T cells are derived from pluripotent stem cells of bone marrow, where T cell precursors first develop in the bone marrow, then move to the thymus for further programming and development, and gradually differentiate into T cells with immune activity in the thymus (1). These T cells are transported by the bloodstream to the lymph nodes, peripheral blood, and immune tissues where they colonize and take their final organ-specific characteristics (1). These peripheral naive T cells can be divided into two major subsets, CD4⁺ and CD8⁺ T cells, based on the CD cluster (CD) on their surfaces. Naive CD4⁺ T cells can differentiate into a variety of forms following antigen stimulation, including T helper (Th1, Th2, Th9, Th17, and Th22) cells, Treg cells, T memory (Tm) cells, and T follicular helper (Tfh) cells (2–4). In general, CD4⁺ T cells primarily perform the tasks of cytotoxicity, accessory immunity, and immune regulation (5). Each subtype specifically has distinct roles, but the differentiation of Th1, Th17, and Treg cells is of special interest because changes in the balance of these cells have been linked to a variety of illnesses, including autoimmune diseases, transplant rejection, and irritability (6, 7). For example, in systemic lupus erythematosus (SLE), excessive differentiation of Th17 cells and reduced differentiation of Treg cells are the main causes of disease development and tissue damage (8). Besides, Th1 and Th17 cells are closely related to the occurrence of this pathological state in acute cellular rejection induced by organ transplantation (9). Hence, it is crucial to restore the equilibrium of different subtypes of CD4⁺ T cells in diseases and stabilize it in healthy organisms.

Glutamine (Gln), a kind of immune regulatory nutrient, is frequently used in large amounts to supply cellular energy and to supply intracellular synthesis of genetic material via glutaminolysis within rapidly proliferating/dividing cells (10). Therefore, the original focus of studies on the effects of glutaminolysis on cells was tumorigenesis. Since immune cells also require substantial proliferation to function after activation, there has been progressively increasing research focusing on glutaminolysis in immune cells in recent years. Immunometabolism mainly investigates the reciprocal influence of immunity and metabolism in physiology and disease, with the ultimate goal of harnessing the distinct metabolic programs of different immune cell populations to treat disease. With the progress of immunometabolism, the role of Gln, considered as an immunomodulatory nutrient, has been gradually unraveled in immune related diseases. For instance, administration of glutaminolysis enzyme inhibitors can increase the acceptance of allografts in a mouse skin transplant model (11). Similar to this, in a mouse psoriasis model, aberrant glutaminolysis activation can cause lesion aggravation by promoting Th17 cell differentiation (12). These studies suggest that glutaminolysis may play a crucial role in immune related diseases and that the generation of these effects seems to be closely related to CD4⁺ T cell differentiation. As a result, manipulating glutaminolysis to reshape CD4⁺ T cell differentiation appears to be an effective intervention for immune-related diseases. In this review, we provide an overview on the role of glutaminolysis in peripheral CD4⁺ T cell differentiation and on the potential points of intervention in the glutaminolysis pathway for the treatment of various diseases.

Glutaminolysis is the process by which cells convert Gln to tricarboxylic acid (TCA) cycle metabolites through the activity of multiple enzymes ( Figure 1 ) (10). To begin with, Gln infiltrates the cytoplasm through amino acid transporters (AATs), which are a type of membrane bound transport proteins that can mediate the transfer of amino acids into and out of cells or organelles. These transporters are mostly sodium ion-dependent neutral AATs, mainly utilizing the concentration gradient of intracellular and extracellular sodium ions to synergistically transport sodium ions and Gln into cells, and then expel excess sodium ions from cells through a sodium ion pump (13). These transporters primarily consist of solute carrier family 1 member 5 (SLC1A5, namely alanine serine and cysteine transporter system 2, ASCT2), SLC38A1 (sodium-coupled neutral amino acid transporters 1, SNAT1) and SLC38A2 (sodium-coupled neutral amino acid transporters 2, SNAT2), with ASCT2 being the most important (14, 15). Subsequently, Gln enters mitochondria via SLC1A5 variant (SLC1A5_var), an AAT that locates on the mitochondrial membrane through its N-terminal targeting signal, then is decomposed into glutamate (Glu) and ammonia under the action of mitochondrial glutaminase (GLS), which is also the rate-limiting step of glutaminolysis (14, 16). GLS is the first enzyme in glutaminolysis, mainly including GLS1, GLS2, and GLS1 splicing isomer (Glutaminase C, GAC) (14). On the one hand, in mitochondria, Glu is then transformed into α-Ketoglutaric acid (α-KG) via glutamate dehydrogenase 1 (GLUD1), glutamic oxaloacetic transaminase 2 (GOT2) and glutamate pyruvate transaminase 2 (GPT2) (10). Specifically, under the catalysis of these three enzymes, Glu not only produces α-KG, but also produces ammonia, aspartate and alanine, respectively (10, 17). The intramitochondrial α-KG can participate in the TCA cycle, supporting the oxidative phosphorylation (OXPHOS) pathway and ATP generation (14). Glu and α-KG produced in the mitochondria are transported out via SLC25A18, SLC25A22 and SLC25A11 on the mitochondrial membrane respectively (14). On the other hand, Glu can also be converted into α-KG in cytoplasm by a group of transaminases, including GOT1, GPT1 and phosphoserine transaminase 1 (PSAT 1) (10). Similarly, GOT1 and GPT1 catalyze Glu to produce aspartate and alanine, respectively (17). PSAT1 is one of the key enzymes in the serine synthesis pathway, and it also produces a portion of α-KG in the pathway of catalyzing serine synthesis (18). Intracellular α-KG can be further catalyzed to generate 2-hydroxyglutarate (2-HG) by isocitrate dehydrogenase (IDH1), a missense mutant metabolizing enzymes (19). In turn, intracytoplasmic α-KG can be regenerated to Glu via GOT1 (10). Cytosolic Glu is involved in the biosynthesis of glutathione (GSH) and non-essential amino acids (NEAAs, e.g. alanine, proline, aspartate, asparagine and arginine) (14). Subsequently, Glu is transported out of the cell via SLC7A11, exchanging with cystine (Cys) (14). Likewise, excess Gln in the cytoplasm exchanges extracellular branched-chain amino acids (BCAAs, e.g. leucine, valine, and isoleucine) through SLC7A5 (LAT1) (20).

As previously mentioned, peripheral naïve CD4⁺ T cells can differentiate into different subtypes following antigen stimulation. Concretely, the first signal of cell activation is specifically obtained by naive CD4⁺ T cells through the interaction of their T cell receptor (TCR) with the antigenic peptide MHC class II (MHC-II) molecular complex that is displayed on the surface of antigen presenting cells (APCs) (21). Then, the second signal of cell activation is produced when these naive CD4⁺ T cells combine with the corresponding ligand (such as B7) on the surface of APCs and the costimulatory molecule (such as CD28) expressed on its surface (21). In response to dual signals, naïve CD4⁺ T cells become activated, immediately after which they need to take up large amounts of Gln and glucose to meet the biosynthesis materials and energy required for proliferation/differentiation (21, 22). Further investigation revealed that this shift was brought about by an increase in SNAT1, SNAT2, and ASCT2 expression when TCR was activated (23, 24). Naive CD4⁺ T cells subsequently differentiate into various subtypes under the influence of various cytokines in the microenvironment. For example, Th0 cells can polarize into Th1 cells when exposed to cytokines like IL-12, whereas Th0 cells can polarize into Th2 cells when exposed to cytokines like IL-4 (25, 26). Studies have shown that even in specific cell differentiation conditions, impairment in glutaminolysis can have a substantial impact on the fate of peripheral naive CD4⁺ T cells during differentiation (27). The underlying mechanism for how glutaminolysis regulates CD4⁺ T cell differentiation is discussed below ( Figure 2 ).

The homeostasis of CD4⁺ T cells is particularly important for the maintenance of organismal health, as several diseases have been linked to aberrant CD4⁺ T cell differentiation. For example, when Th1 and Th17 cells are differentiated excessively while Treg cell differentiation is insufficient, hyper immunological illnesses such as autoimmune diseases, graft rejection, and irritability result (56, 57). Hence, reshaping the disordered CD4⁺ T cell subsets is a simple and promising way to achieve immunotherapy. Specifically, intervention is required in humans to prevent the differentiation of peripheral naïve CD4⁺ T cells into proinflammatory cells in hyper immunological disorders. As previously described, the glutaminolysis pathway is a proper site of intervention to regulate the differentiation of naïve CD4⁺ T cells. In a nutshell, glutaminolysis is primarily separated into two steps: intracellular Gln uptake and progressive degradation. Therefore, from these features, intervention strategies to harness glutaminolysis for immunotherapy can also be developed (see Table 1 for details).

In conclusion, glutaminolysis plays an irreplaceable role during the differentiation of peripheral CD4⁺ T cells. Upon TCR activation, naïve CD4⁺ T cells start taking up Gln in large amounts to promote Th1, Th17 cell differentiation and inhibit Treg cell differentiation through several mechanisms including epigenetic regulation, mTORC1 activation and GSH pathway. After inhibition of glutaminolysis, there was an opposite trend in peripheral naïve CD4⁺ T cell differentiation. Therefore, regulation of peripheral Th1, Th17, and Treg cell differentiation by intervening glutaminolysis in naïve CD4⁺ T cells shows great potential to be exploited in immune-excessive diseases. Besides, in other diseases such as tumor, as an infinitely proliferating cell, it requires a large amount of Gln uptake and decomposition, as do anti-tumor CD4⁺ T cells such as Th1 and Th17 cells. So, although inhibition of Glutaminolysis in tumor cells is effective, it has not completely cleared the tumor lesions, which may be related to the tendency of CD4⁺ T differentiation changes to Treg after Glutaminolysis inhibition. Meanwhile, although there are many studies focusing on glutaminolysis and naïve CD4⁺ T cell differentiation, most of these studies focused on mouse cells in vitro. Studies on human naïve CD4⁺ T cell differentiation are still relatively lacking, and this is perhaps an area in which we deserve to explore in-depth. Moreover, at present, there are a few related reports on the regulation of Th2 cell differentiation by glutaminolysis, and how it regulates Th2 cell differentiation is still unknown, thus it is necessary to carry out related researches. Taken together, we summarized the existing studies, concluding several different interventional strategies for glutaminolysis. These strategies are currently mostly used in tumor diseases, and their application in inflammatory diseases still needs to be experimentally confirmed. Therefore, we expect to be able to guide directions on how to appropriately utilize glutaminolysis for future basic research and clinical applications in inflammatory diseases.

TL, SR, CS drafted manuscript, have contributed equally to this work and should be considered co-first authors. PZ helped in literature searching. HW provided administrative and financial support, manuscript revision and final approval of the manuscript. All authors contributed to the article and approved the submitted version.