, Sapienza–University of Rome, Italy.

Received 29 May, 2009

Accepted 23 April, 2010

Address correspondence and reprint requests to Stefania Uccini, MD, Department of Clinical and Molecular Medicine, II Medical School, Sapienza–University of Rome, Via di Grottarossa 1035, 00189 Rome, Italy (e-mail:

Drs Palmieri and Uccini contributed equally to this article.

Grant support: “Progetti di Ricerca di Ateneo” 2005 to 2007 and “Progetti Coordinati di Ateneo Federato delle Scienze delle Politiche Pubbliche e Sanitarie (SPPS)” 2008.

The authors report no conflicts of interest.

Collapse Box


Objectives: Regulatory T cells (TR cells) play a crucial role in the regulation of intestinal inflammation. To examine the pathogenetic relevance of TR cells in inflammatory bowel disease (IBD), we evaluated their frequency in peripheral blood and inflamed and noninflamed mucosae of pediatric patients with IBD and age-matched controls without IBD; we also characterized the immune profile of the inflammatory infiltrate in the different phases of the disease.

Patients and Methods: Circulating TR cells were investigated on peripheral blood mononuclear cells by fluorescence-activated cell sorting analysis; mucosal TR cells and inflammatory cell populations were investigated by immunohistochemistry on bioptic specimens. FOXP3 messenger RNA expression levels were confirmed using real-time polymerase chain reaction.

Results: FOXP3+ TR cells were significantly increased in the intestinal lesions of patients with active IBD, and returned to normal levels in posttherapy remission phase. At variance, circulating TR cell frequency was elevated in patients with IBD independently of disease activity, as it persisted in the remission phase. A selective imbalance in the frequency of CD4+ T and natural killer cell subsets characterized the abundant inflammatory infiltrate of active intestinal lesions, and also persisted, at a lower level, in noninflamed mucosae of patients in the remission phase.

Conclusions: TR cell frequency is differently regulated in mucosal tissues and at the systemic level during the distinct phases of pediatric IBD. The inactive stage of pediatric IBD is characterized by an incomplete normalization of the immune profile, independently of the clinical efficacy of the therapy. The pediatric, early-onset condition may represent a privileged observatory to dissect the immune-mediated pathogenetic mechanisms at the basis of the disease.

Inflammatory bowel disease (IBD) is a chronic immune-mediated inflammatory condition that can affect almost any portion of the gastrointestinal tract. IBD encompasses 2 apparently distinct disease entities, Crohn disease (CD) and ulcerative colitis (UC). CD is characterized by a transmural, granulomatous inflammation of discontinuously affected intestinal segments, whereas UC, which involves mostly the mucosal but not the deeper layers of the intestinal wall, is restricted to the large intestine, and spreads continuously from distal to proximal segments. Both diseases can follow an unpredictable, often chronic, relapsing course (1–3).

Epidemiological data support the notion that the development of IBD requires an inherited predisposition because it has been demonstrated that first-degree relatives of patients with IBD are much more likely to develop the disease than second-degree relatives (2–5); moreover, recent results obtained by genome-wide association studies have revealed the positive or negative association of distinct gene polymorphisms with 1 or both types of IBD (6,7).

The etiopathogenesis of chronic mucosal inflammation in IBD depends on dysregulated innate and adaptive responses against commensal flora in genetically predisposed individuals (2–5,8). The primary trigger of inflammation in IBD is unknown, and it is probable that a complex interplay of environmental and microbial agents plays an important pathogenetic role (3–5). Recruitment and activation of several immune cell types lead to an increased production of cytokines and other proinflammatory mediators at the mucosal site, which sustain and amplify the inflammatory reaction, and are ultimately responsible for IBD tissue damage. In particular, several subsets of T helper cells display enhanced responsiveness to interleukin (IL)-2, a heightened state of activation, and a higher survival capability in IBD-affected mucosal tissue (5,8–12).

Under physiological conditions, the activation of the mucosal immune system, due to the continuous contact with the environment and with the commensal flora, is kept under control through several tolerance and suppression mechanisms (5).

Regulatory T cells (TR cells) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora, as demonstrated in several experimental models of gut chronic inflammation (13–15). CD4+TR cells are commonly identified as being constitutively CD25bright (16–19). More recently, FOXP3, a member of the forkhead-winged helix family of transcription factors, has been shown to be specifically expressed in “natural” CD4+ CD25+ TR cells, and to play a critical role in the development and suppressive function of this cell subset; genetic defects at the FOXP3 locus cause IPEX, an inflammatory X-linked syndrome accompanied by immune dysregulation, polyendocrinopathy, and entheropathy (14,20). In addition, several recent studies have shown that functionally competent TR cells are characterized by the low expression of CD127 (IL-7 receptor α-chain) surface marker, and that the expression of FOXP3 and the CD127low/neg phenotype highly correlate within the CD4+ CD25bright T-cell population (21–24).

In adult patients affected by IBD, the frequency of FOXP3+ CD4+ TR cells is increased in the lamina propria of inflamed colonic areas compared with normal colon; several studies (17–19,25–27) have also shown that these cells are functional in vitro, leaving unexplained the mechanisms at the basis of their inability to efficiently control mucosal inflammation in vivo. Alterations in the abundance of TR cells in adult patients with IBD also reflect at the systemic level; indeed, TR cells in the peripheral blood of individuals affected by CD or UC have been found to be higher in patients undergoing remission, compared with patients in the active phase of the disease (17,18,25,28,29).

There is a commonly agreed-upon view that the disorder in children has peculiarities in terms of both underlying mechanisms and clinical management (30–32). Moreover, the profile of the immune response and the relative importance of different pathogenic mechanisms likely change during the course of chronic IBD disease (33–35), thus rendering the comprehension of the mechanisms contributing to the onset and perpetuation of the damage more difficult. In this regard, the study of pediatric, early-onset IBD (31,36) could represent a valuable approach and a privileged setting to dissect immune-mediated pathogenetic mechanisms in the relative absence of age-related and/or environmental confounding variables. However, to our knowledge, no information is available on the role of CD4+ TR cells in pediatric patients affected by IBD.

In the present study we examined the frequency of CD4+ TR cells in inflamed and noninflamed mucosal tissue of pediatric patients with IBD and age-matched controls without IBD, and we compared these results with those obtained in the peripheral blood of the same patients, either during the active or in the remission stage of the disease.

Back to Top | Article Outline



We analyzed 91 patients: 19 with a diagnosis of CD, 24 with UC, 5 with undetermined colitis, and 44 age-matched controls without IBD. All of the patients were referred to the Division of Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Sapienza–University of Rome, Italy; they underwent ileocolonoscopy, which was performed with a pediatric videocolonscope (Olympus PCF Q-180, Tokyo, Japan) following conscious sedation with intravenous pethidine (1–2 mg/kg) and midazolam (0.1 mg/kg), or general anesthesia. Definite diagnosis of UC or CD was based on widely agreed clinical, radiological, endoscopic, and histological criteria as well as on the exclusion of infectious and systemic disease, food allergies, and malabsorption syndromes (37). Each patient enrolled in the study was recruited after ethical committee approval and parent's subscription of the informed consent.

The disease severity was graded according to the criteria of the Pediatric Ulcerative Colitis Activity Index (PUCAI) for patients with UC and the Pediatric Crohn Disease Activity Index (PCDAI) for patients with CD, recently validated multi-item measures of clinical and laboratory parameters (PUCAI <10 points: remission, 10–34: mild, 35–64: moderate, ≥65: severe; PCDAI <10 points: remission, 10–30: mild, ≥30: moderate–severe) (38,39).

For patients with CD, 13 had active disease (PCDAI score >10) and 6 were in remission (PCDAI score <10); for patients with UC, 19 had active disease (PUCAI score >10) and 5 were in remission (PUCAI score <10); for the patients with undetermined colitis, 3 were in the active phase and 2 were in remission. The control population was represented by 44 age-matched children investigated for unspecific colitic symptoms in which disorders such as food allergy, malabsorption, and idiopathic inflammation had been excluded. Age mean (ranges) were 14 years (6–18) for patients with CD, 12 years (4–18) for patients with UC, and 10 years (1–17) for controls.

At the time of the study, 42 of 44 controls, 3 of 19 patients with CD, and 5 of 24 patients with UC were not receiving therapy; 4 of 19 patients with CD and 5 of 24 patients with UC were treated with immunosuppressive drugs; 2 of 44 controls, 11 of 19 patients with CD, and 16 of 24 patients with UC were treated with anti-inflammatory drugs; 1 of 19 patients with CD and 1 of 24 patients with UC received anti-TNF therapy.

Back to Top | Article Outline
Histology and Immunohistochemistry

Large-bowel biopsies were taken from macroscopically normal sites of 8 age-matched controls and 12 remission IBD, or from severely affected areas of 13 patients with active IBD (6 CD and 7 UC). All of the samples were formalin fixed and paraffin embedded, and 5-μm sections were stained with hematoxylin and eosin (H/E) for routine histology. Immunohistochemistry was performed on 5-μm paraffin sections after deparaffinization, rehydration, heat-assisted antigen retrieval, and endogenous peroxidase inhibition, by incubation with the following antibodies: FOXP3 (236A/E7, Santa Cruz Biotechnology, Santa Cruz, CA), CD3 (clone PS1, Monosan, Uden, the Netherlands), CD4 (clone 4B12, Monosan), CD8 (clone 4B11, Monosan), CD20 (clone L26, Monosan), CD25 (clone 4C9, Monosan), CD138 (clone MI15, Dako, Dakopatts, Copenhagen, Denmark), CD68 (clone KP1, Dako), and CD56 (clone 123c3D5, Novocastra, Newcastle, UK). The antibodies were used at 1:50/1:100 final dilutions. The antigen retrieval technique was used for the detection of all of the proteins. After repeated washings with phosphate-buffered saline, sections were incubated with avidin-biotin complex kit (ABC-peroxidase, Dako). The reaction product was revealed by 0.2% hydrogen peroxide and 0.6% 3,3-diaminobenzidine (Sigma-Aldrich, St Louis, Mo). Slides were counterstained with Mayer hematoxylin. Negative controls were incubated with isotype-matched nonimmune immunoglobulins and yielded no staining (data not shown). The number of immunostained cells was evaluated using a light microscope at 400 × magnification in 10 fields and reported as mean ± standard deviation in a single high-power field.

Back to Top | Article Outline
Multicolor Immunofluorescence and FACS Analysis

Peripheral blood mononuclear cells were purified by Ficoll-Hypaque (Eurobio; Les Ulis Cedex, France) density gradient. Peripheral blood mononuclear cells were incubated with different antibodies or isotype controls (anti-CD3-PerCP, anti-CD4-PE, immunoglobulin G1 (IgG1)-FITC, anti-CD25-FITC, anti-FoxP3-PE, IgG1-PE Regulatory T Cell Cocktail [consisting of anti-CD4-FITC, anti-CD25-PE-Cy7, and anti-CD127-Alexa-Fluor-647], all purchased from BD Biosciences [San Jose, CA]) for 30 minutes at 4°C. Samples were analyzed with a FACScalibur (BD Biosciences, San Jose, CA) using CellQuest software. CD25 expression has been evaluated after gating on CD3+ CD4+ lymphocytes; CD25bright cells display a level of positivity for CD25 antigen, which is twice that of baseline level, as defined by isotypic control staining. Intracellular staining for FOXP3 was performed according to the manufacturer's instructions, and the analysis was performed on at least 25,000 events collected in the CD4+ lymphocytes gate, identified by side-scatter properties and anti-CD4 staining.

Back to Top | Article Outline
Real-time PCR Analysis

FOXP3 messenger RNA (mRNA) expression was investigated on 11 patients (3 active CD, 3 active UC, 2 remission CD, and 3 remission UC). The expression of FOXP3 in normal mucosa was evaluated in 6 age-matched histologically normal non-IBD samples. Frozen sections of cryopreserved tissues (2 independent mucosa samples for each patient) were used for RNA extraction. Total RNA was obtained using the RNA Fast isolation kit (Molecular System, San Diego, CA), and RNA integrity was assessed by denaturing agarose gel electrophoresis and spectrophotometry. RNA transcripts for FOXP3 were measured by real-time absolute quantitative RT-PCR, based on TaqMan methodology, using the ICycler System (Bio-Rad, Hercules, CA). The specificity of the amplification products was controlled by a melting curve analysis. RNA input was normalized by average expression of the housekeeping gene β-actin in the same samples. Gene-specific primers and probes were FOXP3: forward 5-TTCTCGGTATAAAAGCAAAGTTGT-3, reverse 5-GGCATCGGGTCCTTGTCC-3, probe 5-TGACAGTTTCCCACAAGCCAGGCT-3; β-actin: forward 5-CGGTTCCGCTGCCCTGAG-3, reverse 5-TGGAGTTGAAGGTAGTTTCGTGCAT-3, probe 5-CCACAGGACTCCATGCCCAGGAAGGAA-3.

Triplicates were run for each sample. The expression of FOXP3 mRNA measured in the pool of normal mucosae was considered the unit value, and the results obtained in IBD mucosae are reported as relative levels of FOXP3 mRNA with respect to the unit value.

Back to Top | Article Outline
Statistical Analysis

Differences between groups were analyzed with parametric (analysis of variance and Student t test) and nonparametric tests (Kruskal-Wallis and Mann-Whitney), when appropriate, using statistical software SPSS version 15 (SPSS Inc, Chicago, IL); a level of P < 0.05 was considered significant.

Back to Top | Article Outline


Evaluation of the Immune Infiltrate in Inflamed and Noninflamed Mucosa of Pediatric Patients With IBD

Intestinal biopsies from 8 age-matched controls without IBD, 13 patients with active IBD (6 patients with CD and 7 patients with UC), and 12 pediatric patients with IBD in remission were investigated for histology on H/E tissue sections to confirm the clinical diagnosis. Inflamed mucosae of patients with IBD (either CD or UC) in the acute stage of disease were histologically characterized by a marked increase of inflammatory cell infiltration, including plasma cells, lymphocytes, eosinophils, histiocytes, and mast cells, associated with the presence of crypt abscesses, that is, collections of neutrophils in the glandular lumen (Fig. 1A). The intestinal biopsies of patients with CD or UC undergoing a quiescent or resolving stage of the disease showed the aspect of a posttherapeutic restoring mucosa, being histologically characterized by marked edema with a mild inflammatory infiltrate of the mucosa, irregular branching glands, and nearly total restoration of the mucin content (Fig. 1B). In controls without IBD, the histology showed only a mild inflammation of the lamina propria associated with dilated blood vessels and edema, indicative of a nonspecific, limited inflammatory process (Fig. 1C). Our results show that during the active phase of IBD, either CD or UC, the number of inflammatory cells in the intestinal mucosa, evaluated by morphology on H/E staining, was approximately 4 times higher than that detected in the control biopsies and approximately 2-fold of that observed in the remission stage (Table 1).

Figure 1
Figure 1
Image Tools
Table 1
Table 1
Image Tools

The profile of the inflammatory cells populating the intestinal mucosa, analyzed by immunohistochemistry, showed that the absolute number of all main cell subsets were augmented in active IBD lesions, as compared with mucosa samples obtained from non-IBD controls (Table 1). Results summarized in Table 2 indicate that the markedly increased inflammatory infiltrate in the lamina propria of active IBD mucosa was mainly composed of plasma cells (CD138) (Fig. 2A) and macrophages (CD68) (Fig. 2D), which together accounted for approximately 70% of the entire inflammatory cell population. The lymphocytic population represented <30% of the inflammatory cells, with a marked predominance of T cells over B cells, in a 5:1 ratio (Fig. 3A); interestingly, CD56+ lymphocytes (comprising natural killer [NK] cells and a subset of T lymphocytes) provided a substantial fraction of the total lymphocytes in the inflamed mucosa (Table 2). The main differences in the distribution of the inflammatory cells in active versus controls without IBD were thus restricted to the lymphocyte compartment. In fact, our results show a significative diminution of the percentage of T cells, particularly of the CD4+ subset, and an augmented frequency of CD56+ lymphocytes in inflamed mucosa, as compared with healthy mucosa (Table 2).

Table 2
Table 2
Image Tools
Figure 2
Figure 2
Image Tools
Figure 3
Figure 3
Image Tools

The analysis of noninflamed mucosal samples obtained from patients in the inactive phase of IBD shows that although the percentage of total T cells returns comparable with controls without IBD, a lower frequency of CD4+ T cells and a higher percentage of CD56+ lymphocytes are still detected in noninflamed mucosal tissue (Table 2). These results suggest that, although not characterized by a full-blown inflammation, the inactive stage of IBD still shows signs of an imbalanced immune profile at mucosal site.

We then analyzed the presence of TR cells in the mucosal biopsies of pediatric patients with IBD. As reported in Figure 3D to F, the absolute number of FOXP3+ TR cells was significantly increased in the inflamed mucosa of patients with IBD, being approximately 7 times higher than that detected in the mucosae of patients in remission stage or in controls without IBD (Table 3). The frequency of TR cells, relative either to the percentage of total T cells or to the percentage of CD4+ T lymphocytes, was also significantly increased in IBD inflamed mucosa (Fig. 3A–C, and Table 3), and was comparable between CD and UC samples (data not shown). Real-time PCR analysis confirmed the increased FOXP3 expression in active-stage IBD mucosa biopsies; in fact, FOXP3 mRNA expression closely paralleled the results obtained with immunohistochemistry (Table 4).

Table 3
Table 3
Image Tools
Table 4
Table 4
Image Tools

Collectively taken, our results indicate that the abundant inflammatory infiltrate of the active intestinal lesions of pediatric patients with IBD displays several alterations of the T and NK lymphocyte compartment; moreover, a highly significant increase in the TR cell subset is observed. Our results also show that the remission posttherapy stage is associated with partial normalization in the asset of lymphocyte subsets, concomitantly with a decrease in the frequency of mucosal FOXP3+ regulatory cells.

Back to Top | Article Outline
Evaluation of Circulating TR Cells in Pediatric Patients With IBD

We then analyzed the frequency of TR cells in the peripheral blood of the same cohorts of pediatric patients with IBD and age-matched controls without IBD. The percentage of TR cells, evaluated as CD25bright CD3+ CD4+ lymphocytes (16–19), was significatively higher in patients with IBD as compared with age-matched controls (Fig. 4A). The augmentation of TR cells was observed in patients affected by both forms of IBD (CD and UC) (Fig. 4B), and, by stratifying our patients according to the disease activity, we found that TR cells were significantly elevated in both patients during the active phase of disease and those in remission (Fig. 4C).

Figure 4
Figure 4
Image Tools

The expression of FOXP3 TR cell marker has been reported to be restricted almost exclusively to the CD4+ CD25bright population (21); more recently, functionally competent TR cells, within the CD4+ CD25bright subset, have been shown to selectively express low levels of CD127 (IL-7 receptor α-chain) (22–24). We verified, in a representative group of pediatric patients (12 IBD and 12 age-matched controls), that most of CD4+CD25bright T cells is CD127low/neg and expresses FOXP3, comparably in patients with IBD and controls. A representative cytofluorimetric analysis of FOXP3 and CD127 expression on CD4+ CD25bright T-cell subset is reported in Figure 5.

Figure 5
Figure 5
Image Tools

Moreover, because the inducible expression of CD25 receptor correlates with T-cell activation, we also evaluated the total frequency of CD25+ CD4+ T cells in pediatric patients with IBD. Our results show that the percentage of total CD4+ CD25+ T lymphocytes was also significantly higher in patients with IBD when compared with controls without IBD (Fig. 6A), and that this augmentation, paralleling what was observed for the CD4+ CD25bright subset, comparably occurred in patients with CD and UC (Fig. 6B), independent of disease activity (Fig. 6C). Collectively taken, these results show that the increase of CD4+CD25bright TR cells in pediatric patients with IBD is associated with the augmentation of circulating CD4+ CD25+ recently activated T lymphocytes. A representative cytofluorimetric analysis of CD4+ CD25bright and CD4+ CD25+ T-cell populations in active IBD, inactive IBD, and controls without IBD is reported in Figure 7.

Figure 6
Figure 6
Image Tools
Figure 7
Figure 7
Image Tools
Back to Top | Article Outline


In the present study, we evaluated the frequency of mucosal and peripheral T cells with a regulatory phenotype in distinct stages of pediatric IBD (active disease vs remission); moreover, we have also analyzed the composition of the immune infiltrate in inflamed and noninflamed mucosal tissues of pediatric patients with IBD, in an effort to gain information about the relation between effector cells and the regulatory compartment in the different phases of the disease.

TR cells represent 1 of the most important physiological mechanisms to suppress protective and pathological immune responses in peripheral tissues (14). Their pivotal role in suppressing mucosal immune activation and intestinal inflammation has been shown clearly in several models of gut chronic inflammation (5,6,13–15). Several reports have described the augmented frequency of intestinal and peripheral blood TR cells in adult patients with active IBD and those undergoing remission, respectively, but the role of these cells in the different stages of the disease has not been clarified yet (17–19,25–29).

Our study shows that the majority of the increased inflammatory cell population that infiltrates active mucosal lesions is represented by macrophages and plasma cells, which nevertheless are present in the same proportion as in healthy mucosae and in nonactive intestinal samples. Our results are in agreement with a recent report in which increased densities of macrophages in pediatric IBD colonic biopsies as compared with age-matched controls without IBD are shown (40). On the contrary, the frequency of T and NK lymphocyte subsets with respect to the total immune infiltrate clearly discriminates active IBD lesions from healthy intestinal tissue; in fact, T lymphocytes are proportionally reduced in active lesions, especially the CD4+ subset, whereas lymphocytes expressing the CD56 receptor show a higher frequency.

Interestingly, our analysis shows that the inactive stage of IBD is characterized, at the intestinal level, by an only partially restored immunological equilibrium. In fact, although the total number of inflammatory cells is definitely lower than that in active lesions, they are nevertheless more abundantly present than in non-IBD lamina propria. This quantitative imbalance is accompanied by a skewed immunological profile: Although the total percentage of T cells closely matches that of healthy mucosa, the proportion of CD4+ T cells is lower than normal, and CD56+ lymphocytes are still more represented than in non-IBD mucosal samples. Collectively, these observations suggest that a sort of low-grade inflammation persists in the mucosal tissue of quiescent patients.

Previous observations did not report alterations in the relative frequency of CD4+ T cells in either active or inactive adult IBD mucosal lesions (41), leaving open the possibility that our results reflect a specificity of the pediatric condition. The augmented presence of effector cytotoxic cells expressing CD56 receptor has been previously noted in active and inactive mucosae of adult patients with IBD (42,43) and could play a role in the mechanisms underlying the mucosal damage. In fact, CD56 receptor is expressed in the majority of NK cells, which represent an important component of the innate arm of the immune system, and also play an immunoregulatory role at mucosal sites, by participating in the instruction of the adaptive response (44–46); in addition, CD56 expression marks a T-lymphocyte subset that bears effector-like properties in vivo (44,47) and that is constitutively present in the lamina propria (48).

The increased number of inflammatory cells and the selective imbalance in T-cell subsets and CD56+ lymphocytes are associated with an increased presence of FOXP3+ TR cells in mucosal lesions of active stage IBD (both CD and UC, data not shown), as detected by immunohistochemistry and quantitative mRNA analysis. However, in the inactive stage of the disease, the frequency of FOXP3+ regulatory T lymphocytes returns comparable to what is found in normal lamina propria.

To our knowledge, this is the first description of TR cells in the intestinal inflamed tissues of pediatric patients with IBD. Our results are in agreement with the previously observed augmentation of TR frequency in intestinal specimens of adult patients with active IBD (18,19,25,26). Controversial information is available on the abundance of mucosal TR in adult patients with inactive IBD because it has been reported to be either comparable to (18,19,26) or higher (25) than that in healthy subjects.

Here we show for the first time that peripheral blood CD4+ CD25bright TR cells are significantly augmented in pediatric patients with IBD, with a similar course in the 2 forms of the disease, CD and UC; interestingly, TR augmentation is observed with the same intensity in patients undergoing the active and inactive phases of the disease. These results are in contrast to previous reports indicating that in adult patients with IBD undergoing remission, the frequency of circulating TR cells is higher than that in patients in the active phase of the disease (17,18,25,26,28,29).

It has been demonstrated that the expression of FOXP3, which identifies the TR cell subset, is essentially confined to the population of CD4+ CD25bright T lymphocytes (16–19,21,29). More recently, several studies have shown that functionally competent TR cells are characterized by the low expression of CD127 (IL-7 receptor α-chain) surface marker, and that the expression of FOXP3 and the CD127low/neg phenotype highly correlate within the CD4+ CD25bright T-cell population (21–24). Accordingly, the analysis of these 2 TR markers in our pediatric patients with IBD and controls shows that the majority of peripheral CD4+CD25bright T cells is CD127low/neg and expresses FOXP3.

The effective role of circulating TR cells in dampening mucosal inflammation has not been clarified yet. Previous work in an experimental model of colitis showed that an impaired ability of TR cells to traffic at regional lymph nodes leads to a defective ability to control inflammation (49), whereas other authors found that the inhibition of TR trafficking to the mucosa did not impair their suppressive ability (50).

Our results show that although mucosal TR are markedly augmented in active IBD lesions and return to a frequency comparable to controls in remission phase, they remain persistently elevated in the blood during the inactive stage of the disease. Collectively, these data underscore the difference between the regional environment and the systemic compartment; they also suggest that the circulating and the intestinal pools of TR are differentially regulated and that complex dynamics regulates trafficking and tissue localization of TR cells to inflamed and noninflamed tissues. The functional capability of these cells has not been ascertained; however, previous studies (17–19,25–27) have shown that TR from adult patients with IBD display suppressive ability.

Finally, the augmentation of CD4+ CD25bright TR cells in pediatric patients with IBD is paralleled by a significant elevation of the percentage of CD4+ CD25+ T cells, which include “recently activated” T cells; interestingly, the augmentation of CD25 expression is restricted to the CD4+ T-cell subset, thus ruling out a generalized state of activation of circulating leukocytes in our patients (data not shown). The concurrent augmentation of the “recently activated” CD25+ T-cell population has been noted in some studies (17) but not in others (18,29) performed on adult patients with IBD. The functional capability of these cells and their possible role in the chronic condition of IBD require further investigation.

Our results show alterations in the representativeness of several mucosal (CD4+ and CD56+) and circulating (CD25bright and CD25+ CD4+ T cells) lymphocyte populations in patients undergoing disease remission. This suggests an incomplete normalization of the immune profile, independent of the clinical efficacy of the therapy and with no correlation with the type of pharmacological therapy (data not shown).

Back to Top | Article Outline


Our results highlight novel aspects of the complex modulation of the mucosal and systemic immune system during the course of pediatric IBD. The literature suggests a specificity of the pediatric condition of IBD, in terms of both underlying mechanisms and clinical management (30–32), which depend on a different asset of the immune system. A more pronounced thymopoietic activity in children may support the maintenance of a higher frequency of circulating TR cells during active IBD, which is different from what occurs in adult patients, in whom the recruitment of TR cells at mucosal sites correlates with a depletion of the systemic compartment. A further confirmation of the proposed specificity derives from the recent demonstration that in healthy adults there is a significantly higher colonic mucosal density of CD68+ macrophages as compared with children, and that the abundance of mucosal macrophages in IBD lesions is significantly augmented in pediatric but not in adult patients (40).

Finally, it is also understood that the profile of the pathological immune response is subjected to changes during the course of this chronic condition (33–36), so that the pediatric, early-onset disease could represent a more favorable setting to dissect the immune-mediated pathogenetic mechanisms in the relative absence of age-related and/or environmental confounding variables.

Back to Top | Article Outline


1. Calkins BM, Mendeloff AI. The epidemiology of idiopathic inflammatory bowel disease. In: Kirsner JB, Shorter RG, editors. Inflammatory Bowel Disease. Philadelphia: Williams & Wilkins; 1995. pp. 31–68.

2. Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347:417–429.

3. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998; 115:182–205.

4. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007; 448:427–434.

5. Macdonald TT, Monteleone G. Immunity, inflammation, and allergy in the gut. Science 2005; 307:1920–1925.

6. Cho JH. The genetics and immunopathogenesis of inflammatory bowel disease. Nat Rev Immunol 2008; 8:458–466.

7. Mathew CG. New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nat Rev Genet 2008; 9:9–14.

8. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003; 3:521–533.

9. Brown SJ, Mayer L. The immune response in inflammatory bowel disease. Am J Gastroenterol 2007; 102:2058–2069.

10. Elson CO, Cong Y, McCracken VJ, et al. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol Rev 2005; 206:260–276.

11. Targan SR, Karp LC. Defects in mucosal immunity leading to ulcerative colitis. Immunol Rev 2005; 206:296–305.

12. Cobrin GM, Abreu MT. Defects in mucosal immunity leading to Crohn's disease. Immunol Rev 2005; 206:277–295.

13. Coombes JL, Robinson NJ, Maloy KJ, et al. Regulatory T cells and intestinal homeostasis. Immunol Rev 2005; 204:184–194.

14. Tang Q, Bluestone JA. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 2008; 9:239–244.

15. Huibregtse IL, van Lent AU, van Deventer SJ. Immunopathogenesis of IBD: insufficient suppressor function in the gut? Gut 2007; 56:584–592.

16. Baecher-Allan C, Wolf E, Hafler DA. Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4+ CD25+ T cells. Clin Immunol 2005; 115:10–18.

17. Makita S, Kanai T, Oshima S, et al. CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells. J Immunol 2004; 173:3119–3130.

18. Maul J, Loddenkemper C, Mundt P, et al. Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease. Gastroenterology 2005; 128:1868–1878.

19. Holmén N, Lundgren A, Lundin S, et al. Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity. Inflamm Bowel Dis 2006; 12:447–456.

20. Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked: forkhead box protein 3 mutations and lack of regulatory T cells. J Allergy Clin Immunol 2007; 120:744–750.

21. Shevach EM, DiPaolo RA, Andersson J, et al. The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells. Immunol Rev 2006; 212:60–73.

22. Seddiki N, Santner-Nanan B, Martinson J, et al. Zaunders J, Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006; 203:1693–1700.

23. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006; 203:1701–1711.

24. Hartigan-O'Connor DJ, Poon C, Sinclair E, et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorting of live cells. J Immunol Methods 2007; 319:41–52.

25. Yu QT, Saruta M, Avanesyan A, et al. Expression and functional characterization of FOXP3+CD4+ regulatory T cells in ulcerative colitis. Inflamm Bowel Dis 2007; 13:191–199.

26. Saruta M, Yu QT, Fleshner PR, et al. Characterization of FOXP3+CD4+ regulatory T cells in Crohn's disease. Clin Immunol 2007; 125:281–290.

27. Kelsen J, Agnholt J, Hoffmann HJ, et al. FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease. Clin Exp Immunol 2005; 141:549–557.

28. Yokoyama Y, Fukunaga K, Fukuda Y, et al. Demonstration of low-regulatory CD25high+CD4+ and high-pro-inflammatory CD28-CD4+ T-cell subsets in patients with ulcerative colitis: modified by selective granulocyte and monocyte adsorption apheresis. Dig Dis Sci 2007; 52:2725–2731.

29. Takahashi M, Nakamura K, Honda K, et al. An inverse correlation of human peripheral blood regulatory T cell frequency with the disease activity of ulcerative colitis. Dig Dis Sci 2006; 51:677–686.

30. Bousvaros A, Morley-Fletcher A, Pensabene L, et al. Research and clinical challenges in paediatric inflammatory bowel disease. Dig Liver Dis 2008; 40:32–38.

31. Nieuwenhuis EE, Escher JC. Early onset IBD: what's the difference? Dig Liver Dis 2008; 40:12–15.

32. Bausvaros A, Antonioli DA, Colletti RB, et al. Differentiating ulcerative colitis from Crohn disease in children and young adults: reports of a Working Group of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the Crohn's and Colitis Foundation of America. J Pediatr Gastroenterol Nutr 2007; 44:653–674.

33. Desreumaux P, Brandt E, Gambiez L, et al. Distinct cytokine patterns in early and chronic ileal lesions of Crohn's disease. Gastroenterology 1997; 113:118–126.

34. Arseneau KO, Tamagawa H, Pizarro TT, et al. Innate and adaptive immune responses related to IBD pathogenesis. Curr Gastroenterol Rep 2007; 9:508–512.

35. Kugathasan S, Saubermann LJ, Smith L, et al. Mucosal T-cell immunoregulation varies in early and late inflammatory bowel disease. Gut 2007; 56:1696–1705.

36. Pappa HM, Semrin G, Walker TR, et al. Pediatric inflammatory bowel disease. Curr Opin Gastroenterol 2004; 20:333–340.

37. Carvalho R, Hyams JS. Diagnosis and management of inflammatory bowel disease in children. Semin Pediatr Surg 2007; 16:164–171.

38. Turner D, Otley AR, Mack D, et al. Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study. Gastroenterology 2007; 133:423–432.

39. Loonen HJ, Griffiths AM, Merkus MP, et al. A critical assessment of items on the pediatric Crohn's Disease Activity Index. J Pediatr Gastroenterl Nutr 2003; 36:90–95.

40. Perminow G, Reikvam DH, Lyckander LG, et al. Increased number and activation of colonic macrophages in pediatric patients with untreated Crohn's disease. Inflamm Bowel Dis 2009; 15:1368–1378.

41. Selby WS, Janossy G, Bofill M, et al. Intestinal lymphocyte subpopulations in inflammatory bowel disease: an analysis by immunohistological and cell isolation techniques. Gut 1984; 25:32–40.

42. van Tol EA, Verspaget HW, Peña AS, et al. Normal inflammatory bowel disease mucosa conceals alterations in natural killer cell activity. Scand J Gastroenterol 1992; 27:999–1005.

43. Van Tol EA, Verspaget HW, Peña AS, et al. The CD56 adhesion molecule is the major determinant for detecting non-major histocompatibility complex-restricted cytotoxic mononuclear cells from the intestinal lamina propria. Eur J Immunol 1992; 22:23–29.

44. Lanier LL, Chang C, Azuma M, et al. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J Immunol 1991; 146:4421–4426.

45. Andoniou CE, Coudert JD, Degli-Esposti MA. Killers and beyond: NK-cell-mediated control of immune responses. Eur J Immunol 2008; 38:2938–2942.

46. Münz C. Non-cytotoxic protection by human NK cells in mucosal secondary lymphoid tissues. Eur J Immunol 2008; 38:2946–2948.

47. Pittet MJ, Speiser DE, Valmori D, et al. Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression. J Immunol 2000; 164:1148–1152.

48. Hogan PG, Gibson PR, Hapel AJ, et al. Intestinal lymphokine-activated killer cells in inflammatory bowel disease. J Gastroenterol Hepatol 1991; 6:455–460.

49. Yuan Q, Bromley SK, Means TK, et al. CCR4-dependent regulatory T cell function in inflammatory bowel disease. J Exp Med 2007; 204:1327–1334.

50. Denning TL, Kim G, Kronenberg M. Cutting edge: CD4+CD25+ regulatory T cells impaired for intestinal homing can prevent colitis. J Immunol 2005; 174:7487–7491.


Crohn disease; inflammatory bowel disease; natural killer; regulatory T cells; ulcerative colitis

Copyright 2010 by ESPGHAN and NASPGHAN


Article Tools



Search for Similar Articles
You may search for similar articles that contain these same keywords or you may modify the keyword list to augment your search.

Connect With Us





Visit on your smartphone. Scan this code (QR reader app required) with your phone and be taken directly to the site.