recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .
because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .
previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .
the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .
consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .
this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .
we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .
these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .
no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .
( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .
although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .
thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .
we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .
because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .
the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .
the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard
, it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .
to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .
2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .
we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .
foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .
2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1
was confirmed by genomic pcr ( not depicted ) . when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .
foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .
 foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .
in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) . thus , foxp3 gene ablation in thymic epithelium does not result in autoimmunity .
disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .
( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .
thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .
right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .
( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .
( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .
lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .
( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .
these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .
all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .
although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .
in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies
, we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .
as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .
thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells .
in these experiments , foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .
in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .
the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .
lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .
major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .
these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .
thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .
( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .
( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .
( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .
in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon
, we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .
the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .
3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .
no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .
4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .
the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .
together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .
foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .
( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .
collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .
this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data
are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .
thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .
recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .
because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .
previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .
the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .
consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .
this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .
we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .
these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .
no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .
( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .
although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .
thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .
we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .
because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .
the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .
the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard
, it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .
to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .
2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .
we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .
foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .
2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1
when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .
foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .
 foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .
in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) .
disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .
( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .
thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .
right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .
( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .
( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .
lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .
( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .
these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .
all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .
although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .
in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies
, we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .
as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .
thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells . in these experiments ,
foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .
in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .
the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .
lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .
major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .
these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .
thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .
( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .
( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .
( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .
in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon
, we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .
the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .
3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .
no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .
4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .
the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .
together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .
foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .
( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .
collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .
this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data
are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .
thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .
foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .
b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .
bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .
experimental mice were age and sex matched and housed in specific pathogen - free conditions .
disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .
all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .
510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .
thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .
after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .
thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti 
s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer .
foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .
b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .
bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .
experimental mice were age and sex matched and housed in specific pathogen - free conditions .
disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .
all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .
510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .
thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .
after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .
thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti 
s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer .
 foxp3 is essential for the commitment of differentiating thymocytes to the regulatory cd4 + t ( t reg ) cell lineage . in humans and mice with a genetic foxp3 
 deficiency , absence of this critical t reg cell population was suggested to be responsible for the severe autoimmune lesions . 
 recently , it has been proposed that in addition to t reg cells , foxp3 is also expressed in thymic epithelial cells where it is involved in regulation of early thymocyte differentiation and is required to prevent autoimmunity . here , we used genetic tools to demonstrate that the thymic epithelium does not express foxp3 . furthermore , we formally showed that genetic abatement of foxp3 in the hematopoietic compartment , i.e. in t cells , is both necessary and sufficient to induce the autoimmune lesions associated with foxp3 loss . 
 in contrast , deletion of a conditional foxp3 allele in thymic epithelial cells did not result in detectable changes in thymocyte differentiation or pathology . 
 therefore , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage , whereas there is no role for foxp3 in thymic epithelial cells . 
RESULTS AND DISCUSSION
No detectable Foxp3 protein expression in the thymic epithelium
Disruption of Foxp3 in thymocytes is necessary and sufficient to cause autoimmune syndromes
Distorted thymopoiesis associated with Foxp3 deficiency is secondary to lymphoproliferative autoimmune syndrome
MATERIALS AND METHODS
Mice.
Flow cytometry and immunofluorescence.
Online supplemental material.