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Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8.sup.+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin [alpha]4[beta]7 in the colon but not in the blood, where they were enriched for [alpha]4[beta]7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin [alpha]4[beta]7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

FOXP3+ regulatory T cells (Tregs) are critical for preventing intestinal inflammation. However, FOXP3+ T cells are paradoxically increased in the intestines of patients with the inflammatory bowel disease (IBD) ulcerative colitis (UC) or Crohn's disease (CD). We determined whether these FOXP3+ cells in IBD patients share or lack the phenotype of such cells from patients without IBD. We quantified and characterized FOXP3+ Treg populations, as well as FOXP3- CD4+ T cells, in the lamina propria lymphocytes (LPL) of intestine surgically resected from patients with and without IBD, and in the blood of controls or Crohn's patients with or without disease activity. In all samples, a similar fraction of FOXP3+ cells expressed the \"natural\" Treg (nTreg) marker Helios, suggesting that, in IBD, these cells are not entirely \"induced\" Tregs (iTregs) derived from activated effector T cells. Helios+ and Helios- FOXP3+ T cells demonstrated similar expression of maturation markers, activation markers, and inhibitory molecules between IBD patients and controls, while FOXP3- cells paradoxically expressed more of the inhibitory receptors CD39, CTLA4, and PD-1 in inflamed mucosa. Greater expression of activation markers was also seen in both Helios+ and Helios- Tregs, relative to FOXP3- cells, in both IBD patients and controls, indicating that Tregs are effectively activated by antigen in IBD. Extensive immunophenotyping revealed that Helios+ and Helios- mucosal Tregs exist at a similar frequency, and have a similar expression of inhibitory molecules and activation markers in patients with IBD as in healthy controls.

Although FOXP3 + regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE -/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity. Regulatory T cell (T reg ) maintenance and function require IL-2, yet this cytokine is only present in low levels in vivo. In this study, the authors demonstrate that that T reg use heparanase to access IL-2 bound to heparan sulfate proteoglycans in the extracellular matrix of inflamed brain tissue in mice.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3-IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4*CD62L⁻FoxP3⁻, suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Background/AimsVedolizumab is an anti-α4β7 monoclonal antibody approved for the treatment of inflammatory bowel disease (IBD). This exploratory study aimed to identify biomarkers associated with vedolizumab response.MethodsTwenty-six IBD patients (15 with Crohn’s, 11 with ulcerative or indeterminate colitis) initiating vedolizumab at a single center between 2014 and 2016 underwent sampling of serum and peripheral blood mononuclear cells (PBMCs) before and during vedolizumab therapy. Response was defined as steroid-free improvement in endoscopic score or Harvey–Bradshaw index/simple clinical colitis activity index (reduction greater than 3 or total less than 3). PBMCs were evaluated for immunophenotype and expression of α4β7 integrin on lymphocytes before and during vedolizumab therapy. Serum vedolizumab levels and α4β7 saturation were measured serially after induction.ResultsFourteen out of 26 (54%) patients treated with vedolizumab responded to therapy. Pretreatment α4β7 expression was higher in responders on multiple subsets of T, B, and NK cells, with terminal effector memory (p = .0009 for CD4 and .0043 for CD8) and NK cells (p = .0047) best discriminating between responders and nonresponders. During therapy, log10 serum vedolizumab levels at trough were higher in responders than nonresponders (p = .0007). Conversely, the percentage of effector memory T cells with free α4β7 at trough was lower in responders than nonresponders (p < .0001). However, loss of α4β7 saturation with vedolizumab was more sensitive to low serum vedolizumab in nonresponders.ConclusionsPretreatment α4β7 expression and α4β7 receptor saturation during maintenance therapy were identified as candidate biomarkers for vedolizumab response.

Abstract Background Vedolizumab, an antibody blocking integrin α4β7, is a safe and effective therapy for Crohn’s disease and ulcerative colitis. Blocking α4β7 from binding its cognate addressin MAdCAM-1 on intestinal blood vessel endothelial cells prevents T cells from migrating to the gut mucosa in animal models. However, data supporting this mechanism of action in humans is limited. Methods We conducted a cross-sectional case-control study to evaluate the effect of vedolizumab on intestinal immune cell populations while avoiding the confounding effect of resolving inflammation on the cellularity of the colonic mucosa in treatment-responsive patients. Colon biopsies from 65 case subjects receiving vedolizumab were matched with biopsies from 65 control individuals, similar in disease type, medications, anatomic location, and inflammation. Biopsies were analyzed by flow cytometry and full messenger RNA transcriptome sequencing of sorted T cells. Results No difference was seen between vedolizumab recipients and control individuals in the quantity of any antigen-experienced T lymphocyte subset or in the quality of the transcriptome in any experienced T cell subset. Fewer naïve colonic B and T cells were seen in vedolizumab recipients than control individuals, regardless of response. However, the most striking finding was a marked reduction in CD1c+ (BDCA1+) dendritic cells exclusively in vedolizumab-responsive patients. In blood, these dendritic cells ubiquitously express high levels of α4β7, which is rapidly downregulated upon vedolizumab exposure. Conclusions The clinical effects of vedolizumab reveal integrin α4β7-dependent dendritic cell migration to the intestinal mucosa to be central to inflammatory bowel disease pathogenesis. Lay Summary Vedolizumab had no effect on the number or gene expression of memory T lymphocytes in the colons of recipients relative to control individuals. However, the colons of vedolizumab-responsive patients had distinctly fewer dendritic cells, which in blood express the most integrin α4β7.

Background Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are critical for controlling inflammation in the gastrointestinal tract. There is a paradoxical increase of mucosal FOXP3+ T cells in patients with inflammatory bowel disease (IBD). These FOXP3+ cells were recently shown to include interleukin (IL)-17A-producing cells in Crohn’s disease, resembling Th17 cells implicated in autoimmune diseases. FOXP3 inhibits IL-17A production, but a naturally occurring splice variant of FOXP3 lacking exon 2 (Δexon2) cannot. Aims We hypothesized that IBD patients preferentially express the Δexon2 variant of FOXP3 so the paradoxically increased mucosal Tregs in IBD could represent cells expressing only Δexon2. Methods We used antibodies and primers that can distinguish between the full-length and Δexon2 splice variant of FOXP3 to evaluate expression of these isoforms in human intestinal tissue by immunohistochemistry and quantitative polymerase chain reaction (PCR), respectively. Results No difference in the expression pattern of Δexon2 relative to full-length FOXP3 was seen in ulcerative colitis or Crohn’s disease versus non-IBD controls. By immunofluorescence microscopy and flow cytometry, we also did not find individual cells which expressed FOXP3 protein exclusively in the Δexon2 isoform in either IBD or control tissue. FOXP3+ mucosal CD4+ T cells from both IBD and control specimens were able to make IL-17A in vitro after phorbol myristate acetate (PMA) and ionomycin stimulation, but these cells did not preferentially express Δexon2. Conclusions Our data do not support the hypothesis that selective expression of FOXP3 in the Δexon2 isoform accounts for the inability of copious FOXP3+ T cells to inhibit inflammation or IL-17 expression in IBD.

Successful treatment of inflammatory bowel disease (IBD) with the anti-integrin α4β7 mAb vedolizumab suggests that interaction of this integrin with addressin mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is central to IBD pathogenesis. Although this was presumed to be due to an inhibition of lymphocyte trafficking to the gut, as has been observed in animal models, we report no depletion of CD4 T cells from the colonic mucosa as a consequence of vedolizumab treatment in humans, regardless of efficacy. Likewise, no upregulation of alternative trafficking mechanisms was observed as a consequence of therapy to suggest that this homeostasis is maintained in patients by a mechanistic escape from inhibition. Instead, we explore a role for MAdCAM–integrin interaction as a gut-specific costimulatory signal, demonstrating that it can replace CD28 ligation to activate human T cells in vitro. This activation through integrin α4β7 is mediated through the gut-restricted molecule MAdCAM-1, and it cannot be replicated by matrix molecules or proteins that bind other integrins. A detailed analysis of mRNA expression by human T cell subsets following suboptimal TCR stimulation in the presence or absence of CD28 versus MAdCAM-1 costimulation reveals marked similarity in the effect that these two signals have upon T cells, with temporal or quantitative differences detected in the expression of cytokines associated with Th17 cells or pyogenic inflammation. Thus, we describe an alternative costimulatory pathway for T cells in the intestine, through ligation of integrin α4β7 by MAdCAM-1, which may explain the therapeutic efficacy of vedolizumab and have implications concerning the treatment of IBD.

Ipilimumab is a humanized antibody to CTLA4 and is used to treat cancers refractory to conventional treatment. We treated 21 patients with refractory melanoma or prostate cancer with anti-CTLA4 antibody (ipilimumab), with subsequent development of significant colitis in nine cases. Two of these nine did not respond rapidly to high-dose (2 mg kg⁻¹ day⁻¹) glucocorticoids or infliximab. They required additional immunosuppression, and one ultimately died of opportunistic infection, representing a more refractory course than has previously been described complicating ipilimumab therapy. Both patients had received radiation to the pelvis for prostate cancer less than 1 year prior to receiving ipilimumab. We performed immunohistochemical analysis of colon biopsies from ipilimumab recipients to determine if colitis correlates with depletion of intramucosal FOXP3⁺ regulatory T cells (Tregs), which normally express CTLA4. However, we found no evidence of FOXP3⁺ T cell depletion in any of the nine patients who developed colitis.