
 t - bet ( t - box protein expressed in t cells , also called tbx21 ) was firstly described in 2000 in a report examining the effects of t - bet on the differentiation of t helper 1 ( th1 ) cells . for the past 15 years , many studies have examined the functions of t - bet and have revealed multiple roles for this protein during th cell differentiation , with a focus on the molecular mechanisms involved , the novel functions of this transcription factor in innate immune cells , and t - bet - mediated modulation of inflammatory diseases [ 29 ] .
it has been clarified that t - bet plays a critical role in the coordination of innate immunity and adaptive immunity and that it fulfills an important function in modulating chronic inflammatory diseases , including asthma and inflammatory bowel disease , by controlling a network of highly conserved genetic programs [ 1012 ] .
thus , optimal regulation of t - bet expression and activity seems to be beneficial for preventing or treating chronic inflammation and autoimmune diseases .
although attempts have been made at identifying the small molecules that control the expression and activity of t - bet that affect the t cell - mediated immune response , little progress has been made on this to date .
given the importance of t - bet in the immune regulation , elucidating the functional mechanisms underlying the multiple roles of t - bet would facilitate the development of novel therapeutic interventions for treating chronic inflammatory and autoimmune diseases .
this review summarizes the current state of knowledge about the molecular mechanisms underlying the multiple roles played by t - bet in th cell development .
the t - bet contains an amino - terminus , a t - box domain , and a carboxyl - terminus , which show 82% , 100% , and 79% homology , respectively , between mice ( 530 amino - acid residue protein ) and humans ( 535 amino - acid residue protein ) ( figure 1 ) .
the t - box domain , located between residues 135 and 326 in mouse t - bet , is highly conserved in 18 members of the t - box protein ( tbx ) family [ 13 , 14 ] .
common features shared by t - box proteins include a capacity for dna binding through the t - box domain and transcriptional regulatory activity , which plays a role in controlling the expression of developmental gene in all animal species .
the t - box domain is made up of about 180 amino - acid residues and is both sufficient and necessary for binding to the consensus dna sequence tcacacct [ 1315 ] .
brachyury ( t ) was the first t - box protein to be identified and , in dimeric form , interacts with the major and the minor grooves of dna through hydrophobic interactions and unusual main - chain carbonyl contact with a guanine as a dimer .
tbx1 also binds to the dna sequence as a dimer , whereas tbx2 appears to bind to the same dna sequence as a monomer .
although tbx1 and tbx2 share 61% identity in the t - box domain , the structure of the dna - t - box binding complex appears to be different , because of the low homology among the amino- and carboxyl - terminal regions . the t - box domain in t - bet shows 50% homology with the corresponding domain in brachyury ( t ) , tbx1 , and tbx2
; however , the crystal structure of t - bet bound to the dna sequence remains to be characterized .
t - bet directly binds to the consensus dna sequence within the ifng promoter and activates its transcription .
the t - bet - induced expression of ifng derives th precursor cells to differentiate into th1 effector cells .
while exogenous t - bet overexpression in nave th cells preferentially increases development of th1 cells , t - bet deficiency leads to a failure to produce sufficient ifn- and therefore reduces generation of th1 cells [ 1 , 18 ] .
t - bet expression is substantially increased by stimulation of the t cell receptor ( tcr ) and is augmented by cotreatment with ifn- and il-12 .
ifn- binds to its receptor and induces activation of signal transducer and activator of transcription ( stat ) 1 and transcription of t - bet gene ( tbx21 ) .
subsequently , t - bet directly stimulates the transcription of ifng as well as il12rb2 .
expression of il-12 receptor ( il-12r ) 2 on the cell surface further enhances ifn- production through il-12 and the stat4 signaling pathway , thereby resulting in preferential th1 cell differentiation .
interestingly , enforced t - bet expression can also convert the differentiated th2 cells into th1 cells .
therefore , t - bet is positioned at the crux of the regulatory pathways that induce ifn- in th cells .
this cytokine , an early t cell growth factor , is essential for activation , proliferation , and differentiation of th cells and is abundantly produced upon tcr stimulation .
ectopically introduced t - bet significantly suppresses il-2 production through inhibition of nuclear factor b ( nf-b ) p65 activity , under conditions of both th1 and th2 differentiation . during th1 cell differentiation , il-2 transcription
the t - bet - mediated il-2 inhibition may affect th cell expansion and exquisitely modulate the th1-mediated immune response upon exposure to a pathogenic antigen .
furthermore , exogenous t - bet introduction into th cells suppresses the production of th2 cytokines , such as il-4 , il-5 , and il-13 , via suppression of gata - binding protein-3 ( gata-3 ) .
accordingly , a lack of t - bet induces spontaneous th2 cell development in vitro and in vivo [ 1 , 18 ] .
the th2-suppressive activity of t - bet was also confirmed in the absence of ifn- , indicating that t - bet has a discrete inhibitory function , independent of ifn- stimulation .
recently , many studies have reported that t - bet also modulates other th cell lineages , including th17 , treg , and follicular th ( tfh ) cells , in coordination with many transcription factors , such as the retinoic acid - related orphan receptor-t ( rort ) , runt - related transcription factor 3 ( runx3 ) , and b - cell lymphoma-6 ( bcl6 ) [ 2025 ] .
these findings suggest that t - bet is a transcription factor that is critical for fine - tuning th cell development .
t - bet functions as a multitasking player in the regulation of th cell differentiation .
however , the molecular mechanisms that underlie the stimulatory and inhibitory activity of t - bet in regulating target gene expression remain to be clarified .
many multitasking proteins are known to undergo posttranslational protein modifications and to determine cell fates by exerting direct stimulatory and indirect inhibitory activity on target gene expression [ 26 , 27 ] .
antibody - based detection of t - bet proteins in western blots results in multiple bands , suggesting the posttranslational modification of t - bet in tcr - triggered th cells .
tyrosine phosphorylation of t - bet protein occurs primarily during the early stages ( days 2 to 3 ) of th cell development , upon tcr engagement , and declines afterwards . treating th cells with the tyrosine phosphatase inhibitor pervanadate enhances the tyrosine phosphorylation of t - bet .
t - bet is mainly localized in the nucleus , and the nuclear tyrosine kinase il2-inducible tyrosine kinase ( itk ) was identified as the responsible upstream tyrosine kinase .
itk deficiency prevents tyrosine phosphorylation of t - bet in th cells after stimulation with tcr and il-12 .
mutational research has revealed that tyrosine residue 525 ( y525 ) is the relevant phosphorylation site and that phosphorylation at this site plays an important role in the interaction with gata-3 .
although t - box domain in t - bet is important for dna and protein - protein interaction , tyrosine phosphorylation of y525 is prerequisite for the suppression of gata-3-mediated th2 cell differentiation .
blockade of y525 phosphorylation abrogates the interaction with and suppression of gata-3 , resulting in impairment of th2 suppression .
furthermore , another nuclear tyrosine kinase , c - abl , induces phosphorylation of t - bet at tyrosine residues 219 , 265 , and 304 in mouse t - bet . a deficiency in c - abl as well as mutation of t - bet at these residues ( y219/265/304f mutants ) leads to a failure to increase ifn- induction and to suppress th2 cytokine production , due to the loss of phosphorylation at these tyrosine residues .
this , in turn , results in the aggravation of allergic lung inflammation in vivo .
these findings suggest that itk- and c - abl - induced tyrosine phosphorylation of t - bet is essential for the modulation of th2 cell development and the allergic immune response .
although t - bet - mediated suppression of th2 cell development is impaired by mutation of y525 and the absence of c - abl kinase , il-2 suppression is retained with a y525-mutant t - bet , suggesting the existence of an additional regulatory mechanism for il-2 modulation .
interestingly , the appearance of multiple bands of t - bet protein on western blots could be eliminated by the addition of calf intestinal phosphatase , which predominantly eliminates phosphorylation at serine / threonine residues .
mass spectrometric analysis then revealed serine 508 ( s508 ) as another phosphorylation site in t - bet .
mutation of s508 abolishes casein kinase- ( ck- ) and glycogen synthase kinase-3 ( gsk-3- ) mediated phosphorylation in t - bet , as well as the il-2suppressive activity of the protein . moreover , s508 phosphorylation is important for the interaction of t - bet with nf-b p65 and for prevention of binding of nf-b p65 to the il2 promoter . in accordance with the function of t - bet as an nf-b p65 inhibitor ,
t - bet - null th1 cells sustain nf-b p65 activity during th1 cell differentiation and thus produce more il-2 .
therefore , it has been suggested that t - bet is a physiological inhibitor of il-2 during th1 cell differentiation , through s508 phosphorylation - dependent suppression of nf-b p65 .
very recently , threonine 302 ( t302 ) was characterized as a novel phosphorylation site in t - bet , although it remains unclear which kinase and phosphatase affect the phosphorylation of this residue . however , restoration of t - bet - null th cells with a t302-mutant t - bet stimulated ifn- production as much as did wild - type t - bet ; however , the mutant failed to suppress il-2 and other th2 cytokines .
further analysis demonstrated that t302 phosphorylation is required for the interaction of t - bet with nuclear factor of activated t cells ( nfat ) and for downregulation of nfat - mediated il-2 and th2 cytokines , such as il-4 , il-5 , and il-13 .
nfat is not crucial for induction of ifn- production and t302-mutant t - bet is able to bind to the ifng promoter ; thus , ifn- production was comparable between wild - type and mutant t - bet .
in other words , mutation of t302 abrogated the t - bet - mediated suppression of il-2 and th2 cytokine production but did not affect the dna - binding and ifn--stimulatory activity of t - bet .
indeed , t302 is located in the dna - binding t - box , as is y304 .
the t - box domain consists of several repeats of -strands and -helices and is involved in both dimerization and dna binding .
muller and herrmann predicted that the -helices h3 and h4 in brachyury ( t ) are important for the direct interaction of this protein with the minor and major grooves of dna .
however , t302 may not be associated directly with the dna grooves , regardless of its phosphorylation status .
it would be interesting to know which upstream kinase and phosphatase regulates t302 phosphorylation and whether t302 phosphorylation affects other protein modifications of t - bet .
t - bet expression is critical for the transcriptional regulation of ifng and for the development of th1 cells , but the means of regulation of t - bet at the protein level is yet to be identified .
jang et al . have recently reported that t - bet undergoes ubiquitination - mediated proteasomal degradation during the later stages of th1 cell differentiation .
of the 16 lysine residues present in mouse t - bet protein , 11 are predominantly located within the t - box domain , and the remaining 5 are located at the carboxyl - terminus ( residues 326 through 530 ) , while no lysine residues are present in the amino - terminus ( residues 1 through 134 ) .
interestingly , lysine residues within the t - box domain are preferentially ubiquitinated upon overexpression of ubiquitin .
further analysis has identified that mutation of lysine 313 ( k313 ) decreases ubiquitination - mediated t - bet degradation and enhances the expression level of t - bet in the nucleus and the cytoplasm . despite the increased levels of the k313 mutant , this mutation completely abrogated t - bet functions involving dna binding , transcriptional activation of ifng , and suppression of il-2 and th2 cytokine production .
the crystal structure of the -helices of the t - box domain bound to dna strongly suggests that the amino group of k313 is associated with the phosphate of a dna base via hydrogen - bond interaction .
in addition , mutation of k313 also leads to failure to suppress il-2 and th2 cytokine production ; however , the interaction with and suppression of gata-3 and nf-b p65 are not altered by mutation of k313 .
interestingly , nfat interaction is abolished in k313-mutant t - bet , which is also strongly associated with an absence of phosphorylation at t302 .
it is not clear yet whether and how k313 regulates t302 phosphorylation and vice versa .
since mosmann et al . discovered th1 and th2 subsets that produce signature cytokines , ifn- , and il-4 , il-5 , and il-13 , respectively , and that modulate inflammatory and allergic immune responses , further studies have identified novel subsets of th cells , such as th17 , tfh , and treg cells [ 11 , 21 , 23 , 24 , 3134 ] .
extensive studies have also characterized the cytokine signaling pathways and transcription factors involved in the regulation of immune responses to pathogens [ 1 , 3554 ] .
importantly , t - bet plays a fundamental role in controlling differentiation of several subsets of th cells and in modulating inflammatory and autoimmune diseases [ 1012 ] .
a deficiency in t - bet spontaneously leads to the development of asthmatic symptoms , which is characterized by increased eosinophil infiltration into the airway , mucus - secreting goblet cell hyperplasia , and chronic airway remodeling with collagen accumulation and proliferative myofibroblasts ; these features are often also seen in asthmatic patients [ 5557 ] .
restoration of t - bet expression shifts the immune balance to a th1 response and prevents and attenuates pathologic lung inflammation in vivo [ 58 , 59 ] .
abrogation of t - bet - induced ifn- production resulted in higher susceptibility to intracellular pathogens in vivo , including mycobacterium tuberculosis , leishmania major , and salmonella typhimurium [ 18 , 60 , 61 ] , emphasizing the importance of ifn- production by t - bet - expressing th1 cells in the defense against bacterial infections .
however , t - bet - deficient mice are resistant to infection by listeria monocytogenes , because inf- production by natural killer cells is both necessary and sufficient for the host defense against l. monocytogenes .
furthermore , t - bet can aggravate the development of inflammatory and autoimmune diseases , including inflammatory bowel disease , experimental autoimmune encephalomyelitis , inflammatory arthritis , and type i diabetes , as these inflammatory diseases are attenuated in the absence of t - bet [ 6 , 56 , 63 , 64 ] .
these findings suggest that fine - tuning of the immune response by modulation of t - bet expression could have beneficial effects for patients with chronic asthma , inflammatory bowel disease , arthritis , multiple sclerosis , and diabetes .
t - bet is a t - box domain - containing transcription factor that is typically involved in developmental regulation but exerts multiple functions in th cell differentiation ; transcriptional activation of ifn--expressing th1 cells , indirect suppression of th2 , th17 , and treg cell development , and fine - modulation of il-2 production in th1 cells .
phosphorylation at y525 plays a role in gata-3 suppression during th2 regulation , phosphorylation at s508 causes nf-b p65 suppression in the context of il-2 regulation in th1 cells , phosphorylation at t302 plays a role in fine - tuning il-2 production , and ubiquitination at k313 plays a role in controlling t - bet protein stability .
thus , posttranslational modification of t - bet facilitates its functional diversity and the complexity of its modulation of cytokine expression ( figure 2 ) .
it is not known whether the various posttranslational modifications occur sequentially or simultaneously , whether one type of protein modification affects other modification , or whether changes in the posttranslational modification of t - bet are related to the development of infectious and chronic inflammatory diseases .
further identification of novel protein modifications related to t - bet functions would provide valuable insights into the development of powerful therapeutic interventions for controlling chronic inflammatory and autoimmune diseases .
 t - bet ( t - box protein expressed in t cells , also called as tbx21 ) was originally cloned as a key transcription factor involved in the commitment of t helper ( th ) cells to the th1 lineage . 
 t - bet directly activates ifn- gene transcription and enhances development of th1 cells . 
 t - bet simultaneously modulates il-2 and th2 cytokines in an ifn--independent manner , resulting in an attenuation of th2 cell development . 
 numerous studies have demonstrated that t - bet plays multiple roles in many subtypes of immune cells , including b cell , dendritic cells , natural killer ( nk ) cells , nk t cells , and innate lymphoid cells . therefore , t - bet is crucial for the development and coordination of both innate and adaptive immune responses . to fulfill these multiple roles , 
 t - bet undergoes several posttranslational protein modifications , such as phosphorylation at tyrosine , serine , and threonine residues , and ubiquitination at lysine residues , which affect lineage commitment during th cell differentiation . 
 this review presents a current overview of the progress made in understanding the roles of various types of t - bet protein modifications in the regulation of cytokine production during th cell differentiation . 
1. Introduction
2. Structure of T-Bet
3. Regulation of Th Cell Differentiation by T-Bet
4. Posttranslational Modification of T-Bet
5. T-Bet in Inflammatory and Autoimmune Diseases
6. Conclusions and Perspectives