The role of microbiota:immune system dysregulation in the etiology of colorectal cancer (CRC) is poorly understood. CRC develops in gut epithelium, accompanied by low level inflammatory signaling, intestinal microbial dysbiosis and immune dysfunction. We examined populations of intraepithelial lymphocytes in non-affected colonic mucosa of CRC and healthy donors and circulating immune memory to commensal bacterial species and yeasts. γδ T cells and resident memory T cells, populations with a regulatory CD39-expressing phenotype, were found at lower frequencies in the colonic tissue of CRC donors compared to healthy controls. Patterns of T cell proliferative responses to a panel of commensal bacteria were distinct in CRC, while B cell memory responses to several bacteria/yeast were significantly increased, accompanied by increased proportions of effector memory B cells, transitional B cells and plasmablasts in blood. IgA responses to mucosal microbes were unchanged. Our data describe a novel immune signature with similarities to and differences from that of inflammatory bowel disease. They implicate B cell dysregulation as a potential contributor to parainflammation and identify pathways of weakened barrier function and tumor surveillance in CRC-susceptible individuals.

INTRODUCTION: Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli gene that results in intestinal adenoma formation and colorectal cancer, necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis. METHODS: Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labeled with anti-IgA/IgG and analyzed by flow cytometry. RESULTS: Proportions of resident memory CD103-expressing CD8+ and γδ T-cell receptor+ intraepithelial lymphocytes were dramatically reduced in both the left and right colon of patients with FAP compared with healthy controls. In lamina propria, T cells expressed less CD103, and CD4+ CD103+ cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA+ peripheral B cells, and CD4 T-cell memory responses to commensal bacteria were increased in FAP. DISCUSSION: Loss of resident memory T cells and γδ T cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this precancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function, and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

Nearly 20% of NET have positive immunohistochemical (IHC) expression of ER and/or PR. Targeting ER/PR may benefit selected NET pts. Multicenter Simon two-stage single-arm phase II trial of tamoxifen 20mg PO daily until progression (PD). Eligible pts had progressive metastatic NET and positive IHC expression of ER and/or PR ≧ 1%. Prior somatostatin analogues (SSA) were required and kept in functioning NET. Main exclusion criteria: aggressive NET requiring cytotoxic therapy, oral anticoagulants, thromboembolism in the last 12 months. Imaging was performed every 12 weeks. Primary endpoint was disease control (DCR) at week 24 by RECIST 1.1. H0 was DCR at week 24 of 50%; H1 was DCR of 70%. Considering a dropout rate of 30%, alpha and beta errors of 5% and 20%, we planned to enroll 23 in the first stage; if ≥ 12 pts reached the primary endpoint, a total of 37 pts would be enrolled. If 23 out of 37 reached DCR at week 24, the trial would be positive. From Feb 2019 to Feb 2022, 88 pts were screened, 23 were eligible and enrolled: median age was 56, 15 (65%) were female, the most common sites were pancreas (11; 47.8%) and small bowel (5; 21.7%), 17 (74%) had nonfunctioning NET, 8 (34.8%), 13 (56.5%) and 2(8.7%) had G1, G2, G3 NET, respectively. At a median follow up of 27 months, 19 pts had PD, 1 died from postoperative complications at week 6, one is on trial, 2 stopped treatment at weeks 24 and 108 without PD. Twelve pts (52%) had DCR at week 24. Median progression-free survival (PFS) was 8 months (interquartile range [IQR]: 3.7 to 12.1). Best response was stable disease in 11 pts and 8 had PD at week 12. Median PFS was 6.1 months (IQR: 3.5 – 8.4) vs 9.4 months (IQR: 3.9 – 14.6), according to ER/PR < or ≥ 30% (logrank p = 0.48). The table describes pts with ER/PR ≥ 30% NET and week of PD. There were no grade ≥ 3 adverse events.Table: 897PPts with ER/PR ≥ 30% NETER/PR %GradeKi67 %Primary siteWeek of PD30/0340Lung2450/026Small bowel360/70215Pancreas6010/90210Pancreas480/80210Pancreas1240/011Small bowel9530/60210Ampulla12 Open table in a new tab Tamoxifen for ER/PR-positive NET pts is safe but offers modest antitumor effects.

Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1β. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines.

Objectives: Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli (APC) gene that results in intestinal adenoma formation and colorectal cancer (CRC), necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis. Methods: Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labelled with anti-IgA/IgG and analyzed by flow cytometry. Results: Proportions of resident memory CD103-expressing CD8+ and {gamma}{delta} T cell receptor+ intraepithelial lymphocytes were dramatically reduced in both left and right colon of patients with FAP compared to healthy controls. In lamina propria, T-cells expressed less CD103 and CD4+ CD103+ cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA+ peripheral B cells and CD4 T-cell memory responses to commensal bacteria were increased in FAP. Conclusions: Loss of resident memory T-cells and {gamma}{delta} T-cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this pre-cancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

Background: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all Gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC). Results: In healthy individuals, Bt OMVs stimulated significant (p<0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p<0.001 and p<0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p<0.01 and p<0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). Conclusions: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients.

Background and Aims The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD]. Methods Resident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed. Results Systemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses. Conclusions A previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an infectious disease endemic to south-east Asia. As B. pseudomallei is antibiotic-resistant, the need for cell-based vaccines and therapies is crucial to managing melioidosis. Dendritic cells (DC) provide the first line of defense to infection and direct downstream immune responses. Using practical volumes of fresh healthy donor blood, we show that heat-killed B. pseudomallei activated and stimulated expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DC. Furthermore, B. pseudomallei- pulsed DC induced activation and proliferation of CD4+ T-cells. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be targeted for future therapies and vaccines.Author Summary Melioidosis is an infectious disease endemic to south-east Asia and northern Australia caused by the bacterium Burkholderia pseudomallei . Melioidosis presents a significant public health threat because it has no effective vaccine or cure, leading to a high mortality rate of between 10-50%. We highlight the possibility of immune-based strategies targeting Burkholderia pseudomallei to better treat and prevent melioidosis. Specifically, we show that dendritic cells-the sentinel cells of the immune system-respond to B. pseudomallei in healthy individuals and in turn can orchestrate downstream protective immune responses. Thus, dendritic cells may be key players in the development of both vaccines and therapeutics for melioidosis as well as other bacteria-driven diseases.

44 Background & Aims: The intestinal microbiota is closely associated with resident memory 45 lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral 46 immunity to the microbiota differ in health versus inflammatory bowel disease (IBD). 47 Methods: Resident memory T-cells (Trm) in colonic biopsies and local antibody responses to 48 intraepithelial microbes were analyzed. Systemic antigen-specific immune Tand B-cell 49 memory to a panel of commensal microbes was assessed. 50 Results: Systemically, healthy blood showed CD4 and occasional CD8 memory T-cell 51 responses to selected intestinal bacteria but few memory B-cell responses. In IBD, CD8 52 memory T-cell responses decreased although B-cell responses and circulating plasmablasts 53 increased. Possibly secondary to loss of systemic CD8 T-cell responses in IBD, dramatically 54 reduced numbers of mucosal CD8 Trm and γδ T-cells were observed. IgA responses to 55 intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 56 ectonucleotidases, characteristic of regulatory T-cells. Cytokines/factors required for Trm 57 differentiation were identified, and in vitro-generated Trm expressed regulatory T-cell 58 function via CD39. Cognate interaction between T-cells and dendritic cells induced T-bet 59 expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal 60 responses. 61 Conclusions: A previously unrecognized imbalance exists between cellular and humoral 62 immunity to the microbiota in IBD, with loss of mucosal T-cell-mediated barrier immunity 63 and uncontrolled antibody responses. Regulatory function of Trm may explain their 64 association with intestinal health. Promoting Trm and their interaction with dendritic cells 65

Background: The intestinal microbiota is altered in inflammatory bowel disease (IBD), but how interactions between microbes and the host immune system differ in health versus IBD is not understood. Large numbers of lymphocytes reside in gut mucosal tissues and play a key role in barrier function and immune surveillance. We have analysed immune cells and microbes at the mucosal surface and compared acquired memory responses to specific commensal bacteria. Methods: Colonic biopsies were obtained from healthy donors, or Crohn's disease and ulcerative colitis patients not receiving corticosteroid or biologic therapies. Intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) were isolated by digestion, phenotyped and counted by flow cytometry. Intraepithelial microbes were labelled with SYBR Green and anti-IgA and analysed by flow cytometry. Commensal bacteria were isolated from the GI tract of healthy donors and identified by 16S rRNA gene sequencing. PBMC were CellTrace VioletTM-labelled and cultured with heat-killed bacteria to determine microbe-specific CD4+/CD8+ T cell and B cell responses after 7 days culture. Results: Numbers of intraepithelial lymphocytes (IEL), of both resident memory CD8+ and γδ T cell subsets, were dramatically reduced (75–95%, p<0.006) in IBD, and there were 70% fewer resident memory type CD4 T cells in the lamina propria in ulcerative colitis (p<0.017). IgA responses to bacteria on the gut epithelium, and circulating memory B cells responsive to certain intestinal bacteria were increased in IBD, while healthy PBMC demonstrated CD4 memory T cell and occasional CD8 T cell responses but few B cell responses. There were fewer CD8 memory T cell responses to commensals in Crohn's disease, correlating with the low numbers of CD8 T cells in IEL. Circulating B cells from IBD donors showed signs of activation including increased proportions of plasmablasts in Crohn's donors and IgA-switched memory B cells. Our data suggest an imbalance between humoral and cellular immunity towards the microbiota in IBD, with enhanced mucosal antibody secretion concomitant with a loss of mucosal T cell-mediated barrier immunity. Conclusions: Strong CD4 T cell memory to commensal bacteria develops in health, contradicting the concept of tolerance to intestinal bacteria. Lack of CD8 T cell immunity and deficiency in IEL in IBD may result in compromised barrier function at the mucosal surface, leading to excessive B cell activation and antibody secretion against the microbiota. Therefore, rather than blanket immunosuppression, stimulation of mucosal T cell immunity may improve barrier function and counteract abnormal T cell inflammation and B cell IgA secretion against intestinal microbes in IBD.

Introduction: The aetiology of Crohn's disease remains unclear, but it is characterised by an exaggerated immune response to mucosal antigen in patients with a genetic predisposition. In the gut, the primary antigen presenting cells, the dendritic cells (DC) and macrophages, may promote either a tolerogenic or an inflammatory T-cell response in response to mucosal antigen and are key to the primary pathophysiology of Crohn's disease. Here, we characterise DC and peripheral blood monocytes (macrophage precursors) in patients with Crohn's disease for identification, activation, homing markers and ongoing cytokine production. Methods: Peripheral blood mononuclear cells were isolated from adults with active luminal Crohn's disease and from healthy controls (HC). Multicolour flow cytometry was performed to assess DC subgroups: myeloid DC (mDC) and plasmacytoid DC (pDC). Additionally, monocytes were identified, and a subgroup analysis was made for comparison with the DC population. Expression of phenotypic markers (including maturation markers, homing markers and pattern recognition receptors) and assessment of on-going cytokine production (including TNFα, IL-6, IL-10) were assessed by intracellular staining. Results and Discussion: In patients with Crohn's disease, DC and monocytes expressed a stronger gut homing profile (CLA β7), whilst cells from HC had greater non-tissue-specific homing (CLAβ7) (Fig. A, B). This suggests that these cells have a predilection for travel to the gut from peripheral blood in Crohn's compared with HC. In examining subgroups of DC, we found in both Crohn's and HC, mDC were gut homing whilst pDC were strongly skin and lymph node homing, suggesting that they may have different roles in Crohn's, with mDC being central to gut inflammation, whilst pDC may be more involved in skin manifestations of disease. Production of pro-inflammatory cytokines was upregulated in Crohn's, with mDC producing high levels of TNFα (Fig. C) and pDC producing high levels of IL-6 (Fig. D), allowing these cells to direct a TH1 and TH17 immune response. The maturity marker CD86, indicating differentiation and ability to present antigen to T cells, was upregulated in mDC compared with pDC in Crohn's disease but not in HC. Additionally, the identified macrophage subgroup showed marked activation with elevated intracellular cytokine production and activation and homing markers in Crohn's compared with HC. Conclusions: This study demonstrates significant differences in maturation and homing marker expression and ongoing pro-inflammatory cytokine production by DC and macrophages in Crohn's disease. There are clearly differences in the roles of different DC subtypes in Crohn's, providing insight into the pathogenesis of this condition. That these changes are seen in cells in the peripheral blood before contact with inflamed gut indicates essential differences between the antigen presenting cell compartment of patients with Crohn's and HC, likely as a result of genetic predisposition. Vitamin D regulates dendritic cell activity and trafficking in Crohn's disease P BHAT, P HENDY,* D REDDI, D BERNARDO, L DURANT, A NOBLE, NR ENGLISH, SC KNIGHT, AL HART* *St Mark's Hospital, and Imperial College, London, UK Hospital Universitario de la Princesa, Madrid, Spain Australian National University Medical School, Canberra, Australia

Introduction: Mannose Binding Lectin (MBL) and MBL-associated serine proteases (MASP) activate the lectin pathway of the complement system and subsequent inflammatory mechanisms.The functional activity of MBL-MASP complex plays a crucial role in antimicrobial host defenses.Recently, we showed that MBL is locally produced by the intestinal epithelial cells and is required for intestinal homeostasis.Low MBL levels have been associated with Crohn's disease (CD).An association between MBL-deficiency and anti-yeast antibodies (ASCA) is observed in Crohn's disease (CD), and this MBL deficiency is frequently associated with a severe phenotype of CD.In the present study, we assessed the role of MBL2 polymorphisms in the modulation of MBL level and activity in CD patients.Methods: MBL levels, MBL-MASP functional activity, ASCA level and single nucleotide polymorphisms in the MBL2 and the NOD2 genes were determined in 69 CD patients and 30 age-and sex-matched healthy donors (HD).Results: MBL2 variant rs5030737 (codon 52) was associated with a low level of MBL and impaired MBL-MASP functional activities in both CD and HD.Furthermore, this variant was also associated with a higher level of ASCA in CD patients (p<0.05).Increased ASCA levels were found in CD patients with stricture formation and penetrating disease complications 42%, and 21% respectively.Besides, NOD2 variant rs2066844, highly associated with susceptibility to CD, was significantly correlated with the impairment of the functional activity of MBL-MASP complex.Conclusion: These results provide evidence that CD patients with severe clinical phenotype have an impairment of MBL-MASP functional activity, and this defect is associated with MBL2 and NOD2 variants.

Introduction Crohn’s disease (CD) is characterised by an exaggerated immune response to mucosal antigen. Dendritic cells (DC) are the primary antigen presenting cells and may promote either tolerogenic or inflammatory T cell responses to mucosal antigens. We characterised homing marker profile and ongoing cytokine production of circulating DC subsets from patients with Crohn’s disease. Methods DC within peripheral blood mononuclear cells from adults with active luminal Crohn’s or from healthy controls (HC) were characterised using flow cytometry. DC were identified as HLA-DR + and negative for markers of other cell lineages (CD3, CD14, CD16, CD19, CD34). Myeloid DC (mDC, CD11c + CD123 − ) and plasmacytoid DC (pDC, CD11c − CD123 + ) were assessed for phenotype (maturation status, homing markers and pattern recognition receptors) and on-going cytokine production by surface and intracellular staining, respectively. Results In patients with Crohn’s (n = 16), a greater proportion of myeloid DC expressed a gut-homing profile (CLA − β7 + , p = 0.0022) compared to controls (n = 10) where most myeloid DC were not tissue-specific (CLA + β7 + , p = 0.0014, Fig C). In Crohn’s and controls, myeloid DC were largely gut-homing (CLA − β7 + , p = 0.003) whilst plasmacytoid DC were strongly skin (CLA + β7 − ) and lymph node (CCR7 + ) homing (p Conclusion The increased myeloid DC expression of gut homing phenotype markers and production of TNFα in Crohn’s compared with controls highlights the central role that DC play in the pathogenesis of Crohn’s disease. Differences between homing markers on myeloid DC (gut homing) and plasmacytoid DC (skin homing) suggest that they may have different roles in different manifestations of Crohn’s, with myeloid DC being central to gut inflammation whilst plasmacytoid DC might be involved in cutaneous Crohn’s disease and the skin sequelae of anti-TNFα therapy. Disclosure of Interest None Declared

Background & Aims: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/− subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. Methods: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. Results: Colonic DC identified were myeloid (mDC, CD11c+CD123−) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3−CCR2−. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. Conclusions: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization. Keywords: CCR2, Dendritic Cells, Distal Colon, Human Gastrointestinal Tract, Proximal Colon, Microbiota

Introduction Dendritic cells (DC) can determine whether the mucosal immune system mounts an inflammatory or regulatory response to antigen and may contribute to the pathogenesis of Crohn’s disease. Vitamin D down-regulates DC inflammatory responses and could prove beneficial as a treatment adjunct in Crohn’s. This study assessed the effect of high dose parenteral vitamin D treatment on circulating DC phenotype and function in patients with active luminal Crohn’s receiving anti-TNFα therapy. Methods Peripheral blood mononuclear cells were isolated from 13 patients with active luminal Crohn’s and suboptimal vitamin D levels prior to and 6 weeks after starting anti-TNFα (infliximab) therapy. Patients with low vitamin D (<50nmol/L) were also given a single high dose of parenteral vitamin D (300,000 international units 1,25(OH)2 vitamin D3). Flow cytometry was used to identify total DC, (HLA-DR+ cells negative for markers of other cell lineages (CD3, CD14, CD16, CD19 & CD34)). DC were further subtyped as myeloid (mDC, CD11c+CD123−). Expression of phenotypic markers (including maturation and homing markers and pattern recognition receptors) and on-going DC cytokine production during 4 hours’ culture were assessed. Results Production of TNFα by myeloid DC was significantly reduced (p = 0.016 Fig C) in those patients who received vitamin D alongside anti-TNFα therapy; without vitamin D treatment, TNFα production by myeloid DC did not decrease significantly after anti-TNFα therapy (p = 0.96 Fig B). There was a significant negative correlation between change in vitamin D level and change in TNFα production by myeloid DC (p = 0.025, correlation coefficient =0.68 Fig D). An increase of serum 25(OH)vitamin D greater than 20 nmol/m was associated with a decrease in myeloid DC TNFα production. Anti-TNFα therapy alone induced a significant upregulation of the skin homing marker cutaneous lymphocyte antigen (CLA) on myeloid DC (p = 0.0055), an effect which was not seen in patients receiving adjunctive vitamin D.Abstract PWE-023 Figure 1 A&B TNFα production by mDC before and after treatment with anti-TNFα therapy in: patients who did not (A) and patients who did (B) receive vitamin D. (C) Correlation of change in vitamin D level and decrease in TNFα production by mDC Conclusion High doses of parenteral vitamin D in patients with Crohn’s promotes anti-TNFα down-regulation of circulating myeloid DC production of TNFα which may influence the subsequent interaction of DC and T cells. TNFα promotes a TH-1/ TH-17 response characteristic of Crohn’s inflammation; thus the ability of vitamin D to further block TNFα production may promote a more regulatory T cell response and improve outcomes when used as an adjunct to anti-TNFα therapy. The down-regulation of skin homing marker CLA by vitamin D may be clinically useful in those patients suffering cutaneous sequelae of anti-TNFα therapy. Disclosure of Interest None Declared