A sensitive and less cytotoxic assay for identification of proliferating T cells based on bioorthogonally-functionalized uridine analogue

F.C Stempels,A.S De Wit,M.S Swierstra,S Maassen,F Bianchi,G Van Den Bogaart,M.V Baranov

doi:10.1016/j.jim.2022.113228

Abstract

Quantitative detection of T cell proliferation is an important readout in immunology research, as it is one of the hallmarks of T cell activation. Fluorescence-based methods for T cell proliferation mostly rely on the usage of probes that non-specifically conjugate to free primary amine groups in cells. Each cell division then results in a two-fold dilution of the probes which is detectable with flow cytometry. However, questions have been raised about cytotoxicity of these dilution-based T cell proliferation probes and they potentially affect T cell activation. An alternative assay relies on the incorporation of the uridine analog BrdU in the DNA of dividing T cells that can be detected with an antibody, but this requires harsh fixation and denaturation conditions. Recently, a new assay for detection of cell proliferation has been developed, based on the incorporation of the bioorthogonally-functionalized uridine analog 5-ethynyl-2′-deoxyuridine (EdU). Goal of this study was to compare the sensitivity and cytotoxicity of the EdU assay with a widely-used dilution-based T cell proliferation probe, CellTrace Far Red. We found that, compared to the dilution-based probe, the EdU-based assay better preserves T cell viability, is more sensitive for detecting T cell proliferation, and allows for better discernable interferon gamma responses.

Highlights

Cell proliferation is an important parameter in immunology as it is a measure for the activation of B lymphocytes (Hodgkin et al, 1996) and T lymphocytes (Hasbold et al, 1998; Marchingo et al, 2016), and for hematopoietic stem cells (Oostendorp et al, 2000)
The peripheral blood leukocytes (PBLs) were loaded with CellTrace before they were mixed with the Monocyte-derived dendritic cells (moDCs) (Fig. 1A)
EdU was added to the complete PBL-moDC cell mixture on the final day and incubated with the cells for just 2 h (Fig. 2A)

Summary

Introduction

Cell proliferation is an important parameter in immunology as it is a measure for the activation of B lymphocytes (Hodgkin et al, 1996) and T lymphocytes (Hasbold et al, 1998; Marchingo et al, 2016), and for hematopoietic stem cells (Oostendorp et al, 2000). The potency of dendritic cells (DCs) to induce T cell proliferation is commonly used for the evaluation of DC function, thereby playing an important role in the development of new DC-based therapies (Tel and de Vries, 2012; Tel et al, 2012). An additional costimulatory molecule on the surface of APCs, CD40, binds to CD40 ligand (CD40L) on the surface of activated T cells, ensuring sustained T cell responses (Howland et al, 2000). CD40-CD40L binding allows bidirectional communication between the APCs and T cells, as CD40L induces effector functions in APCs. For example, CD40 stimulation activates DCs and induces their production of the pro-inflammatory cytokine interleukin (IL)-12, a process called DC licensing (Cella et al, 1996; Stüber et al, 1996). Activated T cells secrete IL2 at earlier division stages (Sojka et al, 2004) and interferon γ (IFN-γ) at later divisions (Hasbold et al, 1999)

Methods

Results

Conclusion