Isolation and expansion of murine γδ T cells from mouse splenocytes

• Published in: Journal of immunological methods 2022-09, Vol.508, p.113322-113322, Article 113322
• Main Authors: Williams, Lindsay, Dery, Kenneth J., Lee, Wen-Hui, Li, Harry, Shively, John E., Kujawski, Maciej
• Format: Article
• Language: English
• Subjects: Anti-tumor response; Cytokines; T cells; γδ T cells
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2022.113322

Elucidation of the function of gamma delta T cells (γδ T cells) requires robust models that show how γδ T cells are commonly involved in inflammation, since very little is known about the factors that promote and control their development and function. There are few studies of murine γδ T cells primarily because these cells have proven difficult to isolate, expand and characterize. Here, we describe a simple method that utilizes key expansion elements to isolate and expand murine CD4−CD8−CD3+ γδ T cells typically found in secondary lymphoid tissues. Expansion of γδ T cells reached 150-fold by day 8 of culture, depended on exogenous IL-2, αCD3, and αCD28, and supported efficient and reproducible in vitro differentiation. These studies showed high production of cytokines IFNγ and Granzyme B, with the novel finding of IL-24 upregulation as well. Expression analysis of expanded γδ T cells, after treatment with IL-2, revealed high levels of Granzyme B, Granzyme D, and IFNγ. Lactate dehydrogenase (LDH) cytotoxicity assays showed that expanded γδ T cells were effective at inducing >90% cytolysis of murine MC38 colon cancer, E0771 breast cancer, and B16 melanoma cells at 10:1 effector to target ratios. These findings indicated that murine γδ T cells can be successfully isolated, expanded, and used to perform preclinical therapy studies. •Spleen-derived murine γδ T cells can be effectively expanded in vitro with the proposed expansion protocol.•These cells show high IFNγ and Granzyme B production along with the novel finding IL-24 production after IL-2/αCD3 exposure.•Expanded γδ T cells are highly cytotoxic against tumor cell lines in vitro.