c57bl/6 ( b6 ) mice and their ly-5.2 congenic variant ( b6.ly-5.2 ) were purchased from the national cancer institute animal facility ( frederick , md ) .
i / ii  deficient mice on a b6 background ( 12th backcross , ci / ii ) were purchased from taconic farms .
the b6 thy-1 congenic variant strain , b6.plthy-1 cy , as well as the mice genetically deficient in the cd4 gene 18 , which were used as class ii recipients , and the natural h-2k and h-2 i - a coisogenic variants of b6 mice , b6.ch-2 ( bm1 ) and b6.ch-2 ( bm12 ) , respectively , which were used as stimulators in the mlr , were purchased from the jackson laboratory .
female mice , between 7 and 10 wk of age , were used in all experiments . the allele - specific anti - ly5.1 ( 104 - 2.1 ) and anti - ly5.2 ( a20 - 7.1 ) mabs ( 19 ; obtained from dr .
u. hammerling , memorial sloan - kettering cancer center ) were produced as ascitic fluid , purified , and conjugated to fitc or biotin in our laboratory .
anti - cd4conjugated microbeads , the vs+ and rs+ separation columns , and the supermacs separation device were purchased from miltenyi biotec and were used per the manufacturer 's instructions . all other antibodies and reagents , as well as
the cytotoxic elimination of cd8 cells by mab + c and separation of the resulting population into cd8 and cd8 by panning , were described previously 9 .
cd4 populations from each subset were then purified to > 99% purity , either by flow cytometry ( fcm ) using a mo - flo sorter ( cytomation ) , or by two sequential immunomagnetic bead sorting steps using the vs+ separation columns and anti - cd4conjugated microbeads ( miltenyi biotec ) , as indicated .
for the cell sorting , cells were stained with mabs against cd4 , cd11c , and mhc class ii , and were sorted to obtain a cd4 class ii cd11c fraction . upon reanalysis , these cells contained < 0.1% cells positive for class ii , cd11b , or cd11c , and were used for exp . 2 in table table table .
cd8cd4 cells were used in the intravenous transfer experiment to confirm the stringency of cell purification , and in the intrathymic  spiking  experiment aimed to test the role of contaminating cells ; the cd8cd4 cells were used for intravenous and intrathymic transfers .
b6 mice were depleted of cd4 cells using two intraperitoneal injections of 100 l ( 500 g ) mab gk1.5 in the form of ascites , 2 d apart .
antibody - injected mice were then rested for 4 wk before they entered the experiment to allow elimination of the antibody .
they were subsequently used as recipients for intrathymic transfers or were also thymectomized and used as recipients for the intravenous transfer .
genetically deficient mice ( cd4 , cii , and ci / ii ) used as recipients were not pretreated before intrathymic transfer .
intrathymic injections were performed as described 2021 , with 0.852  10 thymocyte subsets of the b6.ly-5.2 and/or b6.pl origin injected into each thymic lobe .
the thymectomies were performed using the same surgical approach as for the intrathymic injections , except that vacuum suction was applied to remove the organ .
45 mo after intravenous or intrathymic transfer , spleens and mesenteric lymph nodes ( mlns ) from the recipient mice were used to analyze the phenotype and function of cd4 t cells .
phenotypic analysis was performed by fcm , using anti - cd4 and donor- and recipient - specific ly-5 mabs , using a facscan instrument ( becton dickinson ) equipped with the lysys ii software , as described previously 9 . for functional analysis
, donor - derived cd4 cells were magnetically purified from the pooled spleens of the recipients , using fitc - labeled donor allele 
specific mabs , followed by the anti - fitc  conjugated paramagnetic microbeads and the rs+ separation columns ( miltenyi biotec ) .
10 cells / well were seeded in a flat - bottomed 96-well plate , alone or with 2  10 irradiated ( 30 gy ) nucleated spleen cells of bm1 , bm12 , or b6 origin .
culture supernatants were collected 72 h later and frozen for subsequent il-2 quantification by using the il-2dependent ctll indicator line .
5  10 ctll cells were incubated in a 1:1 dilution of the supernatants for 18 h , the last 6 h in the presence of 1 ci of h - tdr ( new england nuclear ) .
the cells were then harvested , and the degree of incorporation of radioactive thymidine was determined in a beta - counter ( amersham pharmacia biotech ) .
c57bl/6 ( b6 ) mice and their ly-5.2 congenic variant ( b6.ly-5.2 ) were purchased from the national cancer institute animal facility ( frederick , md ) .
i / ii  deficient mice on a b6 background ( 12th backcross , ci / ii ) were purchased from taconic farms .
the b6 thy-1 congenic variant strain , b6.plthy-1 cy , as well as the mice genetically deficient in the cd4 gene 18 , which were used as class ii recipients , and the natural h-2k and h-2 i - a coisogenic variants of b6 mice , b6.ch-2 ( bm1 ) and b6.ch-2 ( bm12 ) , respectively , which were used as stimulators in the mlr , were purchased from the jackson laboratory .
female mice , between 7 and 10 wk of age , were used in all experiments .
the allele - specific anti - ly5.1 ( 104 - 2.1 ) and anti - ly5.2 ( a20 - 7.1 ) mabs ( 19 ; obtained from dr .
u. hammerling , memorial sloan - kettering cancer center ) were produced as ascitic fluid , purified , and conjugated to fitc or biotin in our laboratory .
anti - cd4conjugated microbeads , the vs+ and rs+ separation columns , and the supermacs separation device were purchased from miltenyi biotec and were used per the manufacturer 's instructions . all other antibodies and reagents , as well as
the cytotoxic elimination of cd8 cells by mab + c and separation of the resulting population into cd8 and cd8 by panning , were described previously 9 .
cd4 populations from each subset were then purified to > 99% purity , either by flow cytometry ( fcm ) using a mo - flo sorter ( cytomation ) , or by two sequential immunomagnetic bead sorting steps using the vs+ separation columns and anti - cd4conjugated microbeads ( miltenyi biotec ) , as indicated .
for the cell sorting , cells were stained with mabs against cd4 , cd11c , and mhc class ii , and were sorted to obtain a cd4 class ii cd11c fraction . upon reanalysis , these cells contained < 0.1% cells positive for class ii , cd11b , or cd11c , and were used for exp . 2 in table table table .
cd8cd4 cells were used in the intravenous transfer experiment to confirm the stringency of cell purification , and in the intrathymic  spiking  experiment aimed to test the role of contaminating cells ; the cd8cd4 cells were used for intravenous and intrathymic transfers .
b6 mice were depleted of cd4 cells using two intraperitoneal injections of 100 l ( 500 g ) mab gk1.5 in the form of ascites , 2 d apart .
antibody - injected mice were then rested for 4 wk before they entered the experiment to allow elimination of the antibody .
they were subsequently used as recipients for intrathymic transfers or were also thymectomized and used as recipients for the intravenous transfer .
genetically deficient mice ( cd4 , cii , and ci / ii ) used as recipients were not pretreated before intrathymic transfer .
intrathymic injections were performed as described 2021 , with 0.852  10 thymocyte subsets of the b6.ly-5.2 and/or b6.pl origin injected into each thymic lobe .
the thymectomies were performed using the same surgical approach as for the intrathymic injections , except that vacuum suction was applied to remove the organ .
45 mo after intravenous or intrathymic transfer , spleens and mesenteric lymph nodes ( mlns ) from the recipient mice were used to analyze the phenotype and function of cd4 t cells .
phenotypic analysis was performed by fcm , using anti - cd4 and donor- and recipient - specific ly-5 mabs , using a facscan instrument ( becton dickinson ) equipped with the lysys ii software , as described previously 9 . for functional analysis
, donor - derived cd4 cells were magnetically purified from the pooled spleens of the recipients , using fitc - labeled donor allele 
specific mabs , followed by the anti - fitc  conjugated paramagnetic microbeads and the rs+ separation columns ( miltenyi biotec ) .
10 cells / well were seeded in a flat - bottomed 96-well plate , alone or with 2  10 irradiated ( 30 gy ) nucleated spleen cells of bm1 , bm12 , or b6 origin .
culture supernatants were collected 72 h later and frozen for subsequent il-2 quantification by using the il-2dependent ctll indicator line .
5  10 ctll cells were incubated in a 1:1 dilution of the supernatants for 18 h , the last 6 h in the presence of 1 ci of h - tdr ( new england nuclear ) . the cells were then harvested , and the degree of incorporation of radioactive thymidine was determined in a beta - counter ( amersham pharmacia biotech ) .
to investigate the role of mhc class ii molecules in the final maturation of incompletely mature cd8cd4 thymocytes , these cells were intrathymically transferred into recipients genetically deficient in mhc class ii molecules . to that effect , cd8cd4 and cd8cd4 thymocytes were purified ( > 99% ) from b6.ly-5.2 mice , as described in materials and methods .
the key separation step was achieved by panning , and , in our hands , this was found to be the most reliable method of separating these two functionally nonoverlapping subsets . by fcm ,
1 of this paper , data not shown , and figure 1 in reference 9 ) : both subsets were cd8cd4tcr- by fcm , but the cd8cd4 cells contained more cd69 cells and expressed higher levels of cd24 and somewhat lower levels of cd44 than cd8cd4 thymocytes ( not shown ) . however , as shown previously 9 , most of these molecules are expressed by both subsets in at least partly overlapping fashion .
we observed that the most consistent difference in the phenotype of these two subsets was in the level of expression of cd45rb : the levels observed on cd8cd4 cells were more than twice higher than those on cd8cd4 thymocytes ( mean fluorescence intensity [ mfi ] 151 and 341 , respectively ; fig .
this feature correlates very well with the difference in functional responsiveness of the two subsets and the function of cd45 : the former subset is functionally mature , whereas the latter can not respond with the full spectrum of functional activities 12 . to further confirm the identity of the above purified cell populations ,
, only the cd8cd4 thymocytes can give long - term ( > 1 mo ) repopulation of the peripheral lymphoid organs , whereas the cd8cd4 cells disappear 34 wk after transfer 9 .
results shown in table   confirmed this finding , and demonstrated that the cd8cd4 thymocyte subset isolated in this study was phenotypically and functionally indistinguishable from the previously described cells 912 .
moreover , results of this experiment demonstrate that the cross - contamination and outgrowth of the contaminating cells between the two subsets isolated as described in materials and methods was negligible or nonexistent , as the putative progeny of the cross - contaminating cd8cd4 cells was not detectable 45 mo after injection of purified cd8cd4 cells .
cd8cd4 thymocytes from the same cellular preparation were also injected into the thymi of control class ii cd4 recipients , which express wild - type levels of the mhc class ii molecules ( but lack cd4 t cells , thus facilitating the detection of cd4 progeny of the injected cells ) , or of the cii recipients , which genetically lacked these molecules ( and consequently only had very low cd4 numbers , owing to a severe defect in cd4 t cell positive selection ) . in both types of recipient , injected immature cd8cd4 thymocytes yielded long - lived progeny that populated the peripheral lymphoid organs ( table , exp . 1 ) . therefore , class ii molecules appeared not to be necessary either for the terminal differentiation of cd4 thymocytes or for their survival in the periphery .
again , a caveat to this conclusion was the long time course of our experiment , which was conducive for the outgrowth of a minor population of contaminating cd8 cd4 thymocytes .
although the results shown in table   strongly argue against this possibility , we performed another control experiment in which we deliberately spiked the transferred cd8cd4 cells with thy-1 congenic ( thy-1.1 ) cd8cd4 thymocytes .
when we added 1% cd8cd4 thymocytes to the sorted > 99% pure cd8cd4 thymocytes , we could not detect the progeny of the admixed thy-1.1 cells ( table ) .
the progeny of 10% contaminating cells was detectable , but these cells showed no selective proliferative advantage : their representation remained at or below the 10% level among the transferred thymocytes even after 5 mo in vivo ( table ) .
therefore , the above results can not be accounted for by the contaminating mature thymocytes .
another possibility was that we transferred a substantial number of class ii cells with our thymocytes .
however , cells transferred in exp . 2 ( table ) were sorted to exclude class ii and cd11c cells , and , after sorting , contained no cells positive for class ii , for the dendritic cell ( dc ) marker cd11c , or the macrophage ( m ) marker cd11b ( < 0.1% in exp . 2 , table   [ see legend ] ) .
coupled with the fact that the turnover of class ii cells ( dcs , b cells , and m ) is relatively rapid and certainly complete within 1 mo , it is extremely unlikely that those cells could have promoted the final maturation of cd4 thymocytes in the thymus and/or the periphery .
a possibility also existed that mhc class i molecules may provide critical signals to keep cd4 t cells alive in the peripheral organs . to test this possibility , we used ci / ii mice as recipients for thymocytes .
interestingly , upon such transfer , we recovered more cd4 t cells than when recipients only lacked class ii molecules ( table ) , similar to the results of a recently published study 22 .
it is possible that the complete lack of t cells , observed in ci / ii mice 17 , may be conducive to an even more extensive expansion than the one occurring in cii mice .
indeed , from the spleen only we recovered amounts of cd4 cells that were tenfold higher than those injected intrathymically .
however , one caveat of this experiment is that the injected cd4 cells themselves expressed class i , as they were derived from the class i mice ( this was the only way to unambiguously ascertain the donor origin of these cells ) ; therefore , their contact with each other could have provided the survival signal .
however , at a minimum , we can still reliably conclude that the presence of the mhc molecules on stromal cells or on apcs is not necessary for either the terminal cd4 thymocyte differentiation or the survival of at least some cd4 t cells in the periphery . to test whether the peripheral cd4 progeny of the cd8cd4 thymocytes was functional in the absence of mhc molecules
, we isolated > 99% pure cd4ly-5.2 donor - derived cells from the recipient mice , stimulated them with allogeneic cells in vitro , and measured the il-2 release in response to such stimulation ( table ) .
cd4 cells of donor origin produced il-2 at levels comparable to those produced by unmanipulated peripheral cd4 cells , establishing that these cells were functional and immunocompetent .
the fact that donor - derived cd4ly5 - 2 cells responded vigorously to mhc class ii 
disparate i - a stimulators , but not to the class i  disparate k stimulators ( table , exp .
2 ) clearly demonstrated that their precursors must have been appropriately positively selected on mhc class ii molecules before the point of transfer .
similar results were obtained with the donor - derived cd4 t cells recovered from the ci / ii recipients ( data not shown ) .
the primary objective of this study was to investigate the dependence of the final phase of intrathymic maturation on mhc class ii molecules .
mhc contact must be maintained throughout several discrete stages in t cell development , but none of these has addressed the importance of this contact for terminal maturation of sp cells . as pointed out in our previous publications 912 , the cd8cd4 cells in our studies differ significantly from the cells called cd8cd4 232425 : theirs correspond to the cd8cd4 cells , which bear levels of cd8 lower than those on double - positive ( dp ) thymocytes but clearly detectable by fcm .
the cd8cd4 cells studied here belong to the cd4 lineage and score within the cd4 sp population by fcm : their cd8 levels are undetectable by fcm ( for a full phenotypic difference between our cd8cd4 cells and the cd8 cd4 cells of the other authors , see reference 9 )
. our data resolve the class ii dependence of the last phase of intrathymic maturation of sp cd4 thymocytes unambiguously : no mhc class ii 
this conclusion is further supported by the recent data of hare et al . in the reaggregation organ culture system 26 , showing that thymocytes at or past the cd69 dp stage no longer require contact with class ii molecules to become functional cd4 sp thymocytes . at present
, we can only speculate on the nature of the final maturation signal . among the obvious contenders
are the cytokines , the costimulatory molecules , and/or the matrix  integrin interactions , some of which are currently being investigated .
survival and peripheral homeostasis of t cells were not the key subjects of this study .
perhaps surprisingly , the peripheral progeny of intrathymically transferred cd8cd4 cells was able to survive for > 5 mo in the absence of mhc class ii molecules ( albeit the numbers were 5060% of those in class ii mice 5 mo after the transfer ) .
this survival was not due to the outgrowth of contaminating mature cd8cd4 thymocytes ( table ) nor to the contaminating class ii cells ( which were undetectable in the inoculum ) .
we conclude that mhc class ii molecules are not necessary for the long - term survival of at least some cd4 t cells , but are required for their optimal expansion and homeostasis , since both the percentages and the absolute numbers of cd4 t cells were about twofold higher in the presence of class ii molecules . the issue of whether mhc molecules are required and necessary for the survival of peripheral t cells has recently gained much attention .
although it appears that mhc class i molecules are necessary for the survival of cd8 cells 27282930 , the consensus is more tenuous in the case of cd4 cells 22313233 .
31 using the transfer of fetal thymic lobes into cii mice , concluded that class ii molecules are not essential for survival but affect the half - life of cd4 cells .
22 reported on a model of retroviral reconstitution of class ii expression in cii mice , and found that the half - life in reconstituted mice that expressed no class ii in the periphery was similar to that of takeda et al .
these authors concluded that the lack of class ii molecules curtailed ( but did not abolish ) the survival of cd4 cells , and showed that the lack of class i molecules did not further pronounce this curtailment . in these experiments , the uneven expression of intrathymically injected and retrovirus - driven class ii molecules could have caused a situation in which class ii molecules were not expressed in all the intrathymic compartments relevant to the t cell longevity .
this could have resulted in a paucity of intrathymic signals that regulate the half - life of t cells , thus reducing the numbers of long - lived cells or their half - life . by contrast
, the study of brocker 32 argued for an absolutely essential role of class ii on peripheral apcs in the survival of cd4 t cells , based on the results of transplantation of the apc - depleted class ii thymi into cii mice bearing no transgenes or expressing an mhc class ii transgene on dcs alone . in these experiments , good repopulation and intrathymic differentiation of cd4 t cells
were observed , but no extrathymic cd4 t cells were detected unless the dcs expressed the class ii transgene .
33 showed that cd4 t cells bearing a tcr transgene required self class ii molecules for long - term survival in the periphery .
finally , viret et al . showed that the cd4 t cell repertoire is incompletely maintained when a single peptide is present on the majority of mhc class ii molecules 34 .
our data certainly agree with all of the above studies in that class ii molecules are required for optimal cd4 expansion and the filling up of the peripheral compartment .
31 , also clearly show that a substantial proportion of peripheral cd4 t cells do not require mhc class ii molecules .
34 are also compatible with this notion , since these authors saw a reduction , but not disappearance , of cd4 t cells in mice unable to exchange the class ii 
associated invariant chain peptides for other peptides , owing to a lack of h-2m. however , the period of observation in these experiments was rather short . in distinction to the studies of kirberg
33 , we used polyclonal t cells , and it is quite possible that only some cells from the original inoculum have survived , reflecting the requirement of some , but not all , cells for the particular mhc class ii ligand .
it is possible that in our system , we have selected for only those cells that do not require class ii for peripheral survival , and we are testing this possibility at present .
we are also investigating whether these cells express a diverse repertoire , as suggested by their vigorous response to alloantigens ( table ) . at face value ,
however , these differences can be reconciled if class ii molecules , expressed on hematopoietic cells rather than being essential for cd4 t cell survival , are necessary for the export of selected cd4 cells from the thymus .
if this is the case , both studies would have to be reinterpreted slightly . in our study , then , the transferred cd8cd4 cells either would already have received class ii  mediated signals before transfer , or would have been contaminated with sufficient numbers of class ii cells to provide this signal after transfer .
we believe that the latter possibility is highly unlikely , for two reasons . first , the magnetic bead separation retains most dcs and m on the columns nonspecifically , and since the largest theoretical contamination of the inoculum with cd4 cells was in the range of 600800 cells ( see fig .
1 ) , even assuming all of these cells were mhc class ii , this number appears too small to permit substantial proliferation and export of maturing thymocytes .
second , the presence of class ii , cd11c , or cd11b cells was undetectable among fcm - sorted inoculated thymocytes ( see legend to table ) .
in the experiments of brocker 32 , the class ii dcs would provide their essential export signal intrathymically ( indeed , dcs are well known to reside in the thymus ) , and would also help the cd4 expansion in the periphery
. the first function would be absolutely critical , and its lack would mean that no cd4 t cells could be detected in cii mice grafted with class ii epithelium . finally , with regard to cd4 t cell homeostasis , our study demonstrated that the proliferation of the injected thymocytes can occur in the absence of class ii molecules , as well as in the absence of class i apcs .
interestingly , this proliferation was particularly pronounced in ci / ii mice , paralleling the findings of rooke et al . 22 . at present
, it is unclear whether the sp thymocytes in our study proliferated intrathymically 1011 or whether the proliferation occurred in the periphery , and additional studies will be needed to address this issue .
 the majority ( 70% ) of postselection cd4 + single - positive ( sp ) thymocytes are cd8locd4hi . 
 these cells express very low levels of cd8 , undetectable by flow cytofluorimetric ( fcm ) analysis , but sufficiently high to allow purification by panning . unlike the fully mature cd8cd4hi thymocytes , which account for the remaining 30% of the sp cd4 + thymocytes , cd8locd4hi cells are functionally immature and short - lived unless they receive an unidentified maturation signal from the thymus . in this study , we tested the hypothesis that this signal is provided by a t cell receptor ( tcr)major histocompatibility complex ( mhc ) class ii interaction . using intrathymic transfer , we show that the immature cd8locd4hi cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of mhc class ii ( and class i ) molecules . 
 furthermore , in mice devoid of class ii ( and class i ) molecules , the progeny of cd8locd4hi cells was long - lived and functionally reactive to allogeneic class ii molecules , although their numbers in the spleen and the mesenteric lymph node were 4050% lower than those in class ii+ mice 5 mo after transfer . 
 control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes . 
 these results demonstrate that the final maturation , proliferation , and peripheral survival ( up to 5 mo ) of at least some postselection cd4 + sp cells do not require the tcr  mhc class ii interaction . they also indicate that the tcr  mhc class ii interaction(s ) required for the intrathymic development of long - lived cd4 + sp cells 
 occurs before the cd4hi sp stage of development . 
Materials and Methods
Mice.
Antibodies, Reagents, Cell Separation, and Flow Cytofluorometry.
Intrathymic Injection, Adult Thymectomy, and In Vivo Antibody Depletion.
Phenotypic and Functional Analysis of the Peripheral CD4
Results
Discussion