Background and Aims: We sought to determine whether six commonly used immunosuppressive regimens were associated with lower antibody responses after seasonal influenza vaccination in patients with inflammatory bowel disease [IBD].Methods: We conducted a prospective study including 213 IBD patients and 53 healthy controls: 165 who had received seasonal influenza vaccine and 101 who had not. IBD medications included infliximab, thiopurines, infliximab and thiopurine combination therapy, ustekinumab, vedolizumab, or tofacitinib. The primary outcome was antibody responses against influenza/A H3N2 and A/H1N1, compared to controls, adjusting for age, prior vaccination, and interval between vaccination and sampling.Results: Lower antibody responses against influenza A/H3N2 were observed in patients on infliximab (geometric mean ratio 0.35 [95% confidence interval 0.20-0.60], p = 0.0002), combination of infliximab and thiopurine therapy (0.46 [0.27-0.79], p = 0.0050), and tofacitinib (0.28 [0.14-0.57], p = 0.0005) compared to controls. Lower antibody responses against A/H1N1 were observed in patients on infliximab (0.29 [0.15-0.56], p = 0.0003), combination of infliximab and thiopurine therapy (0.34 [0.17-0.66], p = 0.0016), thiopurine monotherapy (0.46 [0.24-0.87], p = 0.017), and tofacitinib (0.23 [0.10-0.56], p = 0.0013). Ustekinumab and vedolizumab were not associated with reduced antibody responses against A/H3N2 or A/H1N1. Vaccination in the previous year was associated with higher antibody responses to A/H3N2. Vaccine-induced anti-SARS-CoV-2 antibody concentration weakly correlated with antibodies against H3N2 [r = 0.27; p = 0.0004] and H1N1 [r = 0.33; p < 0.0001].Conclusions: Vaccination in both the 2020-2021 and 2021-2022 seasons was associated with significantly higher antibody responses to influenza/A than no vaccination or vaccination in 2021-2022 alone. Infliximab and tofacitinib are associated with lower binding antibody responses to influenza/A, similar to COVID-19 vaccine-induced antibody responses.

Introduction Patients with Inflammatory bowel disease (IBD) receiving anti-TNF or JAK-inhibitor therapy have attenuated responses to COVID-19 vaccination. We aimed to determine how IBD treatments affect neutralising antibody responses against Omicron BA.4/5 and wild-type strain. Methods 68 healthy controls (HC) and 261 patients with IBD were sampled after 3 doses of COVID-19 vaccine. The IBD population was established (>12 weeks therapy) on either infliximab (IFX) (n=43), thiopurine (n=60), thiopurine & IFX combination (n=46), ustekinumab (n=43), vedolizumab (n=46) or tofacitinib (n=23). Pseudo neutralisation assays were performed and the half maximal inhibitory concentration (NT50) of participant sera was calculated. Primary outcome was neutralising responses against wild-type (WT) virus and BA.4/5 variant stratified by immunosuppressive therapy, adjusting for prior infection, vaccine type and age. Results Antibody titres against BA.4/5 were significantly lower than against WT virus in both HCs and IBD groups (figure 1. Geometric Mean Ratio (GMR) [95% CI], 0.11 [0.09, 0.15], P<0.0001 in healthy participants; GMR 0.07 [0.06, 0.08], P<0.0001 in IBD patients). Multivariable models showed that neutralising antibodies against BA.4/5 after three doses of vaccine were significantly lower in IBD patients on IFX (GMR 0.44 [0.20, 0.97], P=0.042), IFX and thiopurine combination (GMR 0.34 [0.15, 0.77], P=0.0098) or tofacitinib (GMR 0.37 [0.15, 0.92], P=0.032), but not in patients on thiopurine monotherapy, ustekinumab or vedolizumab. Breakthrough infection was associated with lower neutralising antibodies against WT (P=0.037) and BA.4/5 (P=0.045). Conclusions A third dose of COVID-19 vaccine based on the WT spike glycoprotein boosts neutralising antibody titres in patients with IBD. However, responses are lower against Omicron BA.4/5, particularly in patients taking anti-TNF or JAK-inhibitor therapy. Breakthrough infections are associated with lower neutralising antibodies and immunosuppressed IBD patients may receive additional benefit from bivalent vaccine boosters which target Omicron variants.

Background:Patients with inflammatory bowel disease (IBD) receiving anti-TNF and JAK-inhibitor therapy have attenuated responses to COVID-19 vaccination. We aimed to determine how IBD treatments affect neutralising antibody responses against the Omicron BA.4/5 variant. Methods:In this multicentre cohort study, we prospectively recruited 340 adults (69 healthy controls and 271 IBD) at nine UK hospitals between May 28, 2021 and March 29, 2022. The IBD study population was established (>12 weeks therapy) on either thiopurine (n = 63), infliximab (n = 45), thiopurine and infliximab combination therapy (n = 48), ustekinumab (n = 45), vedolizumab (n = 46) or tofacitinib (n = 24). Patients were excluded if they were being treated with any other immunosuppressive therapies. Participants had two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccines, followed by a third dose of either BNT162b2 or mRNA1273. Pseudo-neutralisation assays against SARS-CoV-2 wild-type and BA.4/5 were performed. The half maximal inhibitory concentration (NT50) of participant sera was calculated. The primary outcome was anti-SARS-CoV-2 neutralising response against wild-type virus and Omicron BA.4/5 variant after the second and third doses of anti-SARS-CoV-2 vaccine, stratified by immunosuppressive therapy, adjusting for prior infection, vaccine type, age, and interval between vaccination and blood collection. This study is registered with ISRCTN (No. 13495664). Findings:Both heterologous (first two doses adenovirus vaccine, third dose mRNA vaccine) and homologous (three doses mRNA vaccine) vaccination strategies significantly increased neutralising titres against both wild-type SARS-CoV-2 virus and the Omicron BA.4/5 variant in healthy participants and patients with IBD. Antibody titres against BA.4/5 were significantly lower than antibodies against wild-type virus in both healthy participants and patients with IBD (p < 0.0001). Multivariable models demonstrated that neutralising antibodies against BA.4/5 after three doses of vaccine were significantly lower in patients with IBD on infliximab (Geometric Mean Ratio (GMR) 0.19 [0.10, 0.36], p < 0.0001), infliximab and thiopurine combination (GMR 0.25 [0.13, 0.49], p < 0.0001) or tofacitinib (GMR 0.43 [0.20, 0.91], p = 0.028), but not in patients on thiopurine monotherapy, ustekinumab, or vedolizumab. Breakthrough infection was associated with lower neutralising antibodies against wild-type (p = 0.037) and BA.4/5 (p = 0.045). Interpretation:A third dose of a COVID-19 mRNA vaccine based on the wild-type spike glycoprotein significantly boosts neutralising antibody titres in patients with IBD. However, responses are lower against the Omicron variant BA.4/5, particularly in patients taking anti-TNF and JAK-inhibitor therapy. Breakthrough infections are associated with lower neutralising antibodies and immunosuppressed patients with IBD may receive additional benefit from bivalent vaccine boosters which target Omicron variants. Funding:Pfizer.

Background and aims Perianal fistulization is a challenging phenotype of Crohn’s disease (CD). Unravelling the cytokine networks and cellular mediators driving tissue injury in perianal CD (pCD) will help inform much needed novel treatment strategies. Methods The phenotype of different T-cell subsets, including unconventional lineages, such as γδ T-cells, MAIT and iNKT-cells in fistula tract tissue and blood samples of patients with pCD or cryptoglandular perianal fistulas was determined using multiparameter flow cytometry. Transcriptomic profiling of fistula tract tissue was performed by RNA-sequencing. Results CD161 + CD4 + T-cells and CD161 + CD4 - CD8 - iNKT-cells significantly accumulated in fistula tissue and produced interleukin-(IL)22 and IL13. Transcripts associated with epithelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) remodelling, interferon-gamma, JAK-STAT and lymphocyte signalling were enriched in pCD, as well as inhibition of pathways associated with wound closure. IL22-responsive transcripts were significantly enriched in fistulas and network analysis identified IL22-mediated regulation of EMT, ECM and other inflammatory pathways. Conclusion This study provides novel molecular and cellular insights into fistula pathogenesis, identifying IL22 producing lymphocytes as novel immune regulators of EMT and ECM dysregulation in perianal fistulas. Targeting the regulatory networks controlling IL22 producing lymphocytes may offer novel therapeutic strategies in pCD.

Abstract Background Patients with Inflammatory bowel disease (IBD) receiving anti-TNF or JAK-inhibitor therapy have attenuated responses to COVID-19 vaccination. We aimed to determine how IBD treatments affect neutralising antibody responses against the currently dominant Omicron BA.4/5 variants. Methods We prospectively recruited 329 adults (68 healthy controls (HC) and 261 IBD) who had received three doses of COVID-19 vaccine at nine UK centres. The IBD population was established (>12 weeks therapy) on either thiopurine (n=60), infliximab (IFX) (n=43), thiopurine & IFX combination (n=46), ustekinumab (n=43), vedolizumab (n=46) or tofacitinib (n=23). Pseudoneutralisation assays were performed and the half maximal inhibitory concentration (NT50) of participant sera was calculated. The primary outcome was anti-SARS-CoV-2 neutralising response against wild-type (WT) virus and the BA.4/5 variant after the second and third doses of anti-SARS-CoV-2 vaccine, stratified by immunosuppressive therapy, adjusting for prior infection, ethnicity, vaccine type and age. Results Heterologous (two doses adenovirus vaccine, third dose mRNA vaccine) and homologous (three doses mRNA vaccine) vaccination strategies significantly increased neutralising titres against both WT SARS-CoV-2 virus and the BA.4/5 variants in HCs and IBD (fig 1). Antibody titres against BA.4/5 were significantly lower than antibodies against WT virus in both groups (Geometric Mean Ratio (GMR) [95% CI], 0.11 [0.09, 0.15], P<0.0001 in healthy participants; GMR 0.07 [0.06, 0.08], P<0.0001 in IBD patients). Multivariable models showed that neutralising antibodies against BA.4/5 after three doses of vaccine were significantly lower in IBD patients on IFX (GMR 0.44 [0.20, 0.97], P=0.042), IFX and thiopurine combination (GMR 0.34 [0.15, 0.77], P=0.0098) or tofacitinib (GMR 0.37 [0.15, 0.92], P=0.032), but not in patients on thiopurine monotherapy, ustekinumab or vedolizumab. Breakthrough infection was associated with lower neutralising antibodies against WT and BA.4/5 (P<0.05). Conclusion A third dose of COVID-19 vaccine based on the WT spike glycoprotein boosts neutralising antibody titres in patients with IBD. However, responses are lower against the currently dominant variant BA.4/5, particularly in patients taking anti-TNF or JAK-inhibitor therapy. Breakthrough infections are associated with lower neutralising antibodies and immunosuppressed IBD patients may receive additional benefit from bivalent vaccine boosters which target Omicron variants. Financial support was provided as an Investigator Sponsored Research Grant from Pfizer Limited.

Perianal fistulizing Crohn’s Disease (pCD) affects about 25% of patients with Crohn’s Disease (CD). It remains a difficult entity to manage with a therapeutic ceiling of treatment success despite improving medical and surgical management. The refractory nature of the disease calls for an imminent need to better understand its immunopathogenesis and classification to better streamline our treatment options. In this article, we overview the current state of pCD management and discuss where the future of its management may lie.

Abstract Background Robust COVID-19 vaccine-induced antibody (Ab) responses are important for protective anti-viral immunity. Data are urgently needed to determine whether vaccine-induced immunity is impacted by commonly used immunosuppressive drug regimens in IBD. Methods We prospectively recruited 447 adults (90 healthy controls and 357 IBD) at nine UK centres. The IBD study population was established (>12 weeks therapy) on either thiopurine monotherapy (n=78), infliximab (IFX) monotherapy (n=61), thiopurine & IFX combination therapy (n=70), ustekinumab (uste) monotherapy (n=56), vedolizumab (vedo) monotherapy (n=62) or tofacitinib (tofa) monotherapy (n=30). Participants had two doses of either ChAdOx1 nCoV-19, BNT162b2 or mRNA1273 vaccines. The primary outcome was anti-SARS-CoV-2 spike (S1 RBD) Ab concentrations, measured using the Elecsys anti-SARS-CoV-2 spike (S) Ab assay, 53–92 days after second vaccine dose, in participants without prior infection, adjusted by age & vaccine type. Secondary outcomes included proportions failing to generate protective Ab responses (defined cut-off anti-S concentration 15U/mL, which correlated with 20% viral neutralization). Results Geometric mean S Ab concentrations (figure 1) were lower in patients treated with IFX (153U/mL;p<0.0001), IFX and thiopurine combination (109U/mL;p<0.0001), tofa (430U/mL;p<0.0001) and uste (561U/mL;p=0.013) compared to controls (1596U/ml). No differences in S Ab concentrations were found between controls and thiopurine monotherapy-treated patients (1020U/mL;p=0.62), nor between controls and vedo-treated patients (944 U/mL;p=0.69). In multivariable modelling (figure 2), lower S Ab concentrations were independently associated with IFX (FC 0.10 [95% CI 0.07–0.14], p<0.0001), tofa (0.36 [95% CI 0.19–0.69],p=0.002) and uste (0.56 [95% CI 0.31–1.00],p=0.049), but not with thiopurine (0.77 [95% CI 0.54–1.11],p=0.17) or vedo (1.01 [95% CI 0.61–1.68],p=0.96). mRNA vaccines (3.67 [95% CI 2.72–4.96],p<0.0001) and older age (0.82 [95% CI 0.73–0.91],p=0.0003) were independently associated with higher & lower S Ab concentrations respectively. Protective Ab responses were generated by all thiopurine monotherapy, vedo, tofa and healthy control participants, but not by 11% of patients on IFX monotherapy, 13% on thiopurine & IFX combination therapy and 4% on uste. Conclusion COVID-19 vaccine-induced Ab responses are significantly reduced in patients treated with IFX, or tofa, and to a lesser extent with uste. No significant reduction was seen in vedo or thiopurine monotherapy-treated patients. Our data suggest that 3rd primary or booster vaccine doses for IBD patients might be tailored to an individual’s immunosuppressive treatment. Financial support was provided as a Research Grant by Pfizer Ltd.

Background COVID-19 vaccine-induced antibody responses are reduced in patients with inflammatory bowel disease (IBD) taking anti-TNF or tofacitinib after two vaccine doses. We sought to assess whether immunosuppressive treatments were associated with reduced antibody and T-cell responses in patients with IBD after a third vaccine dose. Methods VIP was a multicentre, prospective, case-control study done in nine centres in the UK. We recruited immunosuppressed patients with IBD and non-immunosuppressed healthy individuals. All participants were aged 18 years or older. The healthy control group had no diagnosis of IBD and no current treatment with systemic immunosuppressive therapy for any other indication. The immunosuppressed patients with IBD had an established diagnosis of Crohn's disease, ulcerative colitis, or unclassified IBD using standard definitions of IBD, and were receiving established treatment with one of six immunosuppressive regimens for at least 12 weeks at the time of first dose of SARS-CoV-2 vaccination. All participants had to have received three doses of an approved COVID-19 vaccine. SARS-CoV-2 spike antibody binding and T-cell responses were measured in all participant groups. The primary outcome was anti-SARS-CoV-2 spike (S1 receptor binding domain [RBD]) antibody concentration 28-49 days after the third vaccine dose, adjusted by age, homologous versus heterologous vaccine schedule, and previous SARS-CoV-2 infection. The primary outcome was assessed in all participants with available data. Findings Between Oct 18, 2021, and March 29, 2022, 352 participants were included in the study (thiopurine n=65, infliximab n=46, thiopurine plus infliximab combination therapy n=49, ustekinumab n=44, vedolizumab n=50, tofacitinib n=26, and healthy controls n=72). Geometric mean anti-SARS-CoV-2 S1 RBD antibody concentrations increased in all groups following a third vaccine dose, but were significantly lower in patients treated with infliximab (2736.8 U/mL [ geometric SD 4.3]; p<0.0001), infliximab plus thiopurine (1818.3 U/mL [ 6.7]; p<0.0001), and tofacitinib (8071.5 U/mL [3.1]; p=0.0018) compared with the healthy control group (16 774.2 U/mL [2.6]). There were no significant differences in anti-SARS-CoV-2 S1 RBD antibody concentrations between the healthy control group and patients treated with thiopurine (12 019.7 U/mL [2.2]; p=0.099), ustekinumab (11 089.3 U/mL [2.8]; p=0.060), or vedolizumab (13 564.9 U/mL [2.4]; p=0.27). In multivariable modelling, lower anti-SARS-CoV-2 S1 RBD antibody concentrations were independently associated with infliximab (geometric mean ratio 0.15 [95% CI 0.11-0.21]; p<0.0001), tofacitinib (0.52 [ CI 0.31-0.87]; p=0.012), and thiopurine (0.69 [0.51-0.95]; p=0.021), but not with ustekinumab (0.64 [0.39-1.06]; p=0.083), or vedolizumab (0.84 [0.54-1.30]; p=0.43). Previous SARS-CoV-2 infection (1.58 [1.22-2.05]; p=0.0006) was independently associated with higher anti-SARS-CoV-2 S1 RBD antibody concentrations and older age (0.88 [0.80-0.97]; p=0.0073) was independently associated with lower anti-SARS-CoV-2 S1 RBD antibody concentrations. Antigen-specific T-cell responses were similar in all groups, except for recipients of tofacitinib without evidence of previous infection, where T-cell responses were significantly reduced relative to healthy controls ( p=0.021). Interpretation A third dose of COVID-19 vaccine induced a boost in antibody binding in immunosuppressed patients with IBD, but these responses were reduced in patients taking infliximab, infliximab plus thiopurine, and tofacitinib. Tofacitinib was also associated with reduced T-cell responses. These findings support continued prioritisation of immunosuppressed groups for further vaccine booster dosing, particularly patients on anti-TNF and JAK inhibitors. Copyright (c) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.

Anti tumour necrosis factor (anti-TNF) drugs increase the risk of serious respiratory infection and impair protective immunity following pneumococcal and influenza vaccination. Here we report SARS-CoV-2 vaccine-induced immune responses and breakthrough infections in patients with inflammatory bowel disease, who are treated either with the anti-TNF antibody, infliximab, or with vedolizumab targeting a gut-specific anti-integrin that does not impair systemic immunity. Geometric mean [SD] anti-S RBD antibody concentrations are lower and half-lives shorter in patients treated with infliximab than vedolizumab, following two doses of BNT162b2 (566.7 U/mL [6.2] vs 4555.3 U/mL [5.4], p <0.0001; 26.8 days [95% CI 26.2 – 27.5] vs 47.6 days [45.5 – 49.8], p <0.0001); similar results are also observed with ChAdOx1 nCoV-19 vaccination (184.7 U/mL [5.0] vs 784.0 U/mL [3.5], p <0.0001; 35.9 days [34.9 – 36.8] vs 58.0 days [55.0 – 61.3], p value < 0.0001). One fifth of patients fail to mount a T cell response in both treatment groups. Breakthrough SARS-CoV-2 infections are more frequent (5.8% (201/3441) vs 3.9% (66/1682), p = 0.0039) in patients treated with infliximab than vedolizumab, and the risk of breakthrough SARS-CoV-2 infection is predicted by peak anti-S RBD antibody concentration after two vaccine doses. Irrespective of the treatments, higher, more sustained antibody levels are observed in patients with a history of SARS-CoV-2 infection prior to vaccination. Our results thus suggest that adapted vaccination schedules may be required to induce immunity in at-risk, anti-TNF-treated patients.

Abstract Background Antibody responses following SARS-CoV-2 infection or a single-dose of SARS-CoV-2 vaccine are impaired in patients with inflammatory bowel disease treated with anti-TNF compared to those treated with vedolizumab, a gut-selective anti-integrin α4β7 monoclonal antibody. Here we sought to determine if patients treated with infliximab have attenuated serological and T cell responses and an increased risk of breakthrough COVID-19 infection following primary SARS-CoV-2 vaccination. Methods Anti-spike (S) receptor binding domain (RBD) antibody concentration in 2306 infliximab-treated patients were compared to a cohort of 1045 vedolizumab-treated patients. Our primary outcome was anti-S RBD antibodies 2 to 10 weeks after a second dose of the BNT162b2 or ChAdOx1 nCoV-19 vaccines. Secondary outcomes were anti-spike T cell responses, durability of vaccine responses and risk of breakthrough infections following two doses of vaccine. Results Anti-S RBD antibody concentrations were lower in patients treated with infliximab than in those treated with vedolizumab, following a second dose of BNT162b2 (567.3 U/mL [6.1] vs 4601.1 U/mL [5.3], p <0.0001) and ChAdOx1 nCoV-19 (183.9 U/mL [5.0] vs 789.4 U/mL [3.5], p <0.0001) vaccines (Fig. 1). Vaccination with the BNT162b2 vaccine compared to the ChAdOx1 nCoV-19 was independently associated with a 3.7-fold [95% CI 3.30 – 4.13] higher anti-S RBD antibody concentration (p < 0.0001) (Fig. 2). There were no significant differences in the magnitude of anti-spike T cell responses observed in infliximab- compared with vedolizumab-treated patients after one or two doses of either vaccine. Antibody half-life was shorter in infliximab- than vedolizumab-treated patients following two-doses of BNT162b2 (4.0 weeks [95% CI 3.8 – 4.1] vs 7.2 weeks [95% CI 6.8 – 7.6]) and ChAdOx1 nCoV-19 (5.3 weeks [95% CI 5.1 – 5.5] vs 9.3 weeks [95% CI 8.5 – 10.2], p value < 0.0001). Breakthrough SARS-CoV-2 infections were more frequent (5.8% (202/3467) vs 3.9% (66/1691), p = 0.0032) and the time to breakthrough shorter in patients treated with infliximab than vedolizumab (p = 0.0023) (Fig. 3). Higher anti-S RBD antibody concentrations following a second dose of SARS-CoV-2 vaccine protected against breakthrough SARS-CoV-2 infection: overall, for every 10-fold rise in anti-S RBD antibody level we observed a 0.8-fold reduction in odds of breakthrough infection ([95% CI 0.70 – 0.99], p = 0.035). Conclusion Infliximab was associated with attenuated, less durable vaccine induced anti-S RBD antibody responses and a 50% increase in breakthrough SARS-CoV-2 infection. Further follow-up is required to assess whether third primary doses can mitigate the effects of infliximab on anti-S RBD antibody responses.

Abstract Background Perianal fistulas are a common complication of Crohn’s disease (CD) affecting approximately 25% of patients, often predicting a more complicated disease course. Dysregulated immune responses and epithelial-to-mesenchymal transition (EMT) are hypothesised to contribute to fistulizing disease; however, they have been poorly studied. In this study, we investigated the immune phenotype of patients with perianal fistulizing disease and its relationship with tissue remodelling. Methods Immune cells were isolated from fistula curettage samples (n=31) and paired peripheral blood from patients with perianal Crohn’s (pCD) or idiopathic fistulizing disease. Multiparameter flow cytometry was performed to evaluate lymphocyte populations including invariant natural killer T-cells (iNKTs), gamma-delta (γδ) T-cells, CD161+ mucosal-associated invariant T-cells (MAIT), CD4+ T-cells and CD8+ T-cells. Gene expression profiling of fistula (Crohn’s n=11, idiopathic n=11) and rectal tissue (Crohn’s n=9, idiopathic n=9) was performed by RNA-sequencing. Cellular deconvolution of transcriptomic data using CIBERSORTx was performed to define the cellular phenotype of perianal fistulas. Cytokine treated intestinal epithelial organoids were used to probe the impact of selective cytokines on disease relevant pathways in perianal fistulas, using gene-set enrichment analysis (GSEA). Results Perianal fistulas were characterised by expansion of iNKTs and CD4+ T-cells when compared to peripheral blood. Deeper analysis of the phenotype of these populations revealed enrichment of CD8- CD4- CD161+ iNKTs producing interleukin-22 (IL22), and CD4+ CD161+ T-cells producing interleukin-13 (IL13) and IL22. Surprisingly, pCD and idiopathic fistulas displayed similar immunophenotypes. Fistulas exhibited distinct transcriptional profiles to rectal tissue, although the phenotype of pCD and idiopathic fistulas appeared similar. Pathways related to the extracellular matrix (ECM) and EMT were more activated in fistulas compared to rectum. Gene-set enrichment analysis and cellular deconvolution identified an increase in the abundance of iNKTs, activated memory CD4+ T-cells, activated NK cells and neutrophils in fistula versus rectal tissue. IL13, IL22 and TNFα responsive transcripts were enriched in fistula tissue, and in the case of IL22, was shown to regulate key matrisome components. Conclusion Perianal fistulas are characterised by increased infiltration of CD161+ iNKT cells and CD4+ T-cells, producing IL22 and IL13. These pro-inflammatory cytokines are likely important drivers of ECM dysregulation and tissue remodelling in perianal fistulizing disease.

T‐bet is the lineage‐specifying transcription factor for CD4 + T H 1 cells. T‐bet has also been found in other CD4 + T cell subsets, including T H 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T‐bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T‐bet‐expressing cells using a fluorescent fate‐mapping mouse line. We demonstrate that T‐bet is expressed in a subset of CD4 + T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4 + T cells. Naïve‐like T‐bet‐experienced cells are polarized to the T H 1 lineage, predisposed to produce IFN‐γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage‐specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T‐helper response.

Introduction Perianal fistulas are a common complication of Crohn's disease (CD) affecting approximately 25% of patients, often predicting a more complicated disease course. Dysregulated immune responses and epithelial-to-mesenchymal transition (EMT) have been implicated in fistulizing disease; however, they have been poorly studied. In this study, we investigated the immune phenotype of patients with perianal fistulizing disease and its relationship with tissue remodelling. Methods Immune cells were isolated from fistula curettage samples (n=31) and paired peripheral blood from patients with perianal Crohn's (pCD) or idiopathic fistulizing disease. Multiparameter flow cytometry was performed to evaluate lymphocyte populations including invariant natural killer T-cells (iNKTs), gamma-delta (γδ) T-cells, CD161+ mucosal-associated invariant T-cells (MAIT), CD4+ T-cells and CD8+ T-cells. Gene expression profiling of fistula (pCD n=11, idiopathic n=11)) and rectal biopsies (pCD n=9, idiopathic n=9)) was performed by RNA-sequencing. Cytokine treated intestinal epithelial organoids were used to probe the impact of selective cytokines on disease relevant pathways. Results iNKTs and CD4+ T-cells were expanded in perianal fistulas compared to peripheral blood. Deeper analysis of the phenotype of these populations revealed enrichment of CD8- CD4- CD161+ iNKTs producing IL22, and CD4+ CD161+ T-cells producing IL13 and IL22. Interestingly, pCD and idiopathic fistulas displayed similar immunophenotypes. Fistulas exhibited distinct transcriptional profiles to rectal tissue, although the phenotype of pCD and idiopathic fistulas appeared similar. Pathways related to the extracellular matrix (ECM) and EMT were more activated in fistulas compared to the rectal mucosa. IL13, IL22 and TNFα responsive transcripts were enriched in fistula tissue, and in the case of IL22, was shown to regulate key matrisome components. Conclusions Perianal fistulas are characterised by increased infiltration of CD161+ iNKT cells and CD4+ T-cells, producing IL22 and IL13. These pro-inflammatory cytokines are likely important drivers of ECM dysregulation and tissue remodelling in perianal fistulizing disease and targeting of this axis may represent a novel target in the treatment of fistulizing disease.

Abstract Background Mesenchymal Stem Cells (MSC) demonstrated promising results in patients with refractory perianal fistulising Crohn’s Disease (pCD) in the ADMIRE-CD trial, however their use is limited by treatment cost. Studies of Graft vs. Host Disease (GvHD) patients receiving MSCs for steroid refractory disease have shown that in vitro Peripheral Blood Mononuclear Cell (PBMC) induced MSC apoptosis correlates with clinical response. We conducted a pilot prospective study to investigate the clinical and radiological outcome of MSC treatment and determine whether in vitro MSC apoptosis correlates with clinical response in patients with pCD. Methods Patient eligibility and MSC administration mirrored that in ADMIRE-CD. Prior to treatment, patient PBMCs were isolated and co-cultured with MSCs and the proportion of apoptotic MSCs identified using flow cytometry (Annexin V+, 7AAD- MSCs). Patients were followed up at, 6-monthly intervals and clinical indices including the Perianal Disease Activity Index (PDAI) and Fistula Drainage Assessment (FDA) were recorded in addition to MRI response. Clinical response categories were determined according to ADMIRE-CD as well as new criteria accounting for radiological healing (Table 1). Results Ten patients underwent MSC treatment (M=4, median age, 35, range, 22–73 years). At final follow up (median, 24 months, range, 18–30 months), there was a significant improvement in mean FDA (baseline, 1.3, final follow up, 0.5, p<0.01) but not in PDAI. The apoptosis assay was performed in, 9 patients. A greater proportion of MSC apoptosis was demonstrated when co-cultured with pCD patient PBMCs than with PBMCs from healthy controls (Figure, 1). Patients with a percentage of apoptotic MSCs greater than the group median (22.3%) were classed as apoptosishigh. At final follow up, apoptosishigh patients were also those who demonstrated a response to MSC treatment according to both St Mark’s (Figure, 2) and ADMIRE CD criteria (Figure 3). Conclusion The ability of patient PBMCs to induce the highest proportions of MSC apoptosis correlates with clinical response to MSC treatment in this small sample of patients. An adequately powered prospective study is required to determine the potential use of MSC apoptosis as a biomarker of clinical response.