Optimization of Perforin Detection and Quantification by Flow Cytometry

Abstract

The host is protected against invading pathogens by the immune system. This is done by enlisting a variety of cells and molecules capable of recognizing and eliminating a large variety of antigens. Cytotoxic T lymphocytes (CTLs) eliminate target cells by secretion of granules containing cytotoxic proteins. These granules contain Granzymes and Perforin, which work together to induce apoptosis in the target cell. However, there is still a challenge in accurate detection of Perforin by flow cytometry, which in turn makes it difficult to follow the dynamics of CTL responses. This study aimed at optimizing Perforin detection and quantification using flow cytometry by measuring the ability of different T-cells to rapidly increase Perforin production in a specially selected group of HIV-1 chronically-infected individuals showing enhanced immune response from previous screening. Detection and quantification of Perforin were done on Peripheral Blood Mononuclear Cells (PBMCs) that had been stimulated with Cytomegalovirus (CMV) in the presence of co-stimulatory antibodies. Negative controls constituted PBMCs with co-stimulatory antibodies but no CMV while positive controls used staphylococcal enterotoxin B (SEB), which is a superantigen. Results showed a lower amount of Perforin in the CD107a positive cells which is consistent with degranulation. The CD107a positive cells were also more polyfunctional than their corresponding CD107a negative phenotype, further illustrating the correlation between degranulation and protection. This study therefore showed that it is imperative to include a degranulation marker when assaying for granular proteins like Perforin and Granzymes by flow cytometry.