The cell-mediated cytotoxic response to influenza vaccination using an assay for granzyme B activity

Abstract

The measurement of cytotoxic T lymphocyte (CTL) activity through 51Cr assays is a very labour intensive method for studying cytotoxicity in human CTL due to the necessary preparation of autologous targets for the assay. An assay for granzyme B, one of a family of serine proteinases implicated in the ‘lethal hit’ that leads to target cell lysis, is an alternative simple measure of CTL activation. We measured granzyme B activity using its both preferred and unique substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (BAADT) in peripheral blood mononuclear cells (PBMC) obtained from influenza vaccinated subjects, and stimulated with live virus. We found that granzyme B activity increases in parallel and correlates with cytolytic activity as measured by 51Cr release assays in these virus-stimulated PBMC cultures. The assay was then used to measure the cell-mediated cytotoxic response to influenza vaccination in ten healthy elderly subjects. Peak granzyme B activity (day 6) was measured in lysates of PBMC stimulated with influenza virus, obtained from study participants before and after vaccination. We found a signigicant increase in granzyme B activity from pre-vaccination levels to 4 weeks post vaccination (pre = 2.77 U/mg protein, post = 7.23 U/mg protein, p = 0.002) and a subsequent decline in the activity measured at 12 weeks post vaccination (4.34 U/mg protein, p = 0.0007). Due to its substrate specificity which is unique within the family of serine proteases, this assay is highly specific for granzyme B. The assay also avoids the potential hazard of radioactivity ( 51Cr) in the clinical laboratory and the need for a γ counter. The assay of granzyme B activity, therefore, provides a simple, specific and responsive method for measuring changes in cell-mediated cytotoxic activity resulting from influenza vaccination.