in 1960 nowell first described lymphocyte activation by mitogenic lectins to result in " blastic transformation " and subsequent mitosis , which are complex cellular events that require several days to evolve .
more than 40 years later , we have advanced considerably in our understanding of the cellular and molecular events responsible for these phenomena .
now with the genomic revolution , we are poised on a new threshold of discovery , in that it is finally feasible to begin to identify the genes that are responsible for such complicated cellular processes .
however , it is now necessary to develop an experimental approach that will rapidly and accurately facilitate the discovery process . with the description of lymphocyte - derived growth factor activities in the 1960s and 1970s
, it was realized that activated lymphocytes secrete soluble factors that are critical for subsequent lymphocyte proliferation .
moreover , in 1967 it was demonstrated that monocytes or macrophages serve as necessary accessory cells for the lymphocyte activation process .
then , in the early 1970s several reports indicated that mitogen- or endotoxin - stimulated macrophages release soluble lymphocyte activating factors ( lafs ) that also amplify the activation process .
accordingly , lymphokines and monokines , which collectively came to be known as cytokines , became prime research subjects for those interested in lymphocyte activation .
however , it was not until the 1980s that the cell surface and soluble molecules responsible for the complex cellular changes of lymphocyte activation were identified . with the isolation of the lymphocyte - derived molecule responsible for t cell growth factor ( tcgf ) activity , which came to be known as interleukin 2 ( il-2 ) , and
discovery of il-2 receptors ( il-2r ) , it could be shown that mitogenic lectins and specific antigens triggered an initial activation event , which was analogous to the activation of cell cycle " competence " by serum - stimulated fibroblasts , and resulted in transition from the g0 to the g1 phase of the cell cycle .
however , just as in serum - stimulated fibroblasts , this initial activation event did not result in lymphocyte proliferation .
instead , it appeared that the initial activation of cellular competence triggered by the mitogenic lectins was amplified by the laf activity , which became known later as il-1 , and led to the secretion of the lymphocyte - derived il-2 and the expression of il-2rs .
the subsequent interaction of il-2 with the il-2rs then stimulated blastic transformation and progression through g1 and into the s phase of the cell cycle , followed by mitosis .
furthermore , detailed studies indicated that successful activation of the il-2r is a quantal ( i.e. all - or - none ) event that depends on surpassing a threshold of a finite number of il-2-il-2r interactions .
this threshold corresponds to surpassing the " restriction ( r ) point " in the mid to late g1 phase of the cell cycle , first described by pardee for serum - activated fibroblasts .
accordingly , activation of lymphocyte proliferation was realized to require at least 3 signals , one delivered to the t cell via the lectin , another supplied as a soluble factor by accessory macrophages , and a third contributed by the il-2/il-2r interaction . with the discovery of the t cell antigen receptor ( tcr ) in 1983 [ 20 - 22 ] it was possible to demonstrate that the first signal is delivered via antigenic activation of the tcr .
subsequently , discovery of accessory " co - stimulatory " molecule expression by " helper " t cells ( th ) , revealed that activation solely of the tcr itself was insufficient to fully activate il-2 gene expression and t cell proliferation . instead , in the mid ' 80s it was found that the activation of co - stimulatory molecules like cd28 expressed by t cells via ligands such as b7 expressed by antigen presenting cells ( apc ) , deliver additional signals that synergize with those emanating from the tcr , and result in maximal il-2 gene transcription . as well , cd28 activation stabilizes il-2 mrna transcripts , prolonging il-2 mrna life span .
thus , in addition to soluble macrophage - derived products , cell surface molecules expressed by apcs also contribute to the 2signal attributed to the apcs .
the molecules responsible for the contribution by the soluble macrophage - derived lafs still remain obscure , even today . initially the nomenclature of il-1 and il-2 was created to delineate the sequential action of an assumed single macrophage - derived soluble factor ( il-1 ) , which promoted the production of a single lymphocyte - derived factor ( il-2 ) .
however , the molecular nature of the laf activity was never fully elucidated , even after the isolation and identification of multiple mitogen / endotoxin - stimulated macrophage - derived products , such as il-1 , il-6 , il-12 , il-18 , and tumor necrosis factor - alpha ( tnf- ) .
it is probable that all of these macrophage - derived soluble factors together make up the activity originally ascribed to laf .
accordingly , it is obvious that addition of a polyclonal activating ligand such as phytohemagglutinin to peripheral blood mononuclear cells ( pbmcs ) leads to the activation of soluble macrophage - derived and lymphocyte - derived activating factors , and the almost simultaneous activation of multiple cell surface receptors , signaling pathways and transcription factors that ultimately all contribute to lymphocyte activation .
stimulation of the various receptors expressed by t cells and apcs results in the activation of distinct and also redundant signal transduction pathways , and in the activation of distinct as well as redundant transcription factors .
accordingly , it has been assumed that triggering any of these receptors should result in the expression of distinct as well as redundant cellular genes . until recently the identification of large numbers of genes triggered by distinct ligands has been difficult , owing to the cumbersome methods available for gene identification .
cochran and stiles were among the first to use differential cloning methods to identify pdgf - regulated genes in fibroblasts .
these investigators focused on the expression of immediate / early pdgf - stimulated genes , by activating the cells in the presence of inhibitors of protein synthesis such as cycloheximide , thereby preventing the expression of secondary genes .
subsequently , zipfel and co - workers employed subtractive hybridization to isolate and differentially clone immediate / early genes activated via the t cell antigen receptor in the presence of cycloheximide .
a total of 60 novel tcr - induced immediate / early genes were isolated , but most were not identified at the time , in that dna cloning and sequencing methods were tedious and time consuming .
new differential cloning methods were developed subsequently , including differential display , sulfhydryl labeling and affinity purification of newly - synthesized transcripts , representational difference analysis , and serial analysis of gene expression .
each methodological advance has improved both the sensitivity of detection of differentially expressed genes , as well as the speed with which the genes may be identified .
however , the development of dna microarray technology , which allows rapid interrogation of thousands of genes and the detection of gene expression at frequencies even less than 1 in 100,000 , has now greatly facilitated such experimentation .
this communication represents our initial efforts to use dna microarrays in a reductionist approach to begin to identify the genes expressed as a consequence of triggering each individual ligand during lymphocyte activation . because the il-2/il-2r interaction is discrete molecularly , and leads to a very finite , quantal decision to undergo blastic transformation , dna synthesis and mitosis ,
we have focused our first efforts on identifying il-2-regulated genes expressed by normal human t cells .
responses of pbmcs to il-2 are complex , involving direct effects of il-2 on gene expression by t cells , b cells and nk cells , as well as secondary effects mediated by additional cytokines and transcription factors expressed by these cells in response to il-2 . to identify the maximal number of il-2-regulated genes , pbmcs comprised of ~80% cd3 + lymphocytes , with smaller fractions of monocytes , b cells and nk cells ( table 1 ) were examined .
the cells were pre - activated for 72 hr with anti - cd3 to induce t cell il-2 receptor expression , then washed and rested for 3640 hr , prior to restimulation with il-2 for 4 hr . results are averages  se from 5 independent experiments five independent experiments were performed with pbmcs from different donors to identify il-2-regulated genes represented on the affymetrix u95av2 oligonucleotide array .
a comparison was made between the pre - activated , rested cells , and those restimulated with il-2 for 4 hr .
the expression levels were analyzed using dna - chip analyzer software , with normalization to the average chip signal intensity for each experiment , and a 10percentile value cut - off for ' absent ' genes .
approximately 45% of the ~12,600 probe sets were detected above background . using the criterion of p < 0.05 for the difference of means between the two groups , a total of 316 of these ~5,800 probe sets ( ~5.4% ) were identified as putative il-2-responsive genes .
a complete list of all il-2-responsive genes identified in pbmcs and t cells can be found in the attached file ' il-2-regulated genes ' .
the expression patterns of 19 genes already known to be il-2-regulated , which were represented by 25 distinct probe sets on the array , were used to determine the range of expression parameters that might be most likely to identify bona fide il-2-regulated genes ( table 2 ) . as negative controls ,
glyceraldehyde-6-phosphate dehydrogenase ( gapdh ) and actin were used as examples of genes that do not change expression levels under the experimental conditions used ; their expression levels were indeed constant without or with il-2 treatment ( 1.04  0.06 and 1.06  0.01 fold change , respectively ) . by comparison
, the 19 selected genes showed il-2-induced changes in expression levels , ranging from 2.0  0.3-fold ( il-4r , probe set 404_at ) to > 80-fold ( tnf 36296_at ) .
the subtractive differences in fluorescent intensity ranged from 114  20 ( c - myb 1474_s_at ) to > 1000 ( cr7 1779_s_at )
. four of the genes ( c - myb , cr7 , flt3 ligand , tnf- ) were detected with more than one probe set , and these replicates were in fairly good agreement .
it is worthy of note that there is not a correlation between the subtracted difference and the fold change in gene expression levels .
this is likely due to the fact that there is a wide range in the abundance classes of rna transcripts derived from different genes , from as few as 1 copy per cell to several thousand copies per cell .
the signal intensity of a particular transcript detected on an oligonucleotide array would thus be directly related to this abundance level .
therefore , the subtracted change in signal intensity would be profoundly affected by the absolute number of cellular copies of a given transcript . by comparison ,
expression of known il-2-regulated genes in pbmcs based on the results obtained with known il-2 target genes , the criteria used to identify additional bona fide il-2-regulated genes in these samples were set at  2-fold change , with a subtracted difference in fluorescent intensity  100 , and a significance level of the difference between the means of the two treatment groups of p < 0.05 .
of the 316 probe sets that met the standard of p < 0.05 , 251 ( 79% ) also met the additional criteria of either fold change  2 , or subtracted difference  100 , while only 84 probe sets ( 27% ) met all three criteria .
these 84 probe sets represent 72 unique genes ( 60 il-2-induced genes and 12 il-2-repressed genes ) that can be segregated into 8 functional groups .
figure 1 shows the fold induction and subtracted differences in expression levels of these probe sets . among the 4 apoptosis - regulating genes ,
bcl2 , caspase 3 and drak2 are all induced by il-2 , while the expression of toso is suppressed .
it is noteworthy that the il-2 induction of bcl2 would be expected to promote cellular survival , while the il-2 regulation of the other three genes would be expected to promote apoptosis .
additional il-2-responsive genes include those encoding 6 cell surface proteins , 11 cytokine and chemokine receptors , as well as 15 signal transduction mediators , 6 genes involved in cellular metabolism and biosynthesis , 3 cell cycle regulators and 12 transcription factors .
il-2-suppressed genes include lymphocyte antigen 9 , inositol 1,4,5-trisphosphate 3-kinase b , and cdc25b , as well as transcriptional regulators max - interacting protein 1 , cbp / p300-interacting transactivator , inhibitor of dna binding 3 ( id3 ) , glucocorticoid - induced leucine zipper ( gilz ) , and zinc finger protein 36/erf-2 .
all of the 19 already known il-2-regulated genes ( table 2 ) were also identified in this 72-gene cohort , thereby yielding 53 newly identified il-2-regulated genes expressed by pbmcs after 4 hours of il-2 stimulation .
pre - activated , rested human pbmcs were left untreated or restimulated for 4 hr with il-2 , and rna probes were prepared for hybridization with affymetrix u95av2 arrays .
a total of 84 probe sets were identified that showed fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds , right axis ) , and a significance value of the difference between the means of p < 0.05 .
the probe sets represent 72 unique genes , and can be segregated into 8 functional groups .
the pbmc populations used in our initial experiments included nk cells , monocytes and b cells , as well as t cells .
in addition , cytokines including tnf , tnf and flt3 ligand were induced by il-2 as detected by the arrays , so that the il-2 effects could be mediated by the action of these intermediary cytokines on other cells in the mixed population , or as well , by the 12 transcriptional targets activated by il-2 .
therefore to identify the direct il-2 targets in t cells , we used cd56-depleted , cd2-selected t cells , which were 94  1% cd3cells .
the pbmcs were pre - activated with anti - cd3 for 72 hr , the t cells purified , and rested for 3640 hr prior to restimulation for 4 hr with 1 nm il-2 . in addition , 10 g / ml cycloheximide ( chx ) was included to block protein synthesis , so that only immediate / early il-2-regulated genes were detected .
chx has been shown to both elevate and prolong the expression of many transiently induced transcripts , so that the 4 hr il-2 stimulation in the presence of chx should enable detection of genes induced within minutes as well as hours .
samples from 4 independent experiments with pre - activated , rested cells , or cells treated with chx alone were combined for data analysis , and compared to cells treated with il-2  chx . as depicted in figure 2a , using only the criterion of significance level of p < 0.05 for the difference between the means of the two groups , 449 probe sets were identified as il-2-regulated immediate - early targets in the pure t cells .
of these probe sets , 110 were also detected in the il-2-stimulated pbmcs . as shown in figure 2b , when a second standard was included , either a fold change  2 , or a subtracted difference  100 , 161 probe sets ( 36% ) were expressed as immediately / early genes in purified t cells , and of these , 71 were also il-2-regulated in pbmcs .
however , when all 3 criteria were applied ( figure 2c ) , a total of only 52 probe sets ( 12% ) were found to be immediate / early , il-2-regulated genes in t cells .
twenty - three of these 52 probe sets were among the 84 probe sets identified previously as meeting all 3 criteria in the experiments with pbmcs ( figure 3 ) . by comparison ,
the control genes actin and gapdh showed 1.1  0.1 and 1.3  0.2-fold change , respectively .
( a ) . using the criterion of p < 0.05 for the difference between the means of il-2-treated and untreated groups , a total of 316 il-2-sensitive probe sets were identified in pbmcs ( left circle ) , and 439 in purified t cells ( right circle ) . of these ,
( b ) . when a second standard of either fold change  2 or subtracted difference  100 was included in the analysis , 251 probe sets were identified in pbmcs , and 161 in pure t cells , with 71 in common .
( c ) . when all three criteria were used , 84 probe sets were identified in pbmcs , 52 in t cells , and 23 of these were common to both populations . within each population , the percentages of il-2- suppressed and induced probe sets are indicated .
a total of 23 probe sets met all three expression criteria of fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds and circles , right axis ) , and p < 0.05 in both pbmcs and purified t cells .
therefore , only ~1/3 of the genes discovered to be il-2-regulated in pbmcs are actually immediate / early direct il-2 targets in t cells .
these direct il-2 immediate / early target genes included 4 genes that were suppressed , dsip / gilz , the zinc finger protein 36/erf-2/tis11d , toso , and cdc25b .
notably , of the 16 il-2-induced genes , we had already been identified 4 previously as il-2 targets using differential colony hybridization ( cr3 , cr6 , cr7 , cr8 ) , while the 3 other known il-2 stimulated target genes included tnf , the il-4r , and the anti - apoptotic gene , bcl2 .
thus , only 9 genes were newly identified as il-2-induced immediate / early t cell genes ; including the signaling proteins slp76 and rgs1 , the transcription factors irf4 and xbp1 , and 5 miscellaneous genes ( dkfzp586f1323 , mitofilin , camp - specific phosphodiesterase 4b , cmp - n - acetylneuraminate monooxygenase , and the aryl hydrocarbon receptor ) .
in addition to the 23 probe sets in common with those identified in pbmcs , 29 others were detected as il-2-regulated immediate / early targets in the purified t cells ( figure 2c ) .
these probe sets met all three of the criteria of  2-fold change , subtracted difference  100 , and p < 0.05 in t cells , but were not il-2-regulated in pbmcs .
notably , only three of these were induced , while the remaining 24 were all suppressed in response to il-2 + chx .
a total of 29 additional probe sets met all three expression criteria of fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds and circles , right axis ) , and p < 0.05 in purified t cells , but did not meet all three criteria in pbmcs .
these probe sets represent 27 unique genes . of the remaining 61 probe sets identified in pbmcs ( figure 2c ) , 31 showed changes in expression levels in t cells with p < 0.05 ,
although either the fold change was less than 2.0 , or the magnitude of the change was less than 100 ( table 3 ) .
these 31 probe sets are derived from 27 unique genes , and most likely represent bona fide il-2 targets , since the group includes the genes jab / ssi-1/socs-1 , flt3 ligand , il-2rp55 , caspase 3 , and p78 , which have already been reported as il-2-induced genes .
if these genes are also included , 47 of the 72 unique genes ( 65% ) identified in pbmcs are il-2-regulated immediate / early genes in pure t cells .
t cell expression levels of genes identified in pbmcs similarly , of the 29 probe sets identified as meeting all 3 criteria only in pure t cells , 7 showed differences in expression with p < 0.05 in pbmcs ( table 4 ) . these include linker for activation of t cells , t - cell - specific transcription factor 7 , stat4 , il-7r , pas ser / thr kinase , l - mfa domain - containing protein , and an unknown gene designated dkfzp586d1122 .
notably , all of the other genes ( 22 ) were suppressed by il-2 in the pbmcs , as they were in the t cells , although they did not meet the significance value of p < 0.05 in the pbmcs .
responses of pbmcs to il-2 are complex , involving direct effects of il-2 on gene expression by t cells , b cells and nk cells , as well as secondary effects mediated by additional cytokines and transcription factors expressed by these cells in response to il-2 . to identify the maximal number of il-2-regulated genes , pbmcs comprised of ~80% cd3 + lymphocytes , with smaller fractions of monocytes , b cells and nk cells ( table 1 ) were examined .
the cells were pre - activated for 72 hr with anti - cd3 to induce t cell il-2 receptor expression , then washed and rested for 3640 hr , prior to restimulation with il-2 for 4 hr . results are averages  se from 5 independent experiments five independent experiments were performed with pbmcs from different donors to identify il-2-regulated genes represented on the affymetrix u95av2 oligonucleotide array .
a comparison was made between the pre - activated , rested cells , and those restimulated with il-2 for 4 hr .
the expression levels were analyzed using dna - chip analyzer software , with normalization to the average chip signal intensity for each experiment , and a 10percentile value cut - off for ' absent ' genes .
approximately 45% of the ~12,600 probe sets were detected above background . using the criterion of p < 0.05 for the difference of means between the two groups , a total of 316 of these ~5,800 probe sets ( ~5.4% ) were identified as putative il-2-responsive genes .
a complete list of all il-2-responsive genes identified in pbmcs and t cells can be found in the attached file ' il-2-regulated genes ' .
the expression patterns of 19 genes already known to be il-2-regulated , which were represented by 25 distinct probe sets on the array , were used to determine the range of expression parameters that might be most likely to identify bona fide il-2-regulated genes ( table 2 ) . as negative controls , glyceraldehyde-6-phosphate dehydrogenase ( gapdh ) and actin
were used as examples of genes that do not change expression levels under the experimental conditions used ; their expression levels were indeed constant without or with il-2 treatment ( 1.04  0.06 and 1.06  0.01 fold change , respectively ) . by comparison
, the 19 selected genes showed il-2-induced changes in expression levels , ranging from 2.0  0.3-fold ( il-4r , probe set 404_at ) to > 80-fold ( tnf 36296_at ) .
the subtractive differences in fluorescent intensity ranged from 114  20 ( c - myb 1474_s_at ) to > 1000 ( cr7 1779_s_at ) .
four of the genes ( c - myb , cr7 , flt3 ligand , tnf- ) were detected with more than one probe set , and these replicates were in fairly good agreement .
it is worthy of note that there is not a correlation between the subtracted difference and the fold change in gene expression levels .
this is likely due to the fact that there is a wide range in the abundance classes of rna transcripts derived from different genes , from as few as 1 copy per cell to several thousand copies per cell .
the signal intensity of a particular transcript detected on an oligonucleotide array would thus be directly related to this abundance level .
therefore , the subtracted change in signal intensity would be profoundly affected by the absolute number of cellular copies of a given transcript . by comparison ,
based on the results obtained with known il-2 target genes , the criteria used to identify additional bona fide il-2-regulated genes in these samples were set at  2-fold change , with a subtracted difference in fluorescent intensity  100 , and a significance level of the difference between the means of the two treatment groups of p < 0.05 . of the 316 probe sets that met the standard of p < 0.05 , 251 ( 79% ) also met the additional criteria of either fold change  2 , or subtracted difference  100 , while only 84 probe sets ( 27% ) met all three criteria .
these 84 probe sets represent 72 unique genes ( 60 il-2-induced genes and 12 il-2-repressed genes ) that can be segregated into 8 functional groups .
figure 1 shows the fold induction and subtracted differences in expression levels of these probe sets . among the 4 apoptosis - regulating genes , bcl2 , caspase 3 and drak2
it is noteworthy that the il-2 induction of bcl2 would be expected to promote cellular survival , while the il-2 regulation of the other three genes would be expected to promote apoptosis .
additional il-2-responsive genes include those encoding 6 cell surface proteins , 11 cytokine and chemokine receptors , as well as 15 signal transduction mediators , 6 genes involved in cellular metabolism and biosynthesis , 3 cell cycle regulators and 12 transcription factors .
il-2-suppressed genes include lymphocyte antigen 9 , inositol 1,4,5-trisphosphate 3-kinase b , and cdc25b , as well as transcriptional regulators max - interacting protein 1 , cbp / p300-interacting transactivator , inhibitor of dna binding 3 ( id3 ) , glucocorticoid - induced leucine zipper ( gilz ) , and zinc finger protein 36/erf-2 .
all of the 19 already known il-2-regulated genes ( table 2 ) were also identified in this 72-gene cohort , thereby yielding 53 newly identified il-2-regulated genes expressed by pbmcs after 4 hours of il-2 stimulation .
pre - activated , rested human pbmcs were left untreated or restimulated for 4 hr with il-2 , and rna probes were prepared for hybridization with affymetrix u95av2 arrays .
a total of 84 probe sets were identified that showed fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds , right axis ) , and a significance value of the difference between the means of p < 0.05 .
the probe sets represent 72 unique genes , and can be segregated into 8 functional groups .
the pbmc populations used in our initial experiments included nk cells , monocytes and b cells , as well as t cells .
in addition , cytokines including tnf , tnf and flt3 ligand were induced by il-2 as detected by the arrays , so that the il-2 effects could be mediated by the action of these intermediary cytokines on other cells in the mixed population , or as well , by the 12 transcriptional targets activated by il-2 . therefore to identify the direct il-2 targets in t cells
, we used cd56-depleted , cd2-selected t cells , which were 94  1% cd3cells .
the pbmcs were pre - activated with anti - cd3 for 72 hr , the t cells purified , and rested for 3640 hr prior to restimulation for 4 hr with 1 nm il-2 . in addition , 10 g / ml cycloheximide ( chx ) was included to block protein synthesis , so that only immediate / early il-2-regulated genes were detected .
chx has been shown to both elevate and prolong the expression of many transiently induced transcripts , so that the 4 hr il-2 stimulation in the presence of chx should enable detection of genes induced within minutes as well as hours .
samples from 4 independent experiments with pre - activated , rested cells , or cells treated with chx alone were combined for data analysis , and compared to cells treated with il-2  chx . as depicted in figure 2a , using only the criterion of significance level of p < 0.05 for the difference between the means of the two groups , 449 probe sets were identified as il-2-regulated immediate - early targets in the pure t cells . of these probe sets , 110 were also detected in the il-2-stimulated pbmcs . as shown in figure 2b , when a second standard was included , either a fold change  2 , or a subtracted difference  100 , 161 probe sets ( 36% ) were expressed as immediately / early genes in purified t cells , and of these , 71 were also il-2-regulated in pbmcs .
however , when all 3 criteria were applied ( figure 2c ) , a total of only 52 probe sets ( 12% ) were found to be immediate / early , il-2-regulated genes in t cells .
twenty - three of these 52 probe sets were among the 84 probe sets identified previously as meeting all 3 criteria in the experiments with pbmcs ( figure 3 ) . by comparison ,
the control genes actin and gapdh showed 1.1  0.1 and 1.3  0.2-fold change , respectively .
( a ) . using the criterion of p < 0.05 for the difference between the means of il-2-treated and untreated groups ,
a total of 316 il-2-sensitive probe sets were identified in pbmcs ( left circle ) , and 439 in purified t cells ( right circle ) .
of these , 110 were identified in both cell populations ( overlap between circles ) .
( b ) . when a second standard of either fold change  2 or subtracted difference  100 was included in the analysis , 251 probe sets were identified in pbmcs , and 161 in pure t cells , with 71 in common .
when all three criteria were used , 84 probe sets were identified in pbmcs , 52 in t cells , and 23 of these were common to both populations . within each population ,
a total of 23 probe sets met all three expression criteria of fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds and circles , right axis ) , and p < 0.05 in both pbmcs and purified t cells .
therefore , only ~1/3 of the genes discovered to be il-2-regulated in pbmcs are actually immediate / early direct il-2 targets in t cells .
these direct il-2 immediate / early target genes included 4 genes that were suppressed , dsip / gilz , the zinc finger protein 36/erf-2/tis11d , toso , and cdc25b .
notably , of the 16 il-2-induced genes , we had already been identified 4 previously as il-2 targets using differential colony hybridization ( cr3 , cr6 , cr7 , cr8 ) , while the 3 other known il-2 stimulated target genes included tnf , the il-4r , and the anti - apoptotic gene , bcl2 .
thus , only 9 genes were newly identified as il-2-induced immediate / early t cell genes ; including the signaling proteins slp76 and rgs1 , the transcription factors irf4 and xbp1 , and 5 miscellaneous genes ( dkfzp586f1323 , mitofilin , camp - specific phosphodiesterase 4b , cmp - n - acetylneuraminate monooxygenase , and the aryl hydrocarbon receptor ) .
in addition to the 23 probe sets in common with those identified in pbmcs , 29 others were detected as il-2-regulated immediate / early targets in the purified t cells ( figure 2c ) .
these probe sets met all three of the criteria of  2-fold change , subtracted difference  100 , and p < 0.05 in t cells , but were not il-2-regulated in pbmcs .
notably , only three of these were induced , while the remaining 24 were all suppressed in response to il-2 + chx .
a total of 29 additional probe sets met all three expression criteria of fold change  2 ( columns , left axis ) , subtracted difference  100 ( diamonds and circles , right axis ) , and p < 0.05 in purified t cells , but did not meet all three criteria in pbmcs .
these probe sets represent 27 unique genes . of the remaining 61 probe sets identified in pbmcs ( figure 2c ) ,
31 showed changes in expression levels in t cells with p < 0.05 , although either the fold change was less than 2.0 , or the magnitude of the change was less than 100 ( table 3 ) .
these 31 probe sets are derived from 27 unique genes , and most likely represent bona fide il-2 targets , since the group includes the genes jab / ssi-1/socs-1 , flt3 ligand , il-2rp55 , caspase 3 , and p78 , which have already been reported as il-2-induced genes .
if these genes are also included , 47 of the 72 unique genes ( 65% ) identified in pbmcs are il-2-regulated immediate / early genes in pure t cells .
t cell expression levels of genes identified in pbmcs similarly , of the 29 probe sets identified as meeting all 3 criteria only in pure t cells , 7 showed differences in expression with p < 0.05 in pbmcs ( table 4 ) . these include linker for activation of t cells , t - cell - specific transcription factor 7 , stat4 , il-7r , pas ser / thr kinase , l - mfa domain - containing protein , and an unknown gene designated dkfzp586d1122 .
notably , all of the other genes ( 22 ) were suppressed by il-2 in the pbmcs , as they were in the t cells , although they did not meet the significance value of p < 0.05 in the pbmcs .
lymphocyte activation is initiated by specific antigen / receptor interactions , and aided by accessory molecules .
however subsequently , cytokine and cytokine receptor expression ensue , and the cytokines ultimately drive the clonal expansion and functional differentiation of the antigen - selected lymphocyte populations .
il-2 was initially identified by virtue of its t cell growth factor activity , and il-2 is now recognized as the pivotal cytokine in promoting mature peripheral t cell proliferation . therefore , to initiate the identification of the genes responsible for lymphocyte activation , we chose first to delineate the genetic mechanisms of il-2 action . using oligonucleotide arrays of known genes to identify il-2-regulated genes in human lymphocytes ,
a total of 72 unique genes were found to change expression within 4 hr of il-2 stimulation of pbmcs .
these genes could be segregated into functional groups , including cell surface proteins , cytokine and chemokine receptors , as well as signal transduction mediators , cytokines , genes involved in cellular metabolism and biosynthesis , cell cycle regulators and transcription factors . in order to define the criteria that we used to sort the microarray data in this series of experiments
, we were able to take advantage of previous reports of 19 known il-2-regulated genes . by assessing the characteristics of the changes in expression of these known il-2 targets
, we were able to define a range of both fold - induction and subtracted difference that encompassed all of these genes . when applied to the total data set ,
these criteria were likely to identify additional meaningful il-2-regulated genes . since 19 of these genes
had already been reported to be il-2-induced genes , our dna array experiments identified 53 additional il-2-regulated genes expressed by pbmcs . however , only 20 of these genes were found to be il-2-regulated immediate / early t cell genes .
it is important to discriminate these results from other analyses of t cell activation that have employed antigen receptor activation , as opposed to il-2 .
the tcr and il-2 receptors activate some shared signaling pathways , such as the ras - raf - map kinase and pi-3-kinase pathways .
however , there are differences , such as the jak - stat pathway , which is unique to the cytokines , and the ca++/calcineurin pathway , which is activated by the tcr .
these signaling pathways culminate in the activation of specific transcription factors , so that there must be both shared target genes , as well as genes unique to antigen or il-2 signaling . as tcr signaling induces the expression of il-2 , as well as other cytokines and their receptors , the ultimate t cell response is an amalgam of both antigen- and cytokine receptor - triggered events .
this point is well illustrated by the report of teague et al , who used microarrays to identify murine t cell genes induced in vivo by seb stimulation after 8 and 48 hours .
again , cytokine and cytokine receptor genes were induced during these time intervals , so that the entire spectrum of genes identified included both antigen- and accessory molecule - activated , as well as cytokine - regulated targets .
similarly , ellisen et al   used dna arrays to monitor cona - induced genes in human t lymphocytes , over a time scale ranging from 4 to 48 hours .
inducible genes included il-2 and tnf- , as well as the il-1r , il-2r and il-4r .
thus , at the time points used in this study , many of the genes detected were likely regulated by these cytokines as well as con a. in a similar vein , herblot et al   used a subtractive approach to identify 66 genes induced by il-2 but not il-4 in a murine t cell line .
they further demonstrated that while in vitro , cona could induce expression of the il-2 target genes in t cells from il-2 + /+ mice , the genes were not induced by cona treatment of t cells from il-2-/- mice .
thus , cona induces il-2 and il-2r expression , and the cytokine drives expression of additional specific target genes .
recently , riley et al   and diehn et al   described genes regulated by the tcr and the costimulatory receptors cd28 , icos and ctla-4 , over a time course of 148 hours .
once again , numerous cytokines , including il-2 , are strongly induced within 2 hr , such that a number of the observed regulated genes are likely cytokine - responsive targets .
finally , gonzalez et al   have described murine genes induced after 8 hr il-2 stimulation , which likely include direct targets of il-2 , as well as genes induced via il-2- regulated cytokines .
therefore , to discriminate between the distinct genes induced by each ligand that contributes to the overall response initiated by antigenic stimulation , it is important to use cycloheximide to identify immediate / early genes regulated by the tcr and costimulatory receptors vs. il-2 . in addition , the genes activated by tcr , costimulatory receptors and il-2 must be distinguished from those activated by il-1 , il-6 , il-12 , and tnf- , as well as the genes regulated by the toll - like receptors ( tlr ) , all of which have been identified as mediators of lymphocyte activation events that were previously ascribed to laf .
moreover , as il-2 itself induces the expression of numerous cytokines and receptors , it is important to discriminate the direct il-2 targets from those induced secondarily through these intermediaries .
thus , it is worthy of re - emphasis that of the 72 genes regulated by il-2 in pbmcs , only 20 were found to be bona fide il-2-regulated immediate / early genes in purified t cells .
in addition to imparting positive activating signals that result in cell cycle progression , il-2 has also been found to promote negative feedback phenomena .
thus , it is especially intriguing that il-2 suppresses the expression of many genes , both among pbmcs in the absence of chx , as well as in purified t cells in the presence of chx
. the suppression of the expression of these genes may well be important in mediating the feedback down - regulatory phenomena .
the high - affinity il-2 receptor is comprised of three subunits ,  ,  and  .
the  chain is specific to il-2 , while the  chain is shared among il-4 , il-7 , il-9 , il-15 and il-21 .
the  chain of the il-2r is also a component of the il-15 receptor , in conjunction with a specific il-15r  chain .
the sharing of these receptors by multiple cytokines raises the question of how the ligands can trigger specific responses in the target cells .
several signaling events , including the activation of jak kinases 1 and 3 are shared among the ' common  chain ' cytokines .
other signaling events , such as the activation of shc , gab2 , shp-2 and mapk , are shared among il-2 and 15 , but not the other common  chain cytokine receptors , and tyr 388 of il-2r appears critical for these events . despite these signaling similarities , it is clear that the biological properties of il-2 are unique among the common  chain cytokines , as the other cytokines can not compensate for il-2 in the il-2 knockout mouse . in addition , il-15 appears uniquely critical for the development of nk cells , as evidenced by the phenotypes of il-15   and il-15r    deficient mice .
thus , there must be a means whereby il-2 and il-15 , as well as the other common  chain cytokines , trigger distinct intracellular signaling pathways , and target genes .
therefore , identification of each of the cytokine - specific target genes should shed light on the issue of specificity amongst the common  chain cytokines .
complete classification of the genes expressed as a consequence of distinct ligand / receptor interactions during lymphocyte activation will also provide a powerful tool for the assessment and treatment of immunological disorders .
in addition to defining the target genes of specific cytokines and cell surface ligands , it is possible to further subdivide the genes based on the specific signaling proteins and transcription factors that govern their expression .
for example , through the use of stat5-/- mice , ihle et al have been able to identify those il-2 target genes that require stat5 .
similar approaches with mutant signaling proteins , knockouts , or over - expression systems enable such dissection of the signal transduction pathways leading to specific target genes .
thus , each ligand / receptor pair will ultimately be linked to a specific signature of target genes , derived from the signaling pathways and transcription factors activated .
such information will be invaluable in the diagnosis and treatment of disorders involving aberrancies in these pathways .
the rapidity with which lymphocyte activation genes may now be identified using the methods described in this report opens the way to a molecular genetic definition of the immune response , based upon the categorization of the genes regulated by distinct ligand receptor pairs .
consequently , more precise genetic and molecular diagnoses of immune disorders will soon become available , and hopefully , new treatment regimes may be developed to target the specific molecular nature of each disease .
human peripheral blood mononuclear cells ( pbmc ) were purified from venous blood of healthy donors by density centrifugation over histopaque 1.077 ( sigma ) .
cells were cultured in rpmi1640 , 10%fcs , at a density of 1  10cells / ml , and activated with anti - cd3 ( okt3 , 1:500 ; ortho diagnostics ) .
after 72 hr activation , pbmc were washed and replaced in culture without stimulation for 3640 hr prior to restimulation with 1 nm il-2 . to purify t lymphocytes ,
72 hr okt3-activated pbmc were depleted of cd56 , and positively selected for cd2 , using antibody - conjugated magnetic beads ( anti - cd56 polysciences inc , anti - cd2 dynal ) according to manufacturer 's instructions .
subsequent to purification , t cells were replaced in culture for 3640 hr prior to restimulation with 1 nm il-2 , and/or 10 g / ml cycloheximide .
cell populations were stained with anti - cd3-apc , cd14-fitc , cd19-percp and cd56-pe ( becton dickinson ) , and quantitated by flow cytometry ( facscalibur , becton dickinson ) .
five independent experiments were performed with pbmcs from different donors , and four independent experiments were carried out with purified t cells .
briefly , total cellular rna was prepared with qiagen rneasy reagents , and 5 g samples were used to prepare t7-dt - primed double - stranded cdna ( superscript choice , invitrogen ) .
biotin - labeled crna was prepared by in vitro transcription ( bioarray high - yield t7 , enzo ) , and hybridization cocktails were prepared with 20 g crna per sample .
hybridization , wash , and staining of affymetrix human u95av2 arrays were performed following manufacturer 's instructions .
gene expression values were normalized to the average chip signal intensity for each experiment , and a 10percentile value cut - off was used for ' absent ' genes that did not give signals significantly greater than background . to calculate il-2-induced gene expression changes , the subtracted difference values were determined by subtracting the signal intensity of untreated samples from the values of il-2-treated samples .
standard errors were derived from 5 independent pbmc experiments , and 4 experiments with purified t cells .
the average of the standard errors relative to the mean expression values ( se / mean signal intensity)*100% was 22% for untreated and 15% for il-2-stimulated pbmcs , and 16% and 19% for the unstimulated and il-2-treated purified t cell samples , respectively .
the p - values were determined by testing whether the absolute difference between treatment group means is not zero by unpaired t - test .
human peripheral blood mononuclear cells ( pbmc ) were purified from venous blood of healthy donors by density centrifugation over histopaque 1.077 ( sigma ) .
cells were cultured in rpmi1640 , 10%fcs , at a density of 1  10cells / ml , and activated with anti - cd3 ( okt3 , 1:500 ; ortho diagnostics ) .
after 72 hr activation , pbmc were washed and replaced in culture without stimulation for 3640 hr prior to restimulation with 1 nm il-2 . to purify t lymphocytes ,
72 hr okt3-activated pbmc were depleted of cd56 , and positively selected for cd2 , using antibody - conjugated magnetic beads ( anti - cd56 polysciences inc , anti - cd2 dynal ) according to manufacturer 's instructions .
subsequent to purification , t cells were replaced in culture for 3640 hr prior to restimulation with 1 nm il-2 , and/or 10 g / ml cycloheximide .
cell populations were stained with anti - cd3-apc , cd14-fitc , cd19-percp and cd56-pe ( becton dickinson ) , and quantitated by flow cytometry ( facscalibur , becton dickinson ) .
five independent experiments were performed with pbmcs from different donors , and four independent experiments were carried out with purified t cells .
briefly , total cellular rna was prepared with qiagen rneasy reagents , and 5 g samples were used to prepare t7-dt - primed double - stranded cdna ( superscript choice , invitrogen ) .
biotin - labeled crna was prepared by in vitro transcription ( bioarray high - yield t7 , enzo ) , and hybridization cocktails were prepared with 20 g crna per sample .
hybridization , wash , and staining of affymetrix human u95av2 arrays were performed following manufacturer 's instructions .
gene expression values were normalized to the average chip signal intensity for each experiment , and a 10percentile value cut - off was used for ' absent ' genes that did not give signals significantly greater than background .
to calculate il-2-induced gene expression changes , the subtracted difference values were determined by subtracting the signal intensity of untreated samples from the values of il-2-treated samples .
standard errors were derived from 5 independent pbmc experiments , and 4 experiments with purified t cells .
the average of the standard errors relative to the mean expression values ( se / mean signal intensity)*100% was 22% for untreated and 15% for il-2-stimulated pbmcs , and 16% and 19% for the unstimulated and il-2-treated purified t cell samples , respectively .
the p - values were determined by testing whether the absolute difference between treatment group means is not zero by unpaired t - test .
the results reported here represent almost 20 years of effort . k.a.s . initiated the search for il-2-regulated genes in 1983 , using subtractive hybridization , and reported the first il-2-regulated gene in 1986 .
extended these initial efforts and reported on the identification of 8 immediate / early il-2-induced genes in 1993 .
c.b . conceived and performed the experiments contained in this report , and wrote the manuscript .
provided advice in experimental design and analysis of the results , and contributed to the revisions of the manuscript that eventually resulted in the final version .
il-2 target genes in human pbmcs and t cells this file contains expression data for il-2-regulated probe sets detected on the affymetrix u95av2 array , using samples prepared from human pbmcs and purified t cells .
this work was supported by national institutes of health niaid grants ro1 ai 32031 - 24 and ro1 ai 44207 - 02 .
 backgroundlymphocyte activation culminates in blastogenesis , cell cycle progression , dna replication and mitosis . 
 these complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors . until now , 
 the discovery of the genes regulated by each ligand / receptor pair has been hampered by the technologies available.resultsto identify interleukin-2 ( il-2)-responsive genes , human peripheral blood mononuclear cells ( pbmc ) were pre - activated with anti - cd3 , rested , and restimulated with il-2 for 4 hr . gene expression was analyzed using affymetrix u95av2 oligonucleotide arrays . to determine the most stringent parameters to score a gene as a bona fide il-2 target 
 , the expression of 19 known il-2-regulated genes was examined first . 
 all were induced at least 2-fold , with a difference in fluorescent intensity of  100 at p < 0.05 . 
 an additional 53 unique genes met these criteria . to determine which of these were immediate / early il-2 targets in t cells , 
 purified t cells were stimulated with il-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression . 
 of the 72 genes identified in pbmcs , 20 were detected as immediate / early il-2-regulated genes in purified t cells . 
 in addition , 27 unique genes were il-2-regulated in t cells but not in pbmcs.conclusionsfor a successful reductionist approach to the analysis of gene expression in lymphocyte activation , it is necessary to examine purified cell populations and immediate / early gene expression regulated by each ligand / receptor pair involved . 
 this approach should allow the discovery of genes regulated by all of the ligand / receptor pairs involved in lymphocyte activation . 
Background
Results
Detection of IL-2-regulated genes in peripheral blood mononuclear cells (PBMCs)
Detection of PBMC genes already known to be IL-2-regulated
Detection of additional IL-2-responsive PBMC genes
IL-2-regulated immediate/early genes in T cells
Discussion
Conclusions
Materials and Methods
Cell culture
Microarray analysis
Competing interests
Authors' contributions
Supplementary Material
Acknowledgments