ficoll - isolated mononuclear peripheral blood ( pb ) cells from healthy donors were stained with abs against the lin markers cd3 , cd19 and cd56 , as well as with hla - dr , cd14 , cd11c , cd141 , cd1c , cd303 and the indicated workshop abs ( table 1 ) . pdcs and two distinct myeloid dcs ( cd1c dcs , cd141 dcs ) were defined by multicolour immunofluorescence according to the flow cytometry gate setting strategy shown in figure 1 . all dc subsets are characterized by the absence of the lin markers and cd14 and the presence of hla - dr and cd11c . mdc2 were additionally selected by their absence of cd1c . based on this multigating strategy , pdcs express the key marker cd303 ( bdca-2 ) , whereas the myeloid dc subsets express the key markers cd1c ( bdca-1 ) and cd141 ( bdca-3 ) . as demonstrated in figure 1 , mdc1 and mdc2 express high or medium levels of cd11c , respectively , whereas cd11c is only weakly expressed by pdc . for profiling of dc subsets with workshop abs , plots of cd1c , cd141 or cd303 versus the selected monoclonal antibody ( mab)-defined antigens were compared with the corresponding controls . in figure 2 the profiles demonstrate that cd370 ( clec9a / dngr-1 ) is almost exclusively expressed on cd141 dcs , whereas cd366 ( tim-3 ) and cd101 ( table 1 ) expression is restricted to both myeloid dc subsets , and cd371 ( clec12a , cll , micl ) is expressed on all three dc subsets . il-13-ra2 and vimentin , as well as two ab - defined antigens of unknown targets , show heterogeneous reactivity profiles on either the two myeloid dc subsets ( 10 - 30/il-13-ra2/clone fmu - il-13ra2 - 7 and 10 - 69/clone cmrf-56 ) or on all three dc subsets ( 10 - 55/vimentin / clone sc5 and 10 - 25/clone w4a5 ) ( table 1 ) . this suggests that all three dc populations contain additional subsets with probably unknown function . of the new clustered molecules at the hlda10 conference , cd365 ( tim-1 ) is not expressed on primary dcs and weak cd368 ( clec4d , dectin-3 ) expression on these cells is only verified by one of two submitted abs ( positive with clone 9b9 ( 10 - 78 ) , but not with clone fab2806p ( 10 - 21 ) ) . cd367 ( clec4a , dcir ) and cd369 ( clec7a , dectin-1 ) are weakly expressed on all dc subsets shown by staining with two out of three cd367-reactive abs ( clones fab1748p ( 10 - 13 ) and 9e8 ( 10 - 72 ) ) . in contrast , anti - cd369 abs showed clone - dependent reactivity patterns to peripheral blood dcs . clone ge2 was not reactive to all three analysed dc populations , whereas clone fab1859p ( 10 - 35 ) interacted weakly with cd141 dcs and clone 15e2 ( 10 - 79 ) weakly with pdcs , cd1c and cd141 dcs ; table 1 ) . the reactivity profiles of all available workshop mabs to dc subsets are summarized in table 1 . workshop mabs were additionally analysed for their recognition of mo - dcs , which were obtained after culture of peripheral blood mononuclear cells ( pbmcs ) for 7 days in the presence of 100  ng  ml gm - csf and 20  ng  ml il-4 . after culture , cells were double - stained with anti - cd209 ( dc - sign)-apc and the indicated workshop abs and analysed by flow cytometry . figure 3 shows that cultured dcs reside in the  monocyte ' gate in the dual scatter plot , which was verified by their expression of cd209 . after gating , cells were analysed by plotting cd209 versus workshop mab and compared with appropriate controls . the complete set of new cd molecules clustered at the hlda10 conference ( cd365 - 371 ) is expressed on mo - dcs at varying intensity . however , some abs such as 10 - 1 ( cd369 ) , 10 - 2 ( cd370 ) and 10 - 17 ( cd371 ) did not recognize the epitopes on mo - dcs , although other workshop mabs against these molecules showed a positive staining on these cells . mab against cd273 and cd276 , as well as the ab - defined antigens 58b3 , f9 - 3c2 ( 10 - 39 ) , w4a5 ( 10 - 25 ) and w5c4 ( 10 - 32 ) show unique reactivity patterns ( figure 3 ) . in particular , mab 10 - 61 ( cd273 ) , mab 10 - 69 ( cmrf-56 ) , mab 10 - 39 ( f9 - 3c2 ) and mab 10 - 25 ( w4a5 ) give rise to heterogeneous reactivity patterns , suggesting that cultured dcs do not consist of a homogeneous cell population . the reactivity profiles on mo - dc of all available workshop mab are summarized in table 2 . ficoll - isolated mononuclear peripheral blood ( pb ) cells from healthy donors were stained with abs against the lin markers cd3 , cd19 and cd56 , as well as with hla - dr , cd14 , cd11c , cd141 , cd1c , cd303 and the indicated workshop abs ( table 1 ) . pdcs and two distinct myeloid dcs ( cd1c dcs , cd141 dcs ) were defined by multicolour immunofluorescence according to the flow cytometry gate setting strategy shown in figure 1 . all dc subsets are characterized by the absence of the lin markers and cd14 and the presence of hla - dr and cd11c . mdc2 were additionally selected by their absence of cd1c . based on this multigating strategy , pdcs express the key marker cd303 ( bdca-2 ) , whereas the myeloid dc subsets express the key markers cd1c ( bdca-1 ) and cd141 ( bdca-3 ) . as demonstrated in figure 1 , mdc1 and mdc2 express high or medium levels of cd11c , respectively , whereas cd11c is only weakly expressed by pdc . for profiling of dc subsets with workshop abs , plots of cd1c , cd141 or cd303 versus the selected monoclonal antibody ( mab)-defined antigens were compared with the corresponding controls . in figure 2 the profiles demonstrate that cd370 ( clec9a / dngr-1 ) is almost exclusively expressed on cd141 dcs , whereas cd366 ( tim-3 ) and cd101 ( table 1 ) expression is restricted to both myeloid dc subsets , and cd371 ( clec12a , cll , micl ) is expressed on all three dc subsets . il-13-ra2 and vimentin , as well as two ab - defined antigens of unknown targets , show heterogeneous reactivity profiles on either the two myeloid dc subsets ( 10 - 30/il-13-ra2/clone fmu - il-13ra2 - 7 and 10 - 69/clone cmrf-56 ) or on all three dc subsets ( 10 - 55/vimentin / clone sc5 and 10 - 25/clone w4a5 ) ( table 1 ) . this suggests that all three dc populations contain additional subsets with probably unknown function . of the new clustered molecules at the hlda10 conference , cd365 ( tim-1 ) is not expressed on primary dcs and weak cd368 ( clec4d , dectin-3 ) expression on these cells is only verified by one of two submitted abs ( positive with clone 9b9 ( 10 - 78 ) , but not with clone fab2806p ( 10 - 21 ) ) . cd367 ( clec4a , dcir ) and cd369 ( clec7a , dectin-1 ) are weakly expressed on all dc subsets shown by staining with two out of three cd367-reactive abs ( clones fab1748p ( 10 - 13 ) and 9e8 ( 10 - 72 ) ) . in contrast , anti - cd369 abs showed clone - dependent reactivity patterns to peripheral blood dcs . clone ge2 was not reactive to all three analysed dc populations , whereas clone fab1859p ( 10 - 35 ) interacted weakly with cd141 dcs and clone 15e2 ( 10 - 79 ) weakly with pdcs , cd1c and cd141 dcs ; table 1 ) . the reactivity profiles of all available workshop mabs to dc subsets are summarized in table 1 . workshop mabs were additionally analysed for their recognition of mo - dcs , which were obtained after culture of peripheral blood mononuclear cells ( pbmcs ) for 7 days in the presence of 100  ng  ml gm - csf and 20  ng  ml il-4 . after culture , cells were double - stained with anti - cd209 ( dc - sign)-apc and the indicated workshop abs and analysed by flow cytometry . figure 3 shows that cultured dcs reside in the  monocyte ' gate in the dual scatter plot , which was verified by their expression of cd209 . after gating , cells were analysed by plotting cd209 versus workshop mab and compared with appropriate controls . the complete set of new cd molecules clustered at the hlda10 conference ( cd365 - 371 ) is expressed on mo - dcs at varying intensity . however , some abs such as 10 - 1 ( cd369 ) , 10 - 2 ( cd370 ) and 10 - 17 ( cd371 ) did not recognize the epitopes on mo - dcs , although other workshop mabs against these molecules showed a positive staining on these cells . mab against cd273 and cd276 , as well as the ab - defined antigens 58b3 , f9 - 3c2 ( 10 - 39 ) , w4a5 ( 10 - 25 ) and w5c4 ( 10 - 32 ) show unique reactivity patterns ( figure 3 ) . in particular , mab 10 - 61 ( cd273 ) , mab 10 - 69 ( cmrf-56 ) , mab 10 - 39 ( f9 - 3c2 ) and mab 10 - 25 ( w4a5 ) give rise to heterogeneous reactivity patterns , suggesting that cultured dcs do not consist of a homogeneous cell population . the reactivity profiles on mo - dc of all available workshop mab are summarized in table 2 . we screened abs submitted from different providers to the hlda10 workshop in wollongong , australia , for their reactivity with primary human blood dc subsets as well as with in vitro generated mo - dcs . the abs were either purified , delivered as ascites or directly conjugated with fitc ( fluorescein isothiocyanate ) or pe ( phycoerythrin ) and assigned to consecutive numbers . two new abs cluster to single - span class-1 transmembrane proteins , namely cd365 ( tim-1 ) and cd366 ( tim-3 ) . tim-3 has a critical role in regulating the activities of t cells , macrophages , monocytes , dcs , mast cells , natural killer cells and endothelial cells . whether these molecules are also expressed on human blood dc subsets was not yet investigated . here we could show that cd365 is also weakly expressed on primary blood pdcs , cd1c and cd141 dcs , as well as on mo - dcs . in contrast , cd366 is highly expressed on cd1c and cd141 dcs , as well as mo - dcs , but only near to background levels on pdcs . these data are in contrast to a recent study showing no cd366 expression on mo - dcs from healthy volunteers or patients with different tumours , whereas tumour - associated cd11c dcs express high levels of cd366 . this study did not discriminate between different subsets of tumour - associated cd11c dcs nor analysed peripheral blood dcs . we could show that all four hlda10 abs generated against cd370 ( clec9a ) specifically recognize cd141 dcs , although abs 10 - 45 and 10 - 65 weakly reacted with pdcs , cd1c dcs and in vitro generated mo - dcs . cd370 functions as an endocytic receptor specific for the uptake of degraded components from dead cells and is specifically expressed by cd141 dcs . the reasons for the occasionally decreased performance of workshop abs can be several : first second , the quality of abs varied considerably , some were shipped as purified , some as culture supernatant , some as ascites fluid and others as fitc or pe conjugates . third , many abs could be visualized only by indirect staining in combination with multicolour immunofluorescence analysis . as a result , the weaker binding of abs 10 - 45 and 10 - 65 compared with 10 - 02 and 10 - 09 can be explained by these reasons . this is of particular evidence with regard to abs 10 - 02 and 10 - 65 , as these reagents are derived from the same clone . and finally , different epitopes of the same molecule could be differentially expressed on the cell surface of primary and cultured dcs , resulting in a differential recognition of molecules by abs with the same molecule specificity . cd369 ( clec7a / dectin-1 ) is an another pattern recognition receptor specific for -glucans , components of pathogenic bacteria and fungi and is expressed on dcs . we could validate that the submitted cd370-reactive abs bind to primary blood cd1c dcs and cd141 dcs , as well as to in vitro generated mo - dcs , but not to pdcs . in addition , all submitted abs against the c - type lectins including cd367 ( clec4a / dcir ) , cd368 ( clec4d ) and cd371 ( clec12a / micl ) bound to all dc subsets analysed . cd367 expression is decreased upon maturation , but because of limited amount of abs provided , we were not able to confirm these findings . finally , we identified three abs recognizing targets that are heterogeneously expressed on all primary blood dc subsets including vimentin , ab 10 - 69 and ab w4a5 . although the latter two targets are not yet identified , they appear to be of particular interest because they are able to subdivide the subsets into either further sub - populations or into defined activation stages . in conclusion , using abs submitted to the hlda10 workshop , we could not only confirm the expression patterns of known dc targets but also expand the knowledge of molecules expressed on dcs by either demonstrating new reactivity patterns of known targets or presenting heterogeneous expression profiles of known and unknown targets on dc subsets . peripheral blood was collected from two healthy donors according to the guidelines of the local ethics committee . when fluorochrome - conjugated workshop abs were available , cells were incubated with a mixture of all ab conjugates for 30  min at 4  c . conjugates consisted of the lin markers cd3-pacific blue ( sk7 ) , cd19-pacific blue ( hlb19 ) and cd56-pacific blue ( mem-188 ) , as well as of anti - hla - dr - brilliant violet 510 ( l243 ) , cd14-fitc ( m5e2 ) , cd11c - pe - cy7 ( bu15 ) , cd141-apc ( m80 ) , cd1c - apc - cy7 ( l161 ) and cd303-percp - cy5.5 ( 201  a ) ( purchased from biolegend inc . , san diego , ca , usa ) , and of pe - conjugated workshop abs . after washing with facs buffer ( phosphate - buffered saline , 0.1% nan3 , 1% bovine serum albumin ) , cells were additionally stained for 10  min with 3  m dapi ( 4',6-diamidino-2-phenylindole)at room temperature ( sigma - aldrich , saint louis , mo , usa ) for dead cell exclusion and acquired on a facscanto ii analyser ( heidelberg , germany ) . stored data were analysed using the flowjo software programme ( flowjo , llc , ashland , or , usa ) . the gating and plotting strategy to define pdc , cd1c dcs and cd141 dcs is shown in figure 1 . in the case where only non - conjugated after washing , cells were stained with pe - conjugated goat anti - mouse antisera ( biozol diagnostica gmbh , eching , germany ) . in the next step non - reacting ' mouse ab ( biozol diagnostica gmbh ) to block free binding sites of the secondary - step reagent . in the final step , cells were incubated with the commercially available ab conjugates . for generation of mo - dcs cells were seeded ( 1  10/10  ml ) into 75  cm cell culture flasks ( corning , cambridge , ma , usa ) in serum - free x - vivo 20 medium ( cambrex bio science , verviers , belgium ) . after 2  h of incubation at 37  c/5% co2 , non - adherent cells were removed . the monocytes were cultured in 10  ml rp10 medium ( rpmi-1640 with glutamax - i ) , supplemented with 10% inactivated foetal calf serum and antibiotics ( invitrogen , karlsruhe , germany ) . gm - csf ( 100  ng  ml ; leukine liquid sargramostim ; sanofi , bridgewater , nj , usa ) and il-4 ( 20  ng  ml ; r&d systems , wiesbaden , germany ) were added every second day for 7 days . after washing two times with facs buffer , cells were stained with allophycocyanin - conjugated anti - cd209 ab ( dcn46 ; bd biosciences , heidelberg , germany ) and the indicated workshop abs using the protocol described above . peripheral blood was collected from two healthy donors according to the guidelines of the local ethics committee . when fluorochrome - conjugated workshop abs were available , cells were incubated with a mixture of all ab conjugates for 30  min at 4  c . conjugates consisted of the lin markers cd3-pacific blue ( sk7 ) , cd19-pacific blue ( hlb19 ) and cd56-pacific blue ( mem-188 ) , as well as of anti - hla - dr - brilliant violet 510 ( l243 ) , cd14-fitc ( m5e2 ) , cd11c - pe - cy7 ( bu15 ) , cd141-apc ( m80 ) , cd1c - apc - cy7 ( l161 ) and cd303-percp - cy5.5 ( 201  a ) ( purchased from biolegend inc . , san diego , ca , usa ) , and of pe - conjugated workshop abs . after washing with facs buffer ( phosphate - buffered saline , 0.1% nan3 , 1% bovine serum albumin ) , cells were additionally stained for 10  min with 3  m dapi ( 4',6-diamidino-2-phenylindole)at room temperature ( sigma - aldrich , saint louis , mo , usa ) for dead cell exclusion and acquired on a facscanto ii analyser ( heidelberg , germany ) . stored data were analysed using the flowjo software programme ( flowjo , llc , ashland , or , usa ) . the gating and plotting strategy to define pdc , cd1c dcs and cd141 dcs is shown in figure 1 . in the case where only non - conjugated after washing , cells were stained with pe - conjugated goat anti - mouse antisera ( biozol diagnostica gmbh , eching , germany ) . in the next step non - reacting ' mouse ab ( biozol diagnostica gmbh ) to block free binding sites of the secondary - step reagent . in the final step for generation of mo - dcs , monocytes were purified by plastic adherence of pbmcs . for this purpose , cells were seeded ( 1  10/10  ml ) into 75  cm cell culture flasks ( corning , cambridge , ma , usa ) in serum - free x - vivo 20 medium ( cambrex bio science , verviers , belgium ) . after 2  h of incubation at 37  c/5% co2 , non - adherent cells were removed . the monocytes were cultured in 10  ml rp10 medium ( rpmi-1640 with glutamax - i ) , supplemented with 10% inactivated foetal calf serum and antibiotics ( invitrogen , karlsruhe , germany ) . gm - csf ( 100  ng  ml ; leukine liquid sargramostim ; sanofi , bridgewater , nj , usa ) and il-4 ( 20  ng  ml ; r&d systems , wiesbaden , germany ) were added every second day for 7 days . after washing two times with facs buffer , cells were stained with allophycocyanin - conjugated anti - cd209 ab ( dcn46 ; bd biosciences , heidelberg , germany ) and the indicated workshop abs using the protocol described above .