OP0100 MOLECULAR PROFILING OF PERIPHERAL IMMUNE CELL SUBSETS IN PATIENTS WITH RHEUMATOID ARTHRITIS

Background:Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects 1% of the world’s population. Several key biological functions are dysregulated in RA, manifesting clinically as pain, fatigue, and synovitis, with articular destruction, organ-based comorbidities, and functional decline. Defining immune dysregulation in the peripheral blood of patients (pts) with RA will help inform future work to assess the extent to which immune homeostasis can be therapeutically achieved for these pts.Objectives:To identify baseline molecular characteristics of the peripheral immune system, at the level of individual immune cell subsets, in pts with RA recruited to clinical trials of the oral, selective Janus kinase 1 (JAK1) inhibitor, filgotinib.Methods:Peripheral blood mononuclear cells (PBMC) were collected from 324 pts with moderate to severely active RA, who had an inadequate response to methotrexate ([MTX], FINCH-1;NCT02889796; n=109) or who were MTX naïve (FINCH-3;NCT02886728; n=215). PBMC were also collected from 50 demographically matched healthy volunteers (HV). The Immune Profiler platform was used to sort PBMC into 24 immune cell subsets, then quantify their gene expression and chromatin accessibility using RNA-seq and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. Differentially expressed genes (DEGs) and differentially accessible regions (DARs) were identified among immune cell subsets from pts with RA versus HV. Gene set signature scores of Molecular Signatures Database hallmark pathways were calculated using single sample gene set enrichment analysis (ssGSEA) to examine differences in pathway activity between groups.Results:A total of 14,500 sequencing datasets were generated from the pt and HV immune cell subsets. Among these, over 26,000 DEGs and 220,000 DARs were identified in RA versus HV (false discovery rate <0.05) across the 24 immune cell subsets. DEGs were identified in all immune cell subsets tested and were most pronounced in natural killer (NK) subsets; most DARs were detected in myeloid and NK subsets. ssGSEA revealed differential pathway signaling in RA versus HV across multiple functions at the immune cell subset level. Myeloid subsets from pts with RA often showed elevated pathway activities versus HV whereas B, T and NK subsets showed a general decrease. In particular, monocyte populations from pts with RA versus HV had elevated pathway activities involved in inflammatory response and interleukin-6/Janus kinase/signal transducer and activator of transcription 3 signaling. The B, T and NK subsets showed a general decrease in tumor necrosis factor-α signaling; conversely, monocyte subsets showed an increase. Prior MTX exposure did not have a notable impact on the detected molecular profile.Conclusion:Differences in gene expression, hallmark pathway activity, and chromatin accessibility were identified in RA versus HV at the immune cell subset level. Significant contributions to differences in chromatin accessibility identified in the myeloid and NK cell populations suggest that there are more active regulatory sequences in these cell types that are associated with RA. Further investigations based on these findings may increase understanding of the immune regulatory paradigm in the context of RA.Acknowledgments:This study was funded by Gilead Sciences, Inc. Editorial support was provided by Fishawack Communications Inc and funded by Gilead Sciences, Inc.Disclosure of Interests:Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB, Jinfeng Liu Shareholder of: Gilead Sciences Inc., Roche, Employee of: Gilead Sciences Inc., Luting Zhuo Employee of: Gilead Sciences Inc., Yuan Tian Employee of: Gilead Sciences Inc., Thomas Snyder Employee of: Verily Life Sciences, Charlie Kim Employee of: Verily Life Sciences, Pouya Kheradpour Employee of: Verily Life Sciences, Kat Drake Employee of: Verily Life Sciences, Sam Kim Shareholder of: Gilead Sciences Inc., Employee of: Gilead Sciences Inc., Rachael E. Hawtin Shareholder of: Gilead Sciences Inc., Employee of: Gilead Sciences Inc.

P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease

Background Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition. Results Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.

Leukocyte Localization in a Model of Innate Immune-Driven Colitis

Background and Hypothesis: Inflammatory bowel disease (IBD) is a disabling, chronic gut disorder involving immune dysregulation. Our lab has generated a murine IBD model in which the innate immune system drives inflammation. Innate lymphoid cells (ILCs), an innate immune cell subset that was recently discovered, exhibit many T-helper cell characteristics. ILCs, though few, produce cytokines, thereby significantly impacting tissue through local action in mucosal sites. They express the cell surface markers, CD90, which is unique to ILCs, and CD45, which all leukocyte types express. Colitis prevention in our model via ILC depletion indicates a role for ILCs in IBD. Therefore, we aimed to identify the ILCs’ localization in our murine model. We hypothesized that the ILCs will localize to inflamed areas of the intestinal lamina propria and into the intraepithelial spaces. Experimental Design or Project Methods: Mice expressing TNFAIP3, an inhibitor of NF-kB, were mated with adaptive immunity-lacking mice (RAG1-/-). RAG1-/- x Villin-TNFAIP3 (TRAG) mice had colitis that was 100% penetrant by age 6 weeks. Distal colons excised at age 4 weeks and 8 weeks were used for identifying CD45+ and CD90+ cells in both RAG and TRAG mice intestines via immunofluorescence. Results: We observed differences in the distributions of CD90+ and CD45+ cells within TRAG and RAG mice intestines. Conclusion and Potential Impact: Differences exist in intestinal leukocyte distributions within our models. Altered ILC distribution might reflect an inflammatory state or contribute to IBD pathology. This work may further elucidate ILCs’ role in IBD and as IBD treatment targets.