signal transducer and activator of transcription ( stat ) family proteins are requisite molecules in the development of t helper subsets . stat1 and stat4 are required for the development of th1 cells that secrete ifn and mediate immunity to intracellular pathogens . stat3 promotes th17 development and the secretion of il-17 , a cytokine required for immunity to extracellular bacterial and fungal infections . stat6 is required for the differentiation of th2 and th9 cells , which contribute to extracellular parasite immunity . the in vivo function of stat6 has been defined largely following the generation of gene - deficient mice . stat6 mice develop normally , breed as homozygotes , but have altered immune responses in vivo . in the absence of stat6 , mice are resistant to the development of allergic inflammation , and have exaggerated responses to inflammatory insults resulting in greater immunity to intracellular pathogens , but greater susceptibility to autoimmune inflammation , with differences in phenotype attributed to different mouse lines with targeted stat6 alleles . some of the studies that defined these functions utilized experiments involving adoptive transfer of lymphocytes , or where cells or tumors were adoptively transferred into stat6 hosts . whether cells behave normally in these transfer experiments has not been extensively examined . in experiments to test if deficiency in stat4 or stat6 affected development of ctl specific for non - classical mhc molecules , we observed autoreactivity of stat6 ctl for stat6-expressing cells . the development of stat6-specific reactivity by stat6 cd8 t cells might have important implications for the interpretation of experiments where stat6 cd8 t cells are exposed to stat6 expressing cells in vivo . to examine the responses of balb / c , stat4 and stat6 mice ( h2 , qa1 and mta ) as responders against nzb / b1nj ( nzb ) ( h2 , qa1 and mta ) stimulator cells , we generated ctl lines from each strain . we found no consistent differences in the ability of the stat4 or stat6 cd8 t cells to develop responder ctl against qa1 or mta antigens , compared with balb / c t cells ( fig . however , we did observe that stat6 anti - nzb ctl could lyse target cells from stat4 and balb / c mice ( fig . in contrast , balb / c and stat4 responders did not lyse balb / c , stat4 or stat6 cells ( fig . the ability of stat6 ctl to lyse these target cells was unexpected because stat4 , stat6 and balb / c are syngeneic , differing only in the regions adjacent to the targeted stat4 or stat6 alleles . ctl lines generated from secondary mlcs were assayed against stat6 and stat6 con a blast ( cab ) target cells in a standard cr release assay . ( a ) balb / c , ( b ) stat4 and ( c ) stat6 anti - nzb responder ctl were assayed against nzb , balb / c , stat4 or stat6 cab target cells . lysis of nzb cells in ( a ) and ( b ) represents mta and/or qa1 restricted responses . stat6 anti - nzb ctl ( c ) lyse nzb , stat4 and balb / c , but not stat6 target cells , indicating lysis of stat6 cells . ( d ) stat6 anti - balb / c , ( e ) stat6 anti - stat4 ctl and ( f ) stat4 anti - stat6 mlc were assayed against balb / c , stat4 , stat6 , h2 , b6.tla or h2 ( b10.riii ) cab target cells . all data are the mean  sd of triplicate assays and are representative of three or more experiments . to confirm these data and to rule out problems due to the use of one specific mouse strain ( nzb ) , we examined responses of balb / c , stat4 and stat6 as stimulators and responders of ctl in similar assays . stat6 anti - balb / c and stat6 anti - stat4 responder ctl also lyse stat4 and balb / c target cells ( fig . 1d and e ) , whereas stat4 anti - stat6 ctl do not lyse stat4 , stat6 or balb / c target cells ( fig . to determine the restricting mhc molecule involved in these responses , we examined mice expressing different mhc haplotypes and found the responses were restricted to h2 . stat6 anti - stat4 or anti - balb / c ctl lysed target cells from h2 ( balb / c , stat4 and nzb ) mouse strains ( figs .  1d and e and  2 ) , but not cells expressing h2 ( b6.tla ) , h2 ( b10.riii ) , or h2 ( c3h / hej and b10.br ) ( figs . 1d and e and 2c ) , suggesting that the ctl were not alloreactive . to determine whether the restricting element(s ) are h2-k , -d and/or -l molecules , we further compared stat6 ctl responses against a / j ( h2-k , -d and -l ) , balb / c ( h2-k , -d and -l ) and balb.dm2 ( h2-k , -d and l null ) target cells using two different stat6 ctl lines . 2a and b ) are recognized by the polyclonal responder ctl . since h2 cells ( c3h.hej or b10.br ) 2c ) , recognition of cells from a / j ( h2-k , -d and -l ) by the 241.44 line shows restriction to h2-d and/or -l ( fig . in contrast , the lack of response of the 240.43 line demonstrates the failure of these cells to recognize h2-d , suggesting the reactivity against another h2 class i molecule , most likely h2-k ( fig . restricted ctl were also able to lyse balb.dm2 cells , which express h2-k and h2-d , but not h2-l , further suggesting that h2-l restriction is not a prominent reactivity in these ctl lines ( fig . thus , stat6 ctl can develop with specificities restricted to h2-k , h2-d or h2-l . stat6   anti - balb / c ctl lines ( a ) 241.44 , ( b  d ) 240.43 were incubated with target cells from h2 , or h2 expressing mouse strains in a standard cr release assay . ctl line 241.44 ( a ) ( h2-d and -l - restricted ) lyses a / j ( h2-k , -d and -l ) target cells , whereas ctl line 240.43 ( b ) ( h2k - restricted ) did not lyse a / j or ( d ) balb.dm2 ( h2kd ) . neither line lysed h2 target cells ( c , data not shown ) . data are the mean  sd of triplicate assays and are representative of two or more experiments . confirming these data , in figure  3 we show that pre - incubating a / j or stat4 target cells with monoclonal antibodies reactive with h2-k , -d and h2-l molecules blocks target cell recognition and lysis by these ctl . lysis of stat4 or a / j target cells is blocked by monoclonal antibody 30 - 5 - 7s that reacts with h2-d or -l but not h2-k or h2-k , confirming restriction of this line to h2-l ( fig .  3a and b ) . in figure  3c , lysis of stat4 targets by the stat6 anti - balb 240.43 line is not blocked by 30 - 5 - 7s , but rather is blocked by the two antibodies which bind h2-k molecules , 34 - 1 - 2s and 34 - 7 - 23s , demonstrating restriction to h2-k . taken together , the data clearly demonstrate that stat6 anti - balb / c or anti - stat4 ctl are restricted to either h2-k ,- d or -l . figure  3.stat6 anti - balb / c or stat4 ctl are h2-k , -d or l restricted . ( a  c ) stat6 anti - balb / c ctl lines 241.44 ( a and b ) , and 240.43 ( c ) were incubated with cab target cells from stat4 ( a and c ) or a / j ( b ) mice and cytotoxicity was measured in a standard cr release assay . the ability of anti - mhc i antibodies to block lysis was tested . data are the mean  sd of triplicate assays and are representative of two or more experiments . since the primary difference between balb / c or stat4 and stat6 mice is the stat6 gene product , we tested whether stat6 ctl recognized peptides derived from the stat6 protein . we tested a stat6 peptide previously shown to be presented by tumor cells and observed the h2-k restricted ctl recognize stat6 target cells incubated with the sywsdrlil peptide ( fig . 4 ) , which fits the h2-k consensus motif of xyx6(i / l / v)l . we additionally tried several stat6 peptides that fit the h2-d or h2-l consensus motifs , ( xgpx(k / r)x3(l / i / f ) and ( xpx6(l / m / f ) , respectively , but none conferred reactivity . stat6 anti - balb / c ctl 240.43 ( left ) or stat6 anti - stat4 ctl 268 ( middle ) were incubated with stat6 cab target cells at an effector : target cell ratio of 12:1 in a standard cr release assay . cytotoxicity was assessed in the absence or presence of 9-mer peptides from h-2k binding peptides from hiv gp160 ( rgpgrafvti , vgnpiilp ) , or stat6 ( sywsdrlil ) . the alloreactive b10 ( h2 ) anti - balb / c ( h2 ) ctl 262 ( right ) did not require peptide for lysis and was not affected by target cell incubation with peptides . data are the mean  sd of triplicate assays and are representative of three or more experiments . these data demonstrate responses by stat6 ctl against h2 cells from stat6-expressing mouse strains including balb / c , nzb and stat4 . the polyclonal ctl populations generated from the stat6 responder spleen cells initially recognized k , -d and -l restricted antigens , although with weekly passage in vitro some populations eventually self - selected for a predominantly h2-k , -d or -l restricted phenotype . use of target cells from recombinant inbred mouse strains and blocking by antisera specific for h2 class i molecules confirms their mhc restriction . the h2-k restricted ctl recognized the stat6-derived peptide sywsdrlil . these data clearly illustrate the importance of running negative control experiments , particularly when working with genetically altered strains of mice . in some situations for example , in evaluations of immune responses within stat6 mice against exogenous antigens or allergens , or in studies of inflammation or hypersensitivity , the results may show no effects easily attributable to altered peptide presentation by mhc class i molecules on stat6 cells . however , it is possible that results could be influenced by subtle changes in reactivity of cells maturing in the stat6 environment and/or by the unusual peptide repertoire transferred on h2 molecules with stat6 cells . such changes in immune responses due to these altered peptide expression might be negligible or difficult to identify and quantitate , but should nonetheless be considered . however , the situation could be more complicated in adoptive transfer experiments . although most adoptive transfer experiments involving stat6 mice use transferred purified cd4 t cells , in experiments where stat6 splenocytes or cd8 t cells are transferred to wild type recipients , it is possible that stat6-specific cd8 t cells could mediate a graft vs. host response , resulting in background inflammation , apart from the specific responses being studied . conversely , when stat6-expressing cells are transferred into stat6 recipients , it is possible that stat6 cd8 t cells reactive against stat6 peptides might eliminate transferred cells , masking some functional activity of the transferred cells . the results from most of these experiments are consistent with a phenotype arising from stat6-deficiency , suggesting that the contribution of anti - stat6 immunity may be modest . the contribution of anti - stat6 ctl responses may be more important in studies of graft rejection . two reports demonstrated that stat6 mice demonstrated shorter graft survival times in models of cardiac transplant with minor histocompatibility differences , and where ctla4-ig was used to block graft rejection . although it is possible that some of this increase is due to the increased propensity of stat6 t cells to develop into inflammatory t cells , it is likely that anti - stat6 immunity may also be contributing to the observed phenotype . the anti - stat6 immunity that develops in mice lacking endogenous stat6 clearly contributes to the increased anti - tumor immunity in stat6 mice . have shown delayed and reduced primary mammary carcinoma growth in stat6 mice using 4t1 , a balb / c mammary carcinoma that is usually malignant , non - immunogenic and metastatic . the authors hypothesize that the deletion of the stat6 gene facilitates development of potent anti - tumor immunity via a cd4-independent pathway . our data suggest that immunization with the h2 stat6-expressing tumor might simply stimulate cd8 ctl specific for h2 restricted antigens from stat6-expressing cells , which lyse the cells regardless of expression of tumor antigens . indeed , jensen et al . demonstrate that enhanced immunity to 4t1 cells in stat6 mice is dependent upon the stat6 peptide used in our studies . similarly , kacha et al . show that effector cells from stat6 mice have increased lytic activity and produce increased levels of ifn in response to the h2 tumor p1.htr . thus , it is not clear that these studies actually demonstrate an effect of stat6 function , but rather may reflect immune responses that are not tolerant to stat6 peptides presented in the context of mhc i. it is important to note that  autoreactivity  to a missing self - protein is not a ubiquitous phenomenon . although there is clearly an anti - stat6 ctl response in stat6 mice , there is no corresponding anti - stat4 ctl response in stat4 mice . it is possible that stat4 peptides do not compete effectively for binding to mhc class i such that no reactive cells are positively selected in the thymus . alternatively , mhc class i binding peptides from stat4 might be homologous enough to other endogenous peptides that reactive t cells are negatively selected in the thymus . the differences between the phenotypes of stat4 and stat6 ctl illustrate that the autoreactive phenomenon described here must be tested empirically for each mutant strain being studied . in summary , our data illustrate the importance of demonstrating maintenance of self - tolerance in mice made defective in a particular gene by homologous recombination . particularly in genes that have immune function , where common techniques involve transplantation and adoptive transfer , it is critical to define the ability of t cells to recognize peptides from the targeted proteins as foreign . failure to do so could result in misinterpretation of how a gene of interest contributes to immune responses . stat4 and stat6 mice were generated and backcrossed for at least 10 generations to balb / c mice as described previously . b10.br , c3h / hej breeder mice and balb / c.dm2 spleen cells were the kind gift of dr james forman . balb / c , c57bl/6j , nzb / b1nj , c3h / hej , b10.br and a / j breeder mice were purchased from the jackson laboratory . all animals were bred and housed in the iusm - evansville animal facility and procedures were approved by the iusm iacuc . where indicated , mice were immunized intraperitoneally with 25  10 spleen cells in 0.5 ml balanced salt solution , rested for at least 7 d , euthanized , and their spleens removed for the in vitro generation of ctl . using specific strain combinations as described , ctl were generated in secondary mlc as described . briefly , 5  10 responder and 5  10 irradiated ( 3000rad ) stimulator spleen cells were incubated for 67 d in rpmi 1640 supplemented with 710% fetal bovine serum , 1% l - glutamine and 50 ng / ml 2-mercaptoethanol at 37c in 7% co2 . some ctl lines were re - stimulated weekly with irradiated stimulator cells in supplemented mishell - dutton medium that also contained t cell growth factors from ( nh4)2so4-precipitated supernatants of phorbol myristic acetate - stimulated el4.il2 cells . generation of concanavalin a ( cona ) blast cells ( cab ) , cr labeling of target cells , the standard 46 h cr release assay , controls , and the method for determination of the % specific lysis of target cells have been described . standard error was computed by propagation of errors as previously described.cr release assays for analysis of peptide binding , peptide blocking and antibody blocking were performed using pre - incubations as previously described . rma - s - d , t2-d , e3 and ltkd cell lines were provided by dr d. marguiles . using anti - cd4 antibody gk1.4 ( pharmingen ) and anti - cd8 antibody yts169.4 ( kindly provided by dr . stat4 and stat6 mice were generated and backcrossed for at least 10 generations to balb / c mice as described previously . b10.br , c3h / hej breeder mice and balb / c.dm2 spleen cells were the kind gift of dr james forman . balb / c , c57bl/6j , nzb / b1nj , c3h / hej , b10.br and a / j breeder mice were purchased from the jackson laboratory . all animals were bred and housed in the iusm - evansville animal facility and procedures were approved by the iusm iacuc . where indicated , mice were immunized intraperitoneally with 25  10 spleen cells in 0.5 ml balanced salt solution , rested for at least 7 d , euthanized , and their spleens removed for the in vitro generation of ctl . using specific strain combinations as described , ctl were generated in secondary mlc as described . briefly , 5  10 responder and 5  10 irradiated ( 3000rad ) stimulator spleen cells were incubated for 67 d in rpmi 1640 supplemented with 710% fetal bovine serum , 1% l - glutamine and 50 ng / ml 2-mercaptoethanol at 37c in 7% co2 . some ctl lines were re - stimulated weekly with irradiated stimulator cells in supplemented mishell - dutton medium that also contained t cell growth factors from ( nh4)2so4-precipitated supernatants of phorbol myristic acetate - stimulated el4.il2 cells . generation of concanavalin a ( cona ) blast cells ( cab ) , cr labeling of target cells , the standard 46 h cr release assay , controls , and the method for determination of the % specific lysis of target cells have been described . standard error was computed by propagation of errors as previously described.cr release assays for analysis of peptide binding , peptide blocking and antibody blocking were performed using pre - incubations as previously described . rma - s - d , t2-d , e3 and ltkd cell lines were provided by dr d. marguiles . flow cytometry analyses of ctl lines was performed as previously described . using anti - cd4 antibody gk1.4 ( pharmingen ) and anti - cd8 antibody yts169.4 ( kindly provided by dr .
the generation of germline gene mutations in mice has been an invaluable tool for experimental biology . 
 however , studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations . 
 we observed that cd8 + t cells from stat6/ mice displayed  autoreactivity  to stat6-expressing cells , associated with specific stat6 peptides binding to mhc class i molecules . 
 these results suggest caution in interpreting experiments where stat6-expressing cells are transferred into stat6/ mice , or where adoptive transfer of stat6/ lymphocytes is performed . 
 our results further highlight additional considerations when studying immune responses involving cell transfer into gene - deficient mice .