Preclinical development of a bispecific TNFα/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.

Flow Cytometry Reveals the Nature of Oncotic Cells.

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability+ve/caspase-3-ve/RIP3+ve/-ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX+ve/PARP-ve, cleaved PARP induced caspase independent apoptosis H2AX-ve/PARP+ve and hyper-activation or parthanatos H2AX+ve/PARP+ve. Oncotic cells with phenotype cell viability+ve/RIP3-ve/caspase-3-ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis.

Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl Dihydrogen Phosphate, for the Treatment of Inflammatory Diseases.

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3.

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.

Aryl Hydrocarbon Receptor Interacting Protein Maintains Germinal Center B Cells through Suppression of BCL6 Degradation.

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.

Preclinical evaluation of EPHX2 inhibition as a novel treatment for inflammatory bowel disease.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn's patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1ß in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.

Reduced CD27-IgD- B Cells in Blood and Raised CD27-IgD- B Cells in Gut-Associated Lymphoid Tissue in Inflammatory Bowel Disease.

The intestinal mucosa in inflammatory bowel disease (IBD) contains increased frequencies of lymphocytes and a disproportionate increase in plasma cells secreting immunoglobulin (Ig)G relative to other isotypes compared to healthy controls. Despite consistent evidence of B lineage cells in the mucosa in IBD, little is known of B cell recruitment to the gut in IBD. Here we analyzed B cells in blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) with a range of disease activities. We analyzed the frequencies of known B cell subsets in blood and observed a consistent reduction in the proportion of CD27-IgD- B cells expressing all Ig isotypes in the blood in IBD (independent of severity of disease and treatment) compared to healthy controls. Successful treatment of patients with biologic therapies did not change the profile of B cell subsets in blood. By mass cytometry we demonstrated that CD27-IgD- B cells were proportionately enriched in the gut-associated lymphoid tissue (GALT) in IBD. Since production of TNFα is a feature of IBD relevant to therapies, we sought to determine whether B cells in GALT or the CD27-IgD- subset in particular could contribute to pathology by secretion of TNFα or IL-10. We found that donor matched GALT and blood B cells are capable of producing TNFα as well as IL-10, but we saw no evidence that CD27-IgD- B cells from blood expressed more TNFα compared to other subsets. The reduced proportion of CD27-IgD- B cells in blood and the increased proportion in the gut implies that CD27-IgD- B cells are recruited from the blood to the gut in IBD. CD27-IgD- B cells have been implicated in immune responses to intestinal bacteria and recruitment to GALT, and may contribute to the intestinal inflammatory milieu in IBD.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

A functional IL1RL1 variant regulates corticosteroid-induced sST2 expression in ulcerative colitis.

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase.

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4ß7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

Proteolytic cleavage and loss of function of biologic agents that neutralize tumor necrosis factor in the mucosa of patients with inflammatory bowel disease.

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. METHODS: ILCs were isolated from colons of Tbx21(-/-) × Rag2(-/-) mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. RESULTS: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. CONCLUSIONS: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells.

A CD3-specific antibody reduces cytokine production and alters phosphoprotein profiles in intestinal tissues from patients with inflammatory bowel disease.

BACKGROUND & AIMS: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. METHODS: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. RESULTS: Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. CONCLUSIONS: We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.

A role for gut-associated lymphoid tissue in shaping the human B cell repertoire.

We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire, a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus, activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals, this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans.

Proinflammatory Vδ2+ T cells populate the human intestinal mucosa and enhance IFN-γ production by colonic αß T cells.

In nonhuman primates, Vγ9Vδ2(+) (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4ß7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4ß7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103(+) and CD103(-) subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103(-) population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4(+) T cells. Instead, phosphoantigen-activated CD103(-) Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αß T cells via an IFN-γ-dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103(+) and CD103(-) subsets that can promote inflammation by colonic αß T cells.