Isolation and in vitro expansion of human γδT lymphocytes: basic approaches

Abstract

T lymphocytes γδ represent a unique subpopulation of T cells that combine features of innate and adaptive immunity, making them promising candidates for cancer immunotherapy. However, the clinical application of these cells is limited by their low abundance in peripheral blood (1–5 % of all lymphocytes) and functional heterogeneity, necessitating the development of effective methods for their isolation and expansion in vitro. Aim. To analyze and summarize current methods for the isolation and expansion of γδT lymphocytes from human blood for research and therapeutic purposes. A review of the scientific literature on approaches to obtaining γδT cells was conducted. Isolation methods (immunomagnetic and fluorescence sorting) and culture strategies were analyzed: stimulation with phosphoantigens (zoledronate), activation with antibodies to CD3, CD28, and γδT-cell receptor, and the use of artificial antigen-presenting cells (based on the K562 line). A search for sources was conducted in the abstract databases PubMed, Google Scholar, and eLibrary. Isolation using magnetic-activated and fluorescence-activated cell sorting allows for the production of pure populations (up to 90–98 %). The most developed methods are for the selective expansion of Vγ9Vδ2 T cells using phosphoantigens, enabling their proportion in culture to reach 90 % after just 10–14 days. Stimulation with antibodies and artificial antigen-presenting cells ensures long-term proliferation (up to several weeks) and multiple expansion (hundreds of thousands of times), making these methods suitable for clinical application. At the same time, it has been shown that methods for expanding rare subpopulations (Vδ1+, Vδ3+) and animal cells remain poorly understood, and long-term cultivation can lead to phenotypic cell exhaustion. Current protocols allow for the efficient production of Vγ9Vδ2 T cells, but require further optimization to preserve functional activity and selectively expand other subpopulations. Promising approaches include genetic engineering (CAR-γδ T cells; CAR – chimeric antigen receptor) and improved culture conditions, including the selection of cytokine combinations and new stimulating molecules, to expand the therapeutic potential of these cells.