T cells are essential components of the adaptive immune system that can recognize and eliminate pathogens or cancer cells. However, during chronic infections or tumorigenesis, T cells are exposed to persistent antigen stimulation and become dysfunctional, a phenomenon known as T cell exhaustion (Wherry et al., 2007). Exhausted T cells display impaired effector functions, characterized by progressive loss of the capacity to produce interleukin-2 (IL-2) and interferon-γ (IFN-γ) (Fuller and Zajac, 2003; Wherry et al., 2003; Fuller et al., 2004), and increased expression of inhibitory receptors, such as programmed cell death protein-1 (PD-1), T cell immunoglobulin and mucin-domain–containing molecule 3 (Tim-3) (Fourcade et al., 2010; Sakuishi et al., 2010), and lymphocyte activation gene 3 (LAG-3) (Barber et al., 2006; Wherry, 2011). These inhibitory receptors, also known as immune checkpoints, act as negative regulators of T cell activation and prevent excessive inflammation and tissue damage. However, they also limit the efficacy of T cell-mediated immunity against chronic infections and tumors, which exploit these pathways to evade immune recognition and elimination (Pardoll, 2012; Sharpe, 2017).

To overcome the problem of T cell exhaustion, numerous efforts have been made to modulate the immune checkpoint pathways and restore T cell functions. One of the successful examples is the use of monoclonal antibodies that block the interaction between the inhibitory receptors and their ligands, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4. These agents have shown remarkable clinical benefits in various types of cancer, but they also have limitations, such as toxicity, resistance, and low response rates (Sharma and Allison, 2015; Topalian et al., 2015; Ribas and Wolchok, 2018). Therefore, there is a need for alternative or complementary approaches to target the immune checkpoint pathways and enhance T cell functions. One appealing approach to identify compounds that can modulate T cell functions is through phenotypic screens based on the desired cellular output in physiological relevant primary T cells as opposed to target-centered screens. This approach can screen a large number of compounds in parallel. However, developing a high-throughput assay with human primary cells to generate reliable and reproducible data is challenging due to a variety of technical difficulties, including cell reagent scale-up and donor-to-donor variations.

Herein, we describe a protocol to generate dysfunctional T cells from human peripheral blood mononuclear cells (PBMCs) and develop a fully automated high-throughput assay in 384-well format to assess immune checkpoint modulators. As illustrated in Fig. 1, PBMCs were maintained with IL-2 and Phaseolus Vulgaris Leucoagglutinin (PHA-L) over 10 days to induce dysfunction. The cell populations were subsequently examined and quantified using flow cytometry. The validated T cells were cryopreserved in liquid nitrogen and ready for assay development upon thawing. To develop the assay, the recovered dysfunctional T cells were initially incubated with testing compounds for 24 h, as the workflow depicted in Fig. 2. Following another 24-h stimulation with anti-CD3 and anti-CD28, cell supernatants were harvested for cytokine measurements (e.g., IL-2 and IFN-γ) using AlphaLISA assays to evaluate the activation of T cell receptor (TCR) signaling. Cell lysates in the same assay plate were used for a viability assay to assess cytotoxicity. Then, we implemented this workflow to assess 15 compounds selected from the literatures based on their known targets and pathways. 2 compounds out of 15 promoted IL-2 and IFN-γ production in dysfunctional T cells upon a stimulation with anti-CD3 and anti-CD28.