Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells.

Mohd Wajid Ali Khan

doi:10.6026/97320630017460

Abstract

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.

Highlights

Cancer is a disease distinguished by abnormal growth of cells, which leads to morbidity and mortality worldwide, with approximately 18.1 million new cases and 9.6 million cancer deaths according to the International Agency for Research on Cancer in 2018 [1]
Optimization of peripheral blood mononuclear cells (PBMCs) Isolation from fresh blood: Proliferated γδ T cell numbers depend on the starting number of cells
PBMCs were centrifuged at different rpm (1400, 1300, 1100, 900, 800 and 600) (Table 1)

Summary

Introduction

Cancer is a disease distinguished by abnormal growth of cells, which leads to morbidity and mortality worldwide, with approximately 18.1 million new cases and 9.6 million cancer deaths according to the International Agency for Research on Cancer in 2018 [1]. Γδ T cells are atypical T cells that can recognize and lyse diverse cancer cells in a non- major histocompatibility complex (MHC) restricted manner, highlighting their potential for cancer immunotherapy. They vigorously contribute to the anti-tumor immune response in many cancers (colon, breast, lymphoma, myeloma, melanoma, lungs, ovary and prostate) [6]. Γδ T cells offer a selective and non-MHC restricted response to phosphoantigens and, indirectly, to aminobisphosphonates [5] They behave as antigen presenting cells (APCs) [10]. We standardized and established a protocol for the proliferation of large numbers of γδ T cells with high purity, using minimum amounts of human blood

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Conclusion