the pt mice , tcr- mice , and tcr-   mice have been described ( 6 , 17 , 18 ) .
tcr- pt mice   were bred in the animal colony of the basel institute for immunology .
breeding of tcr- pt mice was done in the   animal facilities at the hpital necker ( paris , france ) .
the tcr- transgenic mice , with a transgenic tcr specific   for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the   c57bl/6 ( b6 ) background ( 19 ) .
tcr- transgenic pt   mice were bred in the animal colony of the basel institute for immunology .
the following mabs were used   for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl ,   gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) ,   anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego ,   ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ;   pharmingen ) , anti - cd44 ( biotinylated km81 ; american type   culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 ,   fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ;   pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the   tcr- chain of hy - reactive tcr , fluos - conjugated ) .
two- and three - color stainings were performed with fitc- ,   pe- , and biotin - labeled antibodies at optimal concentrations .  
biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .
all   stainings were done in 96-well plates ( 0.5  10 cells per well ) in   20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20   min on ice . between first and second step reagents cells were   washed in pbs plus 2% fcs plus 0.1% sodium azide as was done   after the last step .
data were analyzed on a facscan ( beckton   dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .
for intracellular / extracellular double staining of thymocytes   cells were first incubated with culture supernatant of mab 2.4g2   to block fcrii / iii .
cells were then stained for surface markers   as described above . after washing in pbs ,
cells were fixed in pbs   plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .
cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at   room temperature and washed in pbs .
intracellular staining with   fitc - conjugated antibodies diluted in pbs plus 0.5% saponin   was performed for 20 min at room temperature , followed by two   washing steps in pbs and 2  15 min on a rocking platform in   pbs plus 2% fcs plus 0.5% saponin on ice .
finally , cells were   washed in pbs plus 2% fcs and analyzed on a facscan , using   lysis ii software .
the pt mice , tcr- mice , and tcr-   mice have been described ( 6 , 17 , 18 ) .
tcr- pt mice   were bred in the animal colony of the basel institute for immunology .
breeding of tcr- pt mice was done in the   animal facilities at the hpital necker ( paris , france ) .
the tcr- transgenic mice , with a transgenic tcr specific   for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the   c57bl/6 ( b6 ) background ( 19 ) .
tcr- transgenic pt   mice were bred in the animal colony of the basel institute for immunology .
the following mabs were used   for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl ,   gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) ,   anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego ,   ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ;   pharmingen ) , anti - cd44 ( biotinylated km81 ; american type   culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 ,   fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ;   pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the   tcr- chain of hy - reactive tcr , fluos - conjugated ) .
two- and three - color stainings were performed with fitc- ,   pe- , and biotin - labeled antibodies at optimal concentrations .  
biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .
stainings were done in 96-well plates ( 0.5  10 cells per well ) in   20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20   min on ice . between first and second step reagents cells
were   washed in pbs plus 2% fcs plus 0.1% sodium azide as was done   after the last step .
data were analyzed on a facscan ( beckton   dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .
for intracellular / extracellular double staining of thymocytes   cells were first incubated with culture supernatant of mab 2.4g2   to block fcrii / iii .
cells were then stained for surface markers   as described above . after washing in pbs ,
cells were fixed in pbs   plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .
cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at   room temperature and washed in pbs .
intracellular staining with   fitc - conjugated antibodies diluted in pbs plus 0.5% saponin   was performed for 20 min at room temperature , followed by two   washing steps in pbs and 2  15 min on a rocking platform in   pbs plus 2% fcs plus 0.5% saponin on ice .
finally , cells were   washed in pbs plus 2% fcs and analyzed on a facscan , using   lysis ii software .
in initial experiments , it was determined whether either   the tcr- or the tcr- could be responsible for the   production of cd48 t cells in pt mice by analyzing the cellular composition of thymuses from either pt   tcr- or pt tcr- double mutant mice that   can only produce the  and the tcr- , respectively .  
as shown in table 1 both types of mutant mice contained   cd48 t cells that were further analyzed by cytoplasmic   staining with antibodies specific for tcr- and tcr-   chains .
for this purpose cells were double stained for surface expression of cd4 and cd8 molecules as well as either for cytoplasmic tcr- or tcr- chains by double   fluorescence using cd4 and cd8 antibodies in one color   ( green ) and tcr- or tcr- antibodies in another color   ( red ) . in this analysis
single positive cd48 and cd48   cells show an intermediate fluorescence between that of   cd48 and cd48 thymocytes and cells were gated accordingly into double negative , double positive ( dp ) , and   single positive cells ( fig .
1 shows that 64% of cd48 cells in wild - type   mice expressed tcr- chains , and that due to tcr- selection by the pre - tcr ( 22 ) the vast majority of cd48   cells contained tcr- chains in their cytoplasm . on the   other hand ,
the picture was   different in pt/ mice where , due to the diminution of   rapidly cycling tcr-selected cd484425 cells ( 6 ) ,   only 21% of the cd48 cells were tcr- positive . in   addition , only 39% of the cd48 cells contained tcr-   chains in their cytoplasm indicating that in the pt/   mice the majority of the cd48 cells were generated by a   mechanism that did not involve tcr- selection . the fact   that not all single positive cells in these mice were tcr-   is due to the fact that these cells are in part immature tcr-   single positive cells , on their way from cd48 to cd48   cells .
the tcr- single positive cells had a   mature cd48 phenotype as confirmed by independent   three - color stainings indicating also that these cells expressed tcr- receptors on the cell surface .
these cells   were present in a higher number in pt mice consistent with the notion that the pre - tcr may have a role in   regulating  rearrangement and/or expression ( 23 and unpublished observations ) .
in pt tcr- mice the proportion of tcr-   cd48 and tcr- single positive cells was even further   reduced . when looking at the absolute numbers of various   cell subsets ( table 1 and fig .
1 ) it is clear that there was a   very marked reduction in cell numbers of cd48 thymocytes and more mature cells in pt and pt   tcr- mice , whereas the numbers of cd48 cells   were within the same range .
pt tcr- mice also had reduced numbers of   dp cells but here the picture differed from that in pt   and pt tcr- mice in that all of the cd48   cells were tcr- positive , i.e. , were exclusively generated   through a mechanism that involved tcr- selection .
the   single positive tcr- cells in pt tcr- mice   were exported from the thymi and cd48 as well as   cd48 cells could be detected in lymphnodes of these   mice ( not shown ) .
this excludes the possibility that these   cells belong exclusively to the nk1.1cd4 subset that   exhibits an unusual phenotype ( 24 ) .
the above results were reproducible in the different   mice with marginal deviations in either the percentage of   cells or absolute cell numbers and are schematically presented in fig .
the main message from this analysis is that   the tcr- can generate cd48 cells through tcr-   selection , i.e. , by intracellular or cell - autonomous signaling   only .
in contrast , the tcr- can generate cd48 cells   that are either tcr- or tcr- but all tcr-   through a mechanism that may involve intercellular communication of unknown nature . if the tcr- would   generate a significant number of dp cells by cell - autonomous signaling one might expect to find some tcr- expression in these cells .
however , the fact that the cd48   cells are tcr- negative suggests that these cells are not   selected by cell - autonomous signaling by the tcr-   even though it can not be entirely excluded that tcr-   expression is abruptly switched off in cd48 cells .
the   notion of intercellular communication is in line with experiments that involved transfer of  t cells into thymuses   of rearrangement - deficient mice that resulted in generation   of cd48 cells of host origin ( 25 ) and also with earlier   data by shores et al .
our experiments suggest that in   the latter experiments  but not  t cells promoted the   development of cd48 thymocytes and make the additional point that the generation of dp cells was not due to   an artefact caused by adoptive transfer of cells .  
the fact that in the absence of the pre - tcr the generation of cd48 cells by the tcr- is rather inefficient ,   i.e. , 240  10 versus 2,880  10 in pt tcr-   versus wild - type mice , could depend on the fact that the   tcr- is inefficiently formed in cd48 cells due to the   late tcr- rearrangement and/or the fact that tcr-   can only inefficiently replace the pre - tcr . to analyze this   question in some more detail we studied mice that express   a transgenic tcr- early in development on cd48   cells , i.e. , tcr- transgenic pt mice .
the transgenic   tcr- could indeed overcome the cellular deficiency in   the cd48 compartment as tcr- transgenic pt   mice contained approximately one - half the number of thymocytes found in tcr- transgenic pt mice and   many more than the number found in nontransgenic pt   mice ( fig .
however , there was a subtle difference between tcr- transgenic pt and tcr- transgenic   pt mice in that the latter , but not the former , contained a discrete subset of cd25 cells , indicating that in   spite of the presence of the transgenic tcr- , the pretcr had its role in the exit from this compartment .
this   could be due to the lack of expression of the transgenic   tcr- in a fraction of cells in the cd25 compartment   of the tcr- transgenic , pt mice .
this was in fact   confirmed by cytoplasmic staining : while only nine percent   of cd25 cells in tcr- transgenic pt mice expressed the transgenic tcr- chain the majority of these   cells expressed the transgenic tcr- chain suggesting that   expression of the two transgenes is differentially regulated   ( fig .
in tcr- transgenic pt mice it is the   combined action of the pre - tcr and the tcr- ( mice   that have only a tcr- transgene still exhibit a significantly larger cd25 compartment than tcr- transgenic mice , not shown ) that reduce the number of cd25   cells while in tcr- transgenic pt mice this compartment is bigger in size because of the absence of the pretcr . from these data
it would appear that the tcr-   can at least partially mimic the function of the pre - tcr   and that in normal mice the contribution of the tcr-   to the generation of the cd48 compartment is limited   due to relatively late expression of most tcr- chains ( 1 , 2 ) .    thus , all of the three known tcrs can have a role in   promoting the development of pre  t cells : the tcr-   most likely by intercellular communication that furthers the   development of cd48 cells irrespective of whether or not   they have succeeded in tcr- rearrangement , the tcr-   that depends strictly on intracellular , cell - autonomous signals generated by the tcr- chains and the pre - tcr   that operates by a similar mechanism as the tcr- but is   much more efficient because of the early and abundant expression of the pt gene during the phase of tcr- rearrangement . therefore , only mice that can not produce any   of these receptors will exhibit complete arrest at the cd48   stage of development as evident in rag mice or mice that   are deficient in both tcr- and tcr- chains and therefore , can make neither tcr- , pre - tcr , nor tcr-   ( 14 ) . in normal mice , the contribution of the tcr- in   development of cells of the  lineage appears to be limited based on the fact that the vast majority of cd48 cells   are tcr- and thus are tcr- selected .
likewise , in   normal mice , the contribution of the tcr- to the transition of dn to dp cells may be limited because of the small   number of dp cells in pt tcr- mice .
however ,   in the absence of pt these receptors avoid a severe immunodeficiency by enabling the formation of a significant number of mature  t cells .
it would appear that both the pretcr and the tcr- do not only mediate maturation   but also proliferation since in wild - type mice and pt   tcr- mice the proportion of large cd48 blasts   that are derived from dividing cd48 precursors ( 15 ) is   very similar ( table 2 ) .
there are only slightly fewer blasts   in pt tcr- mice indicating that also the tcr-   generates dividing cd48 cells .   with regard to
the role of the src kinases in early development , our data is consistent with the notion that signaling through the pre - tcr involves both lck and fyn kinases   but is equally consistent with the idea that the fyn kinase is   involved only in signaling through the tcr- or - ,   and thereby responsible for the incomplete developmental   arrest observed in lck mice .
the fact that the tcr-   promotes development much in the same way as the pretcr , i.e. , by cell - autonomous signaling and thereby tcr-   selection , suggests that t cell development may have proceeded in this way before the advent of the pre 
tcr-   chain in evolution and that the pre - tcr had simply the   advantage of making the pairing of a single tcr- chain   with different tcr- chains more effective .
cd48 thymocyte subsets of wild - type and mutant mice mean values were obtained of four ( two for pt/ tcr-/ )   different mice of each genotype from 68-wk - old litter .
intracytoplasmic staining for tcr- ( tcr-ic ) and tcr- ( tcr-ic ) within thymocyte subsets from c57bl/6 ( wt ) , pt mice ( a ) ,   and pt tcr- , pt tcr- mice ( b ) .
total thymocytes were surface stained with pe - conjugated cd4 antibodies , biotinylated cd8   antibodies followed by pe - streptavidin ; cytoplasmic staining was performed with anti - pantcr- or anti  tcr- antibodies .
the percentages of cells and absolute numbers ( in brackets ) are indicated . a schematic overview of various gene - deficient mice and the corresponding defects in t cell development .
percentages indicate the proportion of cells with cytoplasmic tcr-. the thickness of the bars is meant to correlate with the numbers of cells within the various subsets .
comparision of surface phenotype of   thymocytes from tcr- pt vs. tcr-   transgenic mice .
( top ) total thymocytes were double stained for cd4 ( fitc - conjugated anti - cd4 )   and cd8 ( red613-conjugated anti - cd8 ) surface   antigens as described .
tcr- pt transgenic mice contained approximately one - half of the number of thymocytes   found in tcr- transgenic mice ( 2,870  10   vs. 4,720  10 cells ) .
( bottom ) cells were stained   with fitc - conjugated cd4 and cd8 antibodies   in combination with biotinylated cd44 and peconjugated anti - cd25 antibodies .
the expression of cd25 and cd44 was analyzed by   three - color flow cytometry , using electronic gating   to exclude fitc - positive cells .
assessment of transgenic tcr- and tcr- expression by intracytoplasmic   staining . for intracellular / extracellular double staining ,
thymocytes isolated from transgenic   tcr- mice and transgenic   tcr- pt mice were   stained with pe - conjugated   cd25 antibodies and then with   fitc - conjugated t3.70 antibodies specific for the transgenic   tcr- chain of the hy - reactive tcr or fluos - conjugated f23.1 antibodies , specific   for the transgenic tcr- chain of   the hy - reactive tcr .
proportion of cd48 lymphoblasts in wild type and mutant mice percentages of cd48 blasts were determined by facscan using   forward scatter as an index of size .
the various mice were analyzed on   the same day in the same experiment .  
 the development of pre  t cells with productive tcr- rearrangements can be mediated by   each the pre  
 t cell receptor ( pre - tcr ) , the tcr- as well as the tcr- , albeit by distinct mechanisms . 
 although the tcr- affects cd48 precursor cells irrespective of their   rearrangement status by tcr- mechanisms not involving tcr- selection , both the pretcr and the tcr- select only cells with productive tcr- genes for expansion and   maturation . the tcr- appears to be much less effective than the pre - tcr because of the   paucity of tcr- proteins in tcr-positive precursors since an early expressed transgenic   tcr- can largely substitute for the pre - tcr . 
 thus , the tcr- can assume a role not   only in the rescue from programmed cell death of cd4 + 8 + but also of cd48 thymocytes .   in evolution 
 this double function of the tcr- may have been responsible for the maturation of  t cells before the advent of the pre  tcr- chain . 