progenitor t cells must go through distinct check points as they differentiate in the thymus before becoming mature t cells 1 . the most immature cd4cd8 ( dn ) thymocytes 2 can differentiate into cd4cd8 ( dp ) cells only after the expression of tcr  chains , a process termed  selection 34 . the dp thymocytes , which subsequently rearrange the tcr  chain genes and express tcr-/ , are subjected to the positive and negative selection by the specificity of tcr 567 . only positively selected thymocytes can mature into either cd4 or cd8 single positive ( sp ) t cells . in the  selection of pre - t cells , the signaling from tcr-/pre  tcr- ( pt)cd3 complex ( 8 ; pre - tcr ) is shown to play a crucial role 91011 . based on the analysis of pt-deficient mice 10 , it is indicated that pre - t cells drastically expand during  selection , which is essential for the subsequent generation of clonal diversification . however , it remains to be verified whether the  selection process somehow affects the final tcr repertoire of mature t cells . pd-1 is a 55-kd type i transmembrane protein of the immunoglobulin superfamily bearing an immunoreceptor tyrosine - based inhibitory motif ( itim ) in its cytoplasmic region 1213 . in the thymus , pd-1 is selectively expressed in a very minor population of dn thymocytes 14 , while it is also induced on peripheral t and b cells after activation 12 . expression of pd-1 in dn thymocytes is significantly augmented in both normal and recombination activating gene ( rag)-2deficient mice by the injection of anti - cd3 mab , implying that pd-1 might be involved in the  selection process 14 . recently , pd-1deficient mice were found to spontaneously develop autoimmune diseases characterized by lupus - like glomerulonephritis with immune complex deposition and destructive arthritis as they age 16 , and it has been suggested that the pd-1 deficiency results in the breakdown of peripheral tolerance against self - reactive t cells 16 . the possibility that the pd-1 deficiency affects the repertoire of mature t cells also remains open 15 . in the present study , we report that the absence of pd-1 significantly facilitates the transition of thymocytes from dn to dp stage in the tcr transgenic lines as well as in rag-2 mice injected with anti - cd3 mab , suggesting that pd-1 controls the threshold of  selection . on the other hand , efficiency of positive selection for the transgenic t cells in the relevant genetic background mechanisms for the opposing effects of pd-1 deficiency on  and positive selections as well as their possible involvement in the development of lupus - like diseases are discussed . v8 17 , h - y 18 and 2c transgenic 19 , and rag-2 mice 20 were maintained in specific pathogen - free conditions . for genotyping of mice , restricted dnas of tcr transgenic ( v8 , h - y , and 2c ; ecori ) and rag-2 alleles ( ecori plus ecorv ) were probed with the 300-bp psti - saci fragment of v8.2 and the 950-bp psti - ecorv fragment of rag-2 cdna , respectively . the transgenes and the targeted alleles were confirmed by the appearance of additional bands at 2.4 and 1.2 kb , respectively . mabs for pd-1 12 , h - y tcr- ( t3.70 21 ) , 2c tcr-/ clonotype ( 1b2 22 ) , and cd3 ( 145 - 2c11 ) were all purified from ascites and biotinylated if necessary . the following mabs were purchased from pharmingen : anti  mouse cd8-fitc , phycoerythrin - r ( pe)-conjugated anti - cd4 ( rm4 - 5 ) , biotinylated anti mouse cd8 ( 53 - 5.8 ) , and biotinylated anti  mouse tcr- ( gl3 ) . analysis was performed using a facscalibur ( becton dickinson ) and cellquest software ( becton dickinson ) . v8 17 , h - y 18 and 2c transgenic 19 , and rag-2 mice 20 were maintained in specific pathogen - free conditions . for genotyping of mice , restricted dnas of tcr transgenic ( v8 , h - y , and 2c ; ecori ) and rag-2 alleles ( ecori plus ecorv ) were probed with the 300-bp psti - saci fragment of v8.2 and the 950-bp psti - ecorv fragment of rag-2 cdna , respectively . the transgenes and the targeted alleles were confirmed by the appearance of additional bands at 2.4 and 1.2 kb , respectively . mabs for pd-1 12 , h - y tcr- ( t3.70 21 ) , 2c tcr-/ clonotype ( 1b2 22 ) , and cd3 ( 145 - 2c11 ) were all purified from ascites and biotinylated if necessary . the following mabs were purchased from pharmingen : anti  mouse cd8-fitc , phycoerythrin - r ( pe)-conjugated anti - cd4 ( rm4 - 5 ) , biotinylated anti  mouse cd24 ( m1/69 ) , mouse cd8 ( 53 - 5.8 ) , and biotinylated anti  mouse tcr- ( gl3 ) . analysis was performed using a facscalibur ( becton dickinson ) and cellquest software ( becton dickinson ) . we have comparatively analyzed the expression of pd-1 in the thymus of normal and various tcr transgenic mice . as shown in fig . 1 a , pd-1 was expressed in only 1% of total thymocytes of normal mice , which was confined to the dn population as reported previously 14 . pd-1 cells in the dn population showed two peaks , pd-1 and pd-1 , which represented / t cells and / t cells , respectively 14 . in the thymus of v8 transgenic mice , there was a slight yet significant increase in the pd-1 cells ( 1.2 versus 3.8% ) , which mostly reflected the increase in the pd-1 cells in the dn population ( 40 versus 65% ) . in both mice , we then examined the mice transgenic for both tcr  and  chain genes . in the thymus of h - y mice , 20% of total thymocytes and nearly 90% of the dn population were found to express pd-1 with high intensities in both neutral ( h-2 ) and positively selecting ( h-2 ) backgrounds . the thymus of 2c mice in the positively selecting background ( h-2 ) exhibited as much as 63% pd-1 cells , and again nearly 90% of the dn population strongly expressed pd-1 . unlike normal and v8 transgenic mice , significant proportions of dp thymocytes in h - y and 2c mice also exhibited pd-1 , 15 and 50% , respectively ( fig . 1 a ) . however , the cd4/cd8 expression of thymocytes in the tcr-/ transgenic mice was quite diffuse , particularly in 2c mice . to have a better picture of the distribution of pd-1 cells , 1 b , pd-1 cells were present in the cd4 and/or cd8 in addition to dn populations in both h - y ( h-2 ) and 2c ( h-2 ) mice . the vast majority of cd4cd8 dp cells as well as cd4cd8 and cd4cd8 sp populations lacked pd-1 expression . in the female h - y ( h-2 ) transgenic as well as control mice , cd69 thymocytes barely expressed pd-1 and vice versa ( fig . 1 c ) , implying that thymocytes during the positive selection process lacked pd-1 expression . in 2c ( h-2 ) mice , the majority of thymocytes expressed cd69 weakly , including both pd-1 and pd-1 cells ( fig . 1 c ) . in the tcr transgenic mice ,  selection largely proceeds based on the expression of transgenic tcr  chain , either as tcr- homodimer 2324 in v8 transgenic mice or as tcr-/ complex in h - y and 2c transgenic mice . although the studies on pt mice suggest that the three types of pre - tcr complex mentioned above achieve  selection in somewhat distinct fashions 2526 , signaling downstream of the cd3 complex is expected to be similar 11 . our results here have reinforced that pd-1 expression is induced during the  selection process of dn thymocytes , which is variably enhanced in the tcr transgenic mice . it remains to be further analyzed whether pd-1 is also expressed during the later selection processes such as positive and negative selection , particularly in 2c mice ( see below ) . to explore the functions of pd-1 , we crossed v8 and h - y ( h-2 ) transgenic mice with pd-1 mice and analyzed their thymocytes . as summarized in table , the total cell number of thymocytes in v8  pd-1 transgenic mice was significantly increased compared with that in v8  pd-1 transgenic mice ( 1.49  0.17 vs. 0.99  0.3  10 , p < 0.005 ) . the increase was ascribed mostly to dp thymocytes ( 109.82  12.65 vs. 73.51  2.27  10 , p < 0.01 ) , whereas the number of dn thymocytes remained unaffected . essentially similar results were obtained in the h - y ( h-2 ) mice , dp thymocytes being increased selectively in the absence of pd-1 ( 80.04  30.83 vs. 125.11  27.92  10 , p < 0.005 ) . the results strongly suggest that pd-1 negatively regulates the transition of dn thymocytes to the dp stage in the tcr transgenic mice . the numbers of cd4 and cd8 sp thymocytes were increased proportionally to dp thymocytes in both v8  pd-1 and h - y ( h-2 )  pd-1 mice , suggesting that the overall efficiency for the positive selection based on the tcrs of transgenic  chains and endogenous  chains was largely unaffected in the absence of pd-1 . next , to define the effect of pd-1 deficiency on the positive selection for the transgenic tcr-/ , we crossed pd-1 mice with the female h - y ( h-2 ) and 2c ( h-2 ) mice , in which the cd8 cells expressing the transgenic tcr-/ are positively selected . the number of dp thymocytes in female h - y ( h-2 ) mice was significantly increased in the absence of pd-1 , as before ( table ) . in the female h - y ( h-2 )  pd-1 mice , the number of cd8 sp cells was also increased proportionally to both dp and cd4 sp cells ( 1.5-fold ; table ) . in contrast , the number of cd8 sp cells in the female h - y ( h-2 )  pd-1 mice remained the same as in the control female h - y ( h-2 ) mice ( 17.6  7.63 vs. 16  3.89  10 ) in spite of the fact that the numbers of dp and cd4 sp cells were increased in parallel 1.5-fold ( table ) . facs analysis indicated that the relative proportions of both t3.70 ( transgenic tcr- ) and cd24cd8 cells were indeed decreased in the absence of pd-1 ( fig . 2 a ) , indicating that the efficiency of positive selection for the clonotypic t3.70cd8 sp cells was significantly reduced . on the other hand , the proportion of cd4 sp thymocytes , which are positively selected on the basis of diversified tcrs of the transgenic tcr- and endogenous tcr- , was unaffected ( fig . the number of total as well as dp thymocytes was far less and the relative proportion of cd8 sp thymocytes was much higher than in female h - y ( h-2 ) mice ( table , and fig . 1 ) , probably reflecting higher affinity and/or avidity of 2c tcr-/ for the selecting ligands 2728 . in the 2c ( h-2 )  pd-1 mice , a significant reduction of cd8 sp thymocytes was observed in terms of both absolute cell number and relative proportion , whereas the dp population was increased slightly compared with 2c ( h-2 )  pd-1 mice ( table , and fig . furthermore , within the cd4cd8 population , the percentages of those with the mature phenotypes ( cd24 and cd8 ) were significantly reduced in 2c ( h-2 )  pd-1 ( 50% ) than in 2c ( h-2 )  pd-1 mice ( 70% ) ( fig . 2 b ) , suggesting that the reduction of positively selected cd8 sp thymocytes in the former mice was apparently underestimated by the presence of transient cd4cd8 cells . consistently , expression of cd69 , a marker associated with positive selection , was also significantly reduced in both total and dp thymocytes in the absence of pd-1 ( fig . although there was an apparent increase in the cd4 sp thymocytes ( table , and fig . 2 b ) , the vast majority of cd4 sp thymocytes in both groups highly expressed the clonotypic epitope ( 1b2 ) of transgenic tcr-/ ( fig . therefore , it probably reflects the increase in the transitional thymocytes rather than positively selected mature cd4 sp cells , which is consistent with the accelerated  selection in the absence of pd-1 . these results have collectively suggested that the efficiency of positive selection for the cells expressing transgenic tcr-/ is significantly reduced in the absence of pd-1 . we have reported previously that the injection of anti - cd3 mab into rag-2 mice induces significant expansion of dp cells expressing pd-1 14 . since the procedure is considered to mimic the  selection process 29 , we have generated rag-2  pd-1 mice and injected varying amounts of anti - cd3 mab . 50 g of the anti - cd3 mab induced more expansion of the thymocytes in rag-2  pd-1 than in rag-2  pd-1 mice ( 47.1  21.8 vs. 27.3  14.5  10 ; n = 9 , p < 0.05 ) , whereas no significant difference was observed any more by 100 g or more ( 54.5  26.1 vs. 53.1  12.3  10 ; n = 6 , p > 0.05 ) . our preliminary biochemical studies indicate that the cross - linking of pd-1 indeed inhibits the cell activation signals such as ca influx mediated by b cell receptor ( bcr ) cross - linking in a b cell line transfected with pd-1 cdna ( our unpublished data ) . therefore , it is suggested that pd-1 functions as a negatively regulating receptor for antigen stimulation . these results have strongly suggested that pd-1 negatively regulates the  selection process probably by affecting the threshold of pre - tcr / cd3 complex  mediated signaling . it has been further indicated that the efficiency of positive selection for sp thymocytes expressing transgenic tcr-/ , but not for those expressing diversified endogenous tcr  chains , is significantly reduced in the absence of pd-1 . first , reduced efficiency of positive selection in the absence of pd-1 can be an indirect consequence of the accelerated  selection . since pd-1 deficiency is suggested to lower the threshold for pre tcr-/cd3 signaling , the dp thymocytes developed in the absence of pd-1 would include those that were selected by a weaker activation signal that otherwise could not allow them to expand . such dp cells that expanded by suboptimal  selection signaling may not be qualified for positive selection as long as the transgenic tcr-/s are fixed , resulting in the reduced efficiency of the subsequent positive selection . however , these unqualified thymocytes may be rescued by the expression of endogenous tcr  chain to form new tcr-/s with compensatory higher avidity for the selecting ligands , which may explain the unchanged efficiency of positive selection for such thymocytes in the absence of pd-1 . alternatively , pd-1 might directly affect the positive selection process , independently of the effect on the  selection . unlike in normal and v8 transgenic mice , the h - y and 2c mice expressed pd-1 in a portion of dp thymocytes , the cd4cd8 population , which may undergo positive selection 30 . it may then be possible that the deficiency of pd-1 causes the hyperactivation of this population upon interaction with selecting ligands for tcr , resulting in negative rather than positive selection . we consider the latter less likely because the efficiency of the positive selection for cd4 sp thymocytes was unaffected in h - y  pd-1 mice in both neutral and positive selecting backgrounds . if pd-1 directly regulates the positive selection process , the pd-1 deficiency should affect both cd4 sp and cd8 sp thymocytes similarly . in addition , at least in the h - y model , pd-1 is not significantly expressed in the cd69 population ( fig . is strongly suggested to result in the significant alteration of the final t cell repertoire either directly or indirectly . we have reported that b6 mice deficient for pd-1 develop autoimmune diseases , including lupus - like glomerulonephritis and destructive arthritis , as they age , which are greatly accelerated by the additional lpr / lpr mutation 16 . in addition to the dysregulation of peripheral tolerance 16 , it is tempting to speculate that the altered t cell repertoire formation in the absence of pd-1 may also increase the likelihood of the emergence of mature autoreactive t cells and thereby predispose the mice to the systemic autoimmune diseases .
pd-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine - based inhibitory motif , and disruption of the pd-1 gene results in the development of lupus - like autoimmune diseases . in this study 
 , we examined effects of the pd-1 deficiency on the thymocyte differentiation at the clonal level using t cell receptor ( tcr)- ( v8 ) and tcr-/ ( h - y and 2c ) transgenic mice . in these tcr transgenic lines , pd-1 expression in the thymus was variably augmented , but as in the normal mice , confined largely to the cd4cd8 thymocytes . 
 the transgenic mice crossed with pd-1/ mice in the neutral genetic backgrounds exhibited selective increase in the cd4+cd8 + ( dp ) population with little effect on other thymocytes subsets . 
 similarly , the absence of pd-1 facilitated expansion of dp thymocytes in recombination activating gene ( rag)-2/ mice by anti - cd3 antibody injection . on the other hand , h - y or 2c transgenic pd-1/ mice with the positively selecting background showed significantly reduced efficiency for the generation of cd8 + single positive cells bearing the transgenic tcr-/ in spite of the increased dp population . these results collectively indicate that pd-1 negatively regulates the  selection and modulates the positive selection , and suggest that pd-1 deficiency may lead to the significant alteration of mature t cell repertoire .