Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot

Area of Science:

• Immunology
• Virology
• Animal Models

Background:

• Simian immunodeficiency virus (SIV) infection in macaques serves as a crucial model for human immunodeficiency virus-1 (HIV-1) infection.
• Assessing the role of perforin (PFN), a key cytotoxic molecule, in this model has been limited by a lack of specific reagents.

Purpose of the Study:

• To evaluate the cross-reactivity of anti-human perforin monoclonal antibodies (mAbs) with macaque perforin.
• To develop and validate immunoassays for quantifying perforin release and cytotoxic T lymphocyte (CTL) responses in non-human primate models.

Main Methods:

• Monoclonal antibodies (Pf-80, Pf-164, Pf-344) against human perforin were tested for cross-reactivity with macaque perforin using flow cytometry and immunohistochemistry (IHC).
• Perforin capture ELISA and ELISpot assays were developed using these mAbs to quantify perforin release from stimulated peripheral blood mononuclear cells (PBMC).
• The perforin ELISpot assay was employed to quantify antigen-specific CD8+ T cells in SIV/HIV-1 immunized macaques.

Main Results:

• Monoclonal antibodies Pf-164 and Pf-344 demonstrated cross-reactivity with intracellular perforin in cynomolgus and rhesus macaque PBMC and stained lymphoid tissue.
• Developed ELISA and ELISpot assays successfully quantified perforin release from macaque PBMC.
• The perforin ELISpot assay effectively quantified antigen-specific CD8+ T cell responses in immunized macaques.

Conclusions:

• Macaque-reactive perforin monoclonal antibodies and developed immunoassays are valuable tools for studying cytotoxic T lymphocyte responses in SIV/HIV-1 infection models.
• These reagents will facilitate research into infectious diseases and aid in vaccine development using non-human primate models.