Strategy of TCR sequencing by 44 mrna --CCC XXXXX Universal Oligo Forward Primer (Universal Mix) Forward Primer RT st PCR Va/b ~4bp CDR3 N/nDn ~bp Ja/b ~6bp <3bp nd Reverse Primer Ca/b Reverse Primer AAAAA TTTTT TTTTT Adaptor A (bp) Va-CD4 Va-CD8 % Va / TRAV4 % Va / TRAV4 Flow Flow 44 sequencing 44 sequencing Adaptor B Adaptor B Nested PCR Sequenced up to bp ~3bp Adaptor A NKT % NKT 4 3 Flow 44 sequencing
Sequencing of T cells infiltrating islets Reproducibility Mouse Variation Unique / Total Sequence 46 / 364 483 / 986 Rxn vs Rxn vs Islets vs PLN. <. Rxn 3 vs Rxn 4 vs Rxn vs.9.9.89.93.93.93 Rxn 6 vs.3....3 <. Sum of Rxn -6 (Counts) Reproducibility R² =.8968 Rxn (Counts) Mouse (Counts) Mouse Variation R² =.64 3 4 Mouse (Counts) PLN (Counts) 8 6 4 Islets vs PLN R² =.64 3 Islets (Counts)
Common sequences shared by multiple mice Vgene Frequency Frequency (%)... -4 6 3-6-7 3-3 7-4 3-6-6 3-4 4-3 4-8- 3-7-3 8-4- 9-3 4-9- 7-7- 6-7 - 6-4 - 6-6- 9 9-4 7-6 3-3 6-3 - 7-4-3 8 - - 3-9- -3 3-4 4-4 TRAV Vgene Junc)on Jgene 3 4 TRAV* CAVREPNYNVLYF TRAJ 3 8 7 6 TRAV* CAVRLPNYNVLYF TRAJ TRAV* CAVRMPNYNVLYF TRAJ 8 4 46 6 TRAV* CVVGDRGSALGRLHF TRAJ8 3 98 7 87 634 TRAV* CVVVDRGSALGRLHF TRAJ8 7 4 83 TRAV6* CAMREGWTDKVVF TRAJ34 4 3 6 TRAV6D* CAMREGWTDKVVF TRAJ34 6 7 3 3 TRAV6* CAMRDSGGSNAKLTF TRAJ4 6 63 89 8 3 TRAV6* CAMRNSGGSNAKLTF TRAJ4 37 33 Vgene Junc)on Jgene TRAV6D* CAMRDSGGSNAKLTF TRAJ4 8 3 38 7 TRAV4D * CAASANYNVLYF TRAJ 8 93 9 49 33 3 4 6 TRAVD 4* CAASAGGADRLTF TRAJ4 4 4 7 96 436 TRAVD 4* CAASASGGSNYKLTF TRAJ3 8 3 43 7 TRAVD 4* CAASESGGSNYKLTF TRAJ3 47 89 38 8 363 TRAVD 4*4 CAASESGGSNYKLTF TRAJ3 4 TRAVD 4* CAASEGGGSNYKLTF TRAJ3 6 TRAVD 4* CAASKTGANTGKLTF TRAJ 33 6 4 3 TRAVD 4* CAASPGNYKYVF TRAJ4 4 3 TRAV6 6* CALGDQTGGADRLTF TRAJ4 6 8 48 TRAV3 * CAVSGANTGKLTF TRAJ 4 368 343
PCR protocol for npod samples RNA 4 RT reactions cdna cdna cdna cdna 6 PCR rxns 6 PCR rxns 6 PCR rxns 6 PCR rxns Tag 4 PCR products with MID Run on 44
TRAV- TRAJ7 CAVGGAAGNKLTF TRAV- TRAJ CAVTMGSNDYKLSF TRAV-3 TRAJ6 CAMSARGGSYIPTF TRAV3- TRAJ4 CAATPGGYNKLIF TRAV3- TRAJ CAESSSYKLIF TRAV3- TRAJ4 CAENPSGDGGYNKLIF TRAV3- TRAJ4 CAENSGMNYGGSQGNLIF TRAV7 TRAJ9 CATDASVTPLVF TRAV9 TRAJ CALSEARSNDYKLSF TRAV9 TRAJ3 CALSELSGATNKLIF TRAV9 TRAJ4 CALSEAGNYGGSQGNLIF TRAV9 TRAJ CALSEAQGGRRALTF TRAV TRAJ CAVTGGNKLTF TRAV TRAJ CASDSGSARQLTF TRAV TRAJ3 CAVKPPVGGGKLIF TRAV TRAJ9 CAVKSNSGNTPLVF TRAV TRAJ37 CAAYSTGKLIF TRAV TRAJ47 CAALYGNKLVF TRAV3 TRAJ3 CAATPGRSGGYQKVTF TRAV6- TRAJ3 CIVRVENQGGKLIF TRAV6- TRAJ CILGENQAGTALIF TRAV6- TRAJ9 CILNSGNTPLVF TRAV7 TRAJ7 CAGKDTNAGKSTF TRAV38- TRAJ CAFRVDSSYKLIF TRAV38- TRAJ4 CAYFSGTYKYIF TRAV38- TRAJ43 CAYRSGSNNDMRF TRAV4 TRAJ39 CLVGDPSTGGAGNMLTF TRAV4 TRAJ4 CLVGPLMFSGGYNKLIF TRAV4 TRAJ48 CAVSFGNEKLTF TRAV4 TRAJ49 CAEFYF TRAV6 TRAJ6 CALDPSGGSYIPTF TRAV6 TRAJ48 CAVGDPSFGNEKLTF TRAV8-6 TRAJ4 CAPSPGGYNKLIF TRAV8-6 TRAJ4 CAVSDMGTYKYIF TRAV9- TRAJ CALSPDYKLSF TRAV9- TRAJ7 CALSDRANAGKSTF TRAV9- TRAJ4 CALRGSSGYELNF
Frequent TCRs for Insulin B:9-3-reactive cells Alpha chain Vgene Junc)on Jgene Total 3 4 6 7 8 9 3 4 6 7 8 9 3 4 TRAV6 CALRFGSSNTGKLIF TRAJ37 3 3 3 3 4 69 44 3 37 7 39 9 4 48 9 4 6 TRAV8 CVVQRITITMTCAPSDNNNDMRF TRAJ43 9 4 3 9 4 7 7 39 9 9 7 8 9 6 TRAV CVVNKGTNAGKSTF TRAJ7 8 4 3 7 3 9 4 4 3 4 6 4 4 9 6 8 TRAV3/DV6 CAASGGTGGFKTIF TRAJ9 4 9 4 8 9 6 9 3 8 3 3 TRAV38 CAFMSPYGGATNKLIF TRAJ3 7 6 8 7 6 8 6 7 9 TRAV4/DV4 CAMRPMDTGRRALTF TRAJ 3 3 4 6 7 3 6 4 3 4 3 TRAV4/DV4 CAMRGVDTGRRALTF TRAJ 3 4 3 8 3 8 4 3 4 3 3 TRAV4/DV4 CAMIPMDTGRRALTF TRAJ 4 3 7 3 4 3 3 TRAV CAVKFNKFYF TRAJ 9 3 9 6 4 TRAV6 CALVRSTDKLIF TRAJ34 9 9 3 3 4 Beta chain Vgene Junc)on Jgene Total 3 4 6 7 8 9 3 4 6 7 8 9 TRBV9 CASSIVGRAVDEQFF TRBJ 9 9 3 8 6 4 3 4 9 8 8.7 4 4 TRBV CASSERAGGSTDTQYF TRBJ 3 8 8.8 8 7 6 7.8 7 4 7 3 7 4 7 7 TRBV7 9 CASSSIWGEETQYF TRBJ 4 7. 3 8. 6 7 6 6 4 9. 9 TRBV6 /6 3 CASEGAGGNEQFF TRBJ 4.7.4 7.8 6.4.. 6.8 3.9. 9.7 4. 3.4 4 4.4 7.4. 3.7.3 8. TRBV6 /6 3 CASSDSSGGAGTDTQYF TRBJ 3 4. 4. 6.8 4. 3..7 8. 4.8 3.7 3.3.9.9 3.9.9.7.9 8.8 6.8 TRBV8 CASSGTGGGFSYTF TRBJ 3. 4.9.3.8 3..9.9.4. 3..8 4.6..9.8.8.8 6.8 6. 6.6 3.7 TRBV4 CATSDSSGGRETQYF TRBJ 3.4 6.9 7.... 3. 9.9.3. 3.8.9.3.8 3.3.8 TRBV8 CASNSDSTGSWGQPQHF TRBJ 3 3.7 3.6 7.9.3 3.3.9 6.6 8.4 3.3.8 3.4.7.3.. 4.3 TRBV CASSETGTGKPDTQYF TRBJ 3.. 3.4 7.3..8 4..6.4 3. 3.. 6..8.8 TRBV 3 CAISESISPEQFF TRBJ.9 4..7 3.6 4.3 3.9..3.4 3.7.8.4.3.9 4.4 TRBV9 CASSIHGTRNTEAFF TRBJ.9.3 4. 3.3.4 6.7.8.6 3.4.3 4.7 3..9.7..4.3.. TRBV CASSPKSTSGGDNEQFF TRBJ.4 3..8..4.3..3 3.8.9.9..8. 3.6.3.9