Pengfei Zhou held a scholarship from the Natural Sciences and Engineering Research Council of Canada. Editor:B. addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4+CD45RBhighT cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation. Inflammatory bowel disease (IBD) is usually a chronic inflammatory condition associated with alteration of immunoregulatory mechanisms responsible for the control of immune responses to commensal microbiotas and their products (15,34). Under normal conditions, commensal microflora influences the development and function of local and systemic immune responses limiting an overactive inflammatory response (23,38,44). PF-5006739 Conversely, bacterial pathogens or their products can stimulate both innate and acquired immune responses, resulting in overt acute and chronic mucosal inflammation (22). Superantigens (SAgs) are microbial proteins that activate large subsets of T or B lymphocytes. Staphylococcal enterotoxins, toxic shock syndrome toxin 1, streptococcal SAg, andMycoplasma arthritidismitogen are examples of T-cell SAgs (24,26,28). T-cell SAgs bind to the variable region of the T-cell receptor (TCR) or chain and cross-link with the major histocompatibility complex class II molecules (11,13,18,29). Oral administration ofStaphylococcus aureusenterotoxin B (SEB) induces a transient mucosal T-cell activation followed by persistent anergy and deletion of T cells bearing the SEB-reactive V8 TCR for up to 4 weeks after the treatment (33,47). Given the large number of SAg-producing microbial brokers in the gut flora, it is probable that this mechanism involved in regulation of mucosal immune T-cell responses to microbial SAgs is critical to the prevention of commensal bacterium-induced chronic inflammation (32). Furthermore, SAgs have been implicated in immune-mediated diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and IBD (25,26,39,40,50,51). Skewed TCR repertoires have been identified in patients with IBD (5,37,46), and a SAg-like protein derived fromPseudomonas fluorescens, I2, was identified in colonic lesions of over 50% of Crohn’s disease patients in a study (8,10,14). However, the exact mechanism defining how SAg may contribute to PF-5006739 inflammation in the intestinal mucosa is usually unknown. Here we investigated the role of regulatory T (Treg) cells in the effect of orally administered SEB on T-cell subsets and on the development of mucosal inflammation. SCID mice were fed SEB after mice were reconstituted with CD4+CD45RBhighT cells alone or CD4+CD45RBhighT cells together with CD4+CD45RBlowT cells. While feeding SEB had no clinical effect on SCID mice reconstituted with both CD4+CD45RBhighand CD4+CD45RBlowT cells, feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4+CD45RBhighT cells alone. This was associated with activation and growth of SEB-reactive CD4+V8+T cells and prevention of the development of T cells expressing Foxp3. These results suggest that Treg cells modulate effector T-cell responses to enteric bacterium-derived SAgs, preventing excessive activation of mucosal T cells and preserving the normal intestinal structure and function. == MATERIALS AND METHODS == == Mice. == Congenic C.B-17 SCID mice and BALB/c mice were obtained from Harlan (Indianapolis, IN). DO11.10 breeders were purchased from Charles River Laboratories (Wilmington, MA). Rabbit polyclonal to Bub3 Female mice between 8 and 12 weeks of age were used in these studies. All animal experiments were performed in accordance with institutional guidelines as approved by the Animal Care Review Board of McMaster University. All PF-5006739 mice were housed under specific-pathogen-free conditions at the central animal facility at McMaster University. Donor and recipient mice in our colony were routinely screened forHelicobacterspecies contamination by PCR capable of detecting ribosomal sequences common to allHelicobacterspecies and were free of contamination (48). == Isolation and purification of CD45RBhighand CD45RBlowCD4+spleen cells. == CD4+T-cell subsets from the spleens of BALB/c and DO11.10 mice were isolated and sorted as described previously (36). Briefly, single-cell suspensions were depleted of B220+, MAC1+, and CD8+cells PF-5006739 by unfavorable selection using M-450 sheep anti-rat immunoglobulin G-coated Dynabeads (Dynal Biotech, Oslo, Norway). Purified anti-CD8, anti-CD11b, and anti-MAC1 antibodies were obtained from BD PharMingen (Mississauga, Ontario, Canada). CD4+CD45RBhighand CD4+CD45RBlowT-cell fractions were sorted on a FACSVantage SE cell sorter (BD Biosciences, San Jose, CA) under sterile conditions. The purity of each subpopulation was >98%. == Reconstitution of SCID-bg mice with T-cell subsets and SEB treatment. == BALB/c- and DO11.10-derived CD4+CD45RBhighand.