Laurien A. Waaijer

Vedolizumab, a gut-selective integrin antagonist, is an effective therapy for inflammatory bowel disease (IBD), but immune correlates of treatment response remain poorly understood. We performed longitudinal immune profiling of blood and serum from 20 vedolizumab-treated IBD patients (15 Crohn’s disease, 5 ulcerative colitis) to characterize treatment-associated immune changes and identify baseline features associated with clinical response. Samples were collected at baseline and week 14. Immune cell phenotyping was performed using five 10-color flow cytometry panels, while serum proteomics was assessed with the Olink^® 96 Inflammation panel. Age- and sex-matched healthy controls (HCs) were included for comparison. IBD patients could be distinguished from HCs based primarily on immune cell profiles, whereas serum proteomic differences were less pronounced. Vedolizumab treatment induced modest changes in immune cell composition and serum proteins. Clinical responders exhibited shifts in three immune populations and a reduction in CXCL8 and IL-6 levels by week 14. Unsupervised hierarchical clustering of five baseline immune cell populations stratified patients into two groups that broadly corresponded with treatment outcome. Furthermore, non-responders exhibited increased IFNγ and CXCL9 levels. However, these immune cell populations and proteins did not remain significant upon correcting for multiple testing. Taken together, we identified exploratory immune and cytokine patterns associated with vedolizumab response that may support the development of precision medicine approaches in IBD.

Background: The Janus kinase–signal transducer and activator of transcription (JAK/STAT) pathway regulates key cellular processes. Dysregulation of this pathway can lead to chronic immune activation. Although JAK inhibitors are promising, cytokine-specific STAT activation and JAK inhibitor modulation remain poorly understood. To address this knowledge gap, we developed a phospho-flow-based JAK/STAT assay and demonstrated its potential for precision medicine. We illustrate its application using three clinical case studies. Methods: Peripheral blood mononuclear cells (PBMCs) and biopsy-derived single cell suspensions of inflamed and uninflamed tissues were stimulated with either individual cytokines (IL-4, IL-6, IL-7, IL-13 or IFN-α) or a cytokine mixture. The phosphorylation of STAT1, STAT3, STAT5 and STAT6 was quantified by flow cytometry across multiple immune cell subsets. For JAK inhibition studies, PBMCs from healthy controls and patients, and tissue biopsies from patients, were incubated with JAK inhibitors prior to cytokine stimulation. Findings: Cytokines induced phosphorylation of distinct STAT proteins across multiple immune cell subsets. In addition, JAK inhibitors exhibited differential potency across these subsets, resulting in variable modulation of STAT phosphorylation. Guided by these functional readouts, patients received specific JAK inhibitors, resulting in clinical improvement. In two atopic dermatitis patients treated with a JAK1 inhibitor, stimulation-induced STAT1 phosphorylation decreased in the responder but increased in the non-responder. Interpretation: We developed a JAK/STAT assay enabling immune cell subset-specific analysis in both blood- and tissue-derived immune cells. This approach revealed disease-associated signaling abnormalities and differential JAK-inhibitor responses, supporting therapeutic decision-making and precision medicine in immune-mediated diseases.

Profiling the human immune system is essential to understanding its role in disease, but it requires advanced and novel technologies. Spectral flow cytometry (SFM) enables deep profiling at the single‐cell level. It is able to detect many fluorescent parameters within one measurement; therefore, it is vastly useful when patient material is limited. However, designing and analyzing these high‐dimensional datasets remains complex. We optimized a 42‐parameter panel (40 commercially available fluorochromes, one stacked fluorochrome and an autofluorescent (AF) parameter) that enables the identification of innate and adaptive immune cell composition. It is the first 42‐parameter panel that is optimized on peripheral whole blood, and it outperforms other published OMIPs of 40 colors in terms of complexity. With this panel, we are able to identify neutrophils, basophils, eosinophils, monocytes, dendritic cells, CD4 T cells, CD8 T cells, regulatory T cells, mucosal‐associated invariant T (MAIT) cells, γδ T cells, B cells, NK cells, dendritic cells, and innate lymphoid cells (ILCs). Furthermore, with the utilization of co‐stimulatory, checkpoint, activation, homing, and maturation markers, this panel enables deeper phenotyping. Within one measurement, more than 80 distinct immune cell subsets were identified by FlowSOM and annotated manually. In conclusion, with this high‐dimensional SFM panel, we aim to generate immune profiles to understand disease and monitor therapy response.