upon activation by donor alloantigens , recipient naive t cells can differentiate into a variety of graft - destructive , effector t cell or graft - protective , regulatory t cell ( treg ) phenotypes .
these t - cell commitments are determined largely by the texture of the innate immune milieu in which t - cell activation occurs .
a milieu in which transforming growth factor 1 ( tgf1 ) is expressed in the absence of proinflammatory cytokines promotes the commitment of alloactivated t cells into a tissue - protective , forkhead box p3 ( foxp3 ) treg phenotype .
in contrast , a milieu in which proinflammatory cytokines are abundant prevents the generation of foxp3 tregs and instead directs t - cell commitment into the tissue - destructive t helper 1 ( th1 ) , th2 , or th17 phenotypes [ 1 - 4 ] .
unfortunately , a robust expression of proinflammatory cytokines is typical for recently engrafted organ transplants .
the inflamed state , highly detrimental to the treg induction and immunoregulatory function , is a consequence of innate immune activation in response to the ischemia and reperfusion injury .
although conventionally used agents such as corticosteroids are perhaps useful in restricting the early graft inflammation [ 7 - 9 ] , their broad immunosuppressive action , including blocking the expression of tgf1 , does not facilitate tolerance .
hence , we believe that reducing the adverse forms of inflammation but allowing activity of tgf1 and other inhibitory cytokines within a graft may prove to be a key approach for the induction and maintenance of allograft tolerance .
we suspect that the creation of an intra- and peri - graft milieu devoid of proinflammatory cytokines will serve to guide the majority of donor - activated t cells into a tgf1-incited , treg tissue - protective phenotype . indeed
, our own results and the results of others support this concept [ 12 - 15 ] .
therapies that primarily block adverse inflammation , such as alpha1-antitrypsin , anti - interleukin ( il)-6 , or anti - tumor necrosis factor - alpha , have been successfully utilized by our group in tilting the balance of the allograft response toward tolerance , or in restoring tolerance in the non - obese diabetic ( nod ) model of type 1 diabetes , even in nod mice with frank diabetes .
given the availability of therapies ( many of them approved by the us food and drug administration ) that are suitable for blocking the adverse forms of inflammation , rapid translation into the clinic seems possible for the treatment of certain immune - inflammatory disorders .
we are aware that a pure prevention or a blockade of inflammation may not be sufficient to achieve lasting transplant tolerance in humans .
additional lymphocyte - depleting measures are required first of all in order to prevent rejection in pre - sensitized recipients .
nonetheless , we believe that favorably tipping the balance of pro- and anti - inflammatory cytokines in the milieu in which non - depleted t cells re - populate will foster t - cell commitment into a tissue - protective mode and promote tolerance . our current efforts at blocking tim4 ( t - cell immunoglobulin and mucin domain - containing protein 4 ) , a molecule upregulated by antigen - presenting cells exposed to proinflammatory cytokines and toll - like receptor agonists , have proven equally successful in promoting long - term engraftment in preclinical models of transplantation and autoimmunity , although this method is not yet ready for testing in humans .
if confirmed that tim4 blockade leaves t - cell anti - viral responses largely intact , it may be an especially attractive strategy to promote tolerance in patients with chronic viral infections .
induction and maintenance of transplant tolerance require that graft - protective foxp3 tregs efficiently and durably restrain the pool of graft - destructive effector t cells after anti - rejection therapy is withdrawn   ( figure 1 ) . for tolerance to be permanent , the foxp3-dependent treg immunoregulatory phenotype must be stabilized .
a stable expression of foxp3 , the treg lineage specification transcription factor , is required to maintain treg function and thereby maintain transplant tolerance .
loss of foxp3 gene expression , such as that occurring in the inflamed , il-6-rich environments , can destabilize treg molecular phenotype and immunoregulatory function , thereby undermining the maintenance of transplant tolerance . with the loss of immunoregulatory function among these destabilized tregs , rejection occurs as the immunoregulatory restraints upon donor - reactive effector t cells are released . a milieu dominated by anti - inflammatory cytokines or inflammation - dampening agents
promotes the commitment of donor - activated t cells into a regulatory t - cell phenotype and thereby fosters transplant tolerance .
a milieu dominated by proinflammatory cytokines promotes effector t cell generation and rejection of the allograft .
aat , alpha-1-antitrypsin ; apc , antigen - presenting cell ; il , interleukin ; teff , effector t cell ( t helper 1 [ th1 ] , th2 , and th17 ) ; tgf1 , transforming growth factor 1 ; tnf , tumor necrosis factor - alpha ; treg , regulatory t cell .
foxp3 expression in tregs is regulated , in part , by epigenetic modifications of the foxp3 chromosomal locus .
the methylation status of cpg - sensitive residues upstream of the transcriptional start site ( exon1 ) is an important regulator of foxp3 expression .
methylation of these residues represses foxp3 gene expression while complete demethylation is required for optimal foxp3 gene expression .
other epigenetic mechanisms , such as histone methylation and acetylation , also modulate foxp3 stability . of note ,
therapeutic agents that directly foster maintenance of demethylated foxp3 promote robust expression of foxp3-sensitive genes , immunoregulatory t - cell function , and tolerance .
for this purpose , some inhibitors of dna methyltransferases , such as nucleoside analog 5-azacytidine , have been successfully used in experimental studies . perhaps owing to the known toxicity of demethylating agents , formal clinical trials in transplantation
that treg infusions have the potential to prolong allograft survival and induce transplant tolerance is widely acknowledged .
however , the clinical use of tregs for the adoptive transfer into transplant recipients is hindered by the inherent instability of the foxp3-dependent treg phenotype upon exposure of tregs to inflamed environments . as noted , in the proinflammatory milieu ,
foxp3 expression is diminished , inhibitory function is compromised , and some tregs even convert to t effector - like phenotypes [ 22,30 - 32 ] . such treg instability has been associated with the activation of dna methyltransferases and the consequent remethylation of cpg residues .
it therefore seems intuitive that therapeutic strategies able to maintain foxp3 in a demethylated state may be essential for the effective application of treg therapy in the clinic . while inhibitors of dna methyltransferases and of histone deacetylase are not without toxicity , the addition of safe agents that synergize or supplant these drugs as a means to regulate epigenetic expression of foxp3 should prove extremely valuable in the long - standing quest to induce tolerance .
tregs and conventional effector t cells take important cues from their microenvironment , which influences their commitment into regulatory or effector phenotypes . among extracellular stimuli ,
il-2-triggered expression of stat5 ( signal transducer and activator of transcription 5 ) likely plays a major role in stabilizing tregs .
stat5 , activated downstream of il-2 receptor complex signaling , binds to the promoter region of the foxp3 gene and thereby activates its transcription . by enhancing transcription of foxp3
we and others have utilized il-2 , il-2 anti - il-2 complexes , or il-2.ig either alone or in conjunction with rapamycin and other agents to promote tolerance induction .
such therapies have successfully induced transplant tolerance or restored self - tolerance in the nod model of type 1 diabetes .
in addition to activating the stat5 pathway and expression of foxp3 , il-2 signal , if delivered by a long - lived il-2 anti - il-2 complex or il-2.ig , also causes apoptosis of repeatedly activated effector ( but not regulatory ) t cells .
the fact that calcineurin inhibitors such as cyclosporin negatively impact treg function and activation - related effector t - cell apoptosis   may indeed be a result of suppressed il-2 signals .
we have previously shown that cyclosporin does prevent tolerance induced by co - stimulation blockade in a cardiac transplant model .
rapamycin , on the other hand , did not impede the induction of such achieved tolerance and , as we showed later , does promote the generation of induced tregs .
the imbalance of tissue - protective tregs and tissue - destructive th17 cells was recently implicated in the pathogenesis of autoimmunity and certain types of rejection . of note , the commitment of naive t cells to the th17 phenotype ( a subset with aggressive cytodestructive properties ) is inhibited by il-2 , and the expression of stat5 negatively regulates th17 cells .
it is therefore reasonable to posit that the activation of the il-2/stat5 pathway promotes tolerance by favorably tilting the balance between th17 cells and tregs toward treg dominance .
these strategies have centered upon attempts to delete or at least deplete donor - reactive t cells or alter the early events of t - cell activation such as through co - stimulation blockade .
the notion that graft inflammation is the major impediment to transplant tolerance prompts us to further investigate the means of its most efficient therapeutic targeting .
it may not be possible to create transplant tolerance unless the inflammatory milieu in which donor - reactive t cells perceive donor antigen is modified .
adverse inflammation in the peri - transplant period can guide the majority of donor - activated naive t cells into the graft - protective treg mode .
in addition , strategies that foster the expression of treg foxp3 by epigenetic modification may be utilized in conjunction with anti - inflammatory agents to further stabilize tregs and tolerance .
 certain forms of inflammation of an allograft are highly detrimental to the induction and maintenance of transplant tolerance as they foster stable commitment to graft - destructive , not graft - protective , forms of t - cell immunity . 
 hence , a reduction in adverse tissue inflammation may prove crucial in facilitating the induction and maintenance of a long - lasting state of transplant tolerance . 