A hemolytic plaque assay for activated murine T cells

Area of Science:

• Immunology
• Cell Biology

Background:

• Murine spleen cells stimulated with concanavalin A (Con A) release helper and suppressor factors.
• Previous work demonstrated the presence of these soluble mediators in culture supernatants.

Purpose of the Study:

• To produce rabbit antisera against culture supernatants from Con A-activated spleen cells.
• To develop a plaque assay for detecting mitogen-activated T cells and their secreted products.

Main Methods:

• Production of antisera against spleen cell culture supernatants.
• Development of a plaque assay using antibody-coated SRBC, antisera, and complement.
• Characterization of plaque-forming cells (PFC) using immunological markers (Thy-1, Lyt-2.2) and mitogen responses.

Main Results:

• The plaque assay successfully detected secreting T cells (T-PFC).
• T-cell origin of PFC was confirmed by Thy-1 positivity, B cell depletion enrichment, and depletion by anti-T cell antibodies.
• Mitogen dose and incubation time influenced T-PFC response kinetics.
• Antisera recognized surface antigens on T-PFC, suggesting secreted products are cell-surface expressed.
• Antisera showed activity against cytotoxic T cell products.

Conclusions:

• A novel plaque assay enables the detection and analysis of mitogen-activated T cells and their secreted products.
• The assay provides a tool for clonal analysis of lymphokine production.
• Findings suggest secreted T cell products are expressed on the cell surface, enabling potential isolation of specific cell populations.