Jim Xiang ^1,2,3* Scot C. Leary ⁴ Zhaojia Wu ^2,3 Michelle Yu ^2,3

• ¹ Jim Xiang, Saskatoon, Canada
• ² Saskatoon Cancer Centre, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada
• ³ Division of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
• ⁴ Department of Biochemistry, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

The Yin-Yang theory represents a fundamental principle in traditional Chinese medicine (TCM), guiding its diagnosis and treatment of diseases (1). In the latter context, the Yin element refers to reserve, healing, resting or quiescence, passive or inhibitory factors and negative regulation while the Yang element speaks to consumption, working or activeness, growth or promoting factors and positive regulation. The theory posits that a balance between these two opposing forces becomes the premise of a healthy body (1). The "Qi", a special energy flowing in the body, is a driver of the defense system in TCM (1), and is analogous to the protective immune system in Western medicine (WM). However, there is a huge gap between the conceptional connection of TCM and WM.The Yin-Yang theory has been applied historically to interpret immune system biology mostly from the perspective of the counteractive features of these two immune elements or responses. For example, the suppressive CTLA-4 and immunogenic CD28 have been described as the Yin and Yang of T cell co-stimulation (2). Moreover, CD4 + Treg and CD4 + Th1 cells have been considered to belong to Yin and Yang CD4 + T cell subsets, where the former and latter cells contribute to tolerant and active immune responses, respectively (3). More recently, the regulators AMPKα1 and mTORC1 have been proposed to represent Yin and Yang energy sensors, with mTORC1 signaling the availability of nutrients and promoting immune stimuli that support cell proliferation through glycolysis and AMPKα1 signaling a lack of nutrients and inhibiting cell growth through FAO metabolism (4). However, the molecular basis and pathways underpinning the Yin-Yang theory in T cell immunity remain incompletely understood. mTORC1 is an evolutionarily conserved serine/threonine kinase whose ability to sense three major immune signals (i.e. antigen, co-stimulation, and cytokines) and a variety of environmental cues (e.g. growth factors and nutrient status) allows it to act as a master regulator. Its activity is modulated in a phosphorylation-dependent manner by PI3K-Akt signaling. PI3K phosphorylates PIP2 at the cell surface to generate PIP3, which recruit and phosphorylate Akt at T308 through PDK1 (Fig 1A) (5). mTORC2 can also be activated by PI3K through PIP3 and phosphorylated at S473 (Fig 1A ), that confers for a full activation and substrate specificity of Akt (6). The phosphorylation of Akt at T308 triggers mTORC1 signaling, which in turn up-regulates the downstream substrates ribosomal S6K1 and S6K2 and eIF4E to modulate protein synthesis, cell proliferation, metabolism and differentiation (Fig 1A) (7), whereas the phosphorylation of Akt at S473 represses FOXO1 activity through phosphorylation of FOXO1 at T24 (Fig 1A) (8). Autophagy is a self-recycling process in which cellular constituents are degraded by lysosomes to provide essential anabolic building blocks in support of metabolism and homeostasis under stress conditions (9). Flux through autophagy as well as most branches of intermediary metabolism, including mitochondrial FAO, is regulated by the activity of the evolutionarily conserved energy sensor AMPKα1, which is in turn controlled by LKB1, CaMKK2, PKA and TAK-1 (Fig 1A) (9). As such, AMPKα1 is central to CD8 + TM cell formation (Fig 1A) (9). FOXO1 is another well known, key regulator that controls various aspects of cell development (10) and also contributes to CD8 + TM cell differentiation (Fig 1A) (8). Both AMPKα1 and FOXO1 were also described as tumor suppressors because of their inhibitory effect on tumor growth (8,9). Therefore, AMPKα1/FOXO1 and AKT/mTORC1 can be characterized as Yin and Yang master regulators in T cell immunity as well as cell biology (Fig 1A). In modern T-cell biology, two basic features have been well studied; (i) cellular differentiation into a defined phenotype with respect to form and function, and (ii) metabolic fuel preference to provide the energy necessary to support function and sustain homeostasis. These features or characteristics are thus used as a standard manner of defining Yin and Yang cell subsets in immunity and may even be extended conceptually to various tissues or organs of the body. The pro-inflammatory IL-2 and pro-survival IL-7 cytokines ( The theory argues for negative interplay between the two elements. For example, the Yang regulators mTORC1 and AKT interact with the Yin regulators AMPKα1 and FOXO1 via negative feedback loops (10). Our recent data showed that mTORC1 Strong signaling inhibits Yin AMPKα1 and FOXO1 expression in IL-2/TE cells, while mTORC1 Weak -induced expression of AMPKα1 and FOXO1 promotes T cell memory in IL-15/TM cells (13). mTORC1-induced phosphorylation of AMPKα1 at S485 blocks its phosphorylation at T172 in IL-2/TE cells, whereas AMPKα1-mediated phosphorylation of its downstream target TSC2 at S1387 weakens mTORC1 signaling in IL-15/TM cells (Fig 1A) (13,23). In addition, mTORC2-dependent phosphorylation of AKT at S473 phosphorylates FOXO1 at T24, which inhibits FOXO1 by reducing its nuclear localization (Fig 1A) (24). FOXO1 in turn is able to down-regulate mTORC1 signaling via its activation of sestrin, a suppressor of mTORC1 (Fig 1A) (25). Collectively, these reports confirm the negative interplay between the master regulators Yin FOXO1 or AMPKα1 and Yang AKT/mTORC1 (Fig 1A ,1Ed).In support of this principle, the AMPK activator metformin inhibits mTORC1 (26), while the mTORC1 inhibitor Rapa promotes AMPKα1 and FOXO1 activity (Fig 1A ,1Ee) (7,14).The theory also advances that the Yin response contains some Yang elements and vice versa, which facilitates their interconversion under certain conditions (Fig 1Ef ). For example, a basic feature of TM cells is their ability to rapidly proliferate and switch to functional TE cells in a recall response (Fig 1B). This interconversion is facilitated by the maintenance of an AKTdependent, "imprinted" glycolytic potential (27) or a demethylated, epigenetic mark at the IFN-γ promotor (8, 28) inherited from the Yang CD8 + TE precursor, either of which leads to an efficient immediate-early recall response of Yin CD8 + TM cells upon cognate antigen re-encounter.Differentiation of distinct immune cell subsets is accompanied by complementary changes in fuel preference. To date, the Yin and Yang metabolic pathways in all immune cell subsets are regulated by the Yin AMPKα1 for FAO and the Yang HIF-1α for glycolytic metabolism. In contrast, the Yin and Yang transcriptional factors controlling differentiation into a defined form and function may differ across immune cell subsets. For example, Yin GATA3 is stimulated in CD4 + Th2 which use FAO while Yang T-bet is triggered in CD4 + Th1 cells which rely on glycolysis (17,29). Similarly, Yin Foxp3, Smad3 and FOXO1 are activated in FAO reliant CD4 + Treg cells while Yang ROR-γt is stimulated in glycolytic CD4 + Th17 cells (Fig 1C) (17,30).Finally, Yin STAT6 and PPAR-γ are up-regulated in FAO utilizing tolerant M2 cells while Yang STAT1 and NF-κB are stimulated in immunogenic M1 cells relying on glycolysis (Fig 1C) (18). These data illustrate that the above principle can be used to interpret various cell subsets of the immune system and may perhaps be extended to cell subsets in various tissues and organs of the body (Fig 1Eg).The dynamic balance of Yin and Yang keeps the body healthy, and illness may result when there is an upset within or between these two systems (1,31,32). A deficiency in CD4 + Treg cells or inhibitory co-stimulation and a more active Yang TE cell response derived from some strong vaccinations can induce detrimental autoimmune and lymphoproliferative diseases, respectively, (33)(34)(35), while deficiencies in T-cell immunity may underlie infectious and cancerous diseases (Fig 1Eh) (36). Our current knowledge of various immune elements and genes (Fig 1C) emphasizes the importance of the interplay between Yin and Yang molecular pathways that control immune responses or underlie various immune disorders (31,32). Yet additional, integrative studies of large datasets using artificial intelligence or other technological approaches to analyze and interpret various genes and cell subsets are required to further improve our understanding of the molecular bases and pathways of the Yin-Yang theory in T-cell immunity in the context of human health and disease (37,38). Nevertheless, this nascent model provides a new and simple guide with which to categorize genes into Yin and Yang groups, to facilitate the analysis of complex genetic networks and interactions between genetic elements and molecular pathways. Therefore, this model may act as a novel platform for drug innovation to combat various diseases (37,38). Future studies in this direction should further strengthen the conceptional connection between TCM and WM, that will be greatly beneficial to human health.