McElhaney J E, Pinkoski M J, Upshaw C M, Bleackley R C
Department of Medicine, University of Alberta, Edmonton, Canada.
J Immunol Methods. 1996 Mar 28;190(1):11-20. doi: 10.1016/0022-1759(95)00235-9.
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 significant 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 gamma 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.
通过51Cr检测法测量细胞毒性T淋巴细胞（CTL）活性，对于研究人类CTL的细胞毒性而言是一种劳动强度很大的方法，因为该检测需要制备用于检测的自体靶细胞。颗粒酶B是丝氨酸蛋白酶家族的一员，参与导致靶细胞裂解的“致命一击”，检测颗粒酶B是CTL活化的另一种简单方法。我们使用其首选且独特的底物叔丁氧羰基 - 丙氨酸 - 丙氨酸 - 天冬氨酸 - 硫代苄酯（BAADT），测量了从接种流感疫苗的受试者获取的外周血单个核细胞（PBMC）中的颗粒酶B活性，并使用活病毒进行刺激。我们发现，在这些病毒刺激的PBMC培养物中，颗粒酶B活性与通过51Cr释放检测法测得的溶细胞活性平行增加且相关。然后，该检测法用于测量10名健康老年受试者对流感疫苗接种的细胞介导的细胞毒性反应。在接种疫苗前后从研究参与者获取的、用流感病毒刺激的PBMC裂解物中测量颗粒酶B活性峰值（第6天）。我们发现，颗粒酶B活性从接种前水平显著增加至接种后4周（接种前 = 2.77 U/mg蛋白质，接种后 = 7.23 U/mg蛋白质，p = 0.002），随后在接种后12周测得的活性下降（4.34 U/mg蛋白质，p = 0.0007）。由于其在丝氨酸蛋白酶家族中独特的底物特异性，该检测法对颗粒酶B具有高度特异性。该检测法还避免了临床实验室中放射性（51Cr）的潜在危害以及对γ计数器的需求。因此，颗粒酶B活性检测法为测量流感疫苗接种引起的细胞介导的细胞毒性活性变化提供了一种简单、特异且灵敏的方法。
