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Background The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. Methods Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. Results Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. Conclusions The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.

Multiple sclerosis (MS) has been proposed to be an immune-mediated disease in the central nervous system (CNS) that can be triggered by virus infections. In Theiler's murine encephalomyelitis virus (TMEV) infection, during the first week (acute stage), mice develop polioencephalomyelitis. After 3 weeks (chronic stage), mice develop immune-mediated demyelination with virus persistence, which has been used as a viral model for MS. Regulatory T cells (Tregs) can suppress inflammation, and have been suggested to be protective in immune-mediated diseases, including MS. However, in virus-induced inflammatory demyelination, although Tregs can suppress inflammation, preventing immune-mediated pathology, Tregs may also suppress antiviral immune responses, leading to more active viral replication and/or persistence. To determine the role and potential translational usage of Tregs in MS, we treated TMEV-infected mice with ex vivo generated induced Tregs (iTregs) on day 0 (early) or during the chronic stage (therapeutic). Early treatment worsened clinical signs during acute disease. The exacerbation of acute disease was associated with increased virus titers and decreased immune cell recruitment in the CNS. Therapeutic iTreg treatment reduced inflammatory demyelination during chronic disease. Immunologically, iTreg treatment increased interleukin-10 production from B cells, CD4(+) T cells and dendritic cells, which may contribute to the decreased CNS inflammation.

Most therapeutic agents used in clinical practice today were originally developed and tested in animal models so that drug toxicity and safety, dose-responses and efficacy could be determined. Retrospective analyses of preclinical intervention studies using animal models of different diseases demonstrate that only a small percentage of the interventions reporting promising effects translate to clinical efficacy. The failure to translate therapeutic efficacy from bench to bedside may be due, in part, to shortcomings in the design of the clinical studies; however, it is becoming clear that much of the problem resides within the preclinical studies. One potential strategy for improving our ability to identify new therapeutics that may have a reasonable chance of success in well-controlled clinical trials is to identify the most relevant mouse models IBD and pharmacologic strategies that most closely mimic the clinical situation. To begin this process, we present a critical evaluation of the different mouse models and pharmacological approaches that may be used in intervention studies as well as discuss emerging issues related to study design and data interpretation of preclinical studies.

The etiologies of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) have not been fully elucidated. However, there is very good evidence implicating T cell and T cell trafficking to the gut and its associated lymphoid tissue as important components in disease pathogenesis. The objective of this review is to provide an overview of the mechanisms involved in naive and effector T cell trafficking to the gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes and intestine in response to commensal enteric antigens under physiological conditions as well as during the induction of chronic gut inflammation. In addition, recent data suggests that the GALT may not be required for enteric antigen-driven intestinal inflammation in certain mouse models of IBD. These new data suggest a possible paradigm shift in our understanding of how and where naive T cells become activated to yield disease-producing effector cells.

The purpose of the present work was to investigate the interaction of drugs and octanol with hydroxypropyl β- (HPβCD) and γ- (HPγCD) cyclodextrin, sulfobutyl ether β-cyclodextrin (SBEβCD) and randomly methylated-β-cycoldextrin (RMβCD) and to describe the interaction by theoretical models. The poorly soluble steroid drugs progesterone, estrone and prednicarbate were used as model compounds in this study. Hexane and chloroform were also investigated in combination with HPβCD. Octanol formed a complex with all cyclodextrins and the saturation of the aqueous solution with this solvent therefore had a significant effect on the solubilization and extraction potential of cyclodextrins. Hexane had less affinity for cyclodextrins, but the drugs were poorly soluble in this solvent and it could therefore not be used in phase-distribution investigations. Previously we have derived equations that can be used to account for the competitive interaction between two guest compounds that compete for space in the cyclodextrin cavity. These equations were rearranged to calculate the complexation efficacy from phase-solubility data. An equation was derived that obtains intrinsic solubility (S 0) and intrinsic partition coefficient (P) from the slopes of the phase-solubility and phase-distribution profiles. Investigation of the data showed that the results could not be sufficiently explained by the “classical” drug/cyclodextrin complex model that recognizes the possibility of competitive interactions but ignores any contribution from higher order complexes or aggregation of the cyclodextrin complexes. Relative difference in solubilization potential of different cyclodextrins cannot be translated to relative differences in extraction efficacy. Thus, for these three steroid compounds, RMβCD and SBEβCD gave the best solubilization potential whereas the best extraction efficacy was observed with HPγCD.

Cytokine-producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response. In particular, Th1 and Th17 cells have been implicated in the pathogenesis of such diseases as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Quantification of Th1 and Th17 cells can provide important information in research of these diseases and other Th1- and Th17-mediated immune disorders. In vitro stimulation of cells followed by surface and intracellular staining, presented here, has the advantage of detecting the cytokines directly instead of relying exclusively on surrogate surface markers which, although showing enrichment for the effector T cells, are not specific markers for the cytokine-producing cells.

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)– dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c+ dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.

There is a growing body of experimental and clinical data suggesting that the chronic intestinal inflammation observed in the inflammatory bowel diseases (Crohn’s disease and ulcerative colitis) may originate as a dysregulated immune response to enteric bacterial antigens in genetically susceptible individuals.1,2 There is also good evidence to suggest that, in the absence of appropriate regulatory mechanisms, naive T cells become primed and polarized to yield colitogenic effector T helper (Th)1/Th17 cells via their interaction with enteric antigen-loaded dendritic cells within the gutdraining mesenteric lymph nodes and intestinal lamina propria.3 Unfettered generation and expansion of these disease-producing effector cells result in the enhanced production of Th1 and Th17 cytokines (e.g., interferon [IFN]-γ, interleukin [IL]-17, and IL-22) in the case of Crohn’s disease or the overproduction of Th2 cytokines (IL-5 and IL-13) in ulcerative colitis.4 Studies from numerous laboratories using a variety of immune-manipulated and genetically engineered animal models of chronic intestinal inflammation strongly suggest that regulatory T cells (Tregs; CD4+Foxp3+) are important in regulating and suppressing potentially aggressive immune responses toward autoantigens and commensal enteric antigens.5–8 Two major subsets of Tregs have been identified including natural Tregs (nTregs) that are generated within the thymus and induced Tregs (iTregs) that are produced in the periphery from conventional (CD4+Foxp3 −) T cells in a transforming growth factor (TGF)-β- and IL-2-dependent manner.3 Natural Tregs represent approximately 10% to 15% of the peripheral CD4+ T cells in a healthy mouse (

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

s / Biol Blood Marrow Transplant 19 (2013) S129eS149 S141 CD95. We studied peripheral blood from 54 patients with hematologic malignancies >2 years post HSCT (39 with cGVHD; 15 without cGVHD). In patients without cGVHD, BH3 priming was similar in Treg, Tcon and CD8. In contrast, Treg and CD8 were more primed than Tcon in cGVHD when challenged with BIM, BAD, PUMA, BMF and BAD + NOXA peptides. In patients with cGVHD, Treg expressed higher levels of Bim, BclxL and CD95 and lower levels of Bcl-2, but there was no significant difference in Mcl1. Consistent with these observations, Treg were more susceptible than Tcon and CD8 to both mitochondrial and death receptor pathway apoptosis in vitro. Increased Treg susceptibility to apoptosis in cGVHD therefore appears to be mediated primarily by increased expression of CD95 and Bim and lower levels of Bcl-2. Although Treg priming was increased overall in cGVHD, examination of Treg in severe cGVHD actually revealed less priming in this subset. This correlated with increased Treg expression of Bcl-2 and Mcl1. Since the total number of Treg is significantly reduced in severe cGVHD, these findings suggest that the remaining Treg are relatively resistant to the mitochondrial pathway of apoptosis. However, CD95 expression in Treg remains high in severe cGVHD indicating persistent high-level apoptosis mediated through the death receptor pathway. Administration of low dose IL-2 to patients with severe cGVHD led to increased expression of Bcl-2 and less susceptibility to both mitochondrial and death receptor pathway apoptosis. These studies help define the complex and distinct pathways that regulate survival in different T cell subsets, and changes in these pathways that occur in patients with chronic GVHD. These pathways play important roles in the maintenance of T cell homeostasis and targeting these complex pathways can provide new opportunities to promote immune tolerance after allogeneic HSCT.

Introduction: We have previously demonstrated that adoptive transfer of naive CD4+ T cells devoid of lymphocyte function-associated antigen-1-deficient (LFA-1; CD11a/CD18) into recombination activating gene-1 (RAG-1) deficient (RAG−/−) mice fails to induce chronic colitis whereas transfer of wild type (WT) T-cells induces unrelenting and chronic disease. Methods: The objectives of this study were to assess the role of lymphocyte function-associated antigen-1 (LFA-1) in enteric antigen (EAg)-induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon. Results: We found that EAg-pulsed dendritic cells (DCs) induced proliferation of LFA-1-deficient (CD11a−/−) CD4+ T cells that was very similar to that induced using WT T cells, suggesting that LFA-1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a−/− T cells with EAg-pulsed DCs induced the generation of similar amounts of interferon-gamma, interleukin (IL)-4, and IL-10, whereas IL-17A production was reduced ≈2-fold in cocultures with CD11a−/− T cells. Short-term (20–22 hours) trafficking studies demonstrated that while both WT and CD11a−/− T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a−/− T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3–7 days posttransfer, we observed ≈2–3-fold more WT T cells within the MLNs and colon than CD11a−/− T cells, whereas T-cell proliferation (as measured by CFSE dilution) was comparable in both populations. Conclusions: Taken together, our data suggest that LFA-1 is not required for EAg-induced activation of CD4+ T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation. (Inflamm Bowel Dis 2012;)

Most therapeutic agents used in clinical practice today were originally developed and tested in animal models so that drug toxicity and safety, dose–responses, and efficacy could be determined. Retrospective analyses of preclinical intervention studies using animal models of different diseases demonstrate that only a small percentage of the interventions reporting promising effects translate to clinical efficacy. It is becoming increasingly appreciated that the failure to translate therapeutic efficacy from bench to bedside may be due, in part, to selection of an animal model that may not recapitulate the immunopathologic features of the human disease under investigation. This is especially true for preclinical investigations using mouse models of the inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis). One potential strategy for improving our ability to discover new therapeutics that may have a reasonable chance of success in clinical trials is to identify the most immunologically relevant mouse models of human IBD. This chapter presents a critical evaluation of the different mouse models of IBD and discusses their utility in preclinical studies.

The intestinal tract is colonized by more than 100 trillion (1014) microorganisms, with the vast majority residing within the colonic lumen.1,2 The gut-associated lymphoid tissue (GALT; Peyer’s patches [PPs], isolated lymphoid follicles) as well as the gut-draining mesenteric lymph nodes (MLNs) play important roles in mounting mucosal immune responses to invading pathogenic and/or commensal microorganisms while minimizing the tissue inflammation and systemic immune activation that may arise from these protective responses.3–11 Studies from several different laboratories suggest that commensal bacterial antigens continuously gain access to the intestinal interstitium where they are endocytosed by intestinal dendritic cells (DCs) and then transported from the lamina propria and PPs to the draining MLNs.6–8,12 Using carefully regulated, low-level cellular immune responses to these nonpathogenic bacteria, the MLNs provide for both anatomical and functional compartmentalization of the adaptive immune responses, thereby generating a “firewall” to the systemic dissemination of these microorganisms.6,7 The mechanisms by which the cellular elements of the GALT and MLNs mediate these distinct and seemingly opposing functions are only now beginning to be revealed. Although there is good evidence demonstrating that MLNs play a critical role mounting and regulating mucosal immune responses to enteric bacteria, the evidence demonstrating a role for PPs as a major inductive site for adaptive immune responses is somewhat vague and ill-defined.6–8,13 Recent work by Worbs et al13 demonstrate that the MLNs (but not PPs) are required for the induction of systemic tolerance to orally administered luminal antigens. Those investigators convincingly showed that ovalbumin-loaded DCs are transported from the gut interstitium and PPs to the MLNs by the afferent lymphatics, where tolerance to these luminal antigens is induced. There is an accumulating literature suggesting that a break in tolerance to enteric antigens may be responsible for the induction of the chronic intestinal inflammation observed in patients who suffer from the inflammatory bowel diseases (IBDs; Crohn’s disease; ulcerative colitis).14–17 Indeed, the role of commensal bacteria in the pathogenesis of experimental IBD has been well characterized in number of different animal models of chronic gut inflammation.14,16,18 Although the etiology of these chronic inflammatory disorders has not been definitively elucidated, there is accumulating evidence to suggest that chronic gut inflammation arises from a complex interaction among genetic, immune, and environmental factors.16,18,19 A growing body of experimental and clinical data suggest that chronic gut inflammation arises from a dysregulated immune response to enteric bacterial antigens. An immunologically important yet undefined aspect of disease pathogenesis in animal models of IBD is the anatomic location(s) where naive T cells encounter enteric antigens to yield colitogenic effector cells. It has been assumed that naive T cells migrate to the GALT/MLNs, where they are primed and polarized to yield T helper 1 (Th1) and/or Th17 effector cells. These colitogenic effector cells then exit the GALT and/or MLNs by way of the efferent lymphatic vessels, enter into the systemic circulation, and home to the gut where they initiate intestinal inflammation.15–18,20 In reality, there have been few studies that have assessed the role of the GALT and/or MLNs in the induction of chronic gut inflammation. Spahn et al21 demonstrated that dextran sulfate sodium (DSS)-induced colitis is more severe in mice devoid of MLNs, suggesting an overall suppressive function of these gut-draining lymph nodes, whereas Dohi et al22 showed an exacerbated inflammatory infiltrate and Th1 response in PP- and colonic patch-deficient mice exposed to intrarectal trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol. In contrast to these studies, work by Makita et al23 suggests that neither the GALT, MLNs, spleen, nor peripheral lymph nodes were required for the induction (or suppression) of chronic colitis in mice. These investigators intercrossed lymphotoxin-α deficient (LTα−/−) mice with recombinase activating gene-2 deficient (RAG-2−/−) animals to generate LTα−/− × RAG−/− double-deficient progeny that are devoid of all organized lymphoid tissue.23 They found that adoptive transfer of naive or antigen-experienced (activated/memory) CD4+ T cells obtained from the lamina propria of mice with active colitis to LTα−/− × RAG-2−/− recipients induced colonic inflammation; however, the onset and severity of disease was delayed when compared to the disease induced by T-cell transfer to littermate controls (i.e., LTα+/+ × RAG-2−/− recipients). Although compelling, unanswered questions remain. For example, it is unclear how a global defect in LTα during development of lymphopenic RAG−/− mice affects lymphotoxin receptor-β expression and subsequent lymphotoxin signaling events associated with T-cell trafficking, priming, and polarization following adoptive transfer of naive T-cells. Therefore, the objectives of this study were to evaluate, using both surgical as well as genetic approaches, the role of gut-associated and secondary lymphoid tissue in the conversion of naive T-cells to colitogenic Th1/Th17 effector cells and their induction of chronic colitis. We present data demonstrating that neither the spleen, GALT, MLNs nor peripheral lymph nodes (PLNs) are required for the induction of chronic gut inflammation.

Abstract 3006 Background: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges, with acute lung injury being associated with significant morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that the PHI DMOG (Dimethyl oxalyl glycine) may reduce lung injury evolving after murine allogeneic HCT. Methods: 14 week old BALB/c were conditioned with 750 cGy single dose TBI, followed by transplantation of 5×10E6 bone marrow cells and 4×10E6 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. DMOG in PBS was given to both syngeneic and allogeneic recipients at 8mg per mice ip on alternate days for the first two weeks post-transplant and then twice a week till day 50, while syngeneic and allogeneic controls received PBS only. Animals were monitored for clinical GVHD and survival. At day 50 post HCT, gut, liver and lung pathology as well as BAL fluid T cell numbers were assessed by using a scoring system quantifying severity and extent of parenchymal and peribronchial/perivascular infiltrates and flow cytometry, respectively. In addition, T cell expansion was analyzed both in vivo in the spleen on day +7 and in vitro by mixed lymphocyte reaction (MLR). Results: Allogeneic HCT resulted in severe lung pathology in PBS treated animals (mean 118.8±47.19), but was significantly reduced when DMOG was given (36.0±17.99; p < 0.05), and absent in both PBS and DMOG treated syngeneic controls. Consistently, BAL CD4+ and CD8+ T cells in DMOG recipients were decreased by 53.7% and 68.4%, respectively, when compared to PBS controls. Decreased pulmonary injury was associated with a trend towards better overall survival by day 50 (allogeneic DMOG: 59.2% vs allogeneic PBS 40.7%) and all syngeneic recipients survived. GVHD related injury to liver and GI tract at time of analysis was not different between allogeneic groups. Alloreactive proliferative T cell responses in vitro were suppressed in the presence of DMOG at a concentration of 1mM (cpm after 96h: mean 29316±875.9 vs 41735±3085; p < 0.01). In vivo, splenic T cell expansion of CD4+ but not CD8+ T cells was reduced at day +7 as well (p < 0.05). Interestingly, when compared to PBS treated controls reduced mortality of DMOG treated allogeneic HCT recipients could already be seen soon after HCT (day +10: 26.0% vs 11.1%) in association with decreased CD4+ T cell proliferation, while lung injury in this model this early is rather marginal. Conclusion: DMOG treatment decreases the severity of acute noninfectious lung injury and improves overall survival after allogeneic HCT. While impaired early T cell responses likely contribute to improvement of both endpoints, altered recruitment of CD8+ T cells to the lung seems to uniquely affect the development of pulmonary but not gastrointestinal and hepatic injury at later time points. Current studies are ongoing to delineate the effects of DMOG on T cell proliferation and recruitment to different target organs, and to define the role of this novel approach in the prevention and treatment of HCT related complications. Disclosures: No relevant conflicts of interest to declare.

We have recently reported a novel ex vivo method to convert large numbers of CD4+Foxp3- T-cells to induced regulatory T-cells (iTregs; CD4+Foxp3+) via their incubation with plate-bound CD3 mAb, IL-2, TGFβ and retinoic acid (RA) for 4 days. These iTregs possess enhanced immune-suppressive activity in vitro and in vivo when compared to naturally-occurring Tregs (nTregs; CD4+Foxp3+). The objectives of this study were to: a) quantify and compare mRNA transcripts for genes known to be associated with Treg function using flow-purified iTregs and nTregs obtained from Foxp3GFP reporter mice and b) evaluate the stability of iTreg-mediated suppression in vitro and in vivo. We found 8 genes associated with iTregs that were significantly up-regulated when compared with nTregs with CCL4 (132-fold), Granzyme A (18-fold) and Runx1 (14-fold) showing the most dramatic increases. We also observed significant downregulation of 19, Treg-associated genes with IL-18 (600-fold), CD7 (300-fold), and IL-7 (100-fold) showing the most dramatic reductions in iTregs vs. nTregs. In addition, we found that when ex vivo-generated iTregs were “rested” by culturing iTregs for an additional 3 days in the absence of CD3 mAb, TGFβ and RA but in the presence of IL-2, Foxp3 expression, proliferative activity and in vitro suppressive activity were maintained. Unexpectedly, we found that these cells were more potent than nTregs in reversing preexisting colitis in a mouse model of chronic gut inflammation.

The objective of this study was to evaluate the role of PPs, MLNs and spleen in the pathogenesis of chronic colitis induced in mice. For the first series of studies, we transferred sorted, naïve (CD4+CD45RBhigh) T-cells (5x105 cells) into sham-operated RAG-1-/- mice or into RAG-1-/- mice that had their MLNs, spleen or both tissues surgically removed. We found that at 8 wks following T-cell transfer, all four of the surgically-manipulated RAG-1-/- groups developed moderate-severe colitis that was not different among the four groups. Flow cytometric analysis revealed large and similar numbers of IFN-γ producing T-cells within the colonic lamina propria (LP) of all four groups. For the second series of studies we intercrossed lymphotoxinβ-deficient (LTβ-/-) mice with RAG-1-/- animals to generate LTβ-/-xRAG-/- offspring that were devoid of Peyer’s patches and most peripheral lymph nodes. Surprisingly, and in contrast to what has been reported for LTβ-/- mice, we found no evidence of MLNs in the LTβ-/-xRAG-/- mice suggesting that lymphocytes may be required for MLN development in LTβ-/- mice. Adoptive transfer of CD45RBhigh T-cells into splenectomized LTβ-/-xRAG-/- mice or their littermate controls (LTβ+/+x RAG-/-) induced rapid onset of colitis suggesting that neither the PPs, MLNs nor the spleen are required for induction of disease. The mechanisms by which naïve T-cells are converted to colitogenic effector cells in this model are currently under investigation.

The objective of this study was to develop an ex vivo method to generate large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a T-cell-dependent model of chronic colitis. We found that polyclonal activation of CD4+CD25- Foxp3- T-cells in the presence of small amounts of IL-2, TGFβ and all trans retinoic acid (RA) converts >90% of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as induces a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we found that the presence of RA enhances the surface expression of the gut-homing adhesion molecules α4β7 and CCR9. Finally, we observed that these iTregs are significantly more potent at suppressing T-cell activation in vitro and reversing established colitis in vivo. This protocol routinely generates 30-40 million iTregs from one mouse spleen assuming that the spleen yields approximately 10 million CD4+Foxp3- T-cells and that >90% of these T-cells convert to Foxp3-expressing iTregs as well as undergo a 4-fold proliferation during the 4 day incubation period. We conclude that this protocol generates sufficient numbers of iTregs that can be used in vitro and in vivo to assess the therapeutic efficacy and immunological mechanisms responsible for suppression of inflammation in different models of autoimmune disease (Supported by PO143785).