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Specific Stimulation and Multiplex Secretion Analysis of T Cells Using a Suspendable Hydrogel Platform Enables
Monika Kizerwetter1,2, Doyeon Koo3, Citradewi Soemardy3
1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Insights
This study introduces nanovials, a novel technology for analyzing T cell function by measuring both surface markers and cytokine secretion at the single-cell level. This advance aids in understanding T cell responses and developing improved cell therapies.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry revolutionized immune system analysis but cannot measure T cell cytokine secretion.
- This limitation hinders understanding T cell function and developing cell therapies for cancer and autoimmune diseases.
Purpose of the Study:
- To develop and validate a single-cell technology (nanovials) for simultaneous assessment of T cell surface markers and cytokine secretion.
- To enable functional evaluation of T cells and inform the engineering of future cell therapies.
Main Methods:
- Developed protein-conjugated nanovials for stimulating and capturing secretions from primary mouse CD8+ T cells.
- Demonstrated specific and nonspecific T cell stimulation using nanovials.
- Showcased simultaneous capture of multiple cytokine secretions and sorting of T cells based on secretion levels.
- Applied the platform to identify phenotypic profiles of interferon gamma (IFNγ)-secreting T cells.
Main Results:
- Nanovials effectively stimulated T cells and captured cytokine secretions.
- The platform enabled simultaneous multi-cytokine detection and cell sorting based on secretion.
- Phenotypic characterization of IFNγ-secreting T cells was achieved prior to stimulation.
Conclusions:
- The nanovial platform provides a comprehensive method for analyzing single-cell T cell function, including cytokine secretion.
- This technology enhances the understanding of T cell mechanistic correlates.
- Nanovials will aid in the design and engineering of advanced cell therapies.
Abstract:
The ability to characterize and separate cells based on their surface marker expression profiles using flow cytometry revolutionized our understanding of the immune system at the level of single cells. However, the use of surface protein quantification to functionally evaluate T cells following stimulation fails to capture important outcomes of T cell activation, most prominently, cytokine secretion. This key limitation hinders elucidation of the mechanistic correlates of T cell function, which in turn limits our ability to design effective cell therapies for diseases such as cancer and autoimmune disorders. Here, we employ a single-cell microparticle-based technology (termed nanovials) to assess both the cell surface expression profiles and the protein secretion behavior of stimulated T cells. Using protein-conjugated nanovials, we demonstrated the capacity to stimulate ovalbumin-specific primary mouse CD8+ T cells, both nonspecifically by coating anti-CD3 and anti-CD28 antibodies on the particle, and specifically by coating ovalbumin peptide-loaded major histocompatibility complexes and anti-CD28 antibody on the particle. We further showed that nanovials could simultaneously capture secretions of multiple cytokines from single cells and that viable T cells contained within the nanovials could be sorted based on cytokine secretion levels. We applied this platform to characterize the phenotypic profile of resting T cells that would go on to secrete the interferon gamma (IFNγ) cytokine following activation on nanovials by staining cells for surface marker expression prior to on-particle stimulation. Overall, the nanovial platform and assays developed herein help elucidate the functional properties of T cells, which will inform the engineering of future cell therapies.
