stat4 , stat6 mice were generated by mating stat4   and stat6 mice at the third backcross generation to balb / c and   intercrossing the resulting compound heterozygotes . controls   were inbred balb / c and 129/svj mice , both of which gave   comparable results in each of the assays tested . differentiation of t cells   and assay of cytokines were performed as described ( 12 , 17 ) . in   brief , cells were activated with 1 g / ml plate - bound anti - cd3 .   where indicated , il-12 was added at 1 ng / ml , and anti il-4 , and anti  il-12 were added at 10 g / ml ( genzyme   corp . , after 1 wk in culture , cells were   washed and restimulated with plate - bound anti - cd3 for 24 h.   supernatants were harvested , and ifn- and il-4 production   were quantified by elisa . purification of cd4 and cd62l   cells was done by positive selection using magnetic beads ( miltenyi biotec , inc . , auburn , ca ) according to the supplier 's instructions . for the purification of naive cells , pooled spleens and lymph nodes were   depleted before positive selection with 2.4g2 ( anti - cd16/32 ) ,   2.43 ( anti - cd8 ) , and m5114 ( anti  i - a ) coupled to magnetic   beads . after 1 wk , cells were restimulated with plate - bound anti - cd3 for 6 h and plated in dilution on anti ifn-   ( r4/6a2)-coated immobilon - p membranes in a 96-well plate for   18 h. cells were washed away , and bound cytokine was detected   with biotinylated anti ifn- ( pharmingen , san diego , ca ) ,   avidin  alkaline phosphatase ( sigma chemical co. , st . louis ,   mo ) , and a stable bcip / nbt mix ( gibco brl , gaithersburg ,   md ) . spots were developed for 3060 min , washed with distilled   water , and counted under a dissecting microscope . male mice were immunized with 100 g dnp - klh ( calbiochem corp . , la jolla , ca )   emulsified in cfa ( sigma chemical co. ) , at the base of the tail . after 6 d , footpad thickness was measured using a spring - loaded   caliper , and mice were challenged with 50 g dnp - klh in one   foot and pbs in the contralateral appendage . specific   swelling was determined by subtracting nonspecific swelling in   the pbs - injected foot from antigen - induced swelling . at killing , popliteal and inguinal lymph nodes were removed and stimulated   with 500 g / ml dnp - klh at a cell concentration of 10/ml for   ifn- production and 2  10 for proliferation assay . proliferation assays were pulsed with [ h]thymidine for the last 18 h of a   72-h incubation . stat4 , stat6 mice were generated by mating stat4   and stat6 mice at the third backcross generation to balb / c and   intercrossing the resulting compound heterozygotes . controls   were inbred balb / c and 129/svj mice , both of which gave   comparable results in each of the assays tested . differentiation of t cells   and assay of cytokines were performed as described ( 12 , 17 ) . in   brief , cells were activated with 1 g / ml plate - bound anti - cd3 . where indicated , il-12 was added at 1 ng / ml , and anti  ifn- ,   anti  il-4 , and anti  il-12 were added at 10 g / ml ( genzyme   corp . , cambridge , ma ) . after 1 wk in culture , cells were   washed and restimulated with plate - bound anti - cd3 for 24 h.   supernatants were harvested , and ifn- and il-4 production   were quantified by elisa . purification of cd4 and cd62l   cells was done by positive selection using magnetic beads ( miltenyi biotec , inc . , auburn , ca ) according to the supplier 's instructions . for the purification of naive cells , pooled spleens and lymph nodes were   depleted before positive selection with 2.4g2 ( anti - cd16/32 ) ,   2.43 ( anti - cd8 ) , and m5114 ( anti  i - a ) coupled to magnetic   beads . after 1 wk , cells were restimulated with plate - bound anti - cd3 for 6 h and plated in dilution on anti ifn-   ( r4/6a2)-coated immobilon - p membranes in a 96-well plate for   18 h. cells were washed away , and bound cytokine was detected   with biotinylated anti ifn- ( pharmingen , san diego , ca ) ,   avidin  alkaline phosphatase ( sigma chemical co. , st . louis ,   mo ) , and a stable bcip / nbt mix ( gibco brl , gaithersburg ,   md ) . spots were developed for 3060 min , washed with distilled   water , and counted under a dissecting microscope . male mice were immunized with 100 g dnp - klh ( calbiochem corp . , la jolla , ca )   emulsified in cfa ( sigma chemical co. ) , at the base of the tail . after 6 d , footpad thickness was measured using a spring - loaded   caliper , and mice were challenged with 50 g dnp - klh in one   foot and pbs in the contralateral appendage . specific   swelling was determined by subtracting nonspecific swelling in   the pbs - injected foot from antigen - induced swelling . at killing , popliteal and inguinal lymph nodes were removed and stimulated   with 500 g / ml dnp - klh at a cell concentration of 10/ml for   ifn- production and 2  10 for proliferation assay . proliferation assays were pulsed with [ h]thymidine for the last 18 h of a   72-h incubation . to further examine the mechanism by which stat4 and   stat6 regulate the differentiation of th cell subsets , we generated stat4 , stat6 mice by intercrossing compound   heterozygotes . double - deficient mice were produced at   the expected mendelian frequency and were grossly indistinguishable from their control littermates . all lymphoid   organs in stat4 , stat6 mice had normal cellularity and   contained percentages of cd3 , cd4 , cd8 , and b220   cells comparable to that seen in control littermates ( data not   shown ) . spleen cells from control , stat4 , stat6 , and stat4 ,   stat6 mice were activated in vitro with plate - bound   anti - cd3 in the absence of exogenous cytokines or antibodies to determine their developmental potential . after 1 wk   in culture , cells were washed and restimulated with plate - bound anti - cd3 for 24 h. supernatants were harvested ,   and ifn- and il-4 production were quantified by elisa . as expected , lymphocytes from control mice secrete both   ifn- and il-4 when activated in vitro under neutral conditions ( fig . consistent with previous results ( 1214 ,   17 , 18 ) , stat4 lymphocytes produce high levels of il-4   but greatly reduced levels of ifn- , whereas stat6 lymphocytes secrete high levels of ifn- but no il-4 . stat4 ,   stat6 lymphocytes also do not produce il-4 when activated in vitro , supporting the notion that stat6 is essential   for the differentiation of th2 cells . however , in contrast to   stat4 lymphocytes , stat4 , stat6 lymphocytes secrete   levels of ifn- comparable to that produced by control or   stat6 lymphocytes cultured under identical conditions . similar results were seen when cultures were either initiated with purified naive t cells obtained by selection of   cd4 cd62l cells or when cd4 t cells were purified   after the initial 7-d culture period and subsequently restimulated with anti - cd3 for 24 h ( data not shown ) . il-4 signaling has a dominant effect over il-12 stimulation in determining the outcome of th cell differentiation   ( 21 ) , and the results shown in fig . 1 suggest that the presence of il-4 signaling through stat6 may impair the ability   of cells to develop the th1 cell phenotype . to test this hypothesis , we attempted to recapitulate the ifn-secreting   phenotype of stat4 , stat6 lymphocytes by activating   stat6-expressing cells in vitro in the presence of cytokine - specific antibodies to eliminate stat activation . in the absence of exogenous cytokines and antibodies , in vitro activated stat4 , stat6 lymphocytes secrete more ifn-   than stat4 lymphocytes ( figs . 1 and 2 a ) . when stat4   lymphocytes are activated in the presence of anti il-4 , the   levels of ifn- produced are nearly equal to those seen in   cultures of activated stat4 , stat6 lymphocytes ( fig . 2 a ) . similarly , when control cells are activated in the presence of anti  il-4 plus anti  il-12 , the levels of ifn- produced are indistinguishable from those seen in cultures of   activated stat4 , stat6 lymphocytes . these results are   consistent with a recent report demonstrating ifn- production in the presence of neutralizing antibodies to both   il-4 and il-12 ( 23 ) . thus , substantial amounts of ifn-   can be produced by activated cells lacking stat4 as long as   stat6 is not activated in response to il-4 stimulation . ifn- has been suggested to have an important role in   priming cells for subsequent ifn- production , although it   remains controversial whether it does so by a direct or indirect mechanism . ifn- to primary cultures of stat4 , stat6 lymphocytes activated under neutral conditions did not affect the amount of ifn- produced   by these cells after restimulation with anti - cd3 ( fig . this is in contrast to the lower amount of ifn- produced   by control cells after restimulation when anti these results demonstrate   that the ability of stat4 , stat6 lymphocytes to acquire an   ifn-secreting phenotype is not driven by endogenous   ifn- , and are consistent with studies demonstrating that   the role of ifn- in th1 cell differentiation is indirect , upregulating il-12r2 expression so that cells become responsive to il-12 stimulation ( 7 ) . il-12 stimulation of activated t cells has been shown to   prime cells for high levels of ifn- production after subsequent restimulation ( 24 ) . when compared with control   lymphocytes cultured under th1 cell  promoting conditions ( il-12 plus anti il-4 ) , the levels of ifn- produced   by activated stat4 , stat6 lymphocytes were decreased   four- to fivefold ( fig . the amount of ifn- produced by stat4 , stat6 lymphocytes cultured in the presence of il-12 does not increase over that seen when no exogenous cytokines are added ( data not shown ) . the increased   amounts of ifn- seen in cultures of th1 cell  skewed   control lymphocytes relative to those seen in cultures of   stat4 , stat6 lymphocytes could be the result of an increase either in the number of differentiated th1 cells or in   the amount of ifn- produced on a per cell basis . to distinguish between these possibilities , the number of ifn-secreting lymphocytes in these cultures was quantified   by enzyme - linked immunospot ( elispot ) . spleen cells   from control mice were differentiated into th1 cells in   vitro by activation with plate - bound anti - cd3 in the presence of il-12 and anti il-4 while spleen cells from stat4 ,   stat6 , and stat4 , stat6 mice were activated in the   absence of exogenous cytokines and antibodies . after 1 wk   in culture , cd4 t cells were purified and restimulated with   plate - bound anti - cd3 for 6 h. cells were then transferred   in dilution to plates containing anti ifn- ( r4/6a2)- coated immobilon - p membranes at the bottom of the wells   and incubated overnight . in contrast to cultures of stat4   lymphocytes where very few ifn-secreting cells were   detected , large numbers were seen in cultures of th1 cell skewed control and unskewed stat6 lymphocytes ( fig . strikingly , cultures of stat4 , stat6 lymphocytes   had numbers of ifn-secreting cells comparable to that   seen in cultures of th1 cell  skewed control and unskewed   stat6 lymphocytes . the spots produced in wells containing th1 cell  skewed control cells were both larger and   darker than those seen in wells containing stat4 , stat6   lymphocytes , supporting the conclusion that il-12 stimulation and stat4 activation lead to higher levels of ifn-   production on a per cell basis rather than a substantial increase in the number of differentiated th1 cells .   given that ifn-secreting cells could be generated   from stat4 , stat6 mice in vitro , we sought to determine   whether stat4 , stat6 mice could mount a th1 cell response in vivo . to investigate this possibility , we examined   a classic th1 cell  mediated immune response , the delayed - type hypersensitivity ( dth ) reaction . control , stat4 ,   and stat4 , stat6 mice were immunized with 100 g   dnp - klh in cfa . 6 d later , mice were challenged by   footpad injection with either aqueous antigen or pbs as   control . all animals were killed 48 h after challenge , and   draining popliteal and inguinal lymph node cells were removed and restimulated in vitro with antigen . lymphocytes from all three genotypes of mice produced a similar in   vitro proliferative response to antigen ( fig . 4 a ) , demonstrating comparable in vivo generation of antigen - specific   t cells . as expected , the levels of ifn- produced after antigen stimulation in vitro varied among the groups . stat4   lymphocytes produced < 10% of the ifn- secreted by   control cells ( fig . stat4 , stat6 lymphocytes also secreted less ifn- than control cells , although the levels   were fourfold higher than that seen in cultures of stat4   lymphocytes ( fig . finally , footpad thickness was measured before and 48 h after challenge , and specific swelling   was calculated ( fig . 4 c ) . footpad swelling was significantly different in all three groups using the kruskal - wallis   test ( p < 0.005 ) . furthermore , pairwise comparisons using   the wilcoxon - mann - whitney test showed that swelling in   stat4 mice was significantly reduced compared with   control ( p < 0.005 ) and stat4 , stat6 mice ( p < 0.05 ) . thus , functional th1 cells capable of eliciting a dth response can be generated in vivo in stat4 , stat6 mice . our results demonstrate that the mechanisms that regulate the differentiation of th1 and th2 cells are not the   same . previous studies have shown that stat6 is essential for   the development of th2 cells ( 1214 ) , presumably through   the transcriptional regulation of as yet unidentified genes   involved in the differentiation process . as il-4secreting   th2 cells are not generated from stat4 , stat6 lymphocytes , stat6 is required for their generation even in the   absence of stat4 . in contrast , both stat4-dependent and   -independent pathways appear to exist for the development   of ifn-producing th1 cells . il-12 stimulation and stat4   activation lead to the differentiation of th1 cells capable of   secreting large amounts of ifn-. however , in the absence   of il-4 and il-12mediated signals , the natural tendency   of cd4 th cells is to develop into ifn-secreting cells . thus , il-4mediated activation of stat6 , but not il-12 induced activation of stat4 , results in a differentiative signal   to th cells . mechanistically , this would explain the observation that il-4 signaling has a dominant effect over il-12   stimulation in determining the outcome of th cell differentiation ( 21 ) . interestingly , a paradigm whereby the decision between two possible developmental pathways is dictated by the presence or absence of one signaling cascade   occurs in other differentiative processes , including sex determination . sry expression activates a genetic program   which results in male gonadal development ; female gonads   develop in its absence ( 25 ) . although our results suggest that the ability of il-12 to   promote th1 differentiation may be indirect , by priming   for high levels of ifn- production which would inhibit   th2 development and allow the outgrowth of th1 cells ,   they do not minimize the role of il-12 and stat4 activation   in normal th cell development and function . stat4 activation is clearly involved in regulating the production of   ifn- and potentially other factors such as tnf- that are   important in many th1 cell  mediated responses . nevertheless , the demonstration that functional th1 cells can develop in the absence of stat4 has important implications for   the design of therapeutic strategies that aim to alter immune function by targeting stat proteins in vivo . spleen cells from mice with the indicated genotypes were activated with   plate - bound anti - cd3 in the absence of exogenous cytokines or antibodies for 1 wk . supernatants were collected 24 h after restimulation with   plate - bound anti - cd3 , and cytokine levels were quantified by elisa . ( a )   spleen cells from mice with the indicated genotypes were activated with   plate - bound anti - cd3 in the presence of antibodies as indicated for 1 wk . cells were restimulated with plate - bound anti - cd3 , and supernatants   were collected 24 h later to quantify ifn- levels by elisa . ( b ) cells   were activated as in a in the absence ( white bars ) or presence ( black bars ) of   anti ifn-. cells were restimulated with plate - bound anti - cd3 , and supernatants were collected 24 h later to quantify ifn- levels by elisa . spleen cells from   mice with the indicated genotypes were activated with plate - bound anti - cd3 in the presence of exogenous cytokines or antibodies as indicated   for 1 wk . ( a ) cells were restimulated with plate - bound anti - cd3 , and   supernatants were collected 24 h later to quantify ifn- levels by elisa . ( b ) cells were restimulated with plate - bound anti - cd3 for 6 h and then   transferred to plates containing anti ifn- ( r4/6a2)-coated membranes   for 18 h. elispots were developed with a stable bcip / nbt reagent   and counted under a dissecting microscope . ( a ) draining   lymph node cells from immunized mice were stimulated in vitro in the   absence ( white bars ) or presence ( black bars ) of 500 g / ml dnp - klh . cells in round - bottomed 96-well plates were pulsed with [ h]thymidine   for the last 18 h of a 72-h incubation . ( b ) supernatants from dnp - klh stimulated lymph node cells were analyzed for ifn- levels by elisa . ( c ) specific footpad swelling of each mouse was determined by subtracting nonspecific swelling in the pbs - injected foot from the dnp - klh induced swelling . each data point represents one mouse ; horizontal lines   indicate averages , and results are pooled from two independent experiments .
the differentiation of t helper ( th ) cells is regulated by members of the signal transducer and   activator of transcription ( stat ) family of signaling molecules . 
 we have generated mice lacking both stat4 and stat6 to examine the ability of th cells to develop in the absence of these   two transcription factors . 
 stat4 , stat6/ lymphocytes fail to differentiate into interleukin   ( il)-4secreting th2 cells . 
 however , in contrast to stat4/ lymphocytes , t cells from stat4 ,   stat6/ mice produce significant amounts of interferon ( ifn)- when activated in vitro . 
 although stat4 , stat6/ lymphocytes produce less ifn- than il-12stimulated control lymphocytes , equivalent numbers of ifn-secreting cells can be generated from cultures of stat4 ,   stat6/ lymphocytes activated under neutral conditions and control lymphocytes activated   under th1 cell  promoting conditions . 
 moreover , stat4 , stat6/ mice are able to mount an in   vivo th1 cell  mediated delayed - type hypersensitivity response . these results support a model   of th cell differentiation in which the generation of th2 cells requires stat6 , whereas a stat4-independent pathway exists for the development of th1 cells .