systemic lupus erythematosus ( sle ) is an autoimmune disease characterized by immune system dysfunction and the production of autoantibodies that lead to inflammation and tissue damage . t - lymphocytes ( t - cells ) promote inflammation through producing co - stimulatory signals and cytokines that activate dendritic cells and b - cells . activation of auto - reactive t - cells leads to abnormalities in cd4 and cd8 t - cells , which are critical drivers of the b - cell - dependent autoantibody response [ 3,57 ] . based on these previous findings , we speculate that an imbalance in the t - cell subset distribution eventually results in the development of sle . to that end , immunosuppressive or cytotoxic drugs that target t - cells can induce long - term remission in sle patients . in t - cell leukemia patients , long - term maintenance combination chemotherapy has been shown to be an effective , low - cost treatment regimen with limited adverse effects that can control disease in a stable manner over several years . based on these previous findings , we hypothesized that the combination of the cell cycle - specific drug vincristine with the non - cell cycle - specific drug cyclophosphamide would be effective in treating sle via positively affecting their t - cell subset distribution . thus , the aims of the present study were to ( i ) analyze the t - cell subset distribution in sle patients at baseline and ( ii ) determine whether vincristine - cyclophosphamide combination therapy can positively affect their t - cell subset distribution to keep the disease in remission . this study was approved by the ethics committee ( irb ) of the second hospital at shanxi medical college ( taiyuan , china ) . all subjects recruited for this study provided written informed consent prior to their participation . between october 2008 and october 2012 , 30 sle patients were consecutively recruited from the inpatient population of the second hospital at shanxi medical college . all sle patients fulfilled the american college of rheumatology ( acr ) classification criteria for sle . the degree of sle disease activity was assessed using the sle disease activity index ( sledai ) . patients with any other autoimmune disease , those having received immunosuppressive therapies other than glucocorticoid therapy within the past 6 months , or those with wbc counts of less than 3.010/l were excluded . fifteen healthy volunteers matched by race , sex , and age were recruited from the outpatient population of the same hospital during the same time period . the 30 sle patients were subdivided into 2 groups : those with low disease activity (  low activity , sledai9 , n=17 cases ) and those with high disease activity (  high activity , sledai>9 , n=13 cases ) . all sle patients were given vincristine ( 1 mg by intravenous drip ) followed by intravenous cyclophosphamide over the next 24 hours ( 200600 mg depending on sle patient s wbc count ; 200 mg for 3.04.010/l , 400 mg for 4.01010/l , 600 mg for > 1010/l ) . this therapeutic regimen was executed every three weeks , and prednisone therapy was permitted . according to the total duration of vincristine - cyclophosphamide combination therapy , all sle patients were divided into 4 groups : a 3-month group , a 6-month group , a 1224-month group , and > 24-month group . multitest antibodies  cd3 ( fitc ) , cd8 ( pe ) , cd45 ( percp ) , and cd4 ( apc )  were purchased from bd biosciences . peripheral blood samples ( 2 ml ) from each subject were collected in edta - coated tubes at baseline and at various time points ( i.e. , 3 months , 6 months , etc . ) during vincristine - cyclophosphamide combination therapy . for immunofluorescence staining , fresh edta - treated whole blood samples were kept at room temperature immediately after collection , and 50 l was placed in a trucount tube . after incubation at 4c for 30 min in the dark , stained cells were washed with facs buffer ( phosphate - buffered saline ( pbs ) , 0.2% fetal bovine serum ( fbs ) , 0.09% nan3 ) and then incubated once again for 15 min in the dark . negative controls were performed simultaneously using fitc - labelled mouse anti - human igg1 mab and apc - labelled mouse anti - human igg1 mab . all samples were analyzed on a facscanto ( bd bioscience , san jose , ca , usa ) , and the resulting data was analyzed with multiset software . an independent student s t - test was used for comparisons between 2 groups , and 1-way analysis of variance ( anova ) was used for comparisons among 3 or more groups . this study was approved by the ethics committee ( irb ) of the second hospital at shanxi medical college ( taiyuan , china ) . all subjects recruited for this study provided written informed consent prior to their participation . between october 2008 and october 2012 , 30 sle patients were consecutively recruited from the inpatient population of the second hospital at shanxi medical college . all sle patients fulfilled the american college of rheumatology ( acr ) classification criteria for sle . the degree of sle disease activity was assessed using the sle disease activity index ( sledai ) . patients with any other autoimmune disease , those having received immunosuppressive therapies other than glucocorticoid therapy within the past 6 months , or those with wbc counts of less than 3.010/l were excluded . fifteen healthy volunteers matched by race , sex , and age were recruited from the outpatient population of the same hospital during the same time period . the 30 sle patients were subdivided into 2 groups : those with low disease activity (  low activity , sledai9 , n=17 cases ) and those with high disease activity (  high activity , sledai>9 , n=13 cases ) . all sle patients were given vincristine ( 1 mg by intravenous drip ) followed by intravenous cyclophosphamide over the next 24 hours ( 200600 mg depending on sle patient s wbc count ; 200 mg for 3.04.010/l , 400 mg for 4.01010/l , 600 mg for > 1010/l ) . this therapeutic regimen was executed every three weeks , and prednisone therapy was permitted . according to the total duration of vincristine - cyclophosphamide combination therapy , all sle patients were divided into 4 groups : a 3-month group , a 6-month group , a 1224-month group , and > 24-month group . multitest antibodies  cd3 ( fitc ) , cd8 ( pe ) , cd45 ( percp ) , and cd4 ( apc )  were purchased from bd biosciences . peripheral blood samples ( 2 ml ) from each subject were collected in edta - coated tubes at baseline and at various time points ( i.e. , 3 months , 6 months , etc . ) during vincristine - cyclophosphamide combination therapy . for immunofluorescence staining , fresh edta - treated whole blood samples were kept at room temperature immediately after collection , and 50 l was placed in a trucount tube . after incubation at 4c for 30 min in the dark , stained cells were washed with facs buffer ( phosphate - buffered saline ( pbs ) , 0.2% fetal bovine serum ( fbs ) , 0.09% nan3 ) and then incubated once again for 15 min in the dark . negative controls were performed simultaneously using fitc - labelled mouse anti - human igg1 mab and apc - labelled mouse anti - human igg1 mab . all samples were analyzed on a facscanto ( bd bioscience , san jose , ca , usa ) , and the resulting data was analyzed with multiset software . an independent student s t - test was used for comparisons between 2 groups , and 1-way analysis of variance ( anova ) was used for comparisons among 3 or more groups . patient characteristics are summarized in table 1 , and the percentages of t - cell subsets in each experimental group are shown in table 2 . at baseline , the cd3 t - cell percentage was significantly lower in the high activity group than healthy controls ( p=0.004 ) , while there was no significant difference in the cd3 t - cell percentage between the low activity group and healthy controls ( p>0.05 ; figure 1a ) . furthermore , at baseline , the cd4 helper t - cell ( th cell ) percentage was significantly lower in the low activity group than healthy controls ( p<0.001 ) , and the cd4 th cell percentage was significantly lower in the high activity group than the low activity group ( p<0.001 ; figure 1b ) . finally , at baseline , the cd8 suppressor t - cell ( ts cell ) percentage was significantly higher in the low activity group compared with healthy controls ( p<0.05 ) , while there was no significant differences in the cd8 ts cell percentage between the high activity and low activity groups nor between the high activity group and healthy controls ( p>0.05 ; figure 1c ) . correlation analysis was then applied to evaluate the relationship between t - cell subsets , sledai scores , and the complement component c3 at baseline . a significantly negative correlation was observed between the cd3 t - cell percentage and sledai score at baseline ( r=0.471 , p=0.015 ; figure 2a ) . a significantly negative correlation was observed between the cd4 th cell percentage and sledai score at baseline ( r=0.473 , p=0.015 ; figure 2b ) . moreover , a significantly positive correlation was observed between cd4 th cell percentage and c3 at baseline ( r=0.612 , p=0.002 ; figure 2c ) . however , no correlations were observed among cd8 ts cell percentage , sledai score , or c3 at baseline ( p>0.05 ; data not shown ) . the low activity and high activity patients in each treatment group ( i.e. , 3-month , 6-month , etc . ) were pooled together and compared against the high activity baseline group and low activity baseline group in order to simplify the comparative analysis . after 3 months of combination therapy , the cd3 t - cell percentage was significantly higher than the high activity baseline ( p=0.000 ; figure 3 ) . after 6 months of combination therapy , the cd3 t - cell percentages remained significantly higher than the high activity baseline ( p<0.01 ) . the cd3 t - cell percentages across all combination therapy time groups showed no significant differences with the low activity baseline ( p>0.05 ) . after 3 months of combination therapy , the cd4 th cell percentage was significantly higher than both the high activity and low activity baselines ( p<0.05 ) . after 6 months of combination therapy , the cd4 th cell percentage showed no significant difference with both the high activity and low activity baselines ( p>0.05 ) , but did present a non - significant upward trend . after 3 months of combination therapy , the cd8 ts cell percentage showed no significant difference with both the high activity and low activity baselines ( p>0.05 ) . after 6 months of combination therapy , the cd8 ts cell percentage was significantly higher than the high activity baseline ( p<0.05 ) . after 12 months of combination therapy , the cd8 ts cell percentage was also significantly higher than the low activity baseline ( p<0.05 ) . patient characteristics are summarized in table 1 , and the percentages of t - cell subsets in each experimental group are shown in table 2 . at baseline , the cd3 t - cell percentage was significantly lower in the high activity group than healthy controls ( p=0.004 ) , while there was no significant difference in the cd3 t - cell percentage between the low activity group and healthy controls ( p>0.05 ; figure 1a ) . furthermore , at baseline , the cd4 helper t - cell ( th cell ) percentage was significantly lower in the low activity group than healthy controls ( p<0.001 ) , and the cd4 th cell percentage was significantly lower in the high activity group than the low activity group ( p<0.001 ; figure 1b ) . finally , at baseline , the cd8 suppressor t - cell ( ts cell ) percentage was significantly higher in the low activity group compared with healthy controls ( p<0.05 ) , while there was no significant differences in the cd8 ts cell percentage between the high activity and low activity groups nor between the high activity group and healthy controls ( p>0.05 ; figure 1c ) . correlation analysis was then applied to evaluate the relationship between t - cell subsets , sledai scores , and the complement component c3 at baseline . a significantly negative correlation was observed between the cd3 t - cell percentage and sledai score at baseline ( r=0.471 , p=0.015 ; figure 2a ) . a significantly negative correlation was observed between the cd4 th cell percentage and sledai score at baseline ( r=0.473 , p=0.015 ; figure 2b ) . moreover , a significantly positive correlation was observed between cd4 th cell percentage and c3 at baseline ( r=0.612 , p=0.002 ; figure 2c ) . however , no correlations were observed among cd8 ts cell percentage , sledai score , or c3 at baseline ( p>0.05 ; data not shown ) . the low activity and high activity patients in each treatment group ( i.e. , 3-month , 6-month , etc . ) were pooled together and compared against the high activity baseline group and low activity baseline group in order to simplify the comparative analysis . after 3 months of combination therapy , the cd3 t - cell percentage was significantly higher than the high activity baseline ( p=0.000 ; figure 3 ) . after 6 months of combination therapy , the cd3 t - cell percentages remained significantly higher than the high activity baseline ( p<0.01 ) . the cd3 t - cell percentages across all combination therapy time groups showed no significant differences with the low activity baseline ( p>0.05 ) . after 3 months of combination therapy , the cd4 th cell percentage was significantly higher than both the high activity and low activity baselines ( p<0.05 ) . after 6 months of combination therapy , the cd4 th cell percentage showed no significant difference with both the high activity and low activity baselines ( p>0.05 ) , but did present a non - significant upward trend . after 3 months of combination therapy , the cd8 ts cell percentage showed no significant difference with both the high activity and low activity baselines ( p>0.05 ) . after 6 months of combination therapy , the cd8 ts cell percentage was significantly higher than the high activity baseline ( p<0.05 ) . after 12 months of combination therapy , the cd8 ts cell percentage was also significantly higher than the low activity baseline ( p<0.05 ) . dysfunction in t - cell activation and proliferation plays an important part in sle development through promoting lymphocytic organ infiltration and aiding b - cells in producing auto - antibodies , a process which eventually leads to tissue destruction . specifically , changes in the t - cell subset distribution is a hallmark of sle ; for example , mclnerney et al .   showed that 45% of the sle patient population has markedly depressed cd4 th cells levels , and matsushita et al .   showed that sle patients have an increase in cd8 ts cell counts accompanied by a decrease in cd4 th cell counts . here , we analyzed the t - cell subset distributions of both high and low activity sle patients relative to healthy control individuals at baseline ( prior to vincristine - cyclophosphamide combination therapy ) in order to determine whether t - cell subset distributions vary across different levels of sle disease activity . in the high activity group , the percentages of cd3 t - cells and cd4 th cells were significantly lower than healthy controls at baseline ; however , the cd8 ts cell percentage was not significantly different than healthy controls at baseline . in contrast , in the low activity group , the cd3 t - cell percentage was not significantly different than healthy controls at baseline , but the cd4 th cell percentage was significantly lower , while the cd8 ts cell percentage was significantly higher , than healthy controls at baseline . based on this data , significantly negative correlations were observed between the cd3 + t - cell and cd4 + th cell percentages and sledai score at baseline . these combined results indicate that t - cell subset distributions vary across different levels of sle disease activity with higher cd3 t - cell and cd4 th cell percentages favoring lower sle activity . a complex regulatory interplay exists between cd8 ts cells and cd4 th cells in sle . for example , filaci et al . demonstrated that only cd8 ts cells from sle patients in remission and healthy individuals can function as effective suppressors of cd4 th cells , while cd8 ts cells from active sle patients fail to suppress committed cd4 th cells . moreover , cd42h4 th cells  which are suppressor / inducer cd4 th cells that promote cd8 ts cell - based inhibition of b - cells  have been previously shown to have significantly lower proportions in sle patients . based on this previous evidence from sle patients , the lower cd4 th cell percentages observed with higher sle activity in the current study may actually indicate lower cd42h4 th cell levels and , in turn , lower cd8 ts cell - based inhibition of b - cells . if so , this hypothesis would correctly explain the deficient cd8 ts cytotoxic capacity as well as the b - cell hyperactivity observed in highly active sle [ 1619 ] . in order to test this hypothesis , further research should focus on measuring and correlating the levels of cd42h4 th cells , cd8 ts cells , and b - cell autoantibody production as a function of sle activity . with respect to vincristine - cyclophosphamide combination therapy , vincristine blocks t - cell mitosis in metaphase , while cyclophosphamide targets rapidly - proliferating t - cells irrespective of cell cycle phase . therefore , vincristine and cyclophosphamide can be applied in combination to accomplish a synergistic cytotoxic effect . specifically , as vincristine activity begins to decline after a few hours , t - cells that have accumulated in metaphase re - initiate their cell cycle , and cyclophosphamide is then available to kill these t - cells during dna replication [ 2227 ] . here , we found that after the first 3 months of vincristine - cyclophosphamide combination therapy , the cd3 t - cell and cd4 th cell percentages both increased relative to the high activity baseline , which appropriately correlates with reduced sle disease activity as discussed above . after 6 months of combination therapy , the cd3 , cd4 , and cd8 t - cell percentages all increased relative to the high activity baseline . as cd3 + t - cell and cd4 + th cell percentages negatively correlate with sledai , vincristine - cyclophosphamide combination therapy appears to positively affect t - cell subset distribution to control sle disease activity by increasing their cd3 t - cell and cd4 th cell percentages . t - cell subset distributions vary across different levels of sle disease activity with higher cd3 t - cell and cd4 th cell percentages favoring lower sle activity . vincristine - cyclophosphamide combination therapy appears to positively affect the t - cell subset distribution in sle patients to keep the disease in remission by increasing their cd3 t - cell and cd4 th cell percentages . further studies are needed to examine how the distributions of cd4 th , cd8 ts cells , and other lymphocytes are maintained in sle and to investigate the t - cell - specific cytotoxic mechanisms underlying vincristine - cyclophosphamide combination therapy in sle .
backgroundthis study aimed to analyze the t - cell subset distribution in systemic lupus erythematosus ( sle ) patients and determine whether vincristine - cyclophosphamide combination therapy can positively affect their t - cell subset distribution to keep the disease in remission.material/methodsthirteen sle patients with 
 low activity ( sle disease activity index ( sledai)9 ) , 17 sle patients with 
 high activity ( sledai>9 ) , and 15 healthy controls were recruited . 
 sle patients were treated with vincristine - cyclophosphamide combination therapy . 
 cd3 + , cd4 + , and cd8 + t - cell percentages were analyzed by flow cytometry at baseline , 3 months , 6 months , 1224 months , and > 24 months.resultssignificantly negative correlations were observed between the cd3 + and cd4 + t - cell percentages and sledai scores at baseline ( r=0.471 , p=0.015 ; r=0.473 , p=0.015 , respectively ) . 
 a significantly positive correlation was observed between cd4 + t - cell percentage and the complement component c3 at baseline ( r=0.612 , p=0.002 ) . after 3 months of combination therapy , 
 the cd3 + and cd4 + t - cell percentages were significantly higher than the high activity baseline ( p<0.01 , p<0.05 , respectively ) . 
 after 6 months , the cd3 + , cd4 + , and cd8 + t - cell percentages were all significantly higher than the high activity baseline ( p<0.01 , p<0.05 , p<0.05 , respectively).conclusionst - cell subset distributions vary across different levels of sle disease activity with higher cd3 + t - cell and cd4 + th cell percentages favoring lower sle activity . 
 as cd3 + t - cell and cd4 + th cell percentages negatively correlate with sledai , vincristine - cyclophosphamide combination therapy appears to positively affect the t - cell subset distribution in sle patients to keep the disease in remission by increasing their cd3 + t - cell and cd4 + th cell percentages .