Exabis Library

T cells, as part of the adaptive immune system, are a significant driver of inflammation in Crohn’s disease (CD), yet specific T-cell targets are largely unknown. Genetic factors contribute to a small portion of CD risk including several HLA alleles, such as DRB1*07:01 and HLA-DRB1*01:03, associated with CD. The involvement of HLAs suggests that studying specific T cells could lead to new insights into CD development and progression. In this study, we use established immunoSEQ^® technology to profile T-cell receptors (TCRs) and identify TCRs associated with CD and CD characteristics.

We analyzed TCRs from blood of 1,738 CD cases and 4,970 healthy controls. TCRs that were statistically enriched in cases, but not healthy controls (p <0.001), were termed Enhanced Sequences (ES) associated with CD. An independent cohort of 434 CD cases was used for validation. We inferred associations between the ES and 145 common HLA alleles using data from a separate, HLA-typed dataset. We defined ES clusters by correlating TCRs with single amino acid substitutions.

We identified 1,121 CD-associated ES in the exploratory cohort. These were also enriched in CD cases in our validation cohort (Fig 1A). Using intestinal tissue samples from a subset of cases, we found that a median of 14% ES from individual cases were shared between blood and tissue samples (Fig. 1B). ES breadth (ES diversity relative to total TCR diversity) was significantly associated with history of CD-related surgery (Fig 1C, p < 1x10^-15), with stricturing or fistulizing phenotypes (Fig 1D, p < 1x10^-5 for B1 versus B2 or B3), and with ileal or ileocolonic location (Fig 1E, p < 1x10^-7 for L2 versus L1 or L3).

We found that 202 ES formed clusters of similar sequences consisting of 2-23 members (Fig. 2A). We confidently (p < 0.0001) associated 398 ES to a specific HLA allele (Fig 2B), including 134 of the ES assigned to clusters (Fig 2A). Some clusters, including the largest, had no members that could be assigned to an HLA allele, raising the possibility that these ES clusters bind non-canonical HLAs.

Our discovery set of public TCRs associated with CD indicates that the immune system of CD patients responds to a consistent set of antigens. Importantly, CD ES were present in both tissue and blood, demonstrating that evaluating TCRs in blood may be a surrogate of TCRs in tissue. The HLA allele associations of these ES potentially point to new risk factors and disease insights, such as the involvement of DP and DQ alleles. The association of ES frequency with CD characteristics strongly suggested that further examination of these TCRs may impact CD patient care and advance understanding of the pathophysiology of the disease.

In Inflammatory Bowel Disease (IBD), intestinal barrier dysfunction and epithelial cell injury are believed to be associated with activation of the immune system to drive disease-associated inflammation, which together constitute key features of active disease. Existing drugs used to treat IBD induce endoscopic remission and improvements in mucosal healing  in only a minor proportion of patients, driving a critical need for therapies which lead directly to mucosal healing. Furthermore, predicting patients who may benefit from therapeutics that address specific mechanisms of mucosal healing may augment response rates.

We screened proteins, identified from a meta-analysis of healthy human microbiome, in cellular assays and animal models related to mucosal injury, with the goal of identifying novel therapeutics that have the potential to directly induce mucosal healing. The proteins identified were further optimized by protein engineering to increase their stability as well as gastro-intestinal (GI) targeting via oral administration. For this, therapeutic proteins were expressed using a probiotic, Lactococcus lactis (L.lactis), engineered to display the recombinant proteins on the cell surface, and evaluated for activity in DSS- and DNBS-induced models of colitis in mice. Mechanism of action studies using computational and laboratory based methods to analyze gene expression and direct molecular interactions with human proteins, enabled the identification of pathways modulated by the candidate molecules. These pathways were further evaluated for their ability to identify biomarkers in specific patients most suitable for treatment in a precision medicine approach.

We have identified a novel, healthy microbiome-derived protein that demonstrated robust activity in human epithelial injury assays in vitro. The protein reduced intestinal injury related pathology in mice when orally administered to target directly the GI tract. SG-5-00455, the product based on an L.lactis strain expressing the candidate therapeutic protein, reduced pathology scores, inflammation and barrier function related LPS-binding protein levels to levels comparable to those obtained glucagon-like peptide 2 (GLP-2), as well as improving dysregulated tissue repair and fibrosis-associated gene expression and proteins levels.  SG-5-0455 treatment did not result in systemic exposure, driving its therapeutic activity in a GI-localized manner by targeting pathways related to tissue injury and fibrinolysis.

SG-5-00455, through its novel mechanism of action and oral delivery to directly target tissue repair pathways in the GI-tract, offers the potential to address a large critical need in IBD.

Tissue-resident memory T cells (Trm) persist in peripheral tissues and contribute to pathogen clearance and inflammation. Trm can re-enter the circulation (ex-Trm) and give rise to new effector and Trm populations. Skin-derived ex-Trm can be identified in human blood based on co-expression of the residency marker CD103 and cutaneous leukocyte antigen (CLA), a skin-tropism marker. The existence of ex-Trm derived from the gut would have implications for inflammatory bowel disease (IBD) and its treatment targeting the recruitment of circulating gut-homing cells.

Peripheral blood and colonic biopsies were taken from healthy volunteers and patients with IBD (Crohn’s disease or ulcerative colitis). PBMCs and cells isolated from biopsies by enzymatic digestion were analysed by multi-colour flow cytometry.

More than 80% of colonic αβT cells were Trm, as defined by CD69 expression, in health and IBD; there was no significant increase in cells with a non-resident phenotype in inflamed tissue. Few CD4+ Trm co-expressed CD103. In contrast, CD8+ Trm comprised CD103+ and CD103- subsets, and CD69+CD103- cells were significantly reduced in IBD. Increased staining for KLRG1 and the cytotoxicity-associated protein perforin, indicated a more effector-like Trm phenotype in IBD. Putative gut-derived ex-Trm were identified amongst TCRαβ+CD45RA- blood cells as a β7++CD103+ population, indicative of cells expressing both α4β7 and CD103(α_E)β7 integrin complexes.  A separate CD103β7+α4β7 population defined by 1:1 expression of CD103 and β7 contained CLA+ skin ex-Trm. Gut ex-Trm comprised 0.3% of circulating CD8⁺ T cells (range 0.02-1.4%), and 1.2% of CD4⁺ T cells (range 0.3-3%). Gut and skin ex-Trm were phenotypically similar; both expressed the residency associated markers CD9 and CD101 but lacked CD69 expression. Gut ex-Trm were phenotypically distinct from both the traditional gut-trophic population (α4β7+CD103-CD45RA) and from naïve T cells. The proportion of gut ex-Trm did not differ between health and IBD. However, gut-derived ex-Trm were significantly reduced, relative to skin-derived ex-Trm, in Crohn’s disease, but not ulcerative colitis, when compared with health.

A putative gut-derived ex-Trm population can be identified in both healthy and IBD peripheral blood, with IBD-associated changes identified in this population and intestinal Trm. Circulating ex-Trm could link discreet areas of intestinal inflammation in Crohn’s disease and there is a selective loss of the gut ex-Trm population from the blood of these patients. The role of ex-Trm in IBD merits further study.