antigen - activated cd4 t cells differentiate into either th1 or th2 cells , producing proinflammatory or proallergic cytokines , respectively .
the best understood mode of regulating th cell differentiation is the cytokine environment during primary t cell activation , with il-12 associated with th1 development and il-4 with th2 development .
however , it is still uncertain how these cytokines mediate their effect on differentiation 1 .
coffman and reiner 1 recently articulated three competing models , each compatible with the observed effects of cytokines on differentiation . in the instructive model , cytokine
signaling precedes progenitor commitment ; that is , cytokines direct each progenitor towards a defined fate through a program of molecular cues . in the selective model ,
progenitor commitment is independent of cytokines ; that is , progenitor cells stochastically commit to various fates , and cytokines subsequently act by favoring the selective outgrowth of a particular lineage . in a hybrid instructive - selective model ,
the ratio of progenitors initially committing to each fate may be altered by cytokines , but cytokine - driven selective outgrowth of committed cells is retained as a required mechanism of polarization .
an instructive model seemed compatible with known molecular processes in t cells , such as cytokine regulation of transcription factor expression and cytokine - dependent chromatin alterations in the il-4 locus 23 .
further , the specific requirement of the transcription factors signal transducer and activator of transcription ( stat)4 45 and stat6 67 in th1 and th2 development , respectively , was generally interpreted to indicate an instructive role for cytokines in th differentiation .
however , the analysis of stat4- and stat6-deficient t cells was done on bulk populations and not performed specifically to distinguish instructive or selective models of development .
stat4 and stat6 activation could either initiate an instructive program or alternately might simply mediate signals for selective outgrowth of committed th1 or th2 cells .
moreover , certain findings , such as monoallelic cytokine expression 8910 , a role of cell cycle in cytokine regulation 111213 , and stat6-independent th2 development 141516 have provided an impetus to reconsider the issue of instructive versus selective differentiation . the difficulty in distinguishing between these models by previous studies arises from their inability to directly track fates and measure relative outgrowth of individual progenitors .
initial bulk population analysis suggested that th2 development was absolutely il-4 dependent , consistent with either model 67 .
th2 development has recently been reported to occur in stat6-deficient animals 1516 and to be dependent on gata-3 expression independently of il-4 and stat6 17 .
gata-3 expression , although augmented by il-4 through stat6 , can also be achieved independently of il-4 , involving a stat6-independent pathway of autoactivation .
this feature of gata-3 expression allows progenitors to acquire a stable , cytokine - independent phenotype in which gata-3 directs the elaboration of downstream hallmarks of th2 development that occur in response to il-4 .
importantly , ectopic gata-3 expression in stat6-deficient t cells induces all developmental components of the th2 phenotype such as c - maf expression , chromatin remodeling of the il-4 locus , and th2 cytokine gene expression 17 .
further , th2 commitment need not be absolutely il-4 dependent , as other factors , such as tcr / cd28 signaling 18 and il-12 1719 can regulate gata-3 expression .
thus , any signal that can regulate gata-3 expression would also be expected to regulate th2 development . in this study , we have used two recently described technologies , cellular affinity matrix sorting and retroviral tagging , to determine whether gata-3 functions to promote outgrowth of cells that have stochastically committed to il-4 production ( selection ) , or whether gata-3 mediates programmatic alterations in gene expression without affecting outgrowth of uncommitted progenitors ( instruction ) . to accommodate the known effects of gata-3 expression in stat6-deficient t cells ,
the selective model must adopt gata-3 , rather than stat6 , as a mediator of selective outgrowth of th2 cells .
however , in a direct test of this hypothesis , we found no evidence that gata-3 influences selective outgrowth when expressed in stat6-deficient t cells developing under any conditions .
rather , gata-3 expression increased the proportion of il-4committed progenitors , even when developing in the presence of il-12 , a condition predicted by the selective model to inhibit the outgrowth of cells committed to the th2 phenotype .
these data exclude purely selective models of th2 development and provide strong evidence that gata-3 instructs th2 development without inducing selective outgrowth .
in vitro cultures of do11.10 tcr transgenic wild - type and do11.10  stat6 cd4 t cells were stimulated with ova protein under neutralizing conditions in the presence of il-4 ( 11b11 , 10 g / ml ) , il-12 ( tosh , 10 g / ml ) , and ifn- ( h22 , 10 g / ml ) .
7 d after primary activation , live cd4/il-4secreting cells were purified as described previously 17 with the following modifications .
cell cultures were restimulated with pma / ionomycin for 2.5 h and subsequently labeled with the bifunctional ab conjugate specific for cd45 ( 30-f11 ) and il-4 ( bvd6 - 24g2 ) for surface - capture of secreted il-4 .
the cells were then diluted in medium and allowed to secrete il-4 for 30 min at 37c as described 17 .
il-4secreting cd4 t cells were identified by staining with a secondary mab il-4pe ( 11b11-pe ; bd pharmingen ) and mcd4tricolor ( gk1.5-tc ; caltag ) . il-4 and il-4 cells were purified by facs sorting as described 2021 . do11.10 
stat6 cd4 t cells were activated for 36 h with ova under th1 ( rmil-12 [ 10 u / ml ] , and il-4 ) or th2 ( il-12 , and ifn- and/or rmil-4 [ 100 u / ml ] ) conditions .
t cell cultures were then transduced with retroviral culture supernatants derived from the transient transfection of the phoenix - ecotropic retrovirus packaging cell line 19 .
percentages of retroviral - transduced cells were monitored for the expression of retroviral marker proteins green fluorescence protein ( gfp ) and hcd4 by facs analysis .
stat6 cd4 t cells were activated with ova protein under either th1 or th2 conditions and transduced with retroviral vectors as described in the text .
cells were rested in bulk culture to day 7 of activation and cd4gfp cells were purified by facs sorting .
purified t cells were cloned by single - cell deposition cloning by facs sorting into 96-well plates containing 5  10 irradiated balb / c splenocytes , ova protein ( 0.5 mg / ml ) , and rhil-2 ( 50 u / ml ) in 200 l medium ( dmem supplemented with l - glutamine [ 0.2 mm ] , nonessential amino acids [ 10 m each ] , sodium pyruvate [ 100 m ] , -mercaptoethanol [ 50 m ] , and penicillin / streptomycin [ 100 u / ml each ] ) .
cellular outgrowth was monitored between days 1015 of plating , and individual clones were expanded by restimulation with irradiated balb / c splenocytes , ova protein , and rhil-2 ( 50 u / ml ) .
genomic dna derived from independent t cell clones were amplified for the detection of both the control gfp - expressing retrovirus ( gfprv ) and the gata-3-gfp integrated retroviral vectors by a two - step nested pcr technique .
these pcr reactions were performed with the following primers : gfprv , first set : 5-cctacatcgtgacctgggaagccttgg , 5-gttccgctgcctgcaaagggtcgc , nest : 5-ggtcaagccctttgtacaccctaagcctcc , 5-cctaggaatgctcgtcaagaagacagggc ; gata-3-gfp , first set : 5-cctacatcgtgacctgggaagccttgg , 5-ggtgaagagatgaggactggagtggcc , nest : 5-ccctttgtacaccctaagcctccgcc , 5-ggagggtaaacggacagaggcccc . as controls , genomic dna from nontransduced and single - transduced t cells from bulk culture populations
clones used in the study were infected by one type of retrovirus , positive for either gfprv or gata-3-gfp , but not both .
in vitro cultures of do11.10 tcr transgenic wild - type and do11.10  stat6 cd4 t cells were stimulated with ova protein under neutralizing conditions in the presence of il-4 ( 11b11 , 10 g / ml ) , il-12 ( tosh , 10 g / ml ) , and ifn- ( h22 , 10 g / ml ) .
7 d after primary activation , live cd4/il-4secreting cells were purified as described previously 17 with the following modifications .
cell cultures were restimulated with pma / ionomycin for 2.5 h and subsequently labeled with the bifunctional ab conjugate specific for cd45 ( 30-f11 ) and il-4 ( bvd6 - 24g2 ) for surface - capture of secreted il-4 .
the cells were then diluted in medium and allowed to secrete il-4 for 30 min at 37c as described 17 .
il-4secreting cd4 t cells were identified by staining with a secondary mab il-4pe ( 11b11-pe ; bd pharmingen ) and mcd4tricolor ( gk1.5-tc ; caltag ) . il-4 and il-4 cells were purified by facs sorting as described 2021 .
do11.10  stat6 cd4 t cells were activated for 36 h with ova under th1 ( rmil-12 [ 10 u / ml ] , and il-4 ) or th2 ( il-12 , and ifn- and/or rmil-4 [ 100 u / ml ] ) conditions .
t cell cultures were then transduced with retroviral culture supernatants derived from the transient transfection of the phoenix - ecotropic retrovirus packaging cell line 19 .
percentages of retroviral - transduced cells were monitored for the expression of retroviral marker proteins green fluorescence protein ( gfp ) and hcd4 by facs analysis .
do11.10  stat6 cd4 t cells were activated with ova protein under either th1 or th2 conditions and transduced with retroviral vectors as described in the text .
cells were rested in bulk culture to day 7 of activation and cd4gfp cells were purified by facs sorting .
purified t cells were cloned by single - cell deposition cloning by facs sorting into 96-well plates containing 5  10 irradiated balb / c splenocytes , ova protein ( 0.5 mg / ml ) , and rhil-2 ( 50 u / ml ) in 200 l medium ( dmem supplemented with l - glutamine [ 0.2 mm ] , nonessential amino acids [ 10 m each ] , sodium pyruvate [ 100 m ] , -mercaptoethanol [ 50 m ] , and penicillin / streptomycin [ 100 u / ml each ] ) .
cellular outgrowth was monitored between days 1015 of plating , and individual clones were expanded by restimulation with irradiated balb / c splenocytes , ova protein , and rhil-2 ( 50 u / ml ) .
genomic dna derived from independent t cell clones were amplified for the detection of both the control gfp - expressing retrovirus ( gfprv ) and the gata-3-gfp integrated retroviral vectors by a two - step nested pcr technique .
these pcr reactions were performed with the following primers : gfprv , first set : 5-cctacatcgtgacctgggaagccttgg , 5-gttccgctgcctgcaaagggtcgc , nest : 5-ggtcaagccctttgtacaccctaagcctcc , 5-cctaggaatgctcgtcaagaagacagggc ; gata-3-gfp , first set : 5-cctacatcgtgacctgggaagccttgg , 5-ggtgaagagatgaggactggagtggcc , nest : 5-ccctttgtacaccctaagcctccgcc , 5-ggagggtaaacggacagaggcccc . as controls , genomic dna from nontransduced and single - transduced t cells from bulk culture populations were amplified in parallel pcr reactions .
clones used in the study were infected by one type of retrovirus , positive for either gfprv or gata-3-gfp , but not both .
the observation that both il-4 and ifn-producing t cells can emerge under nonpolarizing conditions has been interpreted to suggest that a random , or stochastic process generates the initial phenotype repertoire , motivating the selective model 1911 .
indeed , when naive progenitors are activated under conditions in which il-12 , il-4 , and ifn- are all neutralized , a persistent population of cells was still observed that committed to the production of either il-4 or ifn- ( fig .
1 c ) . by the selective model , the increase in populations developing in fully polarizing conditions ( fig .
b ) arise from the selective outgrowth of cells that stochastically commit even in the absence of these conditions ( i.e. , from the populations in fig
moreover , this interpretation is consistent with persistence of a small il-4producing population even in stat6-deficient mice ( fig .
f ) , which are proposed to represent committed progenitors whose phenotypes are fixed and awaiting selective outgrowth via the effects of polarizing cytokines . however , the instructive and selective models differ in their predictions of how committed cells should respond to subsequent il-4 exposure . in the selective model
, commitment is permanent , with subsequent cytokine exposure altering cell growth , but not differentiation . in the instructive model , commitment results from cytokine - derived signals , which could be delivered even after initial activation .
these predictions can be distinguished by testing responses of committed progenitors to the polarizing effects of il-4 . to test this experimentally
, cells initially committed to an il-4producing phenotype or il-4nonproducing phenotype would need to be separated and analyzed independently for differentiation when exposed to il-4 ( fig .
to separate il-4producing from il-4nonproducing committed cells , we used the novel cellular affinity matrix technology for purifying live cells based on their il-4 secretion 17 .
wild - type and stat6-deficient ( stat6 ) do11.10 naive t cells were activated in the presence of anti 
2 b , a ) , and cd4 t cells were sorted into il-4nonsecreting cells ( fig .
each population was then divided and restimulated either in the presence of il-4 or anti 
il-4 mab ( 11b11 ) for 7 d. upon restimulation , il-4 and ifn- production by individual cells was measured by intracellular staining ( fig .
. 2 b , c ) retained this property at a frequency compatible with differentiated th2 cells ( fig .
the frequency of il-4positive cells in both wild - type and stat6-deficient populations was only slightly increased by exposure to il-4 in secondary culture ( fig .
cells that initially had not produced il-4 and were not exposed to il-4 in secondary culture remained negative for il-4 production later ( fig .
however , wild - type cells that were initially il-4 negative ( fig . 2 b , b ) , and were exposed to il-4 in secondary culture showed a significantly increased frequency of il-4producing cells ( compare fig . 2 b , e to d ) , consistent with redirection to an il-4producing fate .
2 b , compare m to l ) , suggesting that the effect required an intact il-4 signaling pathway .
thus , this effect can not be explained as a continued stochastic generation of il-4producing cells , as the selective model would predict this to also occur in stat6-deficient cells as well as in wild - type cells exposed to anti 
in addition , we observed only a minor population of cells committed to producing ifn- in wild - type cells sorted for being il-4 negative ( fig .
the percentage of this population in wild - type and stat6-deficient cells was not influenced by either the presence or absence of il-4 ( fig .
thus , in these experiments , the expansion of ifn-producing th1 cells was not seen , eliminating its potential influence on inhibiting th2 development in stat6-deficient t cells . in summary ,
il-4 exposure appears to direct th2 commitment even after the initial period predicted by the selective model to generate the stochastic fixed repertoire . due to the kinetics of gene activation ,
the frequency of il-4 staining in activated th2 cells is not 100% , but 3050% , consistent with previous reports 20212223 .
thus , it is still possible to interpret the above results as consistent with the selective model by explaining the apparent redirection of cells in fig .
2 as the il-4dependent outgrowth of th2-committed cells that simply happened to not produce il-4 at the time of sorting . thus ,
to rigorously distinguish these aspects of selection from instruction , it is therefore necessary to track progenitor fates and frequencies as they expand in culture . to do this
, we developed a retroviral - based tagging system in which either a gfp retrovirus or a gata-3expressing retrovirus is used to mark cells by infection during the initial activation ( fig .
instructive and selective models differ in how they predict progenitors will respond to tagging by the gata-3expressing retroviruses . to account for the known effects of gata-3 , the selective model predicts that cells committing to either il-4positive or il-4negative fates should each expand in response to gata-3 , but should not alter their respective fates ( fig .
, the instructive model predicts that , independent of initial commitment , progenitors tagged by gata-3 may undergo redirection of fate toward il-4 production .
the control retrovirus expressing only the gfp tag is not predicted to alter fates in either model .
in essence , the selective model predicts outgrowth of both gata-3expressing th1 and th2 clones , whereas the instructive model predicts that gata-3 infected clones will be strongly skewed to a th2 fate , with marked reduction in the numbers of gata-3infected th1 clones . to eliminate any bias , we blinded this tagging experiment to the identity of the retrovirus by infecting progenitors with a mixture of control and gata-3 retroviruses , both expressing gfp as the retroviral marker .
the identity of the retrovirus infecting each clone was revealed by pcr only at the end of the experiment .
stat6-deficient progenitors were infected with gfp and gata-3 retroviruses and activated in either th1 or th2 conditions . on day 7 , single retrovirally infected cd4 cells were directly cloned into nonselecting conditions ( i.e. , in the absence of il-12 , il-4 , or ifn- ) .
clones were expanded and characterized for il-4 and ifn- expression by elisa to assign phenotype ( fig .
3 b ) , and only then was the identity of the infecting retrovirus determined .
the majority of clones exhibit cytokine production that is near to one of the cytokine axes , consistent with polarization observed in bulk populations .
the phenotype of each clone was assigned based on its cytokine production being above or below the diagonal , after which the retrovirus identity was determined ( fig . 3 b , c  f ) .
stat6-deficient cells that were infected with control gfp retrovirus were strongly skewed to the th1 phenotype , regardless of initial activation conditions ( fig .
the low number of il-4producing clones is compatible with the observed frequency of il-4positive cells in bulk populations ( fig .
however , stat6-deficient clones infected with the gata-3 retrovirus were markedly skewed to the th2 fate , even in those clones whose progenitors were activated under th1-inducing conditions ( fig .
further , there was no apparent difference in the numbers of clones obtained under various conditions , arguing against any gross differences in selective outgrowth .
thus , the results of this retroviral tagging experiment are inconsistent with predictions of the selective model described above . to try to accommodate for this last experiment ,
the selective model could be slightly revised to propose that the growth - promoting effects of gata-3 can only be exerted in cells that are also committed to il-4 production , and not in other cells . in this revised selective model
, the decreased number of gata-3infected th1 clones would be due to the inability of gata-3 to promote outgrowth of non  il-4producing cells , rather than from the conversion of their phenotype from th1 to th2 fate .
although it may be conceivable to construct a selective mechanism to restrict the effects of gata-3 in this way , importantly this possibility can be tested using a dual - marker retroviral system to simultaneously track both fate and expansion of committed cells over time .
we carried out this approach using unique markers to distinguish two retroviruses , with the control retrovirus expressing hcd4 , and gata-3 retrovirus expressing gfp . in these experiments ( fig .
4 ) , stat6-deficient progenitors were infected with a mixture of control retrovirus and gata-3 retrovirus in the same cultures , and activated under either th1 or th2 conditions .
cells were expanded together in bulk culture , and il-4 production by individual cells was analyzed on day 7 and on day 14 after secondary stimulation , allowing direct measurement of the frequency and phenotype of cells over time . on day 7 and day 14 , cells were activated with pma / ionomycin for 4 h and stained for intracellular production of il-4 .
b ) , and the percentage of il-4positive cells in each gate was measured by intracellular il-4 staining ( fig . 4 c ) . last , the proportion of each retrovirally infected population is expressed as a percentage of the whole population ( fig . 4 d ) .
infection by the control virus had no effect on il-4 production by cells over time , either in the th1- or th2-activated conditions ( fig . 4 c ) , as expected .
in contrast , infection by the gata-3 retrovirus ( gfp - positive cells ) markedly increased the percentage of il-4positive cells between day 7 and day 14 .
this effect occurred in both th1 and th2 conditions , and these data were highly reproducible in replicate experiments ( table ) .
importantly , the percentage of il-4producing cells within the gata-3 population increased even though the total percentage of gata-3infected cells remains constant over time , consistent with a continuing developmental effect .
finally , the prediction of the revised selective model is not observed ; that is , we observe a constant ratio of cells infected with control or gata-3 retrovirus over time in both conditions , rather than an increase in the proportion of gata-3expressing cells as predicted by the selective model .
first , we observed an apparent redirection of fate caused by exposure of previously non  il-4producing cells to an il-4producing phenotype , inconsistent with the selective model .
second , we observed that gata-3 strongly skewed the frequency of individual clones that commit to il-4 production but did not exert selective outgrowth of clones already committed to the th1 fate , as would be predicted by the selective model .
finally , using dual - tag retroviral infection , we directly measured the fate and expansion of distinctly committed cells , which revealed an increasing frequency of il-4 production in the absence of any apparent increase in size of this committed population . taken together , these results strongly support an instructive interpretation of recent findings regarding the transcriptional regulation of gata-3induced th2 development .
the initial production of il-4 by small numbers of stat6-deficient t cells is consistent with either the stochastic expression of gata-3 in a small percentage of t cells or with the regulated expression of gata-3 by additional factors , including the tcr / cd28 signaling pathway , as described recently 18 .
however , it is possible that il-4 could exert some selective / growth - promoting effects on committed th2 cells that may not completely involve stat6-dependent gata-3 regulation 24 .
for example , we observed a 10% increase in the proportion of il-4producing cells in both wild - type and stat6-deficient th2 cells after secondary exposure to il-4 ( fig .
2 b , f to g and n to o ) . however , this observation may be consistent with either instructive or selective models ; instructive , if il-4 could augment expression of gata-3 or other transcription factors through stat6-independent pathways , which we have not excluded ; selective , if il-4 could alter cell survival or proliferation through a stat6-independent pathway such as insulin receptor substrate ( irs)-1 25 .
regardless , our results exclude models exclusively requiring selection to explain gata-3induced th2 development , and strongly support the existence of an instructive component in this process .
 although interleukin ( il)-12 and il-4 polarize naive cd4 + t cells toward t helper cell type 1 ( th1 ) or th2 phenotypes , it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate . to distinguish these instructive and selective models , we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral - based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels . 
 we observe il-4dependent redirection of phenotype in cells that have already committed to a non  il-4producing fate , inconsistent with predictions of the selective model . 
 further , retroviral tagging of naive progenitors with the th2-specific transcription factor gata-3 provided direct evidence for instructive differentiation , and no evidence for the selective outgrowth of cells committed to either the th1 or th2 fate . 
 these data would seem to exclude selection as an exclusive mechanism in th1/th2 differentiation , and support an instructive model of cytokine - driven transcriptional programming of cell fate decisions . 
Introduction
Materials and Methods
Sorting of Live IL-4secreting Cells by Cellular Affinity Matrix Technology.
Retroviral Transduction.
T Cell Cloning.
Identification of GFP-expressing Retrovirus and GATA-3-GFPtransduced T Cell Clones by PCR.
Results and Discussion