primary sjgren 's syndrome ( pss ) is a common systemic autoimmune disease characterized by inflammation and consequential destruction of exocrine glands . in the last decades , large amount of studies confirmed that b cell activation plays a crucial role in the pathogenesis of pss through antigen presentation , autoantibody production , and secretion of several proinflammatory factors . the extension and structural arrangement of the infiltrations vary between wide limits ; even ectopic germinal centres may develop . these structures are characterized by in situ autoantibody production and high expression of homing and retentive chemokines and adhesion molecules . it was reported that the number of ectopic gcs in salivary glands correlates with the severity of inflammation and anti - ssa / ssb autoantibody production [ 1 , 2 ] . additionally , formation of ectopic gcs in glandular tissues carries a higher risk of developing b cell lymphoma in pss . the proliferation and differentiation of b cells in gcs highly depend on their collaboration with follicular helper t ( tfh ) cells , which are generated from peripheral naive cd4 t cells in the t cell zone of lymphoid organs . the appropriate interaction of activated b cells and tfh cells is crucial for the generation of extrafollicular short - lived low - affinity plasma cells , high - affinity memory b cells , and long - lived plasma cells . recent investigations shed light on altered tfh profiles in various autoimmune conditions , suggesting the important role of tfh cells and il-21 cytokine secretion in autoreactive b cell activation and autoantibody production . in labial salivary gland biopsies of pss patients , tfh cell markers ( cd84 , pd-1 , and bcl-6 ) were detected in the lymphocytic infiltrations , especially , in more organized lymphoid structures . in peripheral blood , elevated cd4cxcr5icospd-1tfh - like cell percentages were reported in pss , which showed strong association with anti - ssa and/or anti - ssb autoantibody positivity . moreover , patients with higher tfh cell proportions had elevated il-21 serum levels . as part of immune responses , il-21 is produced by activated cd4 t cells and natural killer ( nk ) t cells . il-21-activated b cells produce il-6 , which further activates b cells in an autocrine manner and contributes to tfh cell differentiation and autoantibody production . recently , lindner et al . revealed that il-21 induces b cells to produce and secrete the active form of the cytotoxic serine protease granzyme b ( grb ) and gain immune regulatory properties by limiting t cell proliferation by a grb - dependent degradation of t cell receptor -chain . in systemic lupus erythematosus , these observations raise the possibility that beside the key role of tfh cells in b cell activation , the interaction of il-21-producing invariant nkt ( inkt ) cells with b cells and grb production may also play a potential role in the pathogenesis of pss . in our study , we investigated the expression of il-21 receptor ( il-21r ) and grb of b cell subsets ; we assessed the il-21 expression of inkt cells and determined the distribution of transitional , mature , and primarily memory b cell subsets in the peripheral blood of pss patients . we enrolled 32 pss patients in the study , who visited our outpatient clinic for systemic autoimmune diseases in the division of clinical immunology of the university of debrecen for a regular medical check - up between april and june 2014 . exclusion criteria included other autoimmune , allergic , and malignant diseases , pregnancy , immunosuppressive or immunomodulating medications , and ongoing infections . twenty - four age- and sex - matched healthy individuals served as controls . among pss patients , 18 suffered from extraglandular manifestations ( egms ) , while 14 had glandular symptoms only . the distribution of egms of pss patients was as follows : polyarthralgia / polyarthritis n = 16 , raynaud 's phenomenon n = 7 , informed written consent was obtained from the subjects , and the study has been approved by the ethics committee of the university of debrecen . all experiments carried out were in compliance with the declaration of helsinki . data on subjects enrolled in the study are summarized in table 1 . for the evaluation of intracellular grb production of cd19cd5 and cd19cd5 b cell , peripheral blood mononuclear cells ( pbmcs ) were isolated from heparinized venous blood sample by ficoll - histopaque ( sigma - aldrich , st . cells were then harvested and washed twice and cultured in modified rpmi 1640 medium with glutamax - i ( life technologies corporation , carlsbad , ca , usa ) supplemented with 100  u / ml penicillin , 100  ng / ml streptomycin , and 10% heat - inactivated fetal calf serum ( fcs ) ( life technologies ) . cells were stimulated at 1  10  cells / ml concentration using 6,5  g / ml anti - human b cell receptor ( bcr ) antibody ( jackson immunoresearch , west grove , pa , usa ) and 50  ng / ml recombinant human il-21 ( r&d systems , inc . , minneapolis , mn , usa ) for 18 hours at 37c in an atmosphere containing 5% co2 , based on the protocol described previously by hagn et al . . the transport of de novo synthesized cytokines from the golgi apparatus was inhibited by 10  g / ml brefeldin - a ( sigma - aldrich ) . next , cells were stained with fluorescein isothiocyanate- ( fitc- ) conjugated anti - cd5 and phycoerythrin - cyanine ( pc)5-labelled anti - cd19 antibodies ( bd biosciences , san diego , ca , usa , and beckman coulter inc . , miami , fl , usa ) for 30  min at 4c . the cells then were fixed and permeabilized with intraprep permeabilization reagent ( beckman coulter ) according to the manufacturer 's instructions , and intracellular grb were stained with phycoerythrin- ( pe- ) conjugated anti - grb antibody ( bd biosciences ) . for the evaluation of il-21r expression , anti - cd5-fitc , anti - cd19-pc5 , and anti - il-21r- ( cd360- ) pe antibodies were used ( bd biosciences , beckman coulter and biolegend , san diego , ca , usa ) , and mean fluorescence intensity ( mfi ) was determined . mouse immunoglobulin ( ig ) g1 antibodies were used as isotype control throughout the experiments . the measurements were performed on a facscalibur flow cytometer ( becton dickinson , franklin lakes , nj , usa ) . cd19 , cd19cd5 , and cd19cd5 b cells were quantified as their percentage in the entire lymphocyte population . grb positivity and il-21r expression levels were quantified within the investigated b cell subpopulations . in order to determine inkt cells from heparinized blood samples , the following monoclonal antibodies to cell surface markers were used : fitc - conjugated anti - cd3 and pe - conjugated 6b11 ( bd biosciences ) . samples were processed according to the coulter q - prep protocol and system ( beckman coulter inc . ) . briefly , cells from 50  l of whole blood were stained with 10  l of each monoclonal antibody . after 20  min incubation , red blood cells were haemolysed and leucocytes were washed in phosphate - buffered saline ( pbs ) supplemented with bovine serum albumin ( bsa ) ( 10  mg / l ) and sodium - azide ( 2  mg briefly , isolated pbmcs were cultured at a concentration of 2  10/ml in modified rpmi 1640 medium with glutamax - i ( life technologies ) supplemented with 100  u / ml penicillin , 100  ng / ml streptomycin , and 10% heat - inactivated fetal calf serum ( life technologies ) . cells were stimulated using 25  ng / ml phorbol - myristate - acetate ( sigma aldrich ) and 1  g / ml ionomycin ( sigma aldrich ) for 6 hours at 37c in an atmosphere containing 5% co2 . the transport of de novo synthesized cytokines from the golgi apparatus was inhibited by 10  g / ml brefeldin - a ( sigma aldrich ) . after stimulation , cells were stained with anti - cd3-fitc and 6b11-pe antibodies ( bd biosciences ) for 30  min at 4c . the cells then were fixed and permeabilized with intraprep permeabilization reagent ( beckman coulter inc . ) according to the manufacturer 's instructions , and intracellular cytokines were stained with allophycocyanin- ( apc- ) conjugated anti - il-21 antibody ( biolegend ) . the cd36b11 inkt cells were quantified as their percentage in the entire lymphocyte population , while il-21 inkt cells were determined as their proportions in the population of cd36b11 inkt cells . pbmcs were isolated from heparinized venous blood sample by ficoll - histopaque ( sigma - aldrich ) density - gradient centrifugation . cells were then harvested and washed twice and stained for 20  min at 4c using specific antibodies . to identify transitional , mature , and primarily memory b cell subpopulations , cells were stained with the combination of the following monoclonal antibodies : pc5-labelled anti - cd19 ( beckman coulter ) , alexa fluor 488-labelled anti - cd24 ( biolegend ) , and pc7-labelled anti - cd38 ( bd biosciences ) . after 30  min incubation , leucocytes were washed in phosphate - buffered saline ( pbs ) supplemented with bovine serum albumin ( bsa ) ( 10 mouse immunoglobulin ( ig ) g1 antibodies were used as isotype control throughout the experiments . samples were processed according to the coulter q - prep protocol and system ( beckman coulter inc . , measurements were performed on a coulter fc500 flow cytometer ( beckman coulter inc . ) . cd19cd24cd38 ( transitional ) , cd19cd24cd38 ( mature ) , and cd19cd24cd38 ( primarily memory ) b cells were quantified as their percentage in the cd19 lymphocyte population . autoantibodies were determined by enzyme - linked immunosorbent assay ( elisa ) technique with autostat ii kits ( hycor biomedical , indianapolis , in , usa ) , according to the manufacturer 's instructions . the spss ver . 20.0 ( spss inc . , chicago , il , uda ) was used for statistical analysis . to assess the distribution of the data kolmogorov - smirnov test was used . in cases of normal distribution , we determined mean  standard deviation ( sd ) values and used two - sample t - test for statistical evaluation of the experimental data . in cases of distributions different from normal , median , minimum , and maximum values were calculated , and the mann - whitney u test was used . when the strength of the linear relationship between two variables was evaluated , pearson 's correlation coefficient was used , while in cases of nonnormal distribution , spearman 's correlation coefficient was applied . we found no significant differences in peripheral blood cd19 , cd19cd5 , and cd19cd5 b cell percentages between pss patients , subgroups of patients , and healthy controls ( data not shown ) . when grb expressions of these b cell subsets were investigated , in the whole cd19 b cell population , significantly enhanced grb expression was observed in the whole group of pss patients ( median ( min  max ) : 5.225 ( 0.4128.1)% versus 3.69 ( 0.047.29)% , resp . , p = 0.0121 ) and in all subgroups of patients ( pss glandular : median ( min  max ) : 5.505 ( 0.4128.1)% versus 3.69 ( 0.047.29)% , resp . , p = 0.0261 ; pss egms : median ( min  max ) : 4.735 ( 0.7322.34)% versus 3.69 ( 0.047.29)% , resp . , p = 0.0486 ) , compared to the values measured in the healthy controls ( figure 1 ) . when we divided cd19 b cell population on the basis of their cd5 positivity , we found that only cd19cd5 b cells expressed grb significantly higher in each group of pss patients , compared to control values ( pss : median ( min  max ) : 4.59 ( 1.0426.45)% versus 2.8 ( 0.186.13)% , resp . , p = 0.0086 ; pss glandular : median ( min  max ) : 6.81 ( 1.626.45)% versus 2.8 ( 0.186.13)% , resp . , p = 0.0101 ; pss egms : median ( min  max ) : 4.03 ( 1.0423.91)% versus 2.8 ( 0.186.13)% , resp . we observed significantly enhanced il-21r expression on cd19 b cells in the whole group of pss patients ( mean  sd : 3.434  0.929 mfi versus 2.961  0.585 mfi , resp . , p = 0.0288 ) and in subgroup of patients with egms , compared to values measured in healthy individuals ( mean  sd : 3.578  0.901 mfi versus 2.961  0.585 mfi , resp . when we divided cd19 b cell population on the basis of their cd5 positivity , we found that only cd19cd5 b cells expressed il-21r significantly higher in the whole group of pss patients ( mean  sd : 3.677  0.894 mfi versus 3.145  0.564 mfi , resp . , p = 0.0114 ) and in the subgroup of patients with egms , compared to control values ( mean  sd : 3.834  0.872 mfi versus 3.145  0.564 mfi , resp . moreover , we found positive correlation between the percentages of grb positivity and the level of il-21r expression within both cd19 b cells ( r = 0.6261 , p = 0.0001 ) and cd19cd5 b cells ( r = 0.5949 , p = 0.0003 ) in pss ( figures 3(a ) and 3(b ) ) . we observed no significant differences in the peripheral blood inkt percentages between pss patients and controls ( data not shown ) . however , in the pss patient population , we found significantly elevated percentages of il-21-producing inkt cells within inkt population , compared to the values determined in healthy individuals ( median ( min  max ) : 2.81 ( 0.129.02)% versus 1.16 ( 0.112.1)% , resp . the distribution of b cells was also evaluated in the study ( figure 4 ) . the percentages of cd19cd24cd38 transitional b cells were significantly higher in pss patients compared to healthy control ( median ( min  max ) : 5.21 ( 1.6827.83)% versus 3.19 ( 0.5713.4)% , resp . , p = 0.0079 ) . regarding the subgroups of patients , subjects suffering from egms had also elevated percentages ( median ( min  max ) : 5.44 ( 3.4127.83)% versus 3.19 ( 0.5713.4)% , resp . , p = 0.0038 ) , while values of patients with glandular symptoms did not differ significantly from the results of healthy individuals . the proportions of cd19cd24cd38 mature b cells were significantly elevated in the whole group of pss patients ( mean  sd : 52.241  15.245% versus 39.628  13.958% , resp . , p = 0.0021 ) and in both of pss subgroups ( pss glandular : mean  sd : 50.885  17.618% versus 39.628  13.958% , resp . , p = 0.0343 ; pss egms : mean  sd : 53.296  13.555% versus 39.628  13.958% , resp . , p = 0.0026 ) , compared to control values . on the contrary , the frequency of cd19cd24cd38 primarily memory b cells were significantly decreased in all groups of pss patients , compared to controls ( pss : mean  sd : 24.166  13.712% versus 35.083  13.887% , resp . , p = 0.0046 ; pss gland : mean  sd : 23.798  14.422% versus 35.083  13.887% , resp . , p = 0.0214 ; pss egms : mean  sd : 24.452  13.551% versus 35.083  13.887% , resp . , p = 0.0165 ) . additionally , we found a positive correlation between the percentages of grb positive cd19 b cells and mature b cells ( r = 0.4022 , p = 0.0224 ) , while a negative correlation between the percentages of grb positive cd19 b cells and primarily memory b cells ( r = 0.4565 , p = 0.0086 ) ( figures 5(a ) and 5(b ) ) . similarly , positive correlation was observed between the levels of il-21r expression of cd19 b cells ( r = 0.4935 , p = 0.0041 ) , while negative correlation was revealed between the levels of il-21r expression of cd19 b cells and primarily memory b cells ( r = 0.4696 , p = 0.0067 ) ( figures 5(c ) and 5(d ) ) . thirteen individuals were positive for anti - ro / ssa and/or anti - la / ssb antibody among pss patients . formerly , we reported higher level of circulating il-21 in pss and found increased circulating tfh cell percentages in the disease . in the present study , we observed elevated il-21 expression of inkt cells , which , along with the increased il-21r expression of b cells , may contribute to the enhanced b cell activation in pss . interestingly , b cells expressing enhanced il-21r on surface were typically positive for surface antigen cd5 . furthermore , when we divided patients into two groups based on the presence or absence of egms , significant increase in expression of il-21r was found only in the group of egm patients , compared to the control values . this observation is in line with our previous results ; namely , significantly elevated ratio of peripheral tfh cells was observed only in pss patients with egms and not in those without egms ; furthermore , higher il-21 cytokine concentrations were associated with the presence of egms . regarding the il-21 overproduction of inkt cells , we did not find marked difference between the subgroups of patients . basically , il-21 stimulation on b cells may have at least three outcomes . in the absence of both b cell receptor ( bcr ) engagement and t cell help , it induces b cell apoptosis . in the presence of cd40 ligation and either a bcr or a toll - like receptor signal , il-21 stimulation fundamentally promotes b cell activation and differentiation processes into memory and plasma cells . however , the combination of il-21 stimulation and bcr engagement in the absence of cd40 ligation enables b cells to produce and secrete the active form of grb   ( figure 6 ) . besides its important and well - known function in antiviral immune responses , grb secretion plays a potential role in the regulation of autoimmune responses as well , through different mechanisms . the spectrum of functions exhibited by grb includes antigen processing , matrix degradation , cleavage , and activation of inflammatory cytokines and immunoregulatory effects [ 13 , 14 ] . consequently , grb - producing b cells may acquire a regulatory function and effects on other immune cells ; furthermore , leakage of grb in the cytoplasm may also result in the apoptotic demise of autoreactive b cells . based on our observations , grb expression of b cells , especially in cd19cd5 ones , is significantly increased in pss regardless of the presence of egms . importantly , we observed a clear positive correlation between grb production and il-21r expression on cd19 b and cd19cd5 b cells as well . additionally , we also found further associations with the altered distribution of transitional , mature , and primarily memory b cells . our observations , namely , elevated mature b cell proportions and decreased primarily memory b cell percentages , are in line with former observations   pointing at the hyperactivated state of b cells in pss patients . in our study , we revealed that not only il-21r expression but also grb production on cd19 b cells correlates positively with the frequency of cd19cd24cd38 transitional and cd19cd24cd38 mature b cell subsets and correlates negatively with cd19cd24cd38 primarily memory b cells . based on our results , we assume that , parallel with the pathologic b cell activation and enhanced autoantibody production driven by the expansion of tfh cells and their pronounced il-21 expression , grb production is mainly induced in cd5 b cell subsets in pss , which could be part of an increased counter - regulatory reaction , presumably compensating the derailed , disproportional immune responses . we believe that further investigation on the activation and function of grb - producing regulatory b cells will open new avenues to understand the b cell operation and autoimmune processes in pss ; furthermore , the modulation of these cells could be a potentially powerful element of the novel therapeutic selection in the disease .
recently , we reported elevated proportions of circulating follicular t helper cells and higher levels of interleukin- ( il- ) 21 in primary sjgren 's syndrome ( pss ) . 
 interaction of invariant natural killer t ( inkt ) cells with b cells and granzyme b ( grb ) production may be also important in pss . 
 thirty - two pss patients and 24 healthy controls were enrolled in our study . 
 we investigated the expression of intracellular grb and il-21 receptor ( il-21r ) of cd19+cd5 + and cd19+cd5 b cells ; furthermore , we determined the il-21 expression of inkt cells as well . 
 we also assessed the proportion of transitional ( cd19+cd24highcd38high ) , mature ( cd19+cd24intcd38int ) and primarily memory ( cd19+cd24highcd38 ) b cells . 
 cd5 + but not cd5 b cells showed elevated grb and il-21r expression in pss ; additionally il-21 expression of inkt cells was also elevated . 
 the ratios of transitional and mature b cells were elevated in pss , while primarily memory b cell percentages were decreased , which correlated with grb and il-21r expression of cd19 + b cells . 
 our results suggest that enhanced il-21r expression of cd19+cd5 + b cells and production of il-21 by inkt cells may play an important role in the pathogenesis of pss by regulating cd19+cd5 + b cell functions and increasing grb production , presumably leading to a counter - regulatory effect in the disease .