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  chain signaling of il-2 and il-15 , and tgf increases foxp3 expression . however , certain cytokines like as tnf- have a controversial role in ttreg generation [ 2 , 3 ] . on the other hand ,
ptreg generation requires stimulation in an anti - inflammatory milieu , a process where dendritic cells are critically involved [ 3 , 4 ] .
according to cytokine production , tregs have been further classified ; for instance , th3 cells are characterized by tgf production ; tr1 cells produce il-10 , and tr35 cells produce il-35 .
while the expression of the transcription factor foxp3 can be transient in humans , this factor is associated with a suppressive function in mice .
several action mechanisms by which treg cells control the immune response have been reported : ( 1 ) inhibition by immunoregulatory cytokines such as tgf , il-10 , and il-35 ; ( 2 ) inhibition by cytolysis of effector cell by producing granzyme and perforin ; ( 3 ) metabolic interruption , including an inhibition of the proliferative response via il-2 receptor , camp - mediated metabolic inhibition , and immunomodulation mediated by the a2 adenosine receptor ; ( 4 ) interaction with dendritic cells that modulates their function and maturation .
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 [ 5 , 6 ] .
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 [ 7 , 8 ] .
il-10 inhibits the production of inflammatory cytokines such as il-12 , causing a decrease in the th1 response and in inf- production ; it also promotes the phagocytic activity , increasing the removal of cellular debris at the inflammation site .
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-b translocation ( figure 1 ) [ 10 , 11 ] . as shown in figure 1
, il-10 exerts its biologic function by activating jak1 and tyk2 , both proteins associated to stat1 , stat3 , and in certain cases stat5 ( figure 1 ) [ 10 , 12 ] . in rats ,
the activation of the il-10 receptor in dendritic cells has been observed to promote the jak1-tyk2-stat3 pathway .
when activated , this pathway inhibits dendritic cell maturation , decreasing the expression of mhc ii , cd11b / c , cd80 , and cd86 . in monocytes , il-10 has been demonstrated to induce the expression of the socs3 suppressor gene , which has influence on ifn - induced tyrosine phosphorylation in stat1 . on the other side , in a lps - induced inflammation model
, il-4 has been observed to promote c - maf expression in activated macrophages ; c - maf binds the promoter of the il-10 gene , favoring its production ( figure 1 ) [ 14 , 15 ] . 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-b - induced factor myd88 , resulting in the inhibition of the il-1 , il-6 , and tnf- cytokines ( figure 1 ) .
moreover , it has been observed that il-10 promotes stat3 phosphorylation and its translocation to the nucleus in treg cells , mediating a further il-10 production ( figure 1 ) .
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 ) family includes several structurally and functionally related proteins acting as multifunctional factors in a wide range of biological processes .
tgf is a 25 kda protein and a multifunctional cytokine , given its different effects on the cell [ 18 , 19 ] . during secretion
, tgf undergoes proteolysis by an endopeptidase , which cleaves the peptide bond linking the mature factor and the latency - associated peptide ( lap ) . for tgf to be activated
this process can be triggered by a number of factors , like temperature and ph changes .
three tgf isoforms are known in humans , 1 , 2 , and 3 , each coded in a different chromosome .
functional tgf receptors are types i and ii ( tgf ri and tgf rii ) , and nonfunctional receptors include types iii , iv , and v. tgf ri and rii bind tgf-1 and tgf-3 with greater affinity than tgf-2 .
tgf ri and tgf rii are responsible for the biological effects of tgf-1 in mammal cells . however , tgf riii is also capable of binding tgf-1 .
while tgf riii has no role in signal transduction , it has been suggested that it controls the availability of tgf-1 in the local extracellular microenvironment and regulates its active presentation to the functional receptors [ 21 , 22 ] . in the extracellular space , tgf binds tgf riii , which then recruits tgf rii and phosphorylates itself ; alternatively , tgf can bind directly to membrane - anchored tgf rii and induce the attraction of tgf ri and its ensuing phosphorylation ( figure 2(a ) ) .
both tgf ri and tgf rii have a cysteine - rich extracellular region , a transmembrane region , and a cytoplasmic region ; the latter includes a serine - threonine kinase domain .
there is a functional dependence between tgf rii and tgf ri , since tgf ri requires tgf rii to bind the ligand , while tgf rii requires tgf ri for a functional signaling .
when tgf binds tgf rii , the latter phosphorylates tgf ri in the serine- and glycine - rich domain near the transmembrane region .
the activation of the ligand - receptor complex allows the direct interaction of smad proteins with the kinase domain of the type - i receptor , recruiting smad2 and smad3 and phosphorylating them , thus activating the canonical pathway and forming a complex with smad4 .
this protein acts as a convergence node for the signaling pathways induced by members of the tgf superfamily ( figure 2(a ) ) .
the active complex formed by smad2 , smad3 , and smad4 is translocated to the nucleus , where it acts as transcriptional coactivator and regulates the transcription of several tgf-responder genes [ 21 , 23 ] .
tgf activates tak-1 , a kinase of serine and threonine residues of the map kinase family , by the noncanonical pathway .
ras , a member of the small g proteins , has also been involved in signaling .
certain mapks , including those kinases regulated by extracellular signals such as erk-1 and erk-2 and stress - activated kinases such as jnk and pi3k , are also activated in some cell types ( figure 2(a ) ) .
a number of mechanisms have been proposed : ( i ) it suppresses effector t cell differentiation ; ( ii ) it promotes the differentiation of nave t cells into regulatory t or th17 cells ; ( iii ) it inhibits t and b cell proliferation ; ( iv ) it inhibits the activity of macrophages , dendritic cells , and nk . the inhibitory activity of tgf on treg cells is due to the high lap / tgf expression in treg membrane . unlike t cells , monocytes and dendritic cells express tgf receptors , thus allowing for cell - to - cell interaction .
tgf has been demonstrated as necessary to generate tolerogenic dendritic cells ( dcs ) by inducing ido , an enzyme that inhibits t cell proliferation . as another relevant suppressive mechanism ,
tgf inhibits the production of il-2 ( figure 2(b ) ) . the cis - actin protein , which suppresses il-2 production ,
is called tob ; this protein binds smad2 , blocking the activation of runx 1/3 .
tgf modulates cell proliferation by controlling the expression of cell cycle - regulating factors , including cyclin - dependent kinases ( cdks ) like p15 , p21 , and p27 and cell cycle promoters like cmyc , cyclin d2 , cdk2 , and cyclin e. tgf also promotes t nave cell differentiation into treg cells by inducing foxp3 expression , or into th17 cells in the presence of il-6 ( figure 2(c ) ) .
foxp3 then induces the transcription of inhibitory cytokines such as tgf ( figure 2(c ) ) . on the other hand , th17 differentiation from nave t cells
signaling via il-6 activates stat3 , which induces the expression of ror. ror then induces the transcription of il-17 ( figure 2(c ) ) .
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 .
il-35 has two known functions : to suppress the proliferation of t helper cells and to promote the conversion of nave t cells into highly suppressor treg cells ( itr35 ) .
recently , this cytokine was demonstrated to be capable of inducing the conversion of b lymphocytes into b regulatory cells .
the il-35 receptor is formed by the il-12r2 and gp130 subunits ; it can be heterodimeric or homodimeric .
while gp130 is expressed in practically all cells , il-12r2 is expressed predominantly in activated t lymphocytes , nk cells , and to a lesser extent dcs and b cells .
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 , causing nave t cells to convert into il-35-producing ( itreg ) cells , suppressing cell proliferation , blocking the shift to a th1 profile , and mediating il-10 production .
treg cells are able to release the immunomodulatory cytokines il-10 , tgf , and il-35 .
this action may target several immune cells expressing receptors for such cytokines . this way ,
several immune cells in different stages ( activated , effector , or resting ) could be affected by the receptor - ligand interaction .
the effect of cytokines is probably not only local , since they could spread by the bloodstream throughout the body .
thus , these cytokines play a role in the polarization of immune response during various pathologies .
regulatory t ( tregs ) cells produce a serine protease called granzyme b , which allows them to induce apoptosis in effector t cells [ 2832 ] . 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 3(a ) ) .
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 3(b ) ) [ 3337 ] .
additionally , granzymes can bind to the cell surface in a way that granzyme recruitment is stimulated by perforin - mediated membrane damage ( figure 3(c ) ) .
once within the target cell , granzyme b could induce apoptosis by caspase - dependent or independent mechanisms , as discussed elsewhere [ 3840 ] .
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 .
il-2 receptor is expressed by t lymphocytes , nk cells , b cells , macrophages , and monocytes ; however , only t lymphocytes are capable of producing this cytokine .
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 .
alpha chain ( il-2r ) has a very short cytoplasmic domain and does not participate in signal transduction ; however , it is required to increase the affinity of il-2 to its receptor . on the other hand , beta ( il-2r ) and gamma ( il-2r ) chains play a crucial role in signal transduction .
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 4(a ) ) [ 3 , 43 ] .
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 ) .
additionally , the high cd25 expression in treg cells has been observed to favor adenylyl cyclase 7 activation and camp accumulation .
the differential expression of cd25 and adenylyl cyclase in treg cells is controlled by foxp3 .
tregs show a low camp degradation rate due to a diminished expression of the phosphodiesterase 3b enzyme in this cell subpopulation .
recent findings indicate that camp concentration is controlled by mir-142 - 3p , a microrna that regulates foxp3 transcription activity , and thus the expression of adenylyl cyclase .
while mir-142 - 3p inhibits the production of adenylyl cyclase 9 in conventional t cells , this effect is not seen in treg cells since the foxp3 transcription factor negatively regulates mir-142 - 3p expression and keeps the ac9/camp pathway active .
treg cells transfer camp to target cells by intercellular communications called gap junctions ( figure 4(b ) ) . in mammal cells ,
communicating junctions are used for the bidirectional traffic of ions , metabolites , and other molecules weighing less than 1  kd .
the increase in intracellular camp activates protein kinase a ( pka ) , since camp binds to the pka regulating subunit , activating it by releasing its catalytic subunit .
on the cell membrane inner face , phosphorylation of tyrosine kinase c - src ( csk ) by pka increases its activity ; then , csk phosphorylates and inactivates the lymphocyte - specific protein tyrosine kinase ( lck ) , an important protein in the proximal activation of t cell receptors . a number of signaling pathways can be regulated by pak .
for instance , the camp response element - binding ( creb ) protein is phosphorylated by pak at serine 1343 , which prevents it from forming a complex with the csk binding protein ( cbp ) and from binding to camp response elements ( cre ) ; these can be found in genes coding for t cell receptors or in other genes involved in t cell activation .
additionally , pak regulates the activity of the nuclear factor in activated t cells ( nf - at ) .
when nf - at is phosphorylated by pak , binding sites for another protein called 14 - 3 - 3 are created .
this new complex decreases nf - at transcription activity ( figure 4(b ) ) [ 4749 ] . when cells are in resting state , nf-b is found in the cytoplasm , complexed with its inhibitor , ib , which prevents it from translocating to the nucleus . during cell activation ,
ib is phosphorylated by an ib kinase , which induces the nf-b / ib complex to split apart .
conversely , when pka is phosphorylated , its catalytic subunit ( pka - c ) binds to the nf-b / ib complex , stabilizing it and keeping it inactive ( figure 4(b ) ) .
nf-b regulates the transcription of a large number of genes , including those coding for the proinflammatory cytokines tnf- il-1b , il-6 , il-8 , vegf , and metalloproteases ( mmp1 , mmp2 , mmp3 , and mmp13 ) . 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 4(b ) ) .
cd39 and cd73 are ectoenzymes , highly expressed on the surface of treg cells ( figure 4(c ) ) .
cd39 is a nucleoside triphosphate diphosphohydrolase-1 ( ntpdase 1 ) that degrades atp into amp .
the expression of cd39 in tregs is regulated by the foxp3 transcription factor , and its catalytic activity is enhanced by tcr compromise . in turn ,
extracellular amp is rapidly degraded into adenosine by cd73 , an ecto-5-nucleotidase bonded to the membrane of treg cells ( figure 4(c ) ) .
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 4(c ) ) .
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-b / p65 and then suppressing the expression of proinflammatory cytokines like tnf- ( figure 4(c ) ) [ 51 , 5355 ] .
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- ; it promotes as well the production of anti - inflammatory cytokines like il-10 ( figure 4(c ) ) .
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 [ 52 , 55 ] .
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 .
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- , a potent inducer of indoleamine 2,3-dioxygenase ( ido ) .
ido degrades tryptophan in a metabolic pathway starting with tryptophan oxidation to n - formylkynurenine , immediately followed by hydrolysis to kynurenine .
this initial product follows various degradation pathways whose final products are kynurenic acid , 3-hydroxykynurenine , anthranilic acid , 3-hydroxyanthranilic acid , and quinolinate ( figure 5(a ) ) [ 5961 ] .
once tryptophan is degraded by ido , a signaling cascade starts . since there is a local tryptophan deficit , the level of transfer rna without amino acid load increases , and as a result the general control nonderepressible kinase 2 ( gcn2 ) is activated , and it hyperphosphorylates the translation initiation factor ( eif2 ) , leading to a suppression in protein synthesis .
the union of the ternary complex formed by eif2 , gtp , and the initiation transfer rna for methionine ( met - rnai ) to the 40s ribosome subunit is inhibited , preventing the release of the binary complex eif2/gtp and inhibiting the eif2-promoted gtp / gdp exchange .
the result is a cell cycle arrest in the g1 phase and thus anergy of effector t cells ( figure 5(a ) ) [ 60 , 62 , 63 ] .
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 [ 64 , 65 ] ; this truncates the bid protein , producing a small proapoptotic fragment called truncated bid ( tbid ) .
this active fragment can translocate to the mitochondria , causing the release of cyt c by interaction with bax , another member of bcl-2 family . in the cytosol ,
cyt c interacts with the proteins apaf1 and caspase-9 in the presence of atp , building the apoptosome , a catalytic complex that starts the caspase cascade activation , leading to the digestion of structural proteins , degradation of chromosomal dna , and phagocytosis of apoptotic bodies ( figure 5(a ) ) [ 66 , 67 ] .
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 . on the other side , t cell apoptosis due to oxygen free radicals produced by kynurenine and
3-hydroxykynurenine has been observed , leading as well to changes in the oxidant - antioxidant balance and promoting oxidative stress .
oxidative stress causes a global collapse of mitochondrial function , reducing energy production and therefore contributing to cell death [ 64 , 69 ] .
another immunosuppression mechanism involves kynurenine binding to the aryl - hydrocarbon receptor ( ahr ) , a transcription factor expressed in cell populations like t cells , resulting in a shift to a t regulatory cell phenotype ( figure 5(a ) ) [ 70 , 71 ] .
lymphocyte - activation gene 3 ( lag3 , cd223 ) is a cell surface molecule expressed in tregs .
structurally , lag3 is homologue to the cd4 receptor and binds to mhc ii with a significantly higher affinity than cd4 [ 7375 ] .
the lag3-mhc ii union induces a signaling cascade starting with plc2 and p72syk phosphorylation and the activation of pi3k / akt , p42/44erk , and p38mapk ( figure 5(b ) ) .
the latter kinase has been involved in the maturation process of dendritic cells ; these exhibit an increase in the expression of costimulatory molecules and a decrease in antigen capture [ 76 , 77 ] .
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 fcr. the inhibitory signaling started by coengagement of mhc with lag3 along with cd4 and tcr could be mediated by several components , including ship-1 , which may not be recruited by themselves by the cd4 or tcr signaling pathways .
ship-1 has been labeled as a nf-b negative regulator and as a negative factor in cell activation .
additionally , the conjoint union of ctla-4 and lag3 promotes a tolerogenic phenotype in dcs ( figure 5(b ) ) .
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 ,
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   and helminthes , and they also modulate the immune response in noninfectious pathologies such as tumors . knowing the modulation molecular mechanisms and their effect according the pathological situation could help in identifying the therapeutic targets allowing an effective immune response .
 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 . 
1. Introduction
2. Molecular Mechanisms of Treg-Mediated Suppression
3. T Regulatory Cells Inhibit the Immune Response through Cytokine Production
4. Treg Cell Regulation Mechanism by Cytolysis of Effector Cells through Granzyme and Perforin
5. T Regulatory Cells Inhibit the Immune Response by Metabolic Interruption
6. Cell-Cell Interaction between Tregs and Dendritic Cells
7. Conclusion