hepatitis c virus ( hcv ) infects over 180 million people worldwide and exhibits a remarkable propensity toward chronic hepatitis , liver cirrhosis and hepatocellular carcinoma1 . chronic hcv infection is also associated with b cell lymphoproliferative disorders2 , including most notably mixed cryoglobulinemia ( mc ) and b cell lymphomas . in chronically hcv - infected individuals , the frequencies of cytotoxic t lymphocytes ( ctl ) are relatively low ; similarly , the proliferative capacity as well as effector functions of hcv - specific cd4/cd8 t cells are impaired , and the production of th1-type cytokines ( i.e. , il-2 and ifn- ) is dramatically suppressed38 . t regulatory ( tr ) cells , including cd4cd25 as well as cd8cd25 cell populations , suppress effector / memory t cell responses that are critical in clearance of pathogens , thus playing a pivotal role in the establishment and maintenance of chronic infections such as hcv916 . understanding the mechanisms underlying t cell dysregulation in hcv infection has also been advanced since the identification of program death-1 ( pd-1 ) , an important inhibitory pathway in regulation of t cell receptor ( tcr ) signaling1720 . we have previously demonstrated that hcv core protein differentially regulates t and b lymphocyte functions through pd-1 and socs-1 negative signaling pathways2127 . we have recently observed that subjects with hcv - associated lymphoma ( hcv - l ) exhibit relatively higher pd-1 expression and more intense t cell suppression than those with non - hcv - associated lymphoma ( non - hcv - l ) or hcv infection alone without lymphoma ( hcv alone ) or healthy subjects ( hs ) [ yao , et al . the relationship between pd-1 pathway and tr cells in regulation of t cell responses in the setting of hcv - l , however , remain unknown . in this report , we describe relatively higher numbers of cd4cd25 and cd8cd25 tr cells as well as higher levels of pd-1 expression on these tr cells in the peripheral blood of subjects with hcv - l compared to those from non - hcv - l or hcv alone or hs . interestingly , pd-1 appears to play a role in regulating tr cell number as well as function in suppressing t cell responses in the setting of chronically hcv - infected subjects with b cell lymphoma . as an initial approach to characterize the role of tr cells in regulation of t cell responses , we first determined the relative number of cd4cd25 and cd8cd25 tr cell populations in the peripheral blood of subjects with hcv - l , and compared with those in non - hcv - l , hcv alone , or hs . as shown in fig . 1a , both cd4cd25 and cd8cd25 tr cells were found to be increased in the subjects with hcv - l when compared to those non - hcv - l , hcv alone , or hs . similar results were obtained in repeated experiments using different antibody conjugates to compare cd4cd25 tr cell numbers . since the transcription factor foxp3 has been accepted as a specific marker for tr cells2829 , intracellular foxp3 expression was analyzed in cd4cd25 tr obtained from a subject with hcv - l , three hcv alone , and a hs . as shown in figure 1b , cd4cd25 tr cells were found to be higher in the subject with hcv - l compared to hcv subjects without lymphoma and a hs . correspondingly , foxp3 expression was found to be highest in the subject with hcv - l , followed by hcv alone , and the lowest in the hs . these results confirm the finding of an increased tr cell component in the setting of hcv - l . since cd25 ( il-2r  chain ) is an early t cell activation marker , higher levels of cd25 expression on cd4 t cells might simply be due to t cell activation rather than secondary to the development of tr cells in subjects with hcv - l . to further elucidate this concern , we stimulated pbmc isolated from hcv - l , hcv alone , and hs with pha for 24 h , and then evaluated cd69 expression , a marker of t cell activation , on cd4cd25 tr cell populations . as shown in figure 1c , cd69 expression on cd4cd25 t cell populations in the setting of hcv - l was lower than in subjects with hcv alone or hs , suggesting that the increased cd25 marker is secondary to the increased number of tr rather than t cell activation . additionally , cd69 expressions were found to be lower on cd4cd25 t cell populations as well as on cd8cd25 t cells and hcv - specific cd8 t cells in hcv - l subjects when compared with hcv alone or hs , which is consistent with the finding that t cell activation is more severely suppressed in the setting of hcv - associated lymphoma . it has been previously reported that pd-1 is highly expressed on cd4cd25 tr cells3031 , although its role in regulating tr cell number and activity remains less clear . here we measured pd-1 expression on cd4cd25 and cd8cd25 tr cells obtained from the subjects with hcv - l , non - hcv - l , and hcv alone without lymphoma by three - color - labeling facs analysis and also found this high level of expression to be the case on tr cells in all groups , i.e. , cd4cd25 and cd8cd25 tr cells exhibit relative higher levels of pd-1 expression versus cd4cd25 and cd8cd25 effector t cells , with the highest pd-1 level in the setting of hcv - l ( fig . because we had found increased numbers of tr cells and higher level of pd-1 expression on these cells in the setting of hcv - l , we next examined the possibility that the pd-1 pathway regulates tr cells ability to inhibit t cell activation and proliferation . to this end , we treated the pbmc isolated from an hcv - l subject with anti - pdl-1 or control antibody overnight , followed by incubation with hcv peptides ex vivo for 5 days , and then examined the total number of cd4cd25 tr cells and cd69 expression on cd4 t cells . 2a , compared with treatment by the control antibody , blocking the pd-1 pathway by treating the cells with pdl-1 antibody reduced the total number of cd4cd25 tr cells ( top ) , and increased cd69 expression ( bottom ) on the suppressed cd4 t cells . these results suggest that blocking the pd-1 pathway may regulate tr cell development and rescue t cell activation in the setting of hcv - l . to determine the effect of blocking the pd-1 pathway on t cell proliferation in hcv - l , we next pre - incubated cfse - labeled pbmc with anti - pdl-1 or control antibody followed by stimulation with either anti - cd3/cd28 or autogeneic healthy pbmc for 5 days . t cell proliferation as examined by cfse dilution was analyzed by flow cytometry after double staining and gating on t cell populations . 2b , the proliferating t cells ( shown in the m1 and m2 gates ) were detected more frequently in the setting of anti - pdl-1 treatment compared to the control antibody - treated cells when pbmc were stimulated with anti - cd3/cd28 antibodies ( upper panel ) . this difference was more significant when the cells were stimulated with autogeneic healthy pbmc ( lower panel ) , in which case 47% and 23% of proliferating t cells were detected in the m1 and m2 gates , respectively , upon blocking with pd-1 signaling versus 28% and 22% cells detected with control antibody treatment . correspondingly , the resting t cells in m3 gating following anti - pd - l1 treatment were observed at 29% , while 48% were detected in the control group . these results suggest that blocking the pd-1 pathway may rescue impaired t cell function in hcv - l . even though blocking pd-1 signaling can reduce the number of tr cells and rescue the suppressed t cell activation and proliferation ex vivo , the effect on rescuing t cell function by pd-1 blocking may be secondary to the reduced tr cells in the bulk pbmc or the result of directly blocking pd-1 signaling on effector t cells , or both . thus , we further examined the role of cd4cd25 tr cells in suppressing t cell proliferation by mixed lymphocyte culture ( mlc ) . to this end , we separated the cd25 cells and cd25cells from a subject with hcv - l , incubated with cfse - labeled healthy pbmc , and analyzed by cfse dilution to determine the ability of these two cell populations from the subject to suppress healthy pbmc proliferation by mlc . 2c , healthy pbmc co - cultured with cd25 tr cells from the hcv - l subject proliferated poorly when compared to those co - cultured with cd25 cells ( upper panel ) . repeated experiments using effector cells from another healthy subject were similar ( lower panel ) . these results suggest that tr cells functionally suppress autogeneic healthy t cell proliferation . because in vitro depletion of cd25 cells results in increased responsiveness of the hcv - specific effector cells911 , it has been suggested that induction of tr cells play a causal role in the establishment of chronic hcv infection . to further elucidate the role of tr cells and pd-1 in suppressing t cell proliferation , we compared the proliferative ability of cd25-depleted cells versus bulk pbmc from a subject with hcv - l , in the presence of anti - pdl-1 or control antibody , by cfse dilution and flow cytometry analysis . anti - pdl-1 proliferated better compared to bulk pbmc treated with anti - pd - l1 or cd25-depleted cells treated with the control antibody . the least proliferation was observed in the setting of bulk pbmc treated with the control antibody ( fig 2d , lower right panel ) . these results suggest that both tr and pd-1 signaling play a role in suppressing t cell proliferation in the setting of hcv - l . although several biomarkers have previously been recognized to play a role in defining tr cells in disease conditions3031 , the discovery of high expression of pd-1 in high number of tr cells in the setting of hcv - l represents a novel finding , most notably in that the function of these cells appears to be regulated via pd-1 signaling . these findings suggest that the pd-1 pathway is involved in the regulation of tr cells as a means of suppressing t cell responses in the context of chronically hcv - infected subjects with b cell lymphoma . it remains unclear as to whether the increased tr cells are a consequence of or contribute to the development of hcv - l . an institutional review board ( irb)-approved protocol at james h. quillen va medical center and east tennessee state university ( johnson city , tn ) has contributed to a database for the storage of blood samples from hcv - infected individuals . three hcv subjects with b cell lymphoma , three with non - hcv - associated b cell lymphoma , three hcv - infected individuals without lymphoma , and three healthy subjects as normal controls are included in this study . all hcv subjects , either genotype 1a or 1b , with hcv rna levels range from 97,765~50,000,000 iu / ml , were selected prior to peg - interferon and ribavirin treatment . hcv - l or non - hcv - l subjects were diagnosed by clinical and histological features , confirmed by immunochemistry and flow cytometry , and were post - antitumor therapy for at least six months . human peripheral blood mononuclear cells ( pbmc ) were isolated from the peripheral blood of subjects by ficoll density centrifugation with lympholyte - h ( cedarlane labs , ontario , canada ) . if indicated , cd25 t cells were further purified or deleted from isolated pbmc by incubation with a magnetic beads - conjugated anti - cd25 antibody , followed by positive or negative selection per the manufacturer s instruction ( miltenyi biotec . , auburn , ca ) . cells were cultured as described previously25 . to determine cell surface marker expression on t lymphocytes , 1  10 purified pbmcs were stimulated with either 1 g / ml pha ( sigma , saint louis , mi ) , or 10 g hcv peptides ( hla - a-0201/ns3/1073 - 1081/cingvcwtv ; hla - a-0201/ns3/1406 - 1415/klvalginav ; synthesized by genscript corporation , piscataway , nj ) , for 24 h and washed as described2425 . the following antibody conjugates , purchased from either bd pharmingen or ebioscience , were used in the double or triple staining as described2526 : pe - anti - cd4 , cd25 , cd69 , pd-1 , pd - l1 , foxp3 transcription factor ; fitc - anti - cd4 , cd8 , cd45ra , cd45ro ; and apc - cd25 , pd-1 , pd - l1 . the primary pe- , fitc , apc - isotype controls were used to determine the level of background staining . in triple staining , gated lymphocytes were further gated on cd4 , or cd8 cells ; then analyzed for the expression of cell surface markers in the double positive cell populations . for intracellular foxp3 staining , a human treg staining kit ( ebioscience ) was employed and the staining was performed according to the manufacturer s instruction . pbmcs isolated from subjects were pre - labeled with cfse according to the instructions per celltrace kit ( molecular probes , inc . , eugene , or ) . to determine the role of pd-1 pathway in regulation of t cell proliferation , the cfse - labeled cells were incubated with anti - pd - l1 or a control isotype igg antibody ( 10 g / ml , ebioscience ) overnight , then stimulated the cells with pha ( 1 g / ml , sigma ) or anti - cd3/cd28 or autogenic pbmc for 5 days . after double staining and gating on t lymphocytes , the cell proliferation , represented as csfe dilution from m4/m3 ( resting cells ) to m2/m1 ( proliferating cells ) , was analyzed by flow cytometry with 488 nm excitation and emission filters appropriate for cfse fluorescein . percentage of cells as detected in the m1 , m2 , m3 , and m4 gating was shown on the histogram of the gated t cell populations . an institutional review board ( irb)-approved protocol at james h. quillen va medical center and east tennessee state university ( johnson city , tn ) has contributed to a database for the storage of blood samples from hcv - infected individuals . three hcv subjects with b cell lymphoma , three with non - hcv - associated b cell lymphoma , three hcv - infected individuals without lymphoma , and three healthy subjects as normal controls are included in this study . all hcv subjects , either genotype 1a or 1b , with hcv rna levels range from 97,765~50,000,000 iu / ml , were selected prior to peg - interferon and ribavirin treatment . hcv - l or non - hcv - l subjects were diagnosed by clinical and histological features , confirmed by immunochemistry and flow cytometry , and were post - antitumor therapy for at least six months . human peripheral blood mononuclear cells ( pbmc ) were isolated from the peripheral blood of subjects by ficoll density centrifugation with lympholyte - h ( cedarlane labs , ontario , canada ) . if indicated , cd25 t cells were further purified or deleted from isolated pbmc by incubation with a magnetic beads - conjugated anti - cd25 antibody , followed by positive or negative selection per the manufacturer s instruction ( miltenyi biotec . , auburn , ca ) . to determine cell surface marker expression on t lymphocytes , 1  10 purified pbmcs were stimulated with either 1 g / ml pha ( sigma , saint louis , mi ) , or 10 g hcv peptides ( hla - a-0201/ns3/1073 - 1081/cingvcwtv ; hla - a-0201/ns3/1406 - 1415/klvalginav ; synthesized by genscript corporation , piscataway , nj ) , for 24 h and washed as described2425 . the following antibody conjugates , purchased from either bd pharmingen or ebioscience , were used in the double or triple staining as described2526 : pe - anti - cd4 , cd25 , cd69 , pd-1 , pd - l1 , foxp3 transcription factor ; fitc - anti - cd4 , cd8 , cd45ra , cd45ro ; and apc - cd25 , pd-1 , pd - l1 . the primary pe- , fitc , apc - isotype controls were used to determine the level of background staining . in triple staining , gated lymphocytes were further gated on cd4 , or cd8 cells ; then analyzed for the expression of cell surface markers in the double positive cell populations . for intracellular foxp3 staining , a human treg staining kit ( ebioscience ) was employed and the staining was performed according to the manufacturer s instruction . pbmcs isolated from subjects were pre - labeled with cfse according to the instructions per celltrace kit ( molecular probes , inc . , eugene , or ) . to determine the role of pd-1 pathway in regulation of t cell proliferation , the cfse - labeled cells were incubated with anti - pd - l1 or a control isotype igg antibody ( 10 g / ml , ebioscience ) overnight , then stimulated the cells with pha ( 1 g / ml , sigma ) or anti - cd3/cd28 or autogenic pbmc for 5 days . after double staining and gating on t lymphocytes , the cell proliferation , represented as csfe dilution from m4/m3 ( resting cells ) to m2/m1 ( proliferating cells ) , was analyzed by flow cytometry with 488 nm excitation and emission filters appropriate for cfse fluorescein . percentage of cells as detected in the m1 , m2 , m3 , and m4 gating was shown on the histogram of the gated t cell populations .
t regulatory ( tr ) cells suppress t cell responses that are critical in the development of chronic viral infection and associated malignancies . programmed death-1 ( pd-1 ) also plays a pivotal role in regulation of t cell functions during chronic viral infection . to examine the role of pd-1 pathway in regulating tr cell functions that inhibit t cell responses during virus - associated malignancy 
 , tr cells were investigated in the setting of hepatitis c virus - associated lymphoma ( hcv - l ) , 
 non - hcv - associated lymphoma ( non - hcv - l ) , hcv infection alone , and healthy subjects ( hs ) . 
 relatively high numbers of cd4+cd25 + and cd8+cd25 + tr cells as well as high levels of pd-1 expressions on these tr cells were found in the peripheral blood of subjects with hcv - l compared to those from non - hcv - l or hcv alone or hs . 
 tr cells from the hcv - l subjects were capable of suppressing the autogeneic lymphocyte response , and depletion of tr cells in pbmc from hcv - l improved t cell proliferation . additionally , the suppressed t cell activation and proliferation in hcv - l was partially restored by blocking the pd-1 pathway ex vivo , resulting in both a reduction in tr cell number and the ability of tr to suppress the activity of effector t cells . 
 this study suggests that the pd-1 pathway is involved in regulating tr cells that suppress t cell functions in the setting of hcv - associated b cell lymphoma .