BACKGROUND
janus kinases  are cytoplasmic tyrosine kinases that participate in the signaling of cell surface receptors, particularly cytokine receptors  <cit> .
ligand-cytokine receptor binding induces activation of jaks, which initiate signaling by phosphorylating cytokine receptors and creating docking sites for signaling proteins, known as signal transducers and activators of transcription   <cit> .
jaks catalyze stat phosphorylation to facilitate stat dimerization, transport to the nucleus and ultimately regulate gene expression  <cit> .
of the members of the jak family, jak <dig> has features that make it a potentially attractive target for immunosuppression, since jak <dig> associates with the common gamma  chain, which is shared by receptors of il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig>  <cit> .
moreover, mice and humans with a heritable absence or mutation of jak <dig> express a severe combined immunodeficiency phenotype  <cit> .
therefore selective inhibition of jak <dig> represents an optimal strategy for immunosuppression and the treatment of autoimmune diseases  <cit> .
cp <dig> , a jak <dig> inhibitor that is currently in clinical trials, has been shown to significantly reduce joint inflammation in rheumatoid arthritis   <cit> .
the jak/stat pathways influence cell-fate decisions made by differentiating naïve t cells, helping to control their development into th <dig> th <dig> and th <dig> cells  <cit> .
commitment to the th <dig> lineage requires stat1- and stat4-dependent mechanisms that induce ifn-γ and t-bet expression  <cit> .
on the other hand, differentiation towards the th <dig> developmental pathway requires stat <dig>  <cit> .
stat <dig> has emerged as an important determinant of t cell differentiation towards the inflammatory th <dig> t cell lineage  <cit> .
as the jak/stat pathway plays a pivotal role in t cell differentiation and cytokine signaling in t cells, we postulated that selective inhibition of jaks with cp <dig>  would modulate t cell functions and characteristics.
in this study, we assessed the effects of a pharmacological inhibitor of jak <dig>  cp <dig> , on gene expression and secretion of cytokines by human cd4+ t cells.
we also examined whether cp <dig>  affected the stat activation status in activated cd4+ t cells.
RESULTS
cytokine production by cd <dig> t cells is greatly reduced by cp <dig>
to investigate the potential role of jaks in t cell activation, cd4+ t cells isolated from healthy subjects were stimulated with a cd <dig> monoclonal antibody in the presence of cp <dig>  for  <dig> days.
as shown in figure  <dig>  freshly isolated cd4+ t cells secreted a significant amount of il- <dig> , ifn-γ , il-17a  and il- <dig>  in response to stimulation with the cd <dig> antibody.
cp <dig>  completely abrogated secretion of these cytokines from cd4+ t cells.
meanwhile, cp <dig>  did not affect the secretion of il- <dig> by anti-cd3-stimulated cd4+ t cells .
to confirm these findings, we examined mrna levels of these cytokines in cd4+ t cells using real-time pcr.
stimulation for  <dig> hrs with the cd <dig> antibody induced il- <dig>  and ifn-γ mrna  expression in cd4+ t cells.
the increased ifn-γ mrna levels were down regulated by cp <dig>  , whereas the anti-cd3-stimulated expression of il- <dig> mrna was not affected .
the expression of il- <dig> and il- <dig> mrna was marginally induced after  <dig> hrs of anti-cd <dig> stimulation.
in contrast, when cd4+ t cells were stimulated with the cd <dig> antibody for  <dig> hrs, expression of il- <dig> and il- <dig> mrna was greatly induced .
consistent with the protein data, the induction of il- <dig> and il- <dig> mrna in anti-cd3-stimulated cd4+ t cells was down regulated by cp <dig>  .
we next examined the effects of cp <dig>  on concanavalin a -activated cd4+ t cells.
as shown in figure  <dig>  cp <dig>  inhibited cytokine mrna expression of con a-activated cd4+ t cells.
furthermore, cp <dig>  inhibited granzyme b mrna expressions in anti-cd3-stimulated cd8+ t cells .
effects of cp <dig>  on cd4+ t cell proliferation
next, we examined the effects of cp <dig>  on t cell proliferation.
as shown in figure  <dig>  cp <dig>  pre-treatment significantly inhibited anti-cd3-induced cd4+ t cell proliferation.
we also investigated whether cp <dig>  pre-treatment induces apoptosis in anti-cd3-activated cd4+ t cells.
cp <dig>  pre-treatment did not result in an increase in the percentages of annexin v-positive/pi-negative early apoptotic cells .
cp <dig>  abrogated cd3-induced phosphorylation of stats in cd4+ t cells
the stat transcription factors help mediate the differentiation of cd4+ t cells into each of the t helper  subsets and are involved in lineage-specific expression of cytokines.
to determine whether cp <dig>  affected the phosphorylation of the stats in the t cell activation process, we assessed the phosphorylation-status of stats in anti-cd3-stimulated t cells .
after  <dig> hr of anti-cd <dig> stimulation, phosphorylation of stat <dig>  stat <dig>  stat <dig> and stat <dig> was induced in cd4+ t cells.
these phosphorylation events were inhibited by  <dig> nm cp <dig>  at  <dig> and also at  <dig> hrs.
cd <dig> antibody stimulation did not augment the phosphorylation of stat <dig> after  <dig> hrs, but this was evident after  <dig> hrs.
similarly, cp <dig>  also inhibited the late phase stat <dig> phosphorylation in activated cd4+ t cells.
cp <dig>  did not affect cd3-induced phosphorylation of zap- <dig> in cd4+ t cells
finally, we tested the effect of cp <dig>  on tcr-directed signaling by assessing tcr-associated phosphorylation of zap- <dig>  zap- <dig> phosphorylation was induced in cd4+ t cells after stimulation for  <dig> min with a cd3-antibody, but cp <dig>  exhibited no inhibitory effect on zap- <dig> phosphorylation .
discussion
it is well-established that cytokines play a critical role in regulating autoimmunity and inflammation, since targeting cytokines has been shown to be effective in the treatment of ra  <cit> .
however, targeting intracellular cytokine signaling using protein kinase inhibitors, appears to be a new strategy for immunosuppression  <cit> .
cp <dig> , a jak inhibitor that is currently in clinical trials, has shown significant efficacy in the treatment of ra  <cit> .
however, the precise mechanisms responsible for cp <dig> -mediated immunosuppression have not been elucidated.
naïve cd4+ t cells can differentiate into two major distinct phenotypes, th <dig> and th <dig> cells, which are characterized by polarized cytokine expression patterns  <cit> .
tcr signaling plays an important role in this differentiation by inducing th-specific cytokines  <cit> .
naïve cd4+ t cells are capable of producing ifn-γ and il- <dig> when they are stimulated with peptide/mhc class ii complexes on antigen-presenting cells   <cit> .
the endogenous production of ifn-γ has been shown to be sufficient for th <dig> differentiation, whereas il- <dig> is critical for th <dig> differentiation  <cit> .
in this study, we examined the effects of cp <dig>  on the production of these cytokines from cd4+ t cells activated by cd3-stimulation.
our data indicated that cp <dig> , a jak <dig> inhibitor, abrogated ifn-γ and il- <dig> production from cd4+ t cells trigged a cd <dig> antibody.
jaks are cytoplasmic tyrosine kinases that participate in the signaling of the cytoplasmic receptor of the cγ chain family receptors for il- <dig>  il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig>  <cit> .
ligand receptor-induced activation of jaks initiates signaling by phosphorylation of the cytokine receptors and catalyzes stat phosphorylation, facilitating stat dimerization, transport to the nucleus and gene regulation  <cit> .
therefore, it is possible that cp <dig>  affects the stat activation processes.
th <dig> differentiation depends on signaling through the ifn-γ receptor, the il- <dig> receptor and their downstream transcription factors, stat <dig> and stat <dig>  <cit> .
similarly, mature th <dig> differentiation depends on the il- <dig> receptor and its downstream transcription factor stat <dig>  <cit> .
we demonstrated that cp <dig>  inhibited stat <dig>  stat <dig> and stat <dig> activation, in addition to ifn-γ and il- <dig> production, in tcr-stimulated cd4+ t cells.
these findings suggest that cp <dig>  inhibits the production of th1- or th2-specific cytokines, as well as cytokine receptor signaling.
the jak1-stat <dig> and jak2-stat <dig> signaling pathways are known to be important for th <dig> cell differentiation, but jak <dig> has not previously been implicated in th <dig> cell differentiation  <cit> .
in contrast, jak <dig> is essential for th <dig> differentiation, since it plays an essential role in il- <dig> signaling  <cit> .
our data suggest that cp <dig> , a jak <dig> inhibitor, affected the cd3-induced th1-related activation of jak1-stat <dig> or jak2-stat <dig> in addition to the th2-related activation of jak3-stat <dig>  cp <dig>  was originally believed to be a jak <dig> inhibitor  <cit> , but it has now become clear that this compound also inhibits jak <dig> and jak <dig> with similar ic50-values  <cit> .
therefore, it is possible that cp <dig>  not only affects the induction of th2-related cytokines, but also the induction of th1-related cytokines via jak <dig> and jak <dig>  in addition to jak <dig>
more recently, the il-2-jak3-stat <dig> axis has been demonstrated to regulate th <dig> cell differentiation, suggesting that il-2-mediated jak3-stat <dig> signaling may generically operate in the production of th1-related cytokines  <cit> .
we also demonstrated that the cd <dig> antibody-induced phosphorylation of stat <dig> was down regulated by cp <dig> , suggesting that jak <dig> inhibition by cp <dig>  down regulated stat5-dependent cytokine signaling.
it is particularly interesting to evaluate the contribution of th <dig> cells to the pathogenesis of ra, as il- <dig> is involved in immune-mediated articular damage  <cit> .
the current view of human th <dig> development is that il- <dig> and il- <dig> are key cytokines that initiate differentiation and maintenance of this lineage  <cit> .
th <dig> cells are also major producers of il- <dig>  in addition to il- <dig>  <cit> .
in contrast with mouse t cells, tcr stimulation alone has been shown to be sufficient to induce both il- <dig> in humans t cells  <cit> .
indeed, anti-cd3-stimulation induced secretion of both il- <dig> and il- <dig> from cd4+ t cells in our study.
our data also demonstrated that cp <dig>  almost completely inhibited cd3-induced production of il- <dig> or il- <dig> from human cd4+ t cells.
recent evidence has supported a new role for stat <dig>  in the differentiation of th <dig> cells  <cit> ; stat3-deficient mice fail to produce il- <dig> and restoration of stat <dig> rescues the il- <dig> defect  <cit> .
it has also been reported that activated stat <dig> binds directly to the stat3-binding sites in the promoter of the il- <dig> gene, increasing its expression  <cit> .
these findings indicate that stat <dig> signaling is important for il- <dig> induction and th <dig> differentiation.
in this study, anti-cd <dig> stimulation activated stat <dig> during the late phase of t cell activation , which was blocked by cp <dig> .
this impaired stat <dig> activation may have partially contributed to the suppression of th17-related cytokine production by cp <dig> .
resting t cells require the sequential activation of two signaling pathways to differentiate.
first, antigen stimulation of the tcr/cd <dig> complex leads to the expression of il- <dig> and il-2r.
second, the binding of il- <dig> to its cognate receptor leads to jak/stat activation  <cit> .
in our study, cp <dig>  did not directly affect tcr-mediated signaling, i.e., zap- <dig> activation.
however, cp <dig>  abrogated the subsequent activation of the stats .
these findings suggest that cp <dig>  induces immunosuppression by affecting cytokine receptor-associated jak/stat signaling, which is downstream of the tcr-signaling pathway.
these findings may provide important information concerning the mechanisms of cp <dig> -mediated immunosuppression in the treatment of ra.
in t cell activation, the first signal is triggered by the engagement of the tcr with the antigen after presentation by the antigen-presenting cell , which is then ultimately transmitted by the final signal triggered by cytokine engagement of their receptors on t cells via the jak/stat pathway  <cit> .
in this study, we demonstrated that cp <dig>  abrogated the tcr-triggered production of cytokines by affecting the jak/stat signaling pathway, not the tcr-mediated signaling pathway.
we believe that small molecule inhibitors that target jaks may provide alternative therapeutic strategies for immunosuppression in contrast to classical immunosuppressants, such as calcineurin inhibitors, that target tcr-mediated signaling.
CONCLUSIONS
cp <dig>   <dig> inhibited anti-cd3-induced ifn-γ, il- <dig> and il- <dig> production from cd4+ t cells without affecting il- <dig> production.
cp <dig>   <dig> also abrogated anti-cd3-induced stat <dig>  stat <dig>  stat <dig>  stat <dig> and stat <dig> activation in cd4+ t cells.
modification of the jak/stat pathway using small molecule inhibitors may provide a new strategy for immunosuppression.

 janus kinases are cytoplasmic tyrosine kinases that participate in the signaling of cell surface receptors, particularly cytokine receptors. ligand-cytokine receptor binding induces activation of jaks, which initiate signaling by phosphorylating cytokine receptors and creating docking sites for signaling proteins, known as signal transducers and activators of transcription. jaks catalyze stat phosphorylation to facilitate stat dimerization, transport to the nucleus and ultimately regulate gene expression. of the members of the jak family, jak has features that make it a potentially attractive target for immunosuppression, since jak associates with the common gamma chain, which is shared by receptors of il- il- il- il- il- and il-. moreover, mice and humans with a heritable absence or mutation of jak express a severe combined immunodeficiency phenotype. therefore selective inhibition of jak represents an optimal strategy for immunosuppression and the treatment of autoimmune diseases. cp, a jak inhibitor that is currently in clinical trials, has been shown to significantly reduce joint inflammation in rheumatoid arthritis. the jak/stat pathways influence cell-fate decisions made by differentiating naïve t cells, helping to control their development into th th and th cells. commitment to the th lineage requires stat1- and stat4-dependent mechanisms that induce ifn-γ and t-bet expression. on the other hand, differentiation towards the th developmental pathway requires stat. stat has emerged as an important determinant of t cell differentiation towards the inflammatory th t cell lineage. as the jak/stat pathway plays a pivotal role in t cell differentiation and cytokine signaling in t cells, we postulated that selective inhibition of jaks with cp would modulate t cell functions and characteristics. in this study, we assessed the effects of a pharmacological inhibitor of jak cp, on gene expression and secretion of cytokines by human cd4+ t cells. we also examined whether cp affected the stat activation status in activated cd4+ t cells.  cytokine production by cd t cells is greatly reduced by cp to investigate the potential role of jaks in t cell activation, cd4+ t cells isolated from healthy subjects were stimulated with a cd monoclonal antibody in the presence of cp for days. as shown in figure freshly isolated cd4+ t cells secreted a significant amount of il-, ifn-γ, il-17a and il- in response to stimulation with the cd antibody. cp completely abrogated secretion of these cytokines from cd4+ t cells. meanwhile, cp did not affect the secretion of il- by anti-cd3-stimulated cd4+ t cells. to confirm these findings, we examined mrna levels of these cytokines in cd4+ t cells using real-time pcr. stimulation for hrs with the cd antibody induced il- and ifn-γ mrna expression in cd4+ t cells. the increased ifn-γ mrna levels were down regulated by cp, whereas the anti-cd3-stimulated expression of il- mrna was not affected. the expression of il- and il- mrna was marginally induced after hrs of anti-cd stimulation. in contrast, when cd4+ t cells were stimulated with the cd antibody for hrs, expression of il- and il- mrna was greatly induced. consistent with the protein data, the induction of il- and il- mrna in anti-cd3-stimulated cd4+ t cells was down regulated by cp. we next examined the effects of cp on concanavalin a -activated cd4+ t cells. as shown in figure cp inhibited cytokine mrna expression of con a-activated cd4+ t cells. furthermore, cp inhibited granzyme b mrna expressions in anti-cd3-stimulated cd8+ t cells. effects of cp on cd4+ t cell proliferation next, we examined the effects of cp on t cell proliferation. as shown in figure cp pre-treatment significantly inhibited anti-cd3-induced cd4+ t cell proliferation. we also investigated whether cp pre-treatment induces apoptosis in anti-cd3-activated cd4+ t cells. cp pre-treatment did not result in an increase in the percentages of annexin v-positive/pi-negative early apoptotic cells. cp abrogated cd3-induced phosphorylation of stats in cd4+ t cells the stat transcription factors help mediate the differentiation of cd4+ t cells into each of the t helper subsets and are involved in lineage-specific expression of cytokines. to determine whether cp affected the phosphorylation of the stats in the t cell activation process, we assessed the phosphorylation-status of stats in anti-cd3-stimulated t cells. after hr of anti-cd stimulation, phosphorylation of stat stat stat and stat was induced in cd4+ t cells. these phosphorylation events were inhibited by nm cp at and also at hrs. cd antibody stimulation did not augment the phosphorylation of stat after hrs, but this was evident after hrs. similarly, cp also inhibited the late phase stat phosphorylation in activated cd4+ t cells. cp did not affect cd3-induced phosphorylation of zap- in cd4+ t cells finally, we tested the effect of cp on tcr-directed signaling by assessing tcr-associated phosphorylation of zap- zap- phosphorylation was induced in cd4+ t cells after stimulation for min with a cd3-antibody, but cp exhibited no inhibitory effect on zap- phosphorylation. discussion it is well-established that cytokines play a critical role in regulating autoimmunity and inflammation, since targeting cytokines has been shown to be effective in the treatment of ra. however, targeting intracellular cytokine signaling using protein kinase inhibitors, appears to be a new strategy for immunosuppression. cp, a jak inhibitor that is currently in clinical trials, has shown significant efficacy in the treatment of ra. however, the precise mechanisms responsible for cp -mediated immunosuppression have not been elucidated. naïve cd4+ t cells can differentiate into two major distinct phenotypes, th and th cells, which are characterized by polarized cytokine expression patterns. tcr signaling plays an important role in this differentiation by inducing th-specific cytokines. naïve cd4+ t cells are capable of producing ifn-γ and il- when they are stimulated with peptide/mhc class ii complexes on antigen-presenting cells. the endogenous production of ifn-γ has been shown to be sufficient for th differentiation, whereas il- is critical for th differentiation. in this study, we examined the effects of cp on the production of these cytokines from cd4+ t cells activated by cd3-stimulation. our data indicated that cp, a jak inhibitor, abrogated ifn-γ and il- production from cd4+ t cells trigged a cd antibody. jaks are cytoplasmic tyrosine kinases that participate in the signaling of the cytoplasmic receptor of the cγ chain family receptors for il- il- il- il- il- and il-. ligand receptor-induced activation of jaks initiates signaling by phosphorylation of the cytokine receptors and catalyzes stat phosphorylation, facilitating stat dimerization, transport to the nucleus and gene regulation. therefore, it is possible that cp affects the stat activation processes. th differentiation depends on signaling through the ifn-γ receptor, the il- receptor and their downstream transcription factors, stat and stat. similarly, mature th differentiation depends on the il- receptor and its downstream transcription factor stat. we demonstrated that cp inhibited stat stat and stat activation, in addition to ifn-γ and il- production, in tcr-stimulated cd4+ t cells. these findings suggest that cp inhibits the production of th1- or th2-specific cytokines, as well as cytokine receptor signaling. the jak1-stat and jak2-stat signaling pathways are known to be important for th cell differentiation, but jak has not previously been implicated in th cell differentiation. in contrast, jak is essential for th differentiation, since it plays an essential role in il- signaling. our data suggest that cp, a jak inhibitor, affected the cd3-induced th1-related activation of jak1-stat or jak2-stat in addition to the th2-related activation of jak3-stat cp was originally believed to be a jak inhibitor, but it has now become clear that this compound also inhibits jak and jak with similar ic50-values. therefore, it is possible that cp not only affects the induction of th2-related cytokines, but also the induction of th1-related cytokines via jak and jak in addition to jak more recently, the il-2-jak3-stat axis has been demonstrated to regulate th cell differentiation, suggesting that il-2-mediated jak3-stat signaling may generically operate in the production of th1-related cytokines. we also demonstrated that the cd antibody-induced phosphorylation of stat was down regulated by cp, suggesting that jak inhibition by cp down regulated stat5-dependent cytokine signaling. it is particularly interesting to evaluate the contribution of th cells to the pathogenesis of ra, as il- is involved in immune-mediated articular damage. the current view of human th development is that il- and il- are key cytokines that initiate differentiation and maintenance of this lineage. th cells are also major producers of il- in addition to il-. in contrast with mouse t cells, tcr stimulation alone has been shown to be sufficient to induce both il- in humans t cells. indeed, anti-cd3-stimulation induced secretion of both il- and il- from cd4+ t cells in our study. our data also demonstrated that cp almost completely inhibited cd3-induced production of il- or il- from human cd4+ t cells. recent evidence has supported a new role for stat in the differentiation of th cells; stat3-deficient mice fail to produce il- and restoration of stat rescues the il- defect. it has also been reported that activated stat binds directly to the stat3-binding sites in the promoter of the il- gene, increasing its expression. these findings indicate that stat signaling is important for il- induction and th differentiation. in this study, anti-cd stimulation activated stat during the late phase of t cell activation, which was blocked by cp. this impaired stat activation may have partially contributed to the suppression of th17-related cytokine production by cp. resting t cells require the sequential activation of two signaling pathways to differentiate. first, antigen stimulation of the tcr/cd complex leads to the expression of il- and il-2r. second, the binding of il- to its cognate receptor leads to jak/stat activation. in our study, cp did not directly affect tcr-mediated signaling, i.e., zap- activation. however, cp abrogated the subsequent activation of the stats. these findings suggest that cp induces immunosuppression by affecting cytokine receptor-associated jak/stat signaling, which is downstream of the tcr-signaling pathway. these findings may provide important information concerning the mechanisms of cp -mediated immunosuppression in the treatment of ra. in t cell activation, the first signal is triggered by the engagement of the tcr with the antigen after presentation by the antigen-presenting cell, which is then ultimately transmitted by the final signal triggered by cytokine engagement of their receptors on t cells via the jak/stat pathway. in this study, we demonstrated that cp abrogated the tcr-triggered production of cytokines by affecting the jak/stat signaling pathway, not the tcr-mediated signaling pathway. we believe that small molecule inhibitors that target jaks may provide alternative therapeutic strategies for immunosuppression in contrast to classical immunosuppressants, such as calcineurin inhibitors, that target tcr-mediated signaling.  cp inhibited anti-cd3-induced ifn-γ, il- and il- production from cd4+ t cells without affecting il- production. cp also abrogated anti-cd3-induced stat stat stat stat and stat activation in cd4+ t cells. modification of the jak/stat pathway using small molecule inhibitors may provide a new strategy for immunosuppression. 
the present study was undertaken to assess the effects of cp on cytokine production and cellular signaling in human cd4+ t cells. cd4+ t cells produced il- il- il- il- and ifn-γ in following stimulation with a cd antibody. at the optimal concentration, cp almost completely inhibited the production of il- il- il- and ifn-γ from these activated cd4+ t cells, but only had marginal effects on il- production. therefore, cp -mediated modification of the jak/stat pathway may be a new immunosuppressive strategy in the treatment of autoimmune diseases.
 the new jak inhibitor, cp, has shown efficacy in the treatment of rheumatoid arthritis. the present study was undertaken to assess the effects of cp on cytokine production and cellular signaling in human cd4+ t cells.  cd4+ t cells produced il- il- il- il- and ifn-γ in following stimulation with a cd antibody. at the optimal concentration, cp almost completely inhibited the production of il- il- il- and ifn-γ from these activated cd4+ t cells, but only had marginal effects on il- production. moreover cp inhibited anti-cd3-induced phosphorylation of stat stat stat stat and stat but not the tcr-associated phosphorylation of zap-  therefore, cp -mediated modification of the jak/stat pathway may be a new immunosuppressive strategy in the treatment of autoimmune diseases. cp690550cytokinesjanus kinasesignal transducers and activators of transcriptiont lymphocytes 