Rapid Identification of Immune Cell Regulators by Microfluidic Cell Secretion Profiling

Thursday, October 24, 2024

8:30 AM – 8:45 AM EST

Location: Room 316

Invited Speaker(s)

ZW

Zongjie Wang

Group Leader
Chan Zukcerberg Biohub Chicago
Chicago, Illinois, United States

Co-Author(s)

ML

Mahmoud Labib

Lecturer
University of Plymouth, United States

Last Author(s)

SK

Shana Kelley

Professor, President of CZI biohub Chicago
Northwestern University, United States

Introduction: The effector function of immune cells is largely mediated by a range of cytokines and chemokines secreted by individual cells. For instance, upon antigen recognition, T cells often release cytolytic and proinflammatory cytokines, such as Granzyme B and Interferon γ, which target and destroy the recognized cells. Moreover, the progression of many autoimmune diseases involves the excessive release of cytokines into the local environment, leading to immune system dysregulation. Consequently, conducting single-cell analysis of immune cells based on their secretion behavior is crucial for understanding disease mechanisms and developing appropriate therapeutics.

Various methods have been developed to analyze cell secretion. Intracellular staining, combined with transport inhibitors, is a traditional method for cytokine analysis, but it only indicates intracellular cytokine accumulation rather than actual secretion. Enzyme-Linked Immunosorbent Assay (ELISA) measures actual cell secretion but lacks single-cell sensitivity. As such, these traditional approaches fall short of providing a comprehensive analysis at the single-cell level.

Materials and

Methods: In this work, we introduce a microfluidic approach called 'secretion-enabled cell ranking and enrichment' (SECRE) that allows for high-throughput analysis of cell secretion with single-cell resolution. The core concept of SECRE is to convert the quantity of cytokines secreted by a cell into a corresponding level of fluorescence and/or magnetic labelling, as illustrated in Fig. 1.

With the assistance of the SECRE approach, we conducted a kinome-wide loss-of-function CRISPR screen on human primary CD4+ T cells. We specifically enriched the populations exhibiting low IFNγ secretion for sequencing, enabling a targeted investigation into the genetic underpinnings that modulate IFNγ secretion levels.

Results, Conclusions, and Discussions: We discovered that the top hits identified by the SECRE approach and intracellular flow cytometry are largely different, which supports the notion that cytokine accumulation and cytokine secretion are distinct biological processes. The top hits from SECRE included kinases such as STK10, STK24, LRRK2, and SLK. Notably, most of these kinases are druggable targets that can be modulated using small molecule inhibitors. We further validated the effectiveness of these small molecules in suppressing IFNγ secretion in vitro, suggesting potential therapeutic applications for controlling inflammatory responses.

We further tested the novel application of XMU-MP1, a small molecule inhibitor of SLK, for treating inflammatory bowel disease (IBD). In our study, immunocompetent mice were administered 2% dextran sulfate sodium (DSS) for 8 days to induce IBD. A subset of these mice received daily i.p. injections of XMU-MP1 at a dosage of 1 mg/kg. We monitored the body weight of the animals throughout the treatment period and collected their colons at the endpoint for immunohistological analysis. The results highlighted XMU-MP1 as a potent therapeutic agent for IBD. The treatment with XMU-MP1 resulted in a 50% reduction in IFNγ secretion and played a significant role in preserving intestinal stem cells (ISCs).

Taken together, we designed a new approach to quantitatively measure cell secretion with single-cell resolution and discovered novel genes and drug regulators for IFNγ secretion. Further implementation of SECRE may help us decipher more immunological biology for cancer and autoimmune treatment.

Acknowledgements (Optional): The research reported in this publication was supported by the Canadian Institutes of Health Research (grant number FDN-148415), the Natural Sciences and Engineering Research Council of Canada (grant number 2016-06090), the Province of Ontario through the Ministry of Research, Innovation and Science (grant number RE05-009) and the National Cancer Institute of the National Institutes of Health (grant number 1R33CA204574). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the other funding agencies.