extracellular vesicles ( evs ) are a heterogeneous group of vesicles that can be subdivided based on their size , biogenesis , and molecular composition . using the biogenesis as a classification tool
, evs can be divided into three major groups , namely , exosomes , microvesicles ( mvs ) , and apoptotic bodies [ 13 ] . even though the molecular composition of these three subsets of evs is different , several markers overlap .
so far , identification of a specific marker that with certainty can distinguish or identify the particular ev subset still awaits .
it can be expected that the different ev subsets may cover different biological roles , but the function of evs has also been described to depend both on the cellular source and on the recipient tissue / cell .
nevertheless , it is now recognized that evs are involved in numerous physiological processes , including intercellular communication and delivery of proteins , lipids , and genetic material to recipient cells [ 2 , 3 , 57 ] .
in addition , evs have been associated with the development and progression of different pathological conditions , including cancer and infectious diseases [ 815 ] .
the immunological effects of evs comprise a broad range of mechanisms , including immune activation , immune suppression , and modulation of immune surveillance .
cells from both the innate and the adaptive immune system have been shown to release evs , such as t and b cells , dendritic cells ( dcs ) , macrophages , mast cells , and natural killer ( nk ) cells [ 1624 ] .
the effect of evs is directly related to their molecular composition and several studies have ascribed an immunostimulatory effect of evs to the presence of a very specific molecular content [ 20 , 23 , 2531 ] ; for instance , cd56-positive and perforin - containing evs from nk cells can mediate ev - induced cytotoxicity .
several immunosuppressive effects of evs have also been reported [ 21 , 32 , 33 ] ; for instance , fas ligand ( fas - l ) evs released from regulatory t cells are able to inhibit dc - induced cytotoxic t - lymphocyte ( ctl ) responses .
furthermore , inhibitory roles of evs derived from immature dcs have been observed in relation to transplant tolerance [ 3436 ] .
thus , identification of a specific molecular signature of evs released by immune cells can provide knowledge that can lead to the use of evs in a therapeutic setting .
numerous studies have investigated the effects of evs released from different leukocytes , leading to an incipient understanding of the physiological functions of these evs .
nevertheless , several basic questions concerning specific characteristics , like the protein composition of the different types of leukocyte - derived evs , remain unclear .
the present study investigated the expression of selected immunological lineage - specific markers and selected vesicle - related markers on five major leukocyte subpopulations , namely , cd4 t cells , cd8 t cells , nk cells , b cells , and monocytes .
the expression was determined for leukocytes present in freshly isolated whole blood and on cultured isolated leukocyte subpopulations and compared to the presentation of these markers on the corresponding leukocyte - derived evs .
the ev array , used for phenotyping of evs , is optimized for detection of small evs with a size below 150  nm that present cd9 , cd63 , and/or cd81 , such as exosomes and exosome - like vesicles .
however , as these markers may be present on several types of evs and as the intracellular origin of the characterized evs was not determined , the ev subset investigated in the current study was denoted by small evs ( sevs ) .
blood samples were obtained from ten healthy volunteers at the department of clinical immunology , aalborg university hospital . from each donor two blood samples were collected , one tube containing edta ( k3edta , vacuette , greiner bio - one , austria ) for immediate analysis of noncultured leukocytes and one tube containing cpda ( vacuette , greiner bio - one , austria ) for the vesicle analysis ( plasma ) .
plasma was isolated by centrifugation at 1800  g for 6  min at room temperature ( rt ) , after which the plasma supernatant was aliquoted and stored at 40c until analysis .
buffy coats were obtained from three healthy donors at the department of clinical immunology , aalborg university hospital , and used for isolation of peripheral blood mononuclear cells ( pbmcs ) .
buffy coats were diluted ( 1  :  4 ) in sterile pbs ( spbs ) and pbmcs were isolated by density gradient centrifugation using lymphoprep ( axis - shield , oslo , norway ) .
pbmcs were subsequently washed twice in growth medium ( rpmi1640 ( gibco , life technologies , carlsbad , ca , usa ) , 10% heat - inactivated fetal calf serum ( fsc ) ( gibco ) , 100  u / ml penicillin , and 10  g / ml streptomycin ( amplicon , odense , denmark ) ) and counted in trypan blue and resuspended in isolation buffer ( ca- and mg - free pbs , 2  mm edta , and 0.1% bovine serum albumin ( bsa ) ) . magnetic dynabeads were used for isolation of human cd4 and cd8 t cells according to the manufacturer 's instructions ( dynabeads cd4 positive isolation kit and dynabeads cd8 positive isolation kit , life technologies ) .
briefly , 1  10  cells / ml were mixed with washed beads and incubated for 20  min at 4c with gentle rotation .
the cell suspension was subsequently placed in a magnet for 3 - 4  min .
the supernatant was removed and the bead - bound cells were incubated with detachabeads for 45  min at rt with gentle rotation . the cell suspension was placed in a magnet for 3 - 4  min and the supernatant containing the detached cells was collected .
the detached cells were washed once in growth medium and adjusted to 3  10  cells / ml . from the cd4 depleted pbmcs , b cells were isolated using the dynabeads untouched human b cells kit ( life technologies ) according to the manufacturer 's instructions . from the cd8 depleted pbmcs , monocytes were isolated using the dynabeads untouched human monocytes kit ( life technologies ) according to the manufacturer 's instructions .
human nk cells were isolated from pbmcs by negative selection according to the manufacturer 's instructions ( dynabeads untouched human nk cells kit , life technologies ) .
briefly , 1  10  cells / ml were mixed with the antibody cocktail and incubated for 20  min at 4c followed by washing and incubation with dynabeads for 15  min at 4c while rotating .
the cell suspension was subsequently placed in a magnet for 3 - 4  min .
the supernatant , containing the cells of interest , was removed and centrifuged at 500  g , for 5  min at rt .
the pellet was washed once in growth medium and adjusted to 3  10  cells / ml . for each isolation , the purity of the cells was determined by flow cytometry .
isolated leukocytes were adjusted to 3  10  cells / ml and cultured in either 12-well ( 3  10  cells / well , 1.5  ml / well ) or 24-well plates ( 1.5  10  cells / well , 1  ml / well ) ( nunc , thermo scientific , carlsbad , ca , usa ) for 4448 hours at 37c and 5% co2 .
following incubation , the plates were centrifuged at 600  g for 10  min at rt and the supernatants containing the cell - derived evs were harvested from the plates .
complete protease inhibitor , edta - free ( roche , de , usa ) , was added to the ev - rich supernatants , which were subsequently upconcentrated using amicon ultra 100k spin columns according to the manufacturer 's instructions .
briefly , pbs was added to the harvested supernatants ( total volume of 5  ml ) and the supernatants were subsequently centrifuged at 600  g for 7  min at rt .
the supernatants were transferred to 50  ml spin columns and centrifuged at 3200  g for 10  min at rt . prior to adding the supernatants , the spin columns were washed once in 5  ml pbs ( 3200  g , 10  min , rt ) . following the first centrifugation of the supernatant
, 5  ml of pbs was added to the retention volume and centrifuged again .
the retention volume , containing the upconcentrated supernatants , was harvested and the filter unit was washed in 50100  l pbs . due to varying cell counts between the subpopulations and across the experiments ,
every supernatant was adjusted to a volume corresponding to 5.5  10  cells / ml . no further purification of the evs was performed and the upconcentrated supernatants were aliquoted and stored at 40c until analysis by the ev array .
the cells were harvested in pbs , washed , and resuspended in pbs with 0.5% bsa and 0.09% nan3 , followed by a subsequent surface marker staining . for the ev array ,
the following antibodies were used for capturing : cd11a ( hi111 ) from ab biotech ( san diego , ca , usa ) ; flotillin-1 and tsg101 ( 5b7 ) from abcam ( cambridge , ma , usa ) ; cd3 ( hit3a ) , cd14 ( m5e2 ) , cd16 ( 3g8 ) , cd28 ( l293 ) , cd49d ( l25 ) , and cd56 ( 3g8 ) from bd biosciences ( mountain view , ca , usa ) ; cd41 ( hip8 ) , cd63 ( mem-259 ) , hla - abc ( w6/32 ) , and alix ( 3a9 ) from biolegend ( san diego , ca , usa ) ; icam-1 ( r6.5 ) from ebiosciences ( san diego , ca , usa ) ; cd9 , cd42a , cd81 , and ctla-4 ( anc152.2/8h5 ) from lifespan biosciences ( seattle , wa , usa ) ; annexin v ( af399 ) , cd4 ( 34930 ) , cd8 ( 37006 ) , cd19 ( 4g7 - 2e3 ) , cd37 ( 424925 ) , cd45 ( 2d1 ) , cd80 ( 37711 ) , cd82 ( 423524 ) , cd83 ( hb15e ) , mic a / b ( 159207 ) , tnf ri , and tnf rii from r&d systems ( minneapolis , mn , usa ) ; tlr3 ( 3.7 ) from santa cruz biotechnologies ( dallas , tx , usa ) ; hla - dr / dp / dq ( hb-145 ) from loke diagnostics ( aarhus , denmark ) ; fas ligand ( 10f2 ) from serotec ( oxford , uk ) ; and pd - l1 from sino biological ( beijing , china ) . the following antibodies were used for detection ( biotinylated ) : cd9 , cd63 , and cd81 from lifespan biosciences . for flow cytometry
the following antibodies were used : cd45 apc ( t29/33 ) , cd45 fitc ( t29/33 ) , and cd3 fitc ( ucht1 ) from dakocytomation ( glostrup , denmark ) ; cd3 apc ( ucht1 ) , cd4 fitc ( rpa - t4 ) , cd4 apc - h7 ( sk3 ) , cd8 apc - h7 ( sk1 ) , cd9 percp - cy5.5 ( m - l13 ) , cd14 fitc ( m5e2 ) , cd16 fitc ( 3g8 ) , cd16 pe - cy7 ( 3g8 ) , cd19 apc ( hib19 ) , cd56 apc ( b159 ) , and cd56 pe - cy7 ( b159 ) from bd biosciences ( mountain view , ca , usa ) ; cd63 pe - cy7 ( h5c6 ) from ebiosciences ; and cd81 pe from lifespan biosciences .
in addition , isotype- and fluorophore - matched control antibodies were included . for analysis of noncultured leukocytes , 100  l of freshly drawn whole blood was labeled with fluorescence - conjugated antibodies for 30  min at rt , red blood cells were lysed , and the remaining leukocytes were washed and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .
the cultured leukocytes were labeled with fluorescence - conjugated antibodies for 30  min at rt .
the leukocytes were washed twice in pbs with 0.5% bsa and 0.09% nan3 and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .
cells were analyzed on a facscanto ii flow cytometer ( bd biosciences ) using the bd facsdiva software version 6.1.3 ( bd biosciences ) .
the acquired data files were analyzed by flowjo vx.0.7 ( treestar , ashland , usa ) , first by adding a leukocyte - gate based on morphologic characteristics ( fsc / ssc ) and subsequently by the use of the lineage - specific markers ; cd3 and cd4 for cd4 t cells ; cd3 and cd8 for cd8 t cells ; cd16 and cd56 for nk cells ; cd19 for b cells ; and cd14 for monocytes . for the production of the protein microarray , microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin ( arrayit , sunnyvale , ca , usa ) .
epoxy coated slides ( 75.6  mm  25.0  mm ; schott nexterion , jena , germany ) were used as microarray basis .
the antibodies listed above were diluted in pbs containing 5% glycerol and printed at a concentration of 180200  g / ml . as a positive control , 100  g / ml of biotinylated human igg in pbs with 5% glycerol was printed . as a negative control ,
for catching , visualization , and data analysis , the procedures were performed as previously described . in short
, the slides were incubated with 100  l plasma , prediluted 1  :  10 , or 100  l upconcentrated cell culture supernatant , prediluted 1  :  2 in wash buffer ( pbs with 0.05% tween-20 ) .
after overnight sample incubation and a subsequent wash , the slides were incubated with a cocktail of biotinylated detection antibodies ( anti - human cd9 , cd63 , and cd81 ) , diluted 1  :  1500 in wash buffer .
after incubation , cy5-labeled streptavidin ( invitrogen , frederick , md , usa ) diluted 1  :  1500 was used for detection . prior to scanning , the slides were washed and dried using a microarray high - speed centrifuge ( arrayit ) .
briefly , the intensity of the antibody signal was calculated by subtracting the mean of the background ( without sample / blank ) from the mean of the triplicate antibody spots .
this signal was then divided by the signal from the mean of the triplicate negative spots ( without capture antibody , with sample ) .
leukocytes can be subdivided based on morphological characteristics upon staining into polymorphonuclear cells ( the granulocytes ) and mononuclear cells ( the lymphocytes and the monocytes ) .
the present study investigated the sevs produced by the different subpopulations of mononuclear cells found in peripheral blood and compared the vesicular phenotype to the cellular phenotype .
in addition , sevs present in plasma were phenotyped for the same panel of markers .
the purity of the different subpopulations throughout the three individual experiments was 9799.5% for the cd4 and the cd8 t cells , 8295% for the b cells , 6894% for the monocytes , and 8597% for the nk cells .
the isolated subpopulations of leukocytes were cultured for 4448 hours and the supernatants , containing the leukocyte - produced evs , were investigated for the presence of a panel of selected immunological and ev - related markers using the ev array ( table 1 ) .
it was investigated whether sevs from isolated leukocyte subpopulations presented the lineage - specific markers that define the parent cells .
the results demonstrated that not all lineage - specific markers could be observed on the corresponding sev subpopulations ( figure 1(a ) ) .
for instance , cd19 , which is a well - defined lineage marker for b cells , could not be detected on sevs in supernatant from cultured b cells .
likewise , cd14 , cd16 , cd56 , and cd3 could not be detected on sevs from cultured monocytes , nk cells , and t cells , respectively ( figure 1(a ) ) .
in contrast , cd4 and cd8 , which are lineage markers for two different populations of t cells , were detected on sevs from the supernatants of these two t cell subpopulations .
in addition , sevs from cultured pbmcs presented cd8 , but none of the other markers . aside from the parental lineage - specific markers ,
likewise , cd45 and cd16 could be detected in one of the experiments with sevs from cd4 t cells .
a panel of ev - related markers was assayed on the different subsets of the leukocyte - derived evs ( figure 1(b ) ) .
cd9 was detected on all sev subsets , though at very low levels on sevs from t cells and nk cells .
similarly , cd81 was detected on all sevs but at very low levels on sevs from b cells and nk cells .
cd82 was detected at high levels on sevs from monocytes and pbmcs and at very low levels on sevs from b cells and t cells but was absent on sevs from nk cells .
furthermore , alix was detected at very low levels on sevs from the cultured cd8 t cells .
in contrast , annexin v , tsg101 , and flotillin-1 could not be detected on the leukocyte - derived sevs .
furthermore , the presence of more general immunological markers was investigated on the different sev populations .
a total of 18 markers were chosen based on their relevance for the function of leukocytes ( table 1 ) .
the majority of markers could not be detected on the sev subpopulations using the ev array ( figure 1(c ) ) .
however , cd49d was found on sevs from all the different leukocyte subpopulations and cd41 was detected on sevs from all subpopulations besides sevs from nk cells .
additionally , sevs from cultured cd4 and cd8 t cells presented several of the immunological markers , including cd11a , tlr3 , cd28 , ctla-4 , and the fas - l .
the data presented in the heat maps were from three individual experiments and illustrated a degree of variation between the individuals .
the presence of a natural variation in the phenotype of sevs between healthy individuals was also observed upon phenotyping of the sevs present in plasma from 10 healthy individuals ( figure 2 ) .
plasma sevs presented several of the markers that were detected on the leukocyte - derived sevs .
in addition , plasma sevs presented some lineage - specific markers , including cd3 , cd14 , and cd19 that were not detected on sevs from cultured leukocytes .
furthermore , tnf ri , tlr3 , cd42a , cd80 , and cd83 as well as annexin v , flotillin-1 , and alix were detected on the majority of the plasma sevs .
the cellular expression pattern of cd9 , cd63 , and cd81 was determined for all the leukocyte subpopulations by flow cytometry ( figure 3 ) .
the expression of these vesicle - related markers was investigated for leukocytes present in freshly drawn whole blood ( n = 10 ) as well as for isolated subpopulations of leukocytes following two days of culture ( n = 3 ) .
figures 3(a ) and 3(b ) demonstrate the expression levels in histograms from two representative donors .
none of the leukocyte subsets from whole blood presented cd9 on their surface ( figure 3(a ) ) .
in contrast , both cd63 and cd81 were expressed on all leukocytes , but the expression pattern varied between the different subsets . the lymphocytes , including the cd4 t cells , the cd8 t cells , the b cells , and the nk cells , expressed cd81 at high levels , while they all expressed cd63 at low - to - intermediate levels .
the opposite situation was observed for the monocytes that presented high levels of cd63 on their surface , but only low - to - intermediate levels of cd81 . regarding the cultured leukocytes ,
each subpopulation was isolated from three different donors and cultured for 4448 hours ( figure 3(b ) ) .
the results showed that , in contrast to the freshly isolated leukocytes , the cultured leukocytes presented cd9 on their surface .
for the lymphocyte populations , the expression of cd9 was low , while the monocyte population expressed intermediate levels of cd9 .
all cultured lymphocyte subpopulations expressed cd63 and cd81 at low - to - intermediate levels , while the monocytes expressed high levels of both markers . even though the levels varied , the expression patterns of cd63 and cd81 on the cultured leukocyte subpopulations were similar to the patterns observed on leukocytes present in freshly isolated blood . based on the obtained minimum and maximum mfi values , it was clear that the expression level of the three markers varied between the individuals illustrating a natural variation . in order to investigate the phenotypic homogeneity between the sevs and the parent cells , the cellular expression patterns of selected lineage - specific and vesicle - related markers were compared to the vesicular presentation of these markers ( figure 4 ) . in relation to the selected lineage - specific markers , cd3 , cd14 , cd16 , and cd19
could not be detected on sevs from cultured t cells , monocytes , nk cells , and b cells , respectively , indicating that not all cell surface molecules are transferred to the sev surface .
for cd9 , cd63 , and cd81 , the majority of the subpopulations presented vesicular levels that overall correlated with the cellular expression .
exceptions were observed with cd63 , in which case the cellular expression on monocytes was high , but the vesicular presentation was low .
similarly , the cellular expression of cd63 was intermediate or low for both nk cells and b cells , but no cd63 could be detected on the corresponding sevs .
several studies have reported that evs are released from different leukocyte subsets ; however , a thorough simultaneous investigation of the major leukocyte subsets with focus on lineage - specific markers has not yet been described . in the current study
the phenotype of five different subsets of leukocyte - derived sevs was determined and related to both the phenotype of sevs present in plasma and the cellular phenotype of the leukocyte subpopulations .
the ev array , used for the investigation of the phenotype of sevs , is a protein microarray technique that provides the opportunity to detect and characterize sevs for up to 60 markers in a high - throughput manner and with high sensitivity [ 37 , 38 ] .
one major advantage of the ev array is the ability to phenotype sevs from unpurified material without a requirement for preanalytical purification .
isolation of specific ev subsets is in many cases highly warranted , but for phenotyping of evs using the ev array , crude plasma or cell - free supernatants are applicable . in order to investigate whether lineage - specific proteins present on leukocytes could be detected on the corresponding sevs
, leukocytes were phenotyped for selected lineage - specific markers using flow cytometry , while the sevs were phenotyped using the ev array .
the results showed that none of the included lineage - specific markers could be detected on the sevs released from b cells , nk cells , and monocytes in monocultures ( cd19 , cd56 , and cd14 , resp . ) .
in contrast , sevs from monocultures of cd4 and cd8 t cells did present cd4 and cd45 or cd8 and cd45 , respectively .
none of the t cell - derived sevs presented cd3 . a study by kornek et al . investigated evs from jurkat t cells activated with phytohemagglutinin ( pha ) and observed that the fraction of large evs ( sedimented at 10.000 
g ) only presented low levels of cd3 , while the fraction of sevs ( sedimented at 100.000  g ) presented high levels of cd3 .
regarding cd19 , a study by admyre and coworkers from 2007 showed that evs from a b cell line presented cd19 , which differs from the observations obtained in the study at hand .
similarly , another study has described that evs from resting nk cells present typical nk cell markers like cd56 and fas - l , but not cd16 . in terms of cd14 , a study by aharon et al . observed this marker on large evs ( mvs ) released from a monocytic cell line .
the discrepancy between some of the results may be due to investigations of different preparations of evs and/or may be explained by the fact that the ev - producing cells were different , for example , cells isolated from peripheral blood versus cell lines .
in addition , differences in the activation state of the ev - producing cells are of importance for the outcome . upon stimulation ,
cells change their phenotype and this phenotypic change depends on the specific stimulus given to the cells [ 28 , 42 ] . in the present study , leukocyte subpopulations were isolated and cultured without any activation signal , while several of the other studies investigated the phenotype of the evs released upon adding a stimulus to the cells .
the presented results obtained with sevs released from cultured pbmcs support this phenomenon as these sevs apparently displayed fewer markers than sevs from monocultures .
overall , these data indicate that the phenotype of the vesicle subset is context - dependent
. however , during the current conditions , several of the leukocyte - derived sevs did not present the lineage - specific markers found on the parent cell type ( figure 1(a ) ) .
variations in the molecular content between cells and evs were also observed in a study by hunter et al . that reported significant differences in the presence of mirnas between plasma evs and pbmcs . for comparison , the presentation of lineage - specific markers on plasma sevs was determined .
in contrast to the leukocyte - derived sevs , plasma sevs presented several lineage - specific markers , including cd3 , cd14 , and cd19 .
compared to the evs present in cell supernatants , evs in plasma are a mixture of evs produced by many different cell types .
thus , plasma can act as a liquid biopsy and will present a very heterogeneous ev phenotype that provides information about numerous cellular processes .
the detection of cd3 , cd14 , and cd19 on plasma sevs , but not on sevs from cultured leukocyte subpopulations , emphasizes that the biological context has a great impact on the phenotype of the released evs .
interestingly , some of the sev subsets presented other markers , for example , sevs from cd8 t cells presenting cd16 .
several studies have described the expression of cd16 on a small portion of different types of t cells and even shown a functional role of cd16 on t cells [ 44 , 45 ] .
thus , the observations of cd16 on sevs released from cultured t cells combined with the fact that functional molecules , such as perforin and lytic granules , can be stored inside evs may indicate a specific functionality of these evs , for example , surrogates or extensions of the effector cell in antibody - dependent , cell - mediated cytotoxicity .
apart from the selected lineage - specific markers , the sevs were phenotyped for the presence of 26 different immunological or vesicle - related markers .
regarding the leukocyte - derived sevs , cd49d was the only marker detected on all sev subsets ( figure 1(c ) ) .
cd49d is an 4 integrin that is expressed as a heterodimer on t , b , and nk cells and monocytes as well as on other immune cells .
together with cd29 , cd49d forms the very late antigen-4 ( vla-4 ) that is involved in leukocyte trafficking , adhesion , and extravasation [ 46 , 47 ] .
so far , two studies have reported on the presence of cd49d / vla-4 on evs from b cells [ 48 , 49 ] , and these results can be confirmed by the present study . in terms of plasma sevs ,
surprisingly , cd41 was observed on sevs from t cells , b cells , monocytes , and pbmcs as well as on plasma sevs from half of the healthy donors ( figure 1(c ) ) .
however , according to the protein atlas , cd41 is expressed by pbmcs   and even though the function is unclear , the presence of cd41 on sevs released from these cells , either in monocultures or as pbmcs , is possible .
in addition , sevs from cultured t cells presented an array of other immunological markers , ranging from more general markers , like cd45 , to specific markers like cd28 , and the tlr3 .
moreover , sevs from cd8 t cells presented ctla-4 and the fas - l . even though the mechanisms are quite different , both of these molecules play a role in downregulation of an immune response , either by transmitting an inhibitory signal in activated t cells or by inducing activation - induced cell death , respectively .
the presence of such regulatory molecules on evs suggests that t cell - derived evs can be involved in immune regulation .
the presence of fas - l on evs from t cells has also been observed in other studies [ 5254 ] , but to the best of our knowledge , studies observing ctla-4 on evs from leukocytes have not previously been published . upon evaluating the presence of markers on evs , it is important to consider the natural variation in the molecular composition of the ev pool that exists between healthy individuals as visualized by the ten plasma samples .
thus , the present results are a snapshot providing insight about the current vesicular presentation of selected markers .
the sevs investigated in this study were identified by the presence of cd9 , cd63 , and/or cd81 .
thus , in order to investigate the similarity of the leukocytes and the corresponding sevs , the cellular expression patterns of cd9 , cd63 , and cd81 were determined .
the expression pattern was determined for both leukocytes present in whole blood and cultured leukocyte subpopulations .
the results demonstrated a lack of cd9 on the surface of leukocytes present in freshly drawn whole blood ( figure 3(a ) ) .
in contrast , cd9 was present on every leukocyte subpopulation following two days of culture .
in accordance with the present results , a recently published study also observed cd9 on the surface of isolated populations of cd3 , cd14 , and cd19 cells .
the expression patterns of cd63 and cd81 were quite similar between the two cell preparations but with minor differences in the expressions levels .
overall , these findings are in agreement with previously described results [ 5661 ] . when looking at both the cellular and the vesicular presentation of these markers , it is clear that , for the majority of the cultured subpopulations , the presentation of these markers was comparable . however , for the nk cells , the b cells , and the monocytes , the cellular expression of cd63 was very different from the presentation observed on sevs . a comparison between the presentation of cd9 , cd63 , and cd81 on whole blood leukocytes and plasma evs is a more complex procedure that needs to take into account that plasma evs are a very heterogeneous group of evs that emanate from multiple cell types .
the tetraspanin signals observed on the plasma sevs represent all tetraspanin - positive sevs irrespective of their origin .
thus , the presentation of tetraspanins on plasma sevs can not be expected to correlate with the expression of tetraspanin on leukocytes .
nevertheless , it is clear that the cd63 signals in plasma generally were low , while the cellular expression of cd63 for several of the leukocyte subpopulations is medium to high , indicating that the presentation of cd63 is different from sevs to cells .
overall , the vesicular signal intensities for cd63 were much lower than the intensities observed for cd9 and cd81 , suggesting that cd63 might be a poor marker for sevs in general , which has also been observed in other studies [ 38 , 6264 ] .
the results underline the importance of detecting evs with a cocktail of antibodies against tetraspanins , as detection with a single marker may overlook some subsets of evs .
surface molecules on evs are responsible for the biodistribution and the ligation to target cells .
thus , the protein composition is related to the functionality of evs , why phenotyping of evs can be used to gain knowledge about the functionality . in summary , the presented data regarding the lineage - specific markers and the tetraspanins support the accumulating observations suggesting that the transfer of molecular cargo into evs is tightly controlled .
a tightly regulated sampling of molecules to evs would match with the fact that evs play an important role as systemic regulators , traveling through tissues providing key intercellular communication as well as transfer of biologically active components .
 extracellular vesicles ( evs ) have a demonstrated involvement in modulating the immune system . 
 it has been proposed that evs could be used as biomarkers for detection of inflammatory and immunological disorders . 
 consequently , it is of great interest to investigate evs in more detail with focus on immunological markers . in this study , 
 five major leukocyte subpopulations and the corresponding leukocyte - derived evs were phenotyped with focus on selected immunological lineage - specific markers and selected vesicle - related markers . 
 the leukocyte - derived evs displayed phenotypic differences in the 34 markers investigated . 
 the majority of the lineage - specific markers used for identification of the parent cell types could not be detected on evs released from monocultures of the associated cell types . 
 in contrast , the vesicular presentation of cd9 , cd63 , and cd81 correlated to the cell surface expression of these markers , however , with few exceptions . furthermore , the cellular expression of cd9 , cd63 , and cd81 varied between leukocytes present in whole blood and cultured leukocytes . in summary , these data demonstrate that the cellular and vesicular presentation of selected lineage - specific and vesicle - related markers may differ , supporting the accumulating observations that sorting of molecular cargo into evs is tightly controlled . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion