Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• T cell characterization commonly uses protein detection via flow cytometry.
• Protein analysis can be limited by background noise and kinetic expression differences.

Purpose of the Study:

• To develop and validate a novel host immunity-based platform for infection detection using RNA analysis.
• To leverage RNA detection for enhanced sensitivity and kinetic insights in T cell characterization.

Main Methods:

• Developed a platform for detecting cytokine messenger RNAs (mRNAs) in T cells.
• Utilized single-molecule fluorescence in situ hybridization (smFISH) combined with flow cytometry.
• Stimulated T cells ex vivo with pathogen-specific antigens (Ags).

Main Results:

• RNA detection demonstrated lower background compared to protein detection, enabling higher sensitivity for low-frequency cells.
• mRNA analysis revealed kinetic differences in cytokine expression, offering novel insights into T cell subset programming.
• Successfully detected T cells recognizing mycobacterial and viral Ags in exposed donors, showcasing utility for assessing immunological memory.

Conclusions:

• RNA-based flow cytometry provides a sensitive and informative method for characterizing T cells and detecting infections.
• This approach enhances the study of immune responses and immunological memory.
• The platform offers new avenues for understanding T cell subsets and host immunity.