Although Tregs are not the central theme here, their pivotal role in the broader context of regulatory cells cannot be overlooked in any comprehensive research of immune regulation.

Conventional Tregs have long been recognized for their pivotal role in maintaining immune homeostasis by suppressing inappropriate immune responses (25). This capacity to modulate immune responses is critical not only in preventing autoimmune diseases but also in controlling inflammation and promoting tolerance across various biological systems (13) ( Figure 1 ). Recent studies have expanded our understanding of Tregs functions beyond their traditional roles (31). These cells are now known to engage in several non-immune functions that are crucial for maintaining tissue homeostasis. For instance, Tregs have been implicated in metabolic regulation, particularly in adipose tissues where they influence insulin sensitivity and lipid metabolism (32). Tregs are alos influenced by lipid metabolism, particularly through the PPAR-γ (Peroxisome proliferator-activated receptor-γ) receptor, which is sensitive to lipid interactions that can impair Tregs functionality (33). Moreover, Tregs contribute to the maintenance of stem cell niches, such as those found in the bone marrow, skin, and intestines (34, 35). By modulating the local microenvironment, Tregs can protect stem cells from oxidative stress and promote their quiescence, which is crucial for long-term tissue regeneration (36). This interaction also highlights the broader role of Tregs in tissue repair and regeneration, where they can directly affect tissue cells to promote healing and restoration after injury. Furthermore, Tregs have been shown to facilitate tissue repair by producing growth factors and cytokines that directly interact with tissue cells. These molecules help in the proliferation and function of cells involved in tissue repair, such as epithelial cells in the skin and lung, and satellite cells in muscle tissue (2, 37).

In the context of clinical applications, the expanded understanding of Treg functions opens new avenues for therapeutic interventions aimed at modulating Treg activity. By targeting the non-canonical functions of Tregs, it is possible to develop treatments that enhance their regulatory capabilities, thereby improving outcomes in diseases characterized by inflammation, autoimmunity, or impaired tissue repair. These insights into the multifaceted roles of Tregs underscore their importance not only in immune regulation but also in broader physiological processes, making them a key target for future research and therapeutic development (1, 38).

When discussing regulatory T cells, it is increasingly imperative not to overlook the presence and significance of CD8+ Tregs. These cells represent a unique subset of T cells with critical implications in maintaining immunological tolerance and modulating the immune environment in various diseases, including cancer (39–41). They exhibit a capacity to suppress immune responses, which is pivotal in preventing autoimmunity but can also facilitate tumor immune evasion by weakening antitumor immune attacks (42). Research has elucidated distinct properties and mechanisms of CD8+ Tregs that differentiate them from their CD4+ counterparts (43). Predominantly, CD8+ Tregs exert their regulatory functions through the expression of molecules like Foxp3, CTLA-4, and others which are shared with CD4+ Tregs, yet they also display unique markers such as CD122 and CD28⁻, reflecting their distinct regulatory pathways (44). These cells are found elevated in various cancers, where they contribute to an immunosuppressive microenvironment that promotes tumor growth and survival by inhibiting effective immune surveillance and clearance. Interestingly, these cells not only modulate immune responses through direct cell-cell interactions but also through the secretion of immunosuppressive cytokines and metabolic disruption of effector cells (45–48). The induction of these Tregs can be driven by the tumor-derived factors and by the tumor microenvironment itself, which alters T cell metabolism and differentiation paths, skewing them towards a regulatory phenotype. The profound impact of CD8+ Tregs in cancer suggests that they could serve as a potential target for therapeutic intervention (49). Modulating the function or abundance of these cells could enhance the efficacy of cancer immunotherapies, providing new avenues to improve patient outcomes in oncological treatments. Understanding and manipulating the balance of Treg activity in the tumor context is therefore crucial for developing more effective cancer immunotherapies (50).

Bregs (Regulatory B cells) are a specialized subset of B lymphocytes that play a crucial role in immune regulation and maintaining immune homeostasis. They are known for their ability to suppress immune responses and promote immune tolerance. Researchers have made significant progress in identifying and characterizing Bregs. They have identified specific cell surface markers and functional properties that distinguish Bregs from other B cell subsets, which helps in understanding their unique regulatory functions (51).

Establishing the existence of one or multiple subsets of Bregs has been challenging due to the absence of specific markers that are analogous to the Treg marker FoxP3 (51, 52). The presence of multiple reported phenotypes for Bregs suggests the potential existence of distinct subsets. However, there are common surface markers shared among the proposed Breg subsets, which suggests the possibility that all Bregs originate from a common precursor (53, 54). The observed differences in phenotype could be attributed to the activation of this common Breg precursor in different microenvironments.

Specific markers for Bregs include (1). CD19: It is important to note that CD19 expression alone is not specific to Bregs, as it is also present on other B cell subsets. It is typically used in combination with other markers to define and isolate regulatory B cell subsets more precisely (51). (2). CD1d: CD1d, a molecule involved in lipid antigen presentation, plays a significant role in the context of Bregs. CD1d allows Bregs to directly interact with NKT cells through the presentation of lipid antigens via CD1d. Then Bregs can influence the function and activity of NKT cells (55). (3). CD5: In some studies, CD5 expression on B cells is commonly associated with B1 cells, a subset of B cells involved in innate-like immune responses and regulatory functions (56). CD5⁺ Bregs can produce regulatory cytokines such as IL-10 and could suppress immune responses and promote immune tolerance. While CD5⁺ Bregs have been well-characterized in mice, their presence and significance in human Bregs are still under investigation (57). (4). CD24: High expression of CD24 is often used as a defining feature and a marker for identifying Bregs in various contexts. CD24⁺ Bregs have been shown to possess potent immunosuppressive properties, including the production of regulatory cytokines such as IL-10 and the ability to suppress excessive immune responses. Modulating the function or increasing the numbers of CD24+ Bregs could be explored as a strategy for treating immune-related disorders, including autoimmune diseases and transplantation rejection (58). (5). CD38: Bregs with regulatory properties have been found to exhibit higher levels of CD38 compared to other B cell subsets. This suggests that CD38 can be used as a phenotypic marker to identify Bregs with immunosuppressive potential. CD38-expressing Bregs have been implicated in various disease contexts (59–61). For example, in autoimmune diseases like RA and SLE (systemic lupus erythematosus), decreased numbers or impaired function of CD38+ Bregs have been observed (62, 63). This suggests that CD38-expressing Bregs may play a role in maintaining immune tolerance and preventing excessive inflammatory responses (58). (6). CD27: The relationship between CD27 expression and Bregs is complex and can vary depending on the context and species studied. These CD27+ Bregs are believed to possess regulatory functions and contribute to immune tolerance. CD27 expression on Bregs has been associated with enhanced suppressive activity and the production of anti-inflammatory cytokines such as IL-10 and TGF-β. Some memory B cells with regulatory functions have been identified within the CD27+ B cell subset. These memory Bregs have been implicated in the regulation of immune responses and the maintenance of immune homeostasis (52).

Bregs employ various mechanisms to suppress immune responses and promote immune tolerance ( Figure 2 ). Initially, Bregs are known to produce immunosuppressive cytokines, such as IL-10 and TGF-β. These cytokines play a crucial role in dampening immune responses by inhibiting the activation and function of effector immune cells. IL-10, in particular, is a potent anti-inflammatory cytokine that can inhibit pro-inflammatory cytokine production and immune cell activation (65, 66). Furthermore, Bregs can act as APCs and present antigens to T cells. However, instead of inducing T cell activation, Bregs often promote tolerance by inducing T cell anergy or Treg differentiation (67, 68). Moreover, Bregs have been shown to interact with and promote the generation and function of Tregs which are key mediators of immune tolerance and can suppress immune responses. Bregs can induce the differentiation and expansion of Tregs through cell-cell contact and the production of regulatory cytokines like IL-10 and TGF-β (65, 69, 70). Additionally, Bregs can exert suppressive effects through direct contact with immune cells. For example, Bregs can engage with and inhibit the activation of APCs such as DCs, thereby reducing their ability to stimulate immune responses. Bregs also directly interact with and suppress the function of effector T cells (71). Finally, Bregs can regulate immune responses by modulating the expression of co-stimulatory molecules. They can downregulate the expression of co-stimulatory molecules on APCs, which is essential for efficient T cell activation. By dampening the co-stimulatory signals, Bregs contribute to the suppression of immune responses (72, 73). Apart from IL-10 and TGF-β, Bregs can secrete other anti-inflammatory factors such as IL-35 and granulocyte-macrophage colony-stimulating factor (GM-CSF). These factors can suppress the activity of immune cells and promote immunosuppression (74, 75).

MDSCs are a heterogeneous population of immature myeloid cells with potent immunosuppressive capabilities. They play a crucial role in regulating immune responses in various pathological conditions, including cancer, infections, autoimmune diseases, and chronic inflammation (76–81). MDSCs are characterized by their myeloid origin, early differentiation stage, and ability to suppress immune responses through multiple mechanisms.

MDSCs arise from myeloid progenitor cells in the bone marrow. Under certain pathological conditions, such as cancer or inflammation, the differentiation of these cells is disrupted, leading to the accumulation of immature myeloid cells with suppressive functions in peripheral tissues (82, 83). MDSCs lack mature markers of myeloid cells and often express a combination of markers associated with early myeloid progenitors, including CD11b and Gr-1 in mice (84, 85). In humans, MDSCs are commonly identified by the expression of CD11b and CD33, as well as the lack of markers associated with mature immune cells, such as HLA-DR (86, 87). However, it is essential to note that MDSC phenotypes can vary depending on the context and the specific tissue involved.

MDSCs suppress immune responses through various mechanisms, contributing to immune evasion in cancer and other diseases. To begin with, MDSCs can secrete factors like IL-10, IL-6, IL-1β, TGF-β, and arginase-1, which dramatically increase the rate of accumulation and T cell suppressive activity of MDSC (88–92). Furthermore, MDSCs can trigger apoptosis in activated T cells through the production of reactive oxygen species (ROS) and other pro-apoptotic factors (93–95). Additionally, MDSCs consume and deplete essential nutrients such as arginine, tryptophan and cysteine, restricting their availability for T cell function (96–99). Moreover, MDSCs can impair the function of dendritic cells and macrophages, leading to decreased antigen presentation and diminished T cell activation (100–102) ( Figure 3 ).

MDSCs can be further classified into two main subsets based on their phenotype in mice: Granulocytic MDSCs (G-MDSCs) express high levels of Ly6G and Ly6C and are morphologically similar to neutrophils (103, 104). Monocytic MDSCs (M-MDSCs) cells are characterized by high expression of Ly6C and low expression of Ly6G in mice, resembling monocytes (105, 106). In humans, the classification of MDSC subsets is more complex and remains an area of ongoing research.

MDSCs play a critical role in promoting tumor progression and immune evasion in cancer. Their accumulation is associated with poor prognosis in cancer patients (107–110). Moreover, MDSCs have been implicated in the pathogenesis of various inflammatory and autoimmune diseases, contributing to the dysregulation of immune responses (111–119).

DCs (dendritic cells) are specialized antigen-presenting cells derived from the bone marrow that play a crucial role in initiating and regulating innate and adaptive immune responses. Regulatory or “tolerogenic” DCs are particularly important especially in the context of maintaining self-tolerance in a healthy state. These Dcregs employ various mechanisms to suppress or redirect the responses of naïve or memory T cells. In animal models of autoimmune diseases and transplant rejection, DCregs have demonstrated the ability to induce or restore T cell tolerance (120–124). Moreover, there is compelling evidence indicating that the transfer of DCregs can effectively modulate T cell responses not only in non-human primates but also in human subjects (120, 125). Building upon insights gained from in vitro experiments and animal models, efforts have been made to develop clinical-grade DCregs for the treatment of autoimmune diseases (120, 126–128). Clinical trials in Phase I evaluating the use of regulatory dendritic cell therapy in type-1 diabetes, rheumatoid arthritis and Crohn’s disease have shown promising results, demonstrating the feasibility and safety of this approach (129–132).

DCregs possess unique suppressive mechanisms that allow them to actively regulate immune responses and promote immune tolerance (see Figure 4 ).

ILCregs are a subset of innate lymphoid cells that possess immunosuppressive functions and play a crucial role in maintaining immune homeostasis. While the concept of ILCregs is still relatively new and evolving, their importance in immune regulation is becoming increasingly recognized (see Figure 5 ).

ILCregs have been identified in various tissues and organs, including kidneys and intestines (21, 157). They are characterized by their ability to produce immuno-suppressive cytokines such as IL-10 and TGF-β, which help dampen excessive immune responses and promote tolerance (21). Similar to other ILC subsets, ILCregs can be classified based on the expression of specific transcription factors and surface markers. Though they lacked expression of the Treg transcription factor Foxp3, ILCregs exert their immune-suppressive effects through multiple mechanisms. In addition to secreting immunosuppressive cytokines, they can interact with other immune cells, such as dendritic cells and T cells to modulate their functions (158). ILCregs can inhibit dendritic cell maturation and antigen presentation, leading to decreased activation of effector T cells. They can also directly interact with T cells, promoting the development of regulatory T cells and suppressing the activity of pro-inflammatory T cell subsets (157) ( Figure 6 ).

Unlike the previously described ILCregs, there is also evidence that ILC2s have the capacity to produce IL-10 and may have immunoinhibitory potential (159). Additionally, several pieces of evidence suggest that ILC3s are plastic and can become ILCregs (160). Indeed, the recent findings reveal a regulatory plasticity within all ILC subtypes, and potential crosstalk between DCs and ILCs which should be further investigated in future research.

The study of ILCregs is still in its early stages, and further research is needed to fully understand their ontogeny, functional diversity, and specific roles in different immune contexts. However, their potential as therapeutic targets for immune-mediated diseases and their ability to regulate immune responses make them an exciting area of investigation in immunology.

While NK cells were initially classified as a uniform population of innate lymphocytes, emerging evidence suggests that NK cells comprise diverse subsets with varying functions, distributions, and developmental origins. Human NK cells in peripheral blood can be categorized into at least two functional subsets based on their expression of CD56 and CD16 (161) (Figure 7). CD56^dimCD16⁺ NK cells make up approximately 90% of the total NK cells present in the blood. These cells are highly efficient in killing target cells and secrete lower levels of cytokines. On the other hand, regulatory NK cells, namely CD56^bright CD16^- NK cells, account for less than 10% of total blood NK cells but are enriched in secondary lymphoid tissues (162).

NK cells have been recognized for their ability to carry out effector functions through both direct cytotoxicity and the release of IFN-γ. However, a study conducted by Perona-Wright and colleagues has shed light on an additional role of NK cells in attenuating inflammatory processes (163). They demonstrated that NK cells can dampen inflammation by producing IL-10. Therefore, NK cells possess the capacity to not only exert their cytotoxic and pro-inflammatory activities but also contribute to the regulation and resolution of immune responses. Some of the key functions of regulatory NK cells include: (1). Immunomodulation: Regulatory NK cells can interact with other immune cells, such as T cells and dendritic cells, and modulate their activation and function. They can suppress the proliferation and activation of T cells, thereby regulating the adaptive immune response (164–166). (2). Cytokine Production: Regulatory NK cells produce various immunomodulatory cytokines, including IL-10 and TGF-β (167–171). These cytokines have anti-inflammatory properties and can suppress immune responses. (3). Interaction with Dendritic Cells: Regulatory NK cells can interact with dendritic cells and influence their maturation and antigen presentation. This interaction can impact the activation and differentiation of other immune cells, such as T cells (163, 169). (4). Induction of Tolerance: Regulatory NK cells contribute to the induction of immune tolerance, particularly in the context of transplantation and autoimmunity. They can suppress excessive immune responses and promote immune tolerance to self-antigens (172, 173).

Although regulatory NK cells are a relatively less studied subset compared to conventional NK cells, their role in immune regulation is increasingly recognized. Further research is needed to fully understand their precise mechanisms and contribution to immune tolerance and regulation. It’s important to note that regulatory NK cells are still an area of active research, and their clinical applications are being explored in various ongoing studies and clinical trials. As our understanding of their functions and therapeutic potential advances, regulatory NK cells may become an integral part of personalized and targeted immunotherapies in the future.