ORIGINAL ARTICLE Peripheral Regulatory T Cells and Serum Transforming Growth Factor-b: Relationship with Clinical Response to Infliximab in Crohn’s Disease Antonio Di Sabatino, MD,* Paolo Biancheri, MD,* Silvia Piconese, PhD, † M. Manuela Rosado, PhD, ‡ Sandro Ardizzone, MD, § Laura Rovedatti, MD,* Cristina Ubezio, MD,* Alessandro Massari, MD,* Gianluca M. Sampietro, MD, ¶ Diego Foschi, MD, k Gabriele Bianchi Porro, MD, § Mario P. Colombo, PhD, † Rita Carsetti, MD, PhD, ‡ Thomas T. MacDonald, PhD, FRCPath, FmedSci, k and Gino R. Corazza, MD* Background: CD4 þ Foxp3 þ regulatory T cells (Treg) inhibit T- cell proliferation in vitro and are effective in suppressing colitis in mouse models. Tumor necrosis factor (TNF)-a, which is cen- trally involved in Crohn’s disease (CD) pathogenesis, also impairs Treg function. Here we investigated the influence of anti-TNF therapy on Treg frequency and function in CD. Methods: Twenty CD patients were treated with infliximab administered at weeks 0, 2, and 6. Blood was collected immedi- ately before the first infusion and after 10 weeks. Treg frequency was quantified by flow cytometry. Treg function was measured using a standard coculture assay. Serum levels of transforming growth factor (TGF)-b1 and interleukin (IL)-10 were measured by enzyme-linked immunosorbent assay (ELISA). Results: Pretreatment Treg frequency and serum TGF-b1 levels were significantly higher in nonresponder than responder patients. Clinical improvement in 12 CD patients was associated with a significant increase of Treg frequency after 10 weeks. Treg were functionally active before and after treatment with infliximab, both in responder and nonresponder CD patients. In responder patients the restoration of Treg pool was accompanied by a paral- lel significant increase of serum TGF-b1 and IL-10. No significant change in the elevated Treg or serum TGF-b1 was seen in nonres- ponder patients. Conclusions: This study suggests that there may be a relation- ship between numbers of Treg in the blood, serum TGF-b1, and response to infliximab; however, further prospective studies are needed. (Inflamm Bowel Dis 2010;16:1891–1897) Key Words: Foxp3, IL-10, suppression assay, TNF-a C D4 þ Foxp3 þ regulatory T cells (Tregs) can be defined as T cells able to suppress naı ¨ve T-cell proliferation both in vitro and in vivo. 1 Tregs are characterized by con- stitutively high expression levels of the transcription factor Foxp3, which is responsible for their regulatory activity. 2 Two major Treg populations have been described so far, which have been designated as naturally occurring and induced Treg. 1,3 Naturally occurring Tregs originate in the thymus and subsequently migrate to the periphery where they can prevent inappropriate immune responses. 3 Extra- thymically, naı ¨ve CD4 þ T cells can differentiate into induced Treg when activated by T-cell receptor stimulation in the presence of transforming growth factor (TGF)-b and interleukin (IL)-2. 3,4 Tregs play a crucial role in controlling T-cell homeo- stasis and in modulating the immune response to autoanti- gens, cancer cells, pathogens, and alloantigens. 5,6 Indeed, mice lacking Foxp3 develop a rapid fatal multiorgan auto- immune disease, 7 and impaired function of circulating Treg has been reported in patients with rheumatoid arthritis, 8 psoriasis, 9 systemic lupus erythematosus, 10 and multiple sclerosis. 11 In the gut, Tregs play a crucial role in the maintenance of immunological tolerance, 12–14 and they exert a potent antiinflammatory action in experimental coli- tis. 14,15 Despite retaining their ability to suppress T-cell Additional supporting information may be found in the online version of this article. Received for publication January 25, 2010; Accepted February 1, 2010. From the *First Department of Medicine, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, Fondazione IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy, † Molecular Immunology Unit, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, ‡ B-cell Development Lab, Bambino Gesu ` Pediatric Hospital, Rome, Italy, § Gastroenterology Division, Department of Clinical Sciences, Luigi Sacco University Hospital, Milan, Italy, ¶ Surgery Division, Department of Clinical Sciences, Luigi Sacco University Hospital, Milan, Italy, k Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London, UK. Reprints: Dr. Antonio Di Sabatino, MD, Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Universita ` di Pavia, Piazzale Golgi 5, 27100 Pavia, Italy (e-mail: a.disabatino@smatteo.pv.it) A.D.S. and P.B. contributed equally to the article and are considered joint first authors; T.T.M. and G.R.C. are considered joint last authors. Copyright V C 2010 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.21271 Published online 1 April 2010 in Wiley Online Library (wileyonlinelibrary.com). Inflamm Bowel Dis  Volume 16, Number 11, November 2010 1891