TITLE:
A Sensitive and Simple Method to Assess NK Cell Activity by RT-qPCR for Granzyme B Using Spleen and Blood
AUTHORS:
Fusako Mitsunaga, Shin Nakamura
KEYWORDS:
NK Activation, Functional Foods, Immunostimulatory Effect, mRNA, Frozen Samples
JOURNAL NAME:
Journal of Biosciences and Medicines,
Vol.9 No.3,
March
18,
2021
ABSTRACT: A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.