Supplemental Table 1. Plasmid Construct backbone. Restriction sites. PCR template. HLA DRα pcdna3.1 Zeo+
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1 SupplementalTable1 Plasmid Construct backbone HLA DRα Zeo+ Restriction sites PCR template PCRCloningstrategy Primerpairs(F/R)andpairsofprimersforchimericconstructs(N terminalandc terminal codingregion)weregiven Lapaqueetal.(2009) cdna BamHI HLA DRβ Zeo+ EcoRI NotI cdna Lapaqueetal.(2009) CD8 DRα Neo EcoRI BamHI HLA DRα Lapaqueetal.(2009),designatedCD8 DRA extrainthatstudy CD8 DRβ Zeo+ EcoRI NotI HLA DRβ Lapaqueetal.(2009) K225R Zeo+ EcoRI NotI CD8 DRβ Lapaqueetal.(2009) K225C Neo EcoRI BamHI Δ229 F:CTTCAGGAATCAGTGTGGACACTCTGGATGAGGATCCGAG, R:CAGAGTGTCCACACTGATTCCTGAAGTAGATGAACAG Δ233 Neo EcoRI BamHI CD8 DRβ F:T7seq,R:TCGGATCCTCATGTTGGCTGCAGTCCAGAGT Δ229 Neo EcoRI BamHI CD8 DRβ F:T7seq,R:AGGAATCAGAAAGGACACTCTGGATGAGGATCCGA Δ227 Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCAGTGTCCTTTCTGATTCCTGATTC Δ225 Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCATTTCTGATTCCTGAAGTAGATG RNAK Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCATTTAGCATTCCTGAAGTAGATGAACAGC RAQK Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCATTTCTGAGCCCTGAAGTAGATGAACAGCC ANQK Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCATTTCTGATTAGCGAAGTAGATGAACAGCCCTG RAAK Neo EcoRI BamHI CD8 DRβ F:T7seq,R:CGGGATCCTCATTTAGCAGCCCTGAAGTAGATGAACAGCC HF0 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:CAGACCCAGTACAGCGCCCCCGACTCCACTCAGTGCCTTGCTCTGTGCAGACCC C term: F:GCTGTACTGGGTCTGCTCTTCGCTGGGACAGGGCTGTTCATCGCATTCAGGAATCAGAAAGG ACAC,R:Zeo2 HF1 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq,r: CCCCTGTCCCTAGGAAGAGAGCGCCCAGAACAAAACCCCCAGCTCCACTCAGCATCTTGCTC C term:f:tctcttcctagggacaggggctttcatctacttcaggaatcagaa,r:zeo2 HF2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:GAAGAGCAGGCCCAGAGCAAAGCCCCCGACTCCACTAGCCATCTTGCTCTGTGCAGAC C term:f:tctgggcctgctcttccttgctacagggctgttcatctacgctaggaatcagaaagga CACTCTG,R:Zeo2 HF3 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:CCCAGTCCCAAGGAAAGCCAGGCCCAGCACAAAACCAGCGACTCCACTCAGCATCTTGC C term:f:tttccttgggactgggctggctatctacttcaggaatcagaaagg,r:zeo2
2 SupplementalTable1,continued HF4 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:CAAGGAAGAGCAGGCCAGCCACAAAGCCCCCGACTCCAGCCAGCATCTTGCTCTGTGC C term:f:tggcctgctcttccttggcgctgggctgttcatctacttcag,r:zeo2 HF5 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:ACAGCCCAGTCCCAAGAGCGAGCAGTCCCAGTACAAAAGCCCCGACTCCACTCAGC C term:f:tcttgggactgggctgttcgcttacttcaggaatcagaaaggaca,r:zeo2 HF6 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:TCCCAAGGAAGAGAAGAGCCAGCACAAAACCCCCGACAGCACTCAGCATCTTGCTCTGTG C term:f:tcttctcttccttgggacagctctgttcatctacttcaggaatcag,r:zeo2 M G A Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq,r:gcccagcacgaagccagcggcagcagcagcagccttgctctgtgcagaccc C term:f:ggcttcgtgctgggcctgct,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq,r:cctgtcccaaggaagagagcagcagcagctgcagccccgactccactcagc G L!A L G!A L F!A S 194 AAAAA S 194 AAA S 197 AA S 194 AAAAE R 222 AAA R 222 EAA C term:f:ctcttccttgggacagg,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:CTGATTCCTGAAGTAGATGAACAGAGCTGCTGCTGCTGCTGCCAGGCCCAGCACAAAG C term:f:tctgttcatctacttcaggaatcag,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:t7seq, R:CAGAGTGTCCTTTCTGATTCCTAGCAGCAGCAGCGGCCCCTGTCCCAAGGAAGAGC C term:f:taggaatcagaaaggacactctg,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:CCCCCTCGTGTGCACTGC C term:f:cagtgcacacgaggggggccgctgctgctgctatgctgagtggagtcggg,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:CCCCCTCGTGTGCACTGC C term:f:cagtgcacacgaggggggccgctgctagcaagatgctgagtggagtcg,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:CCCCCTCGTGTGCACTGC C term:f:cagtgcacacgagggggtctgcacaggctgctatgctgagtggagtc,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:CCCCCTCGTGTGCACTGC C term:f:cagtgcacacgaggggggccgctgctgctgagatgctgagtgga,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:GAAGTAGATGAACAGCCCTGTC C term:f:gacagggctgttcatctacttcgcagcagcgaaaggacactctg,r:zeo2 Zeo+ EcoRI XhoI CD8 DRβ N term:f:5 Prim,R:GAAGTAGATGAACAGCCCTGTC C term:f:gacagggctgttcatctacttcgaggcagcgaaaggacactctg,r:zeo2
3 SupplementalTable1,continued K 1 Neo EcoRI BamHI CD8 DRβ F:5 Prim, R:CGGGATCCTCATGCAGCAGCGGCAGCTGCAGCAGCTTTGTAGATGAACAGCCCTGTCCC K 1 Neo EcoRI BamHI CD8 DRβ F:T7seq, R:CGGGATCCTCATGCAGCGGCAGCAGCGGCAGCTTTGAAGTAGATGAACAGCCCTGTC K 2 Neo EcoRI BamHI CD8 DRβ F:5 Prim,R:CGGGATCCTCATGCAGCGGCAGCTGCAGCTTTAGCGAAGTAGATGAACAGCCC K 3 Neo EcoRI BamHI CD8 DRβ F:5 Prim,R:CGGGATCCTCATGCAGCGGCTTTAGCTGCAGCAGCGAAGTAGATGAACAG K 4 Neo EcoRI BamHI CD8 DRβ N term:f:t7seq,r:tcatgcagcggcagcttttgcagcagcgaagtagatgaacagccctgtc C term:f: AGCTGCCGCTGCATGAGGATCCGAGCTCG,R:GATGCAATTTCCTCATTTTATTAG K 5 Neo EcoRI BamHI CD8 DRβ F:5 Prim,R:CGGGATCCTCATGCAGCGGCAGCAGCTTTAGCAGCGAAGTAGATGAACAG K 6 Neo EcoRI BamHI F:5 Prim,R:CGGGATCCTCATGCAGCTTTGGCAGCTGCAGCAGCGA K 5 K 7 Neo EcoRI BamHI CD8 DRβ F:T7seq, R:CGGGATCCTCATGCTTTAGCGGCAGCAGCGGCAGCGAAGTAGATGAACAGCCCTGTC HLA DRα KK 215,219 RR HLA DRβ HLA DRβ wt Zeo+ Hygro+ Hygro+ BamHI BamHI BamHI HLA DRα cdna, DRB1*0303 CD8 DRβ, HLA DRβ F:5 Prim, R:AATGGATCCTTACAGAGGCCCCCTGCGTTCTGCTGCATTGCTTCTGCGCACTCCCCTGATGATG F:CCCCAAGCTTGGCCACCATGGTGTGTCTGAGGCTCCCTG R:AAGGATCCTAGCTCAGGAATCCTCTTGGCTG N term:f:5 Prim,R:CTGTGCAGATTCAATCGATGCTCTCCATTCCACTG C term:f:gagcatcgattgaatctgcacagagcaagatgct, R:TATGGATCCTCAGCTCAAGAGTCCTGTTGGC ThisconstructwasachimerabetweentheDR3βextracellulardomainandthe perimembrane transmembrane cytoplasmictailregionofdr1β(whichwasalsousedinthe CD8 DRβreporters).AuniqueClaIsite(underlinedinprimers)wasintroducedinthenonfoldedaminoacidchainbetweenthefoldedpartoftheIgdomainandthejuxtamembrane region.thisresultedinminimalaminoacidsubstitutions( 191 RS 192 SI)inthatregionand didnotaffectdrfoldingorantigenloading(themodifieddrwasboundbymab16.23 whichonlybindscorrectlyfolded,sds stabledr3dimers;datanotshown)
4 SupplementalTable1,continued HLA DRβ K 1 HLA DRβ K 1 HLA DRβ K 2 HLA DRβ K 3 HLA DRβ K 4 HLA DRβ K 5 HLA DRβ K 6 XhoI K 1 K 1 K 2 K 3 K 4 K 5 HLA DR3 DRβwt HLA DR3 DRβwt K 6 HLA DRβ K 7 K 7 HLA DQβ pcr2.1 TOPO cdna, HeLa.CIITA HLA DRβ DQβ Zeo+ XhoI HLA DRβwt, HLA DQβ PairofuniversalprimersthatboundattheDRβperimembraneregion(F)and3 terminalof themultiplecloningsiteonhygro+(r).insertwasdigestedwithclaiandxhoi (underlined)andinsertedbetweenthesamesitesinhla DR3 DRβ wt.thereverseprimer alsocontainedam13fprimingsiteforscreeningforinsertsbycolonypcr(italics). F:ATAAGCTTCGCCACCATGTCTTGGAAGAAGGCTTTG, R:TACTCGAGTCTCAGGAGTCAGTGCAGAAG universalprimerpairfordqb1,withhindiiiandxhoisites,startand STOPcodonsunderlined C term:f:5 Prim,R:CCAAGCCCAAGGAAGAGCAGGCCCA N term:f:tgctcttccttgggcttggccttatcatccgtcaaa, R:TACTCGAGTCTCAGGAGTCAGTGCAGAAG
5 SupplementalTable1,continued HLA DRβ DQβ Hygro+ BamHI EGFP MARCH8 pegfpc2 EcoRI KpnI ptracer/ MARCH8 HLA DRβ DQβ , HLA DRβwt EGFP MARCH8mut Furtherprimersequences: T7seq: TAATACGACTCACTATAGGG 5 Prim: ACCCACTGCTTACTGGCTTATCG pegfpc2 EcoRI KpnI ptracer/ MARCH8 mut Zeo2: CATGCCTGCTATTGTCTTCCC SupplementalTable1. Listofprimersusedinthegenerationofconstructsusedinthisstudy. N term.:f:5 Prim,R:TCCTTTCTGATTCCTTTGACGGATGATAAGGCC C term:f:catccgtcaaaggaatcagaaaggacactctgg,r:zeo2 F:GCTTGGTACTAATACGACTCACTATAGGGA,R: AGCGGTACCTAATGGTGATGGTGATGATG MARCH8wtclonedfrompTracerconstructtogetherwithV5andHIStags,cutwithEcoRI (onplasmid)andkpni(underlined) F:GCTTGGTACTAATACGACTCACTATAGGGA,R: AGCGGTACCTAATGGTGATGGTGATGATG
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