J Vet Sci.  2023 Jan;24(1):e11. 10.4142/jvs.22210.

A standardized method to study immune responses using porcine whole blood

• Mattoo SuS
• Aganja RP
• Kim SC
• Jeong CG
• Nazki S
• Khatun A
• Kim WI
• Lee SM

• KMID: 2539431
• DOI: http://doi.org/10.4142/jvs.22210

Abstract

Background
Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment.
Objectives
Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses.
Methods
Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV).
Results
The frequency of IFN-γ ⁺ CD3 ⁺ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ ⁺ CD4 - CD8 ⁺ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples.
Conclusions
We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

Keyword

Porcine whole blood assay; in vitro test; PRRSV; cytotoxic T lymphocytes; flow cytometry; ELISA