all mice were housed and bred in a specific pathogen - free facility and used at 412 wk of age .
tcr mice ( 21 ) were   obtained from the jackson laboratory ( bar harbor , me ) .
tcr   mice ( 22 ) were mated with rag2 mice ( 23 ) to generate double - deficient rag2tcr mice .
mice transgenic for a chimeric   protein consisting of the extracellular and transmembrane domains of human cd25 and the cytosolic domain of murine   tcr ( tt , line no .
35 ) ( 24 ) were also bred onto the rag2   background to make ragtt mice .
rag2 and   rag2 mice were injected intraperitoneally with 250 g of affinity - purified anti - cd3 mab ( 145 - 2c11 ) ( 25 ) or with the dose   indicated .
ragtt mice were injected with 250 g anti - tac   mab ( 1ht4 - 4h3 ) .
where indicated , rag2 mice   were injected with 250 g of affinity - purified anti - cd3 mab   on both day 0 and day 8 and then subjected to the coreceptor reexpression assay on day 12 . where indicated , rag2 mice were   radiated with 400 cgy and analyzed 3 wk later ( 26 , 27 ) .
performance of  the   coreceptor reexpression assay on electronically sorted thymocyte   populations has been described previously ( 8) . in brief , single cell   suspensions of thymocytes
were stained with pe - conjugated anti - cd4 mab ( gk1.5 ; becton dickinson , san jose , ca ) and fitc - conjugated anti - cd8 mab ( 53 - 6 - 72 , becton dickinson ) .
stained   thymocytes were electronically sorted by a facstar plus according to the gates indicated in each figure .
sorted cells were washed   extensively with pbs and treated with 0.04% pronase ( calbiochem novabiochem , san diego , ca ) and 100 g / ml dnase i   ( boehringer mannheim , indianapolis , in ) at 37c for 15 min ,   pelleted , and pronase treated for another 10 min at 37c .
cells   were placed in culture for 1216 h of culture at either 4 or 37c ,   after which harvested cells were restained with anti - cd4pe and   anti - cd8fitc . for three - color analysis cells
were also stained   with anti - cd5 ( 53 - 7 - 3 ; pharmingen ) followed by cy-5 avidin   ( caltag , san francisco , ca ) .
dead cells were excluded by electronic gating on both forward light scatter and propidium iodide   intensity .
flow cytometry using three- or four - decade logarithmic amplification as indicated was performed on a facstar plus   and data were analyzed using software designed by the division   of computer research and technology at the national institutes   of health .
all mice were housed and bred in a specific pathogen - free facility and used at 412 wk of age .
tcr mice ( 21 ) were   obtained from the jackson laboratory ( bar harbor , me ) .
tcr   mice ( 22 ) were mated with rag2 mice ( 23 ) to generate double - deficient rag2tcr mice .
mice transgenic for a chimeric   protein consisting of the extracellular and transmembrane domains of human cd25 and the cytosolic domain of murine   tcr ( tt , line no .
35 ) ( 24 ) were also bred onto the rag2   background to make ragtt mice .
rag2 and   rag2 mice were injected intraperitoneally with 250 g of affinity - purified anti - cd3 mab ( 145 - 2c11 ) ( 25 ) or with the dose   indicated .
ragtt mice were injected with 250 g anti - tac   mab ( 1ht4 - 4h3 ) .
where indicated , rag2 mice   were injected with 250 g of affinity - purified anti - cd3 mab   on both day 0 and day 8 and then subjected to the coreceptor reexpression assay on day 12 . where indicated , rag2 mice were   radiated with 400 cgy and analyzed 3 wk later ( 26 , 27 ) .
performance of  the   coreceptor reexpression assay on electronically sorted thymocyte   populations has been described previously ( 8) . in brief , single cell   suspensions of thymocytes
were stained with pe - conjugated anti - cd4 mab ( gk1.5 ; becton dickinson , san jose , ca ) and fitc - conjugated anti - cd8 mab ( 53 - 6 - 72 , becton dickinson ) .
stained   thymocytes were electronically sorted by a facstar plus according to the gates indicated in each figure .
sorted cells were washed   extensively with pbs and treated with 0.04% pronase ( calbiochem novabiochem , san diego , ca ) and 100 g / ml dnase i   ( boehringer mannheim , indianapolis , in ) at 37c for 15 min ,   pelleted , and pronase treated for another 10 min at 37c .
cells   were placed in culture for 1216 h of culture at either 4 or 37c ,   after which harvested cells were restained with anti - cd4pe and   anti - cd8fitc . for three - color analysis cells
were also stained   with anti - cd5 ( 53 - 7 - 3 ; pharmingen ) followed by cy-5 avidin   ( caltag , san francisco , ca ) .
dead cells were excluded by electronic gating on both forward light scatter and propidium iodide   intensity .
flow cytometry using three- or four - decade logarithmic amplification as indicated was performed on a facstar plus   and data were analyzed using software designed by the division   of computer research and technology at the national institutes   of health .
to assess the possibility that dp thymocytes spontaneously terminated cd8 coreceptor synthesis even in the absence of tcr 
cd3 signals , we examined dp thymocytes   from tcr mice by the coreceptor reexpression assay .  
tcr thymocytes can not express conventional  tcr   complexes and , consequently , can not differentiate beyond   the dp stage of development ( 21 ) . to enrich for dp thymocytes that might have committed to the cd4 or cd8   t  cell lineages , we electronically sorted for cd48 and   cd48 transitional cell populations and utilized the coreceptor reexpression assay to determine the coreceptor molecules they were actively synthesizing ( fig .
1 , a and b ) . in   the coreceptor reexpression assay , preexisting surface cd4   and
cd8 coreceptor molecules are removed from the   sorted cells by treatment with low doses of pronase , and the   stripped cells then placed into single cell suspension cultures for 14 h. metabolic activity of cultured cells is inhibited at 4c , so that cell surface coreceptor reexpression   does not occur ( fig . 1 a and b , middle columns ) . however ,   cells cultured at 37c do reexpress the cd4 and/or cd8   coreceptor molecules that they are actively synthesizing .  
indeed , we have previously demonstrated that coreceptor   reexpression in this assay requires active coreceptor transcription and protein synthesis ( 8) . interestingly , virtually   all cd48 sorted cells from tcr dp thymocytes reexpressed both cd4 and cd8 coreceptors and so reappeared   as dp cells ( fig .
identical results were obtained   with cd48 sorted cells from tcr mice ( fig . 1 b , top ) .  
that is , none of the dp thymocytes present in tcr   mice had selectively terminated either cd4 or cd8 coreceptor synthesis , indicating that none had undergone lineage commitment .
thus , these results indicated that lineage   commitment did not occur spontaneously in immature dp   thymocytes but might be dependent upon signals transduced by surface tcr  cd3 complexes .   to assess directly the role of tcr 
cd3 signals in inducing lineage commitment in dp thymocytes , we assessed   dp thymocytes from experimentally induced rag2 mice .  
rag2 mice fail to express any clonotypic tcr chain because they are unable to recombine any tcr gene locus .   as a result ,
rag2 thymocytes are arrested at the cd48   ( double - negative , dn ) stage of development ( 23 ) . however
, rag thymocytes can be induced to differentiate   further into dp cells by either sublethal -irradiation ( 26 ,   27 ) or by injection of anti - cd3 mab ( 28 , 29 ) ( fig .
1 , a   and b , left column ) . induced rag dp thymocytes did not   further differentiate into phenotypically mature t cells as   thymocytes from stimulated rag mice that appeared   cd48 or cd48 ( fig .
1 , a and b , left columns ) were   predominantly precursor cells that spontaneously became   cd48 in overnight culture ( data not shown ; reference   30 ) . even though sublethal -irradiation and anti - cd3   injection both induced generation of rag dp thymocytes ( fig .
that is , sublethal -irradiation did   not detectably stimulate cd3 signal transduction as revealed by the absence of cd5 upregulation , whereas injection of anti - cd3 mab did stimulate cd3 signal transduction and cd5 upregulation ( fig .
2 ) . assessment of cd48   and cd48 sorted cells from -irradiated rag2 mice   by the coreceptor reexpression assay revealed that none had   undergone lineage commitment ( fig .
assessment of cd48 sorted cells from anti - cd3 induced rag2 mice revealed the presence of cd4-committed dp thymocytes that had selectively terminated cd8   coreceptor synthesis , as well as the presence of uncommitted dp thymocytes ( fig .
the cd4-committed thymocytes that were present in anti - cd3induced rag2   mice expressed the phenotype of newly committed thymocytes in that they were hsa , thymic shared antigen   ( tsa)-1 ( data not shown ) .
in contrast , anti - cd3induced   rag2 thymocytes had no cd8-committed cells among   either cd48 or cd48 sorted cell populations ( fig . 1 ,   a and b ) .
thus , these results ( a ) confirm that dp thymocytes do not undergo lineage commitment in the absence of tcr 
cd3 signals , and ( b ) demonstrate that cd3   signals stimulated by anti - cd3 mabs are sufficient to induce dp thymocytes to selectively terminate cd8 coreceptor synthesis and commit to the cd4 lineage , even in   the absence of clonotypic tcr chains .   to determine whether signals transduced by tcr chains   are indispensable for induction of cd4 commitment , we   generated double knockout rag2tcr mice . in vivo   injection of anti - cd3 mabs into ragtcr double   knockout mice induced the generation of dp thymocytes ,   as has been described ( 31 ) , and signaled these cells to upregulate cd5 expression ( fig .
interestingly , we found that   cd48 sorted thymocytes from these anti - cd3injected   mice did contain cd4-committed cells that had selectively   terminated cd8 coreceptor synthesis ( see fig .
, no cd8-committed cells were detected in either   cd48 or cd48 sorted cell populations ( fig .
1 , a and   b ) . these results demonstrate that cd3-transduced signals   can induce cd4 commitment in dp thymocytes in the absence of clonotypic tcr chains and in the absence of   tcr chains . to determine whether signals transduced by tcr   chains were able to induce cd4 commitment , we assessed   lineage commitment in dp thymocytes from rag2 mice   that expressed a chimeric transgenic protein consisting of   the external and transmembrane domains of human cd25   and the cytosolic domains of tcr ( 24 ) .
the extracellular   domain of this transgenic protein is recognized by anti - tac   mab .
injection of anti - tac mab into ragtt mice   induced the generation of dp thymocytes ( fig .
1 a ) , as   previously reported ( 24 ) , and signaled them to upregulate   cd5 expression ( fig .
we found that cd48 sorted   thymocytes from these mice did contain cd4-committed   cells , but did not contain any cd8-committed cells in either cd48 or cd48 sorted cell populations ( see fig .   1 , a and b ) .
these results demonstrate that signals transduced by the cytosolic portion of tcr chains are sufficient to induce dp thymocytes to selectively terminate   cd8 coreceptor synthesis and to undergo cd4 commitment .
next , we wished to evaluate the relationship between   cd5 upregulation and cd4 commitment in anti - cd3 signaled rag2 thymocytes . in vivo injection of a single   dose of either 10 or 250 g of anti - cd3 mab  induced   substantial numbers of dp thymocytes in rag mice when   assayed 8 d later ( fig .
while both injection doses induced differentiaton to the dp stage , we reasoned that only   the high dose might persist long enough in vivo to stimulate   a subsequent cd3 signal after dp thymocytes appeared .
indeed , only dp thymocytes from high dose injected animals   had upregulated cd5 expression , and only dp thymocytes   from high dose injected animals contained cd4-committed thymocytes ( fig .
3 ) . dp thymocytes from low dose injected animals were cd5 and remained uncommitted   ( fig .
we conclude that cd4 commitment requires   cd3 signals in dp thymocytes that are of sufficient intensity to upregulate surface cd5 expression .  
finally , having observed that a single injection of antibody was sufficient to stimulate cd3 or tcr chains to   transduce signals that upregulated cd5 surface expression   and induced cd4 commitment but not cd8 commitment , we assessed whether cd8-committed cells might   appear in rag2 thymi upon antibody restimulation . in   fig . 4 ,
rag2 mice were injected with anti - cd3 mab   on both days 0 and 8 , and then assessed 4 d later on day 12 .  
thymocytes were sorted into cd48 and cd48 populations and then assessed for coreceptor synthesis by the   coreceptor reexpression assay .
we found that cd48 thymocytes contained cd4-committed cells that had selectively terminated cd8 coreceptor synthesis ( fig .
4 , middle   row ) , but neither sorted population contained cd8-committed cells ( fig .
, cd8-committed cells did not appear in rag2 thymi despite a   second antibody injection and despite assessment on day 12   after the initial injection of antibody ( fig .
, we   also failed to detect cd8-committed thymocytes on days   28 and 35 after antibody injection ( data not shown ) .
the present study demonstrates that immature dp thymocytes do not spontaneously terminate synthesis of either   cd4 or cd8 coreceptor molecules .
rather , selective termination of coreceptor synthesis by immature dp thymocytes   requires signals transduced by either cd3 or tcr chains ,   and can occur in signaled dp thymocytes that lack clonotypic tcr chains .
interestingly , cd3-signaled dp thymocytes upregulated cd5 expression and selectively terminated cd8 coreceptor synthesis , but did not selectively   terminate cd4 coreceptor synthesis .
thus , cd4 commitment is induced in dp thymocytes by cd3 signals that are   of sufficient intensity to upregulate cd5 expression .
the results of the present study are not readily compatible with either instructional ( 1 , 2 ) or stochastic / selection   ( 37 ) models of lineage commitment .
that is , the instructional model can not explain the presence of any lineage - committed rag2 thymocytes in response to lineage - neutral cd3 and tcr signals , whereas the stochastic / selection   model can not explain why cd3 and tcr signals only   induced rag2 thymocytes to become cd4 committed   without inducing an equal number to become cd8 committed .
in contrast with these two models of lineage commitment , the present results are concordant with the asymmetric commitment model of lineage commitment ( 8 , 13 ) .  
that is , our results are consistent with the concept that cd4   commitment , unlike cd8 commitment , can occur in the   absence of lineage - specific signals .
importantly , the present   results extend the asymmetric commitment model by demonstrating that cd4 commitment does not occur spontaneously in unsignaled dp thymocytes , but rather is induced   by lineage - neutral signals transduced by cd3 and/or tcr   chains .
importantly , we found that cd3 signaling did not induce all dp thymocytes in the present study to become   cd4 committed , as only a small minority of cd3-signaled   rag2 thymocytes terminated cd8 coreceptor synthesis ,   even though all dp thymocytes had upregulated cd5 surface   expression .
this observation is consistent with our recent   finding that only a small fraction of cd5 dp thymocytes   in normal mice have undergone lineage commitment , with   most cd5 dp thymocytes remaining lineage uncommitted ( 20 ) .
our current perspective is that cd3 signaling   drives cd5 dp thymocytes to become cd5 , at which   point they developmentally await the induction of lineage - specific signals . if lineage - specific signals are generated ,   perhaps by notch proteins ( 12 ) , cd5 dp thymocytes terminate cd4 coreceptor synthesis and become cd8 committed .
but if lineage - specific signals are not generated ,   cd5 dp thymocytes terminate cd8 coreceptor synthesis   and become cd4 committed .
we do not know how long   cd5 dp thymocytes await the appearance of lineage - specific signals before terminating cd8 coreceptor synthesis ,   and we do not know whether there are intrathymic signals   that regulate the timing of this event .
finally , the present results are remarkable in their demonstration that cd3-signaled dp thymocytes could undergo cd4 commitment even in the absence of clonotypic   tcr chains . of course , dp thymocytes express surface   molecules in addition to clonotypic tcr chains that can   stimulate cd3 signaling , such as cd2 , cd5 , thy-1 , and   ly6 ( 3234 ) .
consequently , it is conceivable that engagement of such nonclonotypic molecules by intrathymic ligands   can inefficiently mimic clonotypic tcr chains in their   ability to stimulate cd3 signals that induce cd5 dp thymocytes to become cd5 , and so eventually to become   cd4 committed .
the absence of cd5 dp thymocytes and   cd4-committed cells in tcr mice does not rule out intrathymic signaling by nonclonotypic molecules because   surface cd3 expression is probably too low on tcr   thymocytes to transduce such signals .
indeed , stimulation   of cd3 signals by nonclonotypic receptors may provide one   explanation for the appearance of small numbers of cd4-committed dp thymocytes in mhc - deficient thymi ( 8) .
purified populations of cd48 thymocytes ( a )   and cd48 thymocytes ( b ) were obtained by electronic cell sorting according to the indicated sorting gates superimposed on the starting thymocyte   populations ( left columns ) . sorted thymocyte populations were stripped of surface coreceptor molecules by treatment with low doses of pronase , after   which they were placed in suspension cultures at 4c ( middle panels ) or 37c ( right panels ) for 1216 h and restained for cd4 and cd8 surface expression .  
the coreceptor reexpression assay detects the coreceptor molecules that individual thymocytes synthesized during the 37c culture , and is dependent   upon new transcription and new protein synthesis ( 8) . sorted thymocytes that reexpress both cd4 and cd8 coreceptor proteins are lineage - uncommitted cells ; those reexpressing only cd4 are cd4 committed ; and those reexpressing only cd8 are cd8 committed .
cells cultured at 4c do not reexpress   surface coreceptor molecules so that their cd4cd8 histograms reflect whatever coreceptor molecules that potentially remain after pronase treatment   ( middle columns ) . as we have previously described ( 8) ,
the anti - cd4 mab used to prepare thymocytes for cell sorting minimally interferes with stripping   of surface cd4 molecules by pronase , resulting in a small number of residual cd4 molecules remaining on the cell surface .
consequently , to highlight   changes in coreceptor reexpression during 37c cultures , histogram boxes were drawn based on the 4c profiles of each sorted and pronase - stripped cell   population . the frequency of cells in each box is indicated .
the number of the thymocytes obtained in these experimental mice were the following :   tcr ( 9  10 cells ) , -irradiated rag2 ( 4  10 cells ) , anti - cd3 mab  injected rag2 ( 1.1  10 cells ) , anti - cd3 mab  injected   ragtcr ( 8  10 cells ) , and anti - tac mab 
thymocytes from the indicated experimental mice were stained   with anti - cd5 mab ( solid line ) or an irrelevant antibody ( shaded curve ) .  
cd5 expression on normal b6 thymocytes stained at the same time as a   positive control is also shown for comparison ( dotted line ) .
rag mice received one intraperitoneal injection of either 250 g   or 10 g of affinity - purified anti - cd3 mab .
8 d later , thymocytes   were sorted according to the indicated gates and assessed for lineage   commitment by the coreceptor   reexpression assay ( left panels ) .  
unsorted thymocytes were placed   in culture for 12 h at 4c , at   which temperature thymocyte   phenotype is stable , and then assessed for surface cd5 expression   ( right panels ) .
the dotted line represents cd5 expression on normal b6 thymocytes   stained at the same time as a positive control and is shown for comparison .
the number of thymocytes obtained on day 8 were 11.5  10 cells and   34  10 cells with the injection of 250 g and 10 g , respectively .
cd8-committed thymocytes do not appear in rag2 thymi   even after two injections of anti - cd3 mab .
rag2 mice were injected   with 250 g of affinity - purified anti - cd3 mab on both day 0 and 8 .   on day 12 , thymocytes were harvested , sorted into cd48 and   cd48 populations , and assessed by the coreceptor reexpression assay   for appearance of cd4-committed and cd8-committed cells .
 as a consequence of positive selection in the thymus , immature cd4 + 8 + double - positive ,   [ dp ] thymocytes selectively terminate synthesis of one coreceptor molecule and , as a result ,   differentiate into either cd4 + or cd8 + t cells . 
 the decision by individual dp thymocytes to   terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment . 
 previously , we reported that the intrathymic signals that induced commitment to the   cd4 versus cd8 t cell lineages were markedly asymmetric . 
 notably , cd8 commitment appeared to require lineage - specific signals , whereas cd4 commitment appeared to occur in the   absence of lineage - specific signals by default . 
 consequently , it was unclear whether cd4 commitment , as revealed by selective termination of cd8 coreceptor synthesis , occurred in all dp   thymocytes , or whether cd4 commitment occurred only in t cell receptor ( tcr)cd3-signaled   dp thymocytes . here 
 , we report that selective termination of cd8 coreceptor synthesis does   not occur in dp thymocytes spontaneously . 
 rather , cd4 commitment in dp thymocytes requires signals transduced by either cd3 or  chains , which can signal cd4 commitment even   in the absence of clonotypic tcr chains . 