A Sensitive Microassay System for Lymphocyte-Mediated Cytotoxicity Induced by PHA and PPD

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Abstract
A sensitive in vitro microassay for lymphocyte cytotoxicity and lymphotoxin (LT) modified from the method of Takasugi and Klein has been characterized in detail. The cytotoxicity assay (nonspecific target cells + activator + lymphocytes) was performed by using phytohemagglutinin (PHA) and purified protein derivative (PPD). Human skin fibroblasts were not useful as target cells because of insensitivity, but L cells (mouse fibroblasts, subline 5b) proved satisfactory. The fractional survival rate of target cells incubated with activated lymphocytes, relative to incubation with non-activated lymphocytes, was calculated. This discounted nonspecific lymphocyte toxicity. The sensitivity of four variations of the microassay systems was compared and a “mixture assay” (preincubation of L cells and lymphocytes before addition to the microplate) was found to be most sensitive. Lymphocytes at a concentration of 0.02 × 106/ml (activated by PHA) and 1.0 × 106/ml (activated by PPD) showed nearly 100% target cell destruction. This assay was approximately 50 times more sensitive than the standard incubation of lymphocytes on L cell micromonolayer for 2 days. Cytotoxicity of the assay performed with direct contact of lymphocytes and target cells was constantly higher than cytotoxicity of the culture supernatant. However, cytotoxicity was strongly correlated in the two, indicating that LT or LT-like materials might be the mediator in this system. The population of PPD-sensitive lymphocytes in the peripheral blood of a tuberculin skin test positive donor was calculated to be 2.0%.

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