T regulatory cells play a key role in the control of the immune response, both in health and during illness. While the mechanisms through which T regulatory cells exert their function have been extensively described, their molecular effects on effector cells have received little attention. Thus, this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective.
T regulatory (Treg) cells are a T lymphocyte subpopulation that control the balance between immune activation and tolerance. Treg cells can originate from two main sources: thymus-generated natural Tregs (tTreg) and peripheral inducible Tregs (pTreg), generated during immune priming.
Several factors are required for tTreg generation; these cells are strongly dependent on TCR and CD28 signals and on several cytokines. Cytokines contribute to Treg maintaining via
On the other hand, pTreg generation requires stimulation in an anti-inflammatory milieu, a process where dendritic cells are critically involved [
According to cytokine production, Tregs have been further classified; for instance, Th3 cells are characterized by TGF
Several action mechanisms by which Treg cells control the immune response have been reported: (1) inhibition by immunoregulatory cytokines such as TGF
IL-10 is an 18-kD protein produced by Tr1 cells. While its production is not restricted to this cell line since monocytes, dendritic cells, neutrophils, other T lymphocytes, and B lymphocytes are able to release it, Tr1 cells are the only regulatory cells to produce IL-10 [
IL-10 has been described as the main immunomodulatory cytokine; additionally, it can act as a paracrine or autocrine signal and can be induced by catecholamines [
IL-10 inhibits the production of inflammatory cytokines such as IL-12, causing a decrease in the Th1 response and in INF-
One of the best known molecular mechanisms of IL-10 is the action on effector cells. The costimulatory molecule CD28 is involved in the interaction between effector cells and antigen-presenting cells. By binding its receptor, IL-10 inhibits tyrosine phosphorylation in CD28, inhibiting PI3K/AKT activation, which in turn inhibits the signaling cascade leading to NF-
Interleukin 10 can act on effector cells, dendritic cells, and cytokine-producer T regulatory cells (Tr1). On effector cells, IL-10 exerts an immunosuppressor function. In dendritic cells, IL-10 can favor a tolerogenic phenotype that promotes the production of further IL-10. On Tr1 cells, IL-10 has an activator effect that also favors IL-10 production.
As shown in Figure
By binding its receptor in Tr1, IL-10 induces JAK1 and TYK2 phosphorylation, activating STAT3, which is translocated to the nucleus, promoting SOCS3; in turn, SOCS3 inhibits the NF-
IL-10 exhibits a wide range of biological activities, including immunosuppression, anti-inflammation, and immunomodulation. IL-10 is able to inhibit MHC I expression in B and T cells and also in dendritic cells, all of them involved in the inflammatory response [
The Transforming Growth Factor-beta (TGF
Functional TGF
In the extracellular space, TGF
(a) The union of TGF
TGF
TGF
The inhibitory activity of TGF
As another relevant suppressive mechanism, TGF
TGF
Interleukin 35 (IL-35) is a heterodimeric cytokine in the interleukin 12 (IL-12) family. This cytokine family can be composed of one to five subunits (p19, p28, p35, p40, and another from Epstein-Barr virus gene 3, also called EBI3). IL-35, an immunosuppressor cytokine formed by the p35 and EBI3 subunits, is produced by Treg cells [
The IL-35 receptor is formed by the IL-12R
The IL-35 signaling path has not been completely described yet; however, it is known that the heterodimeric receptor activates STAT1 and STAT4, which induces EBI3 and p30 expression [
Treg cells are able to release the immunomodulatory cytokines IL-10, TGF
Regulatory T (Tregs) cells produce a serine protease called Granzyme B, which allows them to induce apoptosis in effector T cells [
During Treg-effector cell interaction, directed exocytosis from Treg granules to the extracellular space of both cells takes place; these granules contain granzymes and perforins. Once released from the Treg, perforin molecules insert themselves into the lipid membrane of the target cell and polymerize in the presence of calcium ions to form a transmembranal cylinder; each cylinder forms a pore through which granzymes enter the cell (Figure
T regulatory cells can induce the death of effector cells by releasing granzyme and perforin. Granzyme can enter the effector cell through the perforin-mediated channels (a), or it could be endocyted through receptors (b) and perforin damage (c).
Granzyme can also enter the cell by an endocytosis process mediated by the manose-6-phosphate receptor. In this case, granzyme is sequestered in endosomes into the cytosol, and perforin acts to release it (Figure
Once within the target cell, Granzyme B could induce apoptosis by caspase-dependent or independent mechanisms, as discussed elsewhere [
Cytolysis allows Treg cells to act on several immune cell populations by cell-to-cell interaction. This mechanism is highly effective, since Treg cells induce death by apoptosis on effector cells, thus decreasing the number of effector cells and controlling the immune response.
Interleukin 2 (IL-2), chiefly secreted by T cells in response to antigenic stimuli, is the main cytokine for T cell proliferation. The gene coding for this cytokine is located in chromosome 4 [
IL-2 receptor is a complex formed by three subunits: alpha chain (CD25), beta chain (CD122), and gamma chain (CD132); each subunit plays an important role in facilitating the transduction of IL-2-dependent signals [
Treg cells constitutively express high levels of IL-2 alpha chain, having thus a higher affinity to IL-2, and compete for this growth factor with proliferating cells. By depriving proliferating effector cells from IL-2, Treg cells do not only prevent them from continuing the proliferative process but also leave them without a vital cytokine, causing metabolic interruption and cell death (Figure
T regulatory cells can inhibit the immune response by three mechanisms: (a) By competition with effector cells for IL-2. (b) Through cAMP-mediated immunosuppression. (c) By adenosine production via the ectoenzymes CD39 and CD79.
Cyclic adenosine monophosphate (cAMP) is a second messenger, capable of regulating the functional activity of effector cells and antigen-presenting cells. The high cAMP content in Treg cells is due to the 50-fold higher expression of adenylyl cyclase 9 (AC9) [
Treg cells transfer cAMP to target cells by intercellular communications called gap junctions (Figure
When cells are in resting state, NF-
While the main target for cAMP is PAK, cAMP has also been demonstrated to directly activate the exchange protein directly activated by cAMP (EPAC1 and EPAC2). This protein regulates the activation of a GTPase called Rap-1, responsible for activating ERK, thus inhibiting cell proliferation and differentiation (Figure
CD39 and CD73 are ectoenzymes, highly expressed on the surface of Treg cells (Figure
The adenosine resulting from AMP hydrolysis binds four different surface receptor subtypes coupled to Gs proteins, called A1, A2A, A2B, and A3. The A2AR receptor is the main adenosine receptor associated to T and B lymphocytes, NK cells, macrophages, dendritic cells, and granulocytes (Figure
The outcome of stimulating these receptors is an intracellular AMP accumulation; through the cAMP-dependent protein kinase, these signals phosphorylate and activate CREB. The latter binds the nuclear cofactor p300, producing a complex that regulates the expression of several genes in their promoter regions. CREB is able to regulate indirectly the transcription of some inflammatory genes, competing with NF-
AMP can also activate other substrates like EPAC1 and other kinases such as ERK and JNK, altering the expression of proinflammatory genes through transcription factors responsible for synthesizing interleukins like IL-12 and TNF-
The fact that Tregs produce adenosine and respond to it at the same time means that this molecule acts as an autocrine factor to optimize the anti-inflammatory response. It also increases Treg suppressor capacity, inhibits the expression of costimulatory molecules in dendritic cells, and inhibits the activation of effector cells [
Treg-mediated metabolic disruption occurs by competition for IL-2, a growth factor for effector cells. Under these conditions, Treg cells control the immune response in a nonspecific but effective manner, since by consuming IL-2 Treg cells inhibit effector cell proliferation, actually impairing the immune response. On the other hand, immunosuppression mediated by cAMP and the 2A adenosine receptor induce several signaling pathways in effector cells that in turn impact transcription factors, controlling the effector response.
Tregs can interact with DCs through CTLA-4. This molecule has a high affinity by dendritic cell-expressed CD80 and CD86; thus, Tregs compete with effector cells to bind these ligands.
This interaction involves several events, including the production of INF-
T regulatory cells have contact direct with dendritic cells through CTLA-4 (a) and LAG3 (b).
Additionally, IDO exerts an immunosuppressive activity through the metabolites resulting from tryptophan degradation. Although the mechanism is not well understood, 3-hydroxyanthranilic acid and quinolinic acid have been demonstrated to induce apoptosis in Th1 cells by directly activating caspase-8 [
The metabolite 3-hydroxyanthranilic acid has also been reported to induce death in activated T cells by depleting glutathione, one of the main antioxidant molecules in animal cells. A decrease in glutathione promotes a misbalance between ROS production and the antioxidant capacity [
Lymphocyte-activation gene 3 (LAG3, CD223) is a cell surface molecule expressed in Tregs [
The LAG3-MHC II union induces a signaling cascade starting with PLC
Another study demonstrated that, after crosslinking MHC in the presence of LAG3, ITAM-mediated inhibitory signals are induced, involving ERK sequential activation and SHIP-1 recruitment similar to those proposed for FC
Dendritic cells being a key component of the immune response, the action implying cell-to-cell contact with DCs is one of the most important mechanisms for Treg cells. Depending on its phenotype, a DC can activate or control the immune response. When interacting with a Treg cell, a DC acquires a tolerogenic phenotype, which in turn promotes further Treg cell generation, providing a suppressor microenvironment. The competition for DC ligands between effector and Treg cells allows for an additional control mechanism of the immune response.
The action mechanisms of Tregs described above can act together or independently, according to the requirements of the immune system and homeostasis maintenance, or during the progression of various pathological processes. Treg cells have been used as part of an escape mechanism by several pathogens, including viruses [
The authors declare no conflict of interests regarding the publication of this paper.
This review article was supported by CONACYT (CB-2011-01 167278 and FOSISS 2015-1 261455). The authors thank Carlos Castellanos Barba for paper revision.