RF4 - Functional Immune Response to Jak Inhibitors among Patients with Inflammatory Bowel Disease: Developing a Multidimensional, Preclinical Drug Screen

Purpose/Background: Janus Kinase Inhibitors (JAKi) are approved for a multitude of inflammatory conditions including inflammatory bowel disease (IBD). These oral, small molecule inhibitors have varying specificity for the four JAK proteins (JAK1, JAK2, JAK3, and TYK2). Upadacitinib (JAK1i) is approved in Ulcerative Colitis (UC) and Crohn’s Disease (CD), Tofacitinib (JAK1/3i) is approved in UC, and Deucravacitinib (TYK2i) failed to meet efficacy endpoints in clinical trials for UC and is being evaluated in CD. Response rates vary by drug, disease, and patient. Toward understanding the diverse effects of JAKi on immune function, we developed a multidimensional, single-cell, mass cytometry-based functional assay to evaluate peripheral blood immune cell response to JAKi. Fourteen different cell signaling proteins in the STAT, MAPK, PI3K, and NFkB pathways were evaluated in over 15 different major innate and adaptive immune cells and their subtypes.

Methods/Interventions: People over 18-years old not taking steroids were eligible for this IRB-approved study. Whole blood was collected at the time of routine blood work and processed within one hour of collection. We tested blood from 8 healthy controls (mean age 58+/-11 years, 7 female) 4 patients with CD (39+/-8 years, 2 female), and 4 patients with UC (47+/-18, 2 female). Blood was stimulated with IL-2/IL-6 to induce signaling or left unstimulated as baseline comparison. Unstimulated and IL-2/IL-6 stimulated blood was treated with no inhibitor, Upadacitinib, Tofacitinib, or Deucravacitinib. Standard bioinformatic best-practice pathways identified differences in single-cell signaling.

Results/Outcomes: Functional responses were cytokine, JAKi, disease, and cell-type dependent. For example, in patients with CD, Tofacitinib failed to inhibit IL-2/IL-6-induced STAT signaling in CD4+ T-cells and monocytes. In contrast, in controls and UC, Tofacitinib suppressed IL-2/IL-6 induced STAT signaling. Upadacitinib and Deucravacitinib suppressed IL-2/IL-6 induced STAT signaling in all three groups. The CD group also had a unique response in CD8+ T-Cells and NK cells. In the setting of IL-2/IL-6 stimulation, CD8+ T-Cells and NK cells had decreased levels of GranzymeB after treatment with all three JAKi, whereas that decrease was not seen in controls or UC. Deucravacitinib had a distinct response in controls, with broad suppression of multiple signaling pathways in multiple cell types; broad signaling suppression in response to Deucravacitinib was absent in both CD and UC.

Conclusion/Discussion: We have developed a high-dimensional, functional, single-cell assay for evaluation of patient-derived response to JAKi that identified drug, disease, and cell type specific responses. This study provides insight into the differential response of patients with IBD to JAKi. Further, these studies provide proof of concept toward building preclinical screening models of specificity, response, and off target effects to therapeutics.