IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ ghiotto-2010-1 in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results." but does not state when each form(s) were used. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr aniel.olive@inserm.fr International immunology 2010 Only protein-protein interactions imex curation imex curation ghiotto-2010-2 in vitro In vitro facs fluorescence-activated cell sorting FACS Flow cytometry facs antibody detection identification by antibody antibody detection Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr aniel.olive@inserm.fr International immunology 2010 Only protein-protein interactions imex curation imex curation pdcd1_human Programmed cell death protein 1 PDCD1 PD1 protein protein human Homo sapiens Human MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL pd1l1_human Programmed cell death 1 ligand 1 CD274 B7H1 PDCD1L1 PDCD1LG1 PDL1 B7 homolog 1 protein protein human Homo sapiens Human MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET pd1l2_human Programmed cell death 1 ligand 2 Butyrophilin B7-DC PDCD1LG2 B7DC CD273 PDCD1L2 PDL2 protein protein human Homo sapiens Human MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI cd80_human T-lymphocyte activation antigen CD80 CD80 CD28LG CD28LG1 LAB7 Activation B7-1 antigen CTLA-4 counter-receptor B7.1 BB1 protein protein human Homo sapiens Human MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV ctla4_human Cytotoxic T-lymphocyte protein 4 CTLA4 CD152 Cytotoxic T-lymphocyte-associated antigen 4 protein protein human Homo sapiens Human MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN pdcd1-cd274-3 3316983 N/A 3316985 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316986 unspecified role unspecified role prey prey ecd sufficient to bind sufficient binding region sufficient to bind range range range range false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false direct interaction direct interaction false false false 3316983 Table 1, fig 1, 2, Supp fig 2/3 The conformational change model gave the best and reliable fit. Pre-incubation of PD-L1 with three different antibodies against it influenced the interaction outcome. One of the anitbodies (PD-L1.3) disrupted binding, while the ohter two (PD-L1.1 and PD-L1.2) seemed to strengthen it, probably by increasing PD-L1 stability. pdcd1-cd274-5 3316983 N/A 3316985 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos Stoichiometry: 1.0 N/A 3316987 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos Stoichiometry: 1.0 direct interaction direct interaction false false false 3316983 Table 1, fig 1,3, Supp fig 1 pdcd1-pdcd1lg2-3 3316983 N/A 3316986 competitor competitor competitor prey prey purified purified region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind range range range range false N/A 3316985 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos Stoichiometry: 1.0 N/A 3316987 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos Stoichiometry: 1.0 physical association physical association false false false Fig 3A -top PD-L1 can out-compete PD-L2 following saturation of PD-L2 occupancy pdcd1-pdcd1lg2-4 3316983 N/A 3316986 unspecified role unspecified role bait bait region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind range range range range false N/A 3316985 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316987 competitor competitor competitor prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos physical association physical association false false false Fig 3A -middle PD-1 proteins were pre-incubated with PD-L2 and the resulting complexes were injected onto PD-L1-Ig chips;PD-L2 pre-incubation prevented PD-1 binding to PD-L1 in a dose-dependent manner cd274-cd80-6 3316983 N/A 3316988 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc N/A 3316986 unspecified role unspecified role prey prey region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind range range range range false direct interaction direct interaction false false false 3316983 Fig 1A, 3, Table 1 the parameters of PD-L1 binding to CD80 fitted also with the conformational change model. cd274-cd80-7 3316983 N/A 3316988 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc N/A 3316989 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos direct interaction direct interaction false false false 3316983 Fig 1A, Table 1 cd80-cd274-3 3316983 N/A 3316985 competitor competitor competitor prey prey purified purified region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3316988 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc N/A 3316986 unspecified role unspecified role prey prey region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind range range range range false direct interaction direct interaction false false false 3316983 3A bottom cd80-ctla4-2 3316983 N/A 3316989 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316986 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind range range range range false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3316988 ancillary ancillary prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association false false false Supp fig 4-2 CTLA-4 cannot directly bind to PD-L1 chip but strongly bind to CD80/PD-L1 complexes cd80-cd274-4 3316983 N/A 3316989 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316989 competitor competitor competitor prey prey ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316988 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association false false false Fig 3B bottom cd274-cd80-8 3316983 N/A 3316986 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind range range range range false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3316988 unspecified role unspecified role prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc direct interaction direct interaction false false false Supp fig 4-1 cd274-cd80-9 3316983 N/A 3316989 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316986 unspecified role unspecified role prey prey ecd sufficient to bind sufficient binding region sufficient to bind range range range range false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3316988 ancillary ancillary prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association false false false Supp fig 5 CTLA-4 and PD-L1 can bind to human CD80 simultaneously cd274-ctla4-2 3316984 N/A 3316988 unspecified role unspecified role neutral component neutral component c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc N/A 3316986 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos N/A 3316989 unspecified role unspecified role neutral component neutral component region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos Chlorocebus aethiops cos cos physical association physical association false false false Fig 3C top It is not stated if the PD-L1 molecule was full length or ECD only. CTLA-4-Ig can bind to PD-L1 expressing COS cells in presence of CD80 but not PD-1.