pd-1 was   shown to deliver a negative signal after   binding to either of its two ligands , pd - l1 ( b7-h1 ) or pd - l2 ( b7-dc ) in immune responses [ 14 ] . meanwhile ,
pd-1 and its ligands have been reported to play an important role in the etiopathogenesis of murine autoimmune diseases .
spontaneous autoimmune diseases including lupus - like glomerulonephritis , arthritis , and fatal autoimmune dilated cardiomyopathy developed in pd-1 deficient mice of different genetic backgrounds [ 57 ] . in addition
, blockade of pd-1/pd - l pathway could exaggerate or accelerate the development of autoimmune diseases such as diabetes in prediabetic nod mice and experimental autoimmune encephalitis [ 8 , 9 ] .
recently , up - regulated expression of pd-1 molecule and/or its ligands was demonstrated in human diseases including rheumatoid arthritis ( ra ) and inflammatory colitis , indicating that pd-1 and pd-1 ligands were actively participating in the pathogenetic process in human autoimmune diseases [ 10 , 11 ] .
systemic lupus erythematosus ( sle ) is a prototype of human systemic autoimmune disease , characterized by dysregulated activation of both t and b lymphocytes and development of many autoantibodies .
accordingly , pd-1 and pd-1 ligands may play a critical role in the pathogenesis of sle .
the present study thus aimed to investigate the expression and function of pd-1 and its two ligands on peripheral mononuclear cells ( pbmcs ) in patients with sle .
twenty - eight patients who fulfilled at least 4 criteria for the diagnosis of sle set by the american college of rheumatology   were enrolled in the study .
all enrolled patients were not taking corticosteroid and immunosuppressant agents for more than 4 weeks prior to the study .
their peripheral blood was drawn , and pbmc were immediately isolated for flowcytometric analysis and in vitro culture .
flowcytometry with dual staining was performed using fluorescece - conjugatd mabs including fitc - conjugated anti - cd3 , anti - cd19 , and anti - cd14 ( bd biosciences , sna diego ca , usa ) and pe - conjugated anti - pd-1 , anti - pdl1 , and anti - pdl2 ( mih clones , e bioscience , sna diego , ca , usa ) .
briefly , pbmc were prepared from fresh heparinized blood using ficoll - hypaque density gradient separation and adjusted to 5  10 cells / ml . two hundred microliters of cell suspension were incubated simultaneously with 20 l fitc - conjugated mab and 20 l pe - conjugated mab in different combinations on ice in the dark for 30 minutes .
cells incubated with fitc- and pe - conjugated mouse igg were used as isotype controls .
the stained cells were analyzed with a facscalibur ( becton dickinson ) flow cytometer and associated software programs ( cellquest ) . in selected sle patients and normal individuals ,
their pbmcs ( 300 l ) at the concentration of 1  10 cells / ml were cultured in 96-well microtiter plates under the stimulation with pma ( 5 ng / ml ) plus ionomycin ( 1 g / ml ) . for blocking pd-1/pd-1 ligands interaction , we added mab against pd-1 ( mih4 clone ) or pd - l1 ( mih1 clone ) or pd - l2 ( mih18 clone ) in the cultured cell system .
their endotoxin levels were all less than 0.001 ng/g antibody as determined by the lal assay .
the different groups include addition of anti - pd-1 mab ( 1 g / well ) , anti - pd - l1 mab ( 1 g / well ) , anti - pd - l2 mab ( 0.5 g / well ) , or anti - pd - l1 plus anti - pd - l2 . after 48 hours ' culture ,
the supernatants were collected , and cytokines including ifn- , il-2 , il-4 , and il-10 were assayed by elisa kits .
the concentrations of different cytokines in cultured supernatants were measured by commercially available elisa kits ( bender medsystems , vienna , austria ) .
the assay employed the quantitative sandwich enzyme immunoassay technique and was performed as manufacturer 's instruction .
standard or samples were added to react with specific anticytokine mab precoated onto a microplate for 2 hours at room temperature .
after washing , horseradish peroxidase - conjugated polyclonal antibodies against cytokines were added to react with the bound cytokines for 1 hour .
then , substrate solution was added and incubated for 20 minutes , followed by addition of stop solution .
the optical density of each well was immediately determined by microplate reader set to wavelength 450 nm .
fisher 's lsd method was applied to compare the levels of pd-1 and pd-1 ligands - expressing cells between sle and normal controls .
wilcoxon rank sum test was used to compare the increase percentages of cytokine production between patients with sle and normal subjects .
the expression of pd-1 and its two ligands on freshly prepared pbmc from 26 normal controls and 28 patients with sle were shown in table 1 .
one representative facs dot - plot data of pd-1 and pd-1 ligands on t cells , b cells , and monocytes was depicted in figure 1 . in normal controls , pd-1 was barely detectable on t cells ( < 1% ) .
only a very minority of b cells ( mean 2.14% ) and monocytes ( mean 5.78% ) expressed a low level of pd-1 .
in contrast , pd - l1 expression was readily detected on t cells , b cells , and monocytes .
different to pd - l1 , pd - l2 expression was mainly noted on monocytes ( mean 72.12% ) with only a minority of t cells and b cells ( mean < 4% ) expressed pd - l2
. similar expression pattern of pd-1 and pd-1 ligands on each mononuclear cell subset was also observed in patients with sle .
compared with normal controls , patients with sle had significantly increased percentages of pd-1-expressing cd3 t cells ( 1.51  1.12% versus 0.64  0.53% , p < .001 ) and pd-1-expressing cd19b cells ( 5.11  3.91%   versus 2.14  1.67% , p < .005 ) .
regarding pd-1 ligands , sle patients had more pd - l1-expressing cd19b cells ( 20.59  10.24% versus 13.21  1.67% , p < .005 ) and pd - l2-expressing cd14monocytes ( 84.78  12.82%   versus 72.12  26.92% , p <
.005 ) than normal controls . despite slightly increased frequencies of pd-1 and pd-1 ligands expression in some cell populations from sle patients ,
the mean fluorescence intensities of pd-1 and pd-1 ligands expression on the positive cells were not significantly different between patients with sle and normal controls ( data not shown ) .
the result suggests that the expression of pd-1/pd-1 ligands was not impaired in human sle .
we thus designed to investigate the functional effects of blocking pd-1 or pd-1 ligands on the production of various cytokines produced by in vitro cultured pbmc .
the cytokines examined in the study included th1-derived cytokines ( il-2 and ifn- ) and th2-derived cytokines ( il-4 , il-10 ) . in selected patients and normal individuals , 8 in each ,
their freshly prepared pbmcs were cultured and   stimulated with pma plus ionomycin in the absence or presence of anti - pd-1 mab , anti - pd - l1 mab , anti - pd - l2 mab , or anti - pd - l1 plus anti - pd - l2 mabs .
we found that blockage of either pd-1 or pd-1 ligands substantially increased the production of il-2 , ifn- , and il-10   in all cases of pbmc from both sle and normal individuals but has no any effect on il-4 .
as expected , the concentration of cytokines produced by stimulated pbmc in vitro varied widely among different sle patients and normal controls . for further comparison between sle and normal subjects ,
the amplitudes of enhancement in cytokine production were expressed as percentages of increase , which was calculated by the increased amount of cytokine in the presence of blocking ab divided by baseline cytokine concentration in isotype igg control . among the experiments , a few samples of stimulated pbmc had no detectable baseline cytokine .
they were omitted in comparison due to difficulty in calculation of percentages of increase . as shown in table 2
,   blockage of pd-1/pd-1 liagnds pathway greatly increased production of ifn- , which ranged averagely from 134% to 160% in normal controls and from 153% to 203% in sle .
similar to ifn- , il-2 levels were much increased , in which the amplitudes of increased percentages ranged averagely from 156% to 243% in normal control and from 189% to 397% in patient with sle ( table 3 ) . the enhancing effect on il-10
was readily seen too , which ranged averagely from 97% to 126% in normal controls and from 73% to 132% in sle ( table 4 ) .
there were also no significant differences in the increased production of il-2 and il-10 between sle patients and normal subjects .
in addition , it appeared that anti - pd-1 possessed similar effects as anti - pd - l1 or anti - pd - l2 .
meanwhile , there were no synergic effects by anti - pd - l1 plus anti - pd - l2 compared with either alone .
there was no any effect on il-4 effect by blockage of pd-1/pd-1 ligands pathway ( data not shown ) .
the expression of pd-1 and its two ligands on pbmc of normal individuals and patients with sle in the present study was in concordance with previous observation [ 1315 ] .
, pd - l1 is constitutively expressed on t cells , b cells macrophages , and dentritic cells ( dcs ) and further upregulated upon activation .
in contrast , the expression of pd - l2 was regulated more tightly and observed on activated macrophage and dcs .
the expression of pd - l1 is also detected on a wide variety of nonlymphoid cells including vascular endothelial cells , epithelial cells , muscle cells , hepatocytes ,  cells in pancreas , astrocytes in the brain , as well as at sites of immune privilege including placenta and eye [ 1618 ] .
the expression of pd - l1 on nonlymphoid tissue was shown to play a crucial role in the control and maintenance of peripheral t cell tolerance . compared with normal controls , generally patients with sle
had increased expression of pd-1 and pd-1 ligands on t cells , b cells , and monocytes , some of which reached statistically significant differences , because pd-1 could be induced on t cells after activation , and increased activated t cells have already been well documented in patients with sle .  
thus the increased numbers of pd-1-expressing cd3 t cells in sle patients could be due to increased numbers of activated t cells in the periphery of sle patients .
increased expression of pd-1 and/or pd-1 ligands had also been reported in other human autoimmune diseases .
hatachi et al . demonstrated that pd-1 t cells were enriched in ra synovial fluid , and phenotypic analysis suggested that these cells a unique anergic cell subset .
kobayashi et al . showed that the expression of pd-1 in salivary lymphocytes and pd - l1 on ductal and acinar epithelial cells in salivary glands was enhanced in patients with sjogren 's syndrome .
however , decreased pd-1 expression on peripheral cd4 t cells was found in patients with type-1 diabetes in one study .
probably , pd-1/pd-1 ligands pathway plays different roles in systemic versus organ - specific autoimmune diseases .
in addition , one study in lichen planus , a t cell - mediated chronic inflammatory mucocutaneous disease , revealed abundant expression of pd-1 and pd - l1 in infiltrating t cells and macrophages in the subepithelium and substantial pd - l1 on kerotinocytes . the inhibitory function of pd-1 molecule was initially suggested by the studies on pd-1 deficient mice , which exhibited hyperactivation of the immune system and subsequently developed autoimmune diseases .
interestingly , pd-1 deficient mice developed different kinds of autoimmune diseases depending on the genetic background [ 57 ] .
on c57bl/6 background , pd-1 deficient mice developed lupus - like glomerulonephritis and arthritis with deposition of igg , and c3 in the glomeruli , whereas pd-1 deficient mice suffered a fatal dilated cardiomyopathy on balb / c background .
the result of our functional study was in concordance with the concept of the immunosuppressive role of pd-1/pd-1 ligands .
we showed that blockage of pd-1/pd-1 ligands pathway augmented the production of cytokines in stimulated pbmc in vitro , with predominant effects on th-1 derived cytokines ( il-2 and ifn- ) .
regarding th-2 cytokines , we also observed enhancing effect on il-10 though weaker compared with those on il-2 and ifn-. however , there was no effect on il-4 production . blocking pd-1 molecule or either of the two pd-1 ligands demonstrated similar effects on the production of various cytokines
. as previously reported , blockage with both anti - pd - l1 and anti - pd - l2 did not show a significantly synergic effect in our study .
ansari et al . reported that blockade of pd-1 or pd - l1 but not pd - l2 exaggerated diabetes in prediabetic nod mice .
they observed the expression of pd - l1 but not pd - l2 on the  cells , which might explain the reason why the anti - pd - l1 but not anti - pd - l2 antibody exaggerated diabetes , whereas , salama et al . observed the acceleration of eae by the blockade of pd-1 and pd - l2 but not pd - l1 . since pd - l1 but not pd - l2
was highly expressed in the central nervous system , the expression of ligands in target organs might not determine the efficient of each blocking antibody .
blockade of pd-1/pd - l pathway might have an opposite effect on autoimmune diseases in certain condition .
examined the effect of blocking antibodies against pd - l1 and pd - l2 on the colitis model .
antibodies against pd - l1 but not pd - l2 suppressed the development of colitis , indicating the costimulatory function of pd - l1 in the inflammatory response in the gut . according to
the report by loke and allison , the expression of pd - l1 and pd - l2 was   differentially regulated by th1 and th2 cytokines .
since the recent analyses using blocking antibodies against pd - l1 and pd - l2 revealed nonoverlapping functions of these ligands , the differential regulation of pd - l1 and pd - l2 expression might have some biological significance .
in conclusion , the present study found that there were no intrinsically defective expression of pd-1 and pd-1 ligands on pbmc in patients with sle . on the contrary ,
we noted increased expression of pd-1 and/or its ligands in some subsets of pbmc from patients with sle . since pd-1 and pd-1 ligands play a role in regulation of both activation and tolerance of lymphocytes , the findings of our study most likely reflect the activation status of pbmc in lupus patients and they do not rule out a defect in tolerance mediated by pd-1 .
the exact function of increased pd-1 and pd-1 ligands in some subsets of pbmc in the pathogenesis of human sle needs further study .  
 programmed death-1 ( pd-1 ) was shown to deliver an inhibitory signal after binding to its ligands , pd - l1 ( b7-h1 ) or pd - l2 ( b7-dc ) . 
 recently , up - regulated expression of pd-1 molecule and/or its ligands was demonstrated in human diseases including rheumatoid arthritis and inflammatory colitis . 
 the study aimed to investigate the expression and function of pd-1 and pd-1 ligands on circulating t cells , b cells and monocytes from patient with systemic lupus erythematosus ( sle ) . 
 the results showed that patients with sle had significantly increased percentages of pd-1-expressing cd3+t cells and cd19+b cells , pd - l1-expressing cd19+b cells and pd - l2-expressing cd14+b monocytes . in selected sle patients and normal subjects , functional study of pd-1/ pd-1 ligands pathway on the production of cytokines by stimulated pbmc was examined . 
 blockages of pd-1 or pd-1 ligands substantially increased the production of il-2 , ifn- and il-10 , the amplitude of increase roughly ranged from one to three times . 
 there were no significant differences of the enhancing effects on cytokine production by blockage of pd-1/pdl pathway between sle patients and normal subjects . 
 the study indicates that there are no intrinsically defective expression and function of pd-1 and pd-1 ligands on pbmc in patients with sle . 
 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion