MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses.

Although intestinal epithelial cells (IECs) can express major histocompatibility complex class II (MHC II), especially during intestinal inflammation, it remains unclear if antigen presentation by IECs favours pro- or anti-inflammatory CD4+ T cell responses. Using selective gene ablation of MHC II in IECs and IEC organoid cultures, we assessed the impact of MHC II expression by IECs on CD4+ T cell responses and disease outcomes in response to enteric bacterial pathogens. We found that intestinal bacterial infections elicit inflammatory cues that greatly increase expression of MHC II processing and presentation molecules in colonic IECs. Whilst IEC MHC II expression had little impact on disease severity following Citrobacter rodentium or Helicobacter hepaticus infection, using a colonic IEC organoid-CD4+ T cell co-culture system, we demonstrate that IECs can activate antigen-specific CD4+ T cells in an MHC II-dependent manner, modulating both regulatory and effector Th cell subsets. Furthermore, we assessed adoptively transferred H. hepaticus-specific CD4+ T cells during intestinal inflammation in vivo and report that IEC MHC II expression dampens pro-inflammatory effector Th cells. Our findings indicate that IECs can function as non-conventional antigen presenting cells and that IEC MHC II expression fine-tunes local effector CD4+ T cell responses during intestinal inflammation.

NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease.

Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.

Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.

Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.

Inflammatory cues enhance TGFß activation by distinct subsets of human intestinal dendritic cells via integrin αvß8.

Regulation of intestinal T-cell responses is crucial for immune homeostasis and prevention of inflammatory bowel disease (IBD). A vital cytokine in regulating intestinal T cells is transforming growth factor-ß (TGFß), which is secreted by cells as a latent complex that requires activation to function. However, how TGFß activation is regulated in the human intestine, and how such pathways are altered in IBD is completely unknown. Here we show that a key activator of TGFß, integrin αvß8, is highly expressed on human intestinal dendritic cells (DCs), specifically on the CD1c+ but not the CD141+ intestinal DC subset. Expression was significantly upregulated on intestinal DC from IBD patients, indicating that inflammatory signals may upregulate expression of this key TGFß-activating molecule. Indeed, we found that the Toll-like receptor 4 ligand lipopolysaccharide upregulates integrin αvß8 expression and TGFß activation by human DC. We also show that DC expression of integrin αvß8 enhanced induction of FOXP3 in CD4+ T cells, suggesting functional importance of integrin αvß8 expression by human DC. These results show that microbial signals enhance the TGFß-activating ability of human DC via regulation of integrin αvß8 expression, and that intestinal inflammation may drive this pathway in patients with IBD.

CD11c(+) monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23.

In inflammatory bowel diseases, a breakdown in host microbial interactions accompanies sustained activation of immune cells in the gut. Functional studies suggest a key role for interleukin-23 (IL-23) in orchestrating intestinal inflammation. IL-23 can be produced by various mononuclear phagocytes (MNPs) following acute microbial stimulation, but little is known about the key cellular sources of IL-23 that drive chronic intestinal inflammation. Here we have addressed this question using a physiological model of bacteria-driven colitis. By combining conditional gene ablation and gene expression profiling, we found that IL-23 production by CD11c(+) MNPs was essential to trigger intestinal immunopathology and identified MHCII(+) monocytes and macrophages as the major source of IL-23. Expression of IL-23 by monocytes was acquired during their differentiation in the intestine and correlated with the expression of major histocompatibility complex class II (MHCII) and CD64. In contrast, Batf3-dependent CD103(+) CD11b(-) dendritic cells were dispensable for bacteria-induced colitis in this model. These studies reinforce the pathogenic role of monocytes in dysregulated responses to intestinal bacteria and identify production of IL-23 as a key component of this response. Further understanding of the functional sources of IL-23 in diverse forms of intestinal inflammation may lead to novel therapeutic strategies aimed at interrupting IL-23-driven immune pathology.

CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut.

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

CTLA-4 promotes Foxp3 induction and regulatory T cell accumulation in the intestinal lamina propria.

Thymic induction of CD4(+)Foxp3(+) regulatory T (Treg) cells relies on CD28 costimulation and high-affinity T-cell receptor (TCR) signals, whereas Foxp3 (forkhead box P3) induction on activated peripheral CD4(+) T cells is inhibited by these signals. Accordingly, the inhibitory molecule CTLA-4 (cytotoxic T-lymphocyte antigen 4) promoted, but was not essential for CD4(+) T-cell Foxp3 induction in vitro. We show that CTLA-4-deficient cells are equivalent to wild-type cells in the thymic induction of Foxp3 and maintenance of Foxp3 populations in the spleen and mesenteric lymph nodes, but their accumulation in the colon, where Treg cells specific for commensal bacteria accumulate, is impaired. In a T cell-transfer model of colitis, the two known CTLA-4 ligands, B7-1 and B7-2, had largely redundant roles in inducing inflammation and promoting Treg cell function. However, B7-2 proved more efficient than B7-1 in inducing Foxp3 in vitro and in vivo. Our data reveal an unappreciated role for CTLA-4 in establishing the Foxp3(+) compartment in the intestine.

CD103+ GALT DCs promote Foxp3+ regulatory T cells.

There is evidence that Foxp3(+) regulatory T (T(R)) cells contribute to intestinal homeostasis and that deficiencies in this population can lead to chronic intestinal inflammation. Here, we review recent studies that demonstrate that the gut is a site of peripheral generation of T(R) cells. Functionally specialized gut dendritic cell populations promote T(R) cells through a transforming growth factor-beta and retinoic acid-dependent mechanism. Gut-driven T(R) cells may represent a tissue-specific mechanism to broaden the repertoire of T(R) cells focussed on the gut.

Immune therapy in inflammatory bowel disease and models of colitis.

BACKGROUND: A proliferation of animal models has not only improved our understanding of inflammatory bowel disease, it has also formed the basis of new treatment strategies. METHODS: A search was conducted using the National Library of Medicine for articles discussing immune therapies for inflammatory bowel disease. This was supplemented by findings from the authors' own laboratory. RESULTS: An overview of the different animal models is presented. These models are used to highlight the recent human trials of immune therapies. Potential future therapies are also discussed. CONCLUSION: Immune therapies have altered the management of patients with inflammatory bowel disease. In future they will influence not only the indications for surgery but also its timing and outcome.

Control of intestinal inflammation by regulatory T cells.

Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo. Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.

CD4(+) regulatory T cells.

There is now compelling evidence that CD4(+) T cells that specialize in the suppression of immune responses play a key role in the control of immune pathology. Recently, there have been a number of reports that have provided information on the generation of CD4(+) regulatory T cells in the thymus and in the periphery. These cells have also been identified in humans, paving the way for analysis of the function of CD4(+) regulatory T cells in immune-mediated disease.

Control of intestinal inflammation by regulatory T cells.

Regulatory T(Treg)-cell populations have been identified in a number of disease models. In this review we focus on the role of naturally occurring Treg cells in the control of intestinal inflammation. Specifically, we discuss their mechanism of action with particular emphasis on the role of anti-inflammatory cytokines and cell surface molecules.

Regulatory T cells in the control of immune pathology.

It is now well established that regulatory T (T(R)) cells can inhibit harmful immunopathological responses directed against self or foreign antigens. However, many key aspects of T(R) cell biology remain unresolved, especially with regard to their antigen specificities and the cellular and molecular pathways involved in their development and mechanisms of action. We will review here recent findings in these areas, outline a model for how T(R) cells may inhibit the development of immune pathology and discuss potential therapeutic benefits that may arise from the manipulation of T(R) cell function.

CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice.

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.

Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro.

CD4(+)CD25(+) T cells in mice and rats are capable of transferring protection against organ-specific autoimmune disease and colitis and suppressing the proliferation of other T cells after polyclonal stimulation in vitro. Here we describe the existence in humans of CD4(+)CD25(+) T cells with the same in vitro characteristics. CD4(+)CD8(-)CD25(+) T cells are present in both the thymus and peripheral blood of humans ( approximately 10 % of CD4(+)CD8(-) T cells), proliferate poorly in response to mitogenic stimulation and suppress the proliferation of CD4(+)CD25(-) cells in co-culture. This suppression requires cell contact and can be overcome by the addition of exogenous IL-2. CD4(+)CD25(+) cells from thymus and blood were poor producers of IL-2 and IFN-gamma, and suppressed the levels of these cytokines produced by CD4(+)CD25(-) cells. However, CD4(+)CD25(+) PBL produced higher levels of IL-4 and similar amounts of IL-10 as CD4(+)CD25(-) cells. Regulatory CD4(+)CD25(+) T cells have an activated phenotype in the thymus with expression of CTLA-4 and CD122 (IL-2Rbeta). The fact that CD4(+)CD25(+) regulatory T cells are present with a similar frequency in the thymus of humans, rats and mice, suggests that the role of these cells in the maintenance of immunological tolerance is an evolutionarily conserved mechanism.

The transmembrane form of the CX3CL1 chemokine fractalkine is expressed predominantly by epithelial cells in vivo.

Fractalkine (CX3CL1) is synthesized as a type I transmembrane protein. Its unique CX(3)C chemokine domain is attached to a 241-amino acid mucin stalk, a 19-amino acid transmembrane domain, and a 37-amino acid intracellular domain of unknown function. A soluble form of fractalkine can be generated by proteolytic cleavage at the base of the mucin stalk. Novel monoclonal and polyclonal antibodies that specifically recognize only the amino- or carboxyl-terminal ends of the human fractalkine molecule have revealed that epithelial cells are the predominant cell type expressing transmembrane forms of fractalkine in human skin, the tonsil, and the large intestine. Using these specific anti-fractalkine reagents we do not detect high-level expression of fractalkine on endothelial cells in normal or inflamed colon samples obtained from patients with Crohn's disease or ulcerative colitis. In contrast to previous reports we do not detect fractalkine expression by Langerhans cells or immature dendritic cells in mucosal-associated lymphoid tissues in vivo. We show that the reagent used in previous studies, an anti-fractalkine N-terminal peptide antisera, cross-reacts with human CD84. Finally we discuss potential roles for fractalkine in constitutive leukocyte trafficking based on its observed pattern of expression in epithelia.

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo.

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.

Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells.

The severe combined immunodeficiency (SCID) model of colitis shares many features of idiopathic inflammatory bowel disease (IBD) in humans. The SCID model is highly reproducible and easily manipulated, and as such provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. This unit describes a cell transfer system in which SCID mice are reconstituted with CD4(+) CD45RB(high) cells to induce IBD. The CD4(+) cells are isolated by immunomagnetic negative selection using monoclonal antibodies, and are then separated by fluorescent cell sorting into CD45RB(high) and CD45RB(low) cells. The former population reproducibly induces disease in recipients. A support protocol describes methods to monitor disease progression, which is characterized by weight loss, loose stools, and histologically assessed lesions in the colon.

Control of experimental inflammatory bowel disease by regulatory T cells.

A helper T cell type 1-mediated colitis driven by enteric bacteria develops in severe combined immunodeficient mice after transfer of CD45RB(high)CD4(+) T cells. Development of disease can be prevented by cotransfer of the reciprocal CD45RB(low) subset. Analysis of the mechanism of immune suppression transferred by CD45RB(low)CD4(+) cells revealed essential roles for both IL-10 and TGF-beta. These data indicate that a functionally specialized population of regulatory T (Treg) cells exists in normal mice and that these can prevent the development of pathogenic responses toward commensal bacteria. The role of Treg cells in the control of the immune response is discussed.