Immunization is critical for the maintenance of biological homeostasis. It refers to the physiological function by which a body's immune system fights against the invasion of foreign substances and distinguishes internal components from external ones. The immune system is responsible for the body's immune response and immune function. While it produces robust immune responses to attack various antigens, it also negatively regulates or inhibits abnormal immune responses, maintaining a relatively stable immune reactivity (1–3). The immune system consists of three major components, namely the immune organs, immune cells and immunologically active substances. In particular, immune cells are produced, mature and are concentrated in the immune organs. These cells can be divided into phagocytic cells and lymphocytes, which are composed of T and B cells that mature in the thymus and bone marrow, respectively. Immunologically active substances mainly refer to antibodies, lymphokines and lysozyme (4–6).

Physiologically, the tolerance of internal components and the response to ‘non-self’ antigens are under the strict control of the body's immune regulation mechanism. Immune regulation is crucial for the maintenance of the physical environment stability in the human body (7,8). Therefore, dysfunction of immune regulation will result in serious pathological consequences. For instance, if the immune system develops a strong immune attack on the body's own components, autoimmune diseases occur (9,10). The body may also be harmed if the immune system cannot respond adequately to an infection caused by external pathogenic microorganisms. In this case, a weak response can result in severe infection, whereas an excessively strong response can result in allergy (11–18). Therefore, immune regulation determines the occurrence and the strength of an immune response. This elegant and complicated regulation functions in multiple steps in an immune response process.

In 1970, Gershon and Kondo (19) identified CD8⁺ regulatory T cells (CD8⁺ Tregs). Later studies revealed the dual effects of these cells in immune responses. They have been reported to inhibit the immune response to pathogens and the host's inflammation following pathogen infection. However, by weakening the body's immune surveillance on malignant cells, the host can be relieved from autoimmune diseases (20–22). Although CD8⁺ Tregs were recognized >40 years ago, little is known regarding their function in negative regulation (23). Furthermore, recent studies on CD8⁺ Tregs revealed their crucial role in immunology (24–26), while CD8αα⁺ T cell receptor (TCR)αβ⁺ Tregs, a novel subtype of CD8⁺ Tregs, was demonstrated to recognize the major histocompatibility complex class Ib (MHC-Ib) molecules Qa-1 in mice. Tregs only target activated T lymphocytes, and are considered to complement the inhibition function of CD4⁺ forkhead box P3 (FoxP3)⁺ Tregs (27,28). Therefore, further studies on CD8αα⁺ Tregs may provide novel therapeutic strategies for human inflammatory diseases, tumor immunity, transplant tolerance and autoimmune diseases.

In the process of thymic negative selection, only T cell clones with high affinity to autologous antigens are removed (29,30). Therefore, certain T cells with low affinity to autologous antigens are leaked to the peripheral immune system. Under certain conditions, they may be activated and result in autoimmune diseases. Therefore, this process is monitored by a series of peripheral immune tolerance mechanisms, including Tregs with immunosuppressive effects, namely CD4⁺ and CD8⁺ Tregs (31–33).

To date, there is no reliable surface marker that is able to distinguish CD8⁺ Tregs from ordinary CD8⁺ T cells. In different experimental systems, CD8⁺ Treg surface molecules have been demonstrated to differ. For instance, in an experimental autoimmune encephalomyelitis (EAE) model, CD8⁺CD28⁻ Tregs demonstrated an inhibitory effect on the secretion of interferon (IFN)-γ by myelin oligodendrocyte glycoprotein-specific CD4⁺ T cells through cell contact inhibition (34,35). Similarly, CD8⁺CD45R⁺ and CD8⁺CD122⁺ T cells also possess regulatory suppressor functions, including suppression of immune activity following autologous mixed lymphocyte reaction activation (36–38). In comparison, CD8⁺ Tregs induced by plasma-like dendritic cells (DCs) in the ascites of cancer patients are featured the interleukin (IL)-10⁺ C-C motif chemokine receptor 7 (CCR7)⁺ CD45RO⁺CD8⁺ phenotype. In particular, by secreting IL-10, CCR7⁺CD45RO⁺CD8⁺ Tregs inhibit the function of effective T cells that specifically attack tumor antigens (39–41). In addition, immature DCs induce the generation of CD8⁺ Tregs, while FoxP3⁺ antigen-specific CD8⁺CD28⁻ T cells can also be produced in vitro through rendering vascular endothelial cells tolerogenic (42–45). Notably, inhibitory CCR7⁺CD45RO⁺CD8⁺ T cells can originate from human tumor tissues, indicating their potential association with tumor-induced immune tolerance (46,47).

CD8⁺ Tregs in humans are predominantly CD8⁺CD28⁻ Tregs; however, two CD8⁺ Tregs subgroups can be produced by induction in vitro, namely CD8⁺CD28⁺ and CD8⁺CD28⁻ Tregs (48,49). Currently, the majority of studies conducted have investigated CD8⁺CD28⁻ Tregs, and three categories of these cells have been identified. Among them, type I cells have a direct contact with DCs to influence the expression of the costimulatory molecules CD80 and CD86, demonstrating a negative regulatory role. However, the inhibitory function of type II cells is exerted through cytokine secretion, such as IFN-γ and IL-6, while direct contact with antigen-presenting cells (APCs) has not been observed. Finally, type III cells function by secreting IL-10 (50–52).

Common molecules that serve as markers for Tregs include FoxP3, CD25, CD127, CD39 and CD73. Among them, FoxP3 is a member of the fork-like transcription factor family and was first reported in 2001 by Brunkow et al (53). Studies have identified that FoxP3 expression and function are closely correlated with Tregs. FoxP3 is mainly expressed in lymphoid organs and tissues, including in the thymus, spleen and lymph nodes (54–56). In mice, FoxP3 has been reported to be preferentially expressed in CD4⁺CD25⁺ T cells, while its expression in CD8⁺ T cells was limited. By contrast, in humans, FoxP3 can be expressed in both CD4⁺CD25⁺ T cells and CD8⁺ T cells (57,58). However, its expression in CD4⁺ T cells is significantly higher in comparison with that in CD8⁺ T cells. Thus far, FoxP3 has been recognized as the most sensitive marker of Tregs (54–56).

Traditionally, the identification of Tregs mainly relied on CD25 labeling. However, it was later reported that identifying Tregs merely based on CD25 positivity was not accurate (59,60).

CD127, an IL-7 receptor, is downregulated in a subset of CD4⁺ T cells in the peripheral blood. These cells are FoxP3 positive, and CD25 weak positive or negative (61). The combination of CD4, CD25 and CD127 selection generates high purity Tregs, which exhibit a strong signal in functional inhibition tests. The population of Tregs that can be distinguished by CD4 and CD127 expression (including CD25⁺CD4⁺ and CD25⁻CD4⁺ cells) is three times as large as the T cell sub-population that can be selected by CD4⁺CD25^hi (62). As CD127 has been successfully applied to quantify the Tregs of patients, it has been proposed as a marker of human Tregs (63,64).

Studies have also reported that Foxp3⁺ Tregs express the cell surface CD39 and CD73 molecules simultaneously (65,66). When cell damage or apoptosis occurs, intracellular ATP is released, causing increased concentration of extracellular ATP. As the signaling molecules for cell damage, they activate a variety of immune responses. Furthermore, CD39 and CD73 are extracellular enzymes that are expressed by various immune cells, including DCs, B cells and T cells. Notably, they dephosphorylate ATP or AMP, as well as decompose AMP, thus achieving an immunosuppression function and inhibition of T cell inflammatory factors (67–69).

Different types of CD8⁺ Treg subsets can function by secreting various inhibitory cytokines and chemokines, including IL-10, transforming growth factor (TGF)-β, IL-16, IFN-γ and chemokine (C-C motif) ligand 4 (33,70–77). CD8⁺CD28⁻ Tregs render the APCs tolerogenic by upregulating the expression levels of immunoglobulin-like transcript (ILT)3 and ILT4, which then function as cell surface inhibitory receptors. These tolerogenic APCs demonstrate an anti-inflammatory function. The downregulation of costimulatory molecules CD80 and CD86 on APCs by CD8⁺CD28⁻ Tregs also inhibits the immune response of CD4⁺ T cells. In addition, CD80 and CD86 are important for the inhibitory function of CD8⁺CD122⁺ T cells (78–80). Certain subsets of CD8⁺ Tregs exert an inhibitory function by cell contact-dependent mechanisms, in which TGF-β and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) expressed on the cell surface serve key roles (81,82). CD8⁺ Tregs exert a cytotoxic effect against antigen-activated CD4⁺ T cells, and this function depends on the expression of the MHC-Ib molecule Qa-1 in mice (HLA-E in humans) (28,83,84). The aforementioned mechanisms are presented in Fig. 1A-D.

Currently, several issues remain unaddressed regarding the functions of Tregs and the corresponding research methodology (21,23). Firstly, phenotype identification and a functional/mechanistic study of CD8⁺ Tregs have to be further conducted in different experiment systems. In addition, it remains unclear whether different CD8⁺ cell subpopulations originate from common precursor cells. Finally, the application of CD8⁺ Tregs can be further explored in the scenarios of prevention, diagnosis and clinical treatment of diseases.

Thus far, relatively well-studied and well-recognized CD8⁺ Tregs include natural CD8⁺CD28⁻ T cells, as well as the induced CD8⁺CD28⁻, CD8⁺CD25⁺ and CD8⁺CD122⁺ T cells. A systematic analysis on the transcription profile of these CD8⁺ Tregs identified CD25⁺CD28⁻ to be the most common surface marker (5,122). Other surface markers include FoxP3, CD103, CD122 and CTLA-4 (49,123).

Studies on CD8⁺ Tregs are of great value for human disease treatment, and thereby attract increasing researchers into this field. Investigating this group of cells may provide novel ideas for immune regulation and immune intervention. Furthermore, this may elucidate the pathogenesis of associated disease and offer an objective index to their diagnosis, treatment and evaluation. We believe that studies on CD8⁺ Tregs will generate broad application prospects in the fields of inflammatory diseases, autoimmune diseases, tumor immunity and transplantation tolerance.