Cancers are threatening the health of thousands of people around the world and represent a major cause of death; therefore, multiple efforts have been directed to fight against human cancers, and those efforts have led to the development of several approaches to treatment, including chemotherapy, photodynamic therapy, radiotherapy, and immunotherapy [1], [2], [3], [4]. By focusing on the immunotherapy approach, several human immune cells, including natural killer cells, macrophages, dendritic cells, and T cells, have been targeted to stimulate the immune system to fight against the growing tumor [5], [6], [7]. The top-ranked immune cell with a direct cytotoxic effect on cancer cells is CD8+ T cells. That cell can secrete cytotoxic molecules such as perforin and granzymes in addition to effector cytokines, including TNF-α and IFN-γ, where these molecules can directly lyse cancer cells. On the other hand, cancer cells can release specific molecules to fight back against CD8+ T cells and allow for their dysfunction [8], [9].

CD8+ T cell exhaustion is a newly defined state that occurs in chronic stimulation conditions, such as cancers and chronic viral infections [10]. That state has the hallmarks of reduced secretion of effector cytokines and cytotoxic molecules and elevated expression of other molecules such as PD-1, TIGIT, LAG-3, and TIM-3 [11]. These molecules are categorized as immune checkpoints, and ligation of these molecules, for example, PD-1 and PD-L1, with specific ligands on cancer cells, further supports the exhaustion state of the CD8+ T cells [12]. Hence, targeting those molecules with specific antibodies that would inhibit or hinder the ligation between the immune checkpoints and their ligands represents a novel form of cancer immunotherapy that can at least partially reinvigorate CD8+ T cells and restore their cancer-killing ability [13], [14]. These antibodies are named as immune checkpoint inhibitors (ICI). While immune ICI represented a great development in cancer treatment approaches, some patients still resist that treatment [15]. Hence, combining other treatment options, such as chemotherapy or radiotherapy, with immunotherapy is a major approach employed to overcome that resistance [16], [17]. Moreover, a deep understanding of the reasons for CD8+ T cells exhaustion could help researchers to define other immunotherapeutic targets that would act synergistically with ICI. For example, cancer cells can modify the tumor microenvironment through hypoxia nutrient deprivation and activate specific pathways that would finally develop new metabolic pathways in CD8+ T cells that further support their exhaustion and, in turn, resistance to immunotherapeutic drugs [17], [18], [19].

Exposure of the CD8+ T cells to chronic stimulation signals is a major reason for their exhaustion. One of the hallmarks of chronic stimulation in the tumor microenvironment is the elevated levels of reactive oxygen species (ROS) [20], [21]. High ROS levels can deteriorate the mitochondrial shape and function of CD8+ T cells, which further supports their exhaustion. Hence, ROS has been targeted to reduce the oxidative stress on CD8+ T cells and enhance their function [22]. For instance, nicotinamide adenine dinucleotide (NAD) has been defined as a molecule that can relieve CD8+ T cells exhaustion and improve their killing ability through the modulation of ROS. 2-Mercaptoethanol (2-ME) is a molecule with antioxidant activity, through scavenging hydroxyl radicals. Hence, 2-ME was reported to protect the aorta from dissection through the modulation of oxidative stress [23].

Additionally, 2-ME was defined to improve embryonic development through ROS control [24]. It can also interfere with the metabolism, where low-dose administration enhances functional outcomes in animals with pre-existing obesity [25]. Despite its reported antioxidant activity, the effect of 2-ME on cells, especially as a media component, is not well studied. Currently, many studies have applied a repeated stimulation approach to generate exhausted CD8+ T cells in vitro and assess the effect of different drugs or genetic modifications on the behavior of those exhausted CD8+ T cells [26], [27]. Additionally, adoptive cell transfer (ACT) of CD8+ T cells is considered a novel form of cancer immunotherapy where the generation of exhausted CD8+ T cells in vitro and expansion of the cells for ACT require culturing CD8+ T cells in a suitable medium [28], and components of that medium could interfere with the characteristics of the cultured CD8+ T cells. In the current study, we explored the influence of 2-ME as a media component in two models. Firstly, we investigated the effect of 2-ME on the exhausted CD8+ T cells that were generated in vitro through repeated stimulation. Next, we checked the influence of 2-ME on antigen-specific CD8+ T cells that were expanded in vitro before adoptive transfer to tumor-bearing mice. Our study shed light on the importance of the culture media components with a focus on the reagents with antioxidant activity to generate CD8+ T cells with enhanced survival and activity.