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jurkat t3 5  (ATCC)


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    ATCC jurkat t3 5
    Jurkat T3 5, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/j+rt3+t3+5/J%2ERT3-T3%2E5/us12643949-1981-5-7
    Average 96 stars, based on 176 article reviews
    jurkat t3 5 - by Bioz Stars, 2026-10
    96/100 stars

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    Related Articles

    Luciferase:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Article Title: NY-ESO-1 T cell receptors and methods of use thereof
    Article Snippet: .. Jurkat clone J.RT3.T3.5 (ATCC #TIB-153) which lacks its own endogenous TCR was transduced by lentiviruses encoding the human CD8A/B genes, Cignal Lenti AP-1 Luciferase Reporter (Qiagen—SAbiosciences, #CLS-011 L) and NY-ESO-1 specific TCRs derived either from immunization of VelociTTM mice (WO 2016/164492) or from published comparator NY-ESO-1 TCRs (1G4 and clone 113: Li, Y. et al. Nat Biotechnol 2005; 23:349-354). ..

    Cloning:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Polymerase Chain Reaction:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Plasmid Preparation:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Reverse Transcription:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Staining:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Ligation:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Single Cell:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Transcriptomics:

    Article Title: The carbonyl nucleobase adduct M 3 Ade is a potent antigen for adaptive polyclonal MR1-restricted T cells.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Lymphoprep Stemcell Technologies Cat#07851 Recombinant Human IL-2 Peprotech Cat#200-02 Phyto-haemagglutinin (PHA) Remel Cat#R30852801 5-A-RU Toronto Research 900 Chemicals Cat#A629245 Methylglyoxal Sigma-Aldrich Cat#M0252 CellTrace CFSE Thermo Fisher Scientific Cat#C34554 CellTrace Violet Thermo Fisher Scientific Cat#C34557 CellTrace Blue Thermo Fisher Scientific Cat#C34568 CellTrace Far Red Thermo Fisher Scientific Cat#C34572 DAPI Sigma-Aldrich Cat#D9542-5MG Zombie NIR Biolegend Cat#423106 LIVE/DEAD Fixable Blue Thermo Fisher Scientific Cat#L23105 1,1,3,3-Tetraethoxypropane Sigma-Aldrich Cat#T9889 Adenine Sigma-Aldrich Cat#A8626 Brefeldin A Sigma-Aldrich Cat#B6542 Monensin Sigma-Aldrich Cat#M5273 Dasatinib Axon Medchem Cat#1392 Streptactin Magnetic Microbeads IBA Cat#6-5510-050 Metafectene Pro Biontex Cat#T040 Streptavidin-PE Agilent Cat#PJRS25 Streptavidin-APC Agilent Cat#PJ25S-1 Streptavidin-BV421 Biolegend Cat# 405226 Bovine serum Albumin Sigma-Aldrich Cat#A2153 Sodium Azide Sigma-Aldrich Cat#08591 Recombinant human GM-CSF Peprotech Cat#300-03 Accutase Innovative Cell Cat#AT-104 Collagenase IV Worthington Cat#LS004189 DNAse I Sigma-Aldrich Cat#D5025 Hyaluronidase Sigma Aldrich Cat#H6254 Alpha-Galactosylceramide Avanti Polar Lipids Cat#KRN7000 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) Echelon Biosciences Cat#I-M055 Sodium Chloride (NaCl) Sigma-Aldrich Cat#S9888 TRIS Buffer Sigma-Aldrich Cat#PPB023 Glycerol Sigma-Aldrich Cat#G5516 Ammonium Formate Sigma-Aldrich Cat#516961 Acetonitrile Sigma-Aldrich Cat#34851 MR1wt-M3Ade-tetramer De Libero Lab N/A MR1 CD8-null-M3Ade-tetramer De Libero Lab N/A MR1-6-FP tetramer NIH Tetramer Core Facility 33024 MR1-5-OP-RU tetramer NIH Tetramer Core Facility 33021 MR1- M3Ade monomer (twin strep-tag) De Libero Lab N/A Annexin V-PE Biolegend Cat#640908 Critical commercial assays EasySep Biotin Positive Selection Kit Stemcell Technologies Cat#17683 StemSep Human CD14 Positive Selection Kit Stemcell Technologies Cat#14758 Easysep Human T cell enrichment Kit Stemcell Cat#19051 Easysep Human CD19 Positive selection Kit II Stemcell Cat#17854 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Bio-Glo Luciferase Assay System Promega Cat#G7940 NucleoSpin RNA Macherey-Nagel Cat#740955.250 ReliaPrep RNA Cell Miniprep System (10 Preps) Promega Cat#Z6010 SMARTerRACE 5’/3’ Kit Takara Cat#634858 In-Fusion HD Cloning Kit Takara Cat#639649 NucleoSpin Gel and PCR Clean-up Kit Macherey-Nagel Cat#740609.250 NucleoSpin Plasmid Kit Macherey-Nagel Cat# 740588.250 NucleoBond Xtra Midi EF Kit Macherey-Nagel Cat# 740420.50 Bam HI-HF Restriction Enzyme NEB Cat#R3136S XbaI Restriction Enzyme NEB Cat#R0145S CloneAmp HiFi PCR Premix Takara Cat# 639298 SuperScriptIII Reverse Transcriptase Invitrogen Cat#18080-093 BirA biotin protein ligase kit Avidity BirA500 Intracellular Staining Permeabilization Wash Buffer (10x) Biolegend Cat#421002 Fixation Buffer Biolegend Cat#420801 Quick Ligation Kit New England Biolabs Cat#M2200S Deposited data MR1-M3Ade structure This paper RCSB: 8BP6 Single-cell transcriptomics raw data This paper European Genome-Phenome Archive: ega-archive.org/studies/EGAS50000000479, ega-archive.org/datasets/EGAD50000000690 Experimental models: Cell lines Human: A375 ATCC CRL-1619 Human: A375b-MR1 De Libero Lab Di Blasi et al.46 Human: THP-1 ATCC TIB-202 Human: THP-1-MR1 De Libero lab3 N/A Human: J.RT3-T3.5 (TCRb-deficient) ATCC TIB-153 Human: J.RT3-T3.5 (TCRab- and b2M-deficient) De Libero Lab Chancellor et al.23 Human: HEK 293T DSMZ ACC 635 Human: HEL (HEL 92.1.7) ATCC TIB-180 Human: CMY Gift of J. Schwaller, University of Basel CVCL_A602 Human: TF-1 ATCC CRL-2003 Human: KMOE-2 DSMZ CVCL_1332; ACC 37 Human: MEL-JUSO DSMZ CVCL_1403; ACC 74 Hamster: CHO-K1 ATCC CCL-61 Oligonucleotides Custom synthetic gene blocks Integrated DNA Technologies (IDT) N/A GSP SMARTpRACE_TRAC; 5’GATTACGCCAAGC TTTCAGCTGGACCACAGCCGCAGCGTCATG-3’ De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC1; 5’GATTACGC CAAGCTTTCAGAAATCCTTTCTCTTGACCATGGCC De Libero Lab Chancellor et al.23 GSP Primer: SMARTpRACE_TRBC2; 5’GATTACGC CAAGCTTCTAGCCTCTGGAATCCTTTCTCTTGACC De Libero Lab Chancellor et al.23 TRAV1-2_pLenti_Fw; 5’CAGGACCGGTTCTAGAA TGTGGGGAGTTTTCCTTCTTTATG De Libero Lab Chancellor et al.23 TRAC_pLenti_Stop_Rw; 5’-TTGTTGCGCCGGAT CCTCAGCTGGACCACAGCCG De Libero Lab Chancellor et al.23 (Continued on next page) e3 Immunity 58, 1–17.e1–e10, February 11, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER TRBV6-5_pLenti_Fw; CAGGACCGGTTC TAGAATGAGCATCGGCCTCCTGTG De Libero Lab Chancellor et al.23 TRBC1_pLenti_Stop_Rw; 5’TTGTTGCGCCGGAT CCTCAGAAATCCTTTCTCTTGACCATGG De Libero Lab Chancellor et al.23 TRBC2_pLenti_Stop_Rw; 50TTGTTGCGCCGGATC CCTAGCCTCTGGAATCCTTTCTCTTGA De Libero Lab Chancellor et al.23 pLenti_Fw; 5’GTGCAGTAGTCGCCGTGAACG De Libero Lab Chancellor et al.23 pLenti_Rw; 5’CAAAGGCTTGGCCATCGGTCC De Libero Lab Chancellor et al.23 TRAV29_pLenti_for CAGGACCGGT TCTAGAATGGCCATGCTCCTGGG This paper N/A TRBV28_pLenti_for 5’CAGGACCGGTTCTA GAATGGGAATCAGGCTCCTGTGTC This paper N/A TRBV12-3/4_pLenti_for 5’CAGGACCG GTTCTAGAATGGGCTCCTGGACC-3’ This paper N/A TRAV9-2_pLenti_for CAGGACCGGTTC TAGAATGAACTATTCTCCAGGCTTAG This paper N/A TRAV19_pLenti_for 5’CAGGACCGGTTC TAGAATGCTGACTGCCAGCCTG This paper N/A TRAV14_pLenti_for 5’CAGGACCGG TTCTAGAATGTCACTTTCTAGCCTGCTG This paper N/A TRAV17_pLenti_for 5’CAGGACCGGTTCT AGAATGGCTCAGAAGGTAACTCAAGC This paper N/A TRBV19_pLenti_for 5’CAGGACCGGTTC TAGAATGAGCAACCAGGTGCTCTG This paper N/A TRAV8_pLenti_for 5’CAGGACCGGTTCT AGAATGCTCCTGCTGCTCGTCCC This paper N/A TRBV5-3/6/7_pLenti_for 5’CAGGACCGGTTC TAGAATGGGCCCCGGGCTCCTCTGCTGG This paper N/A Recombinant DNA lentiCas9-Blast Addgene Cat#52962 pET23d Novagen Cat#69748-3 scMR1 BirA in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 scMR1 twin-streptag in pET23d De Libero Lab Human b2M Uniprot ID: P61769 Human MR1 Uniprot ID: Q95460 pMD2.G Addgene RRID:Addgene_12259 pMDLg/pRRE Addgene RRID:Addgene_12251 pRSV-REV Addgene RRID:Addgene_12253 pLenti_G7C2_TRAV17 This paper N/A pLenti_G7C2_TRBV19 This paper N/A pLenti_AC1A4_TRAV8 This paper N/A pLenti_AC1A4_TRBV5 This paper N/A pLenti_AC1B76_TRAV29 This paper N/A pLenti_AC1B76_TRBV28 This paper N/A pLenti_DGB129_TRAV29 This paper N/A pLenti_DGB129_TRBV12 This paper N/A pLenti_QY1A16_TRAV9 This paper N/A pLenti_QY1A16_TRBV6 This paper N/A pLenti_AVA34_TRAV19 This paper N/A pLenti_AVA34_TRBV12 This paper N/A pLenti_AVA46_TRAV14 This paper N/A pLenti_AVA46_TRBV6 This paper N/A pLenti_MRC25_TRAV1 De Libero Lab Chancellor et al.23 (Continued on next page) Immunity 58, 1–17.e1–e10, February 11, 2025 e4

    Derivative Assay:

    Article Title: NY-ESO-1 T cell receptors and methods of use thereof
    Article Snippet: .. Jurkat clone J.RT3.T3.5 (ATCC #TIB-153) which lacks its own endogenous TCR was transduced by lentiviruses encoding the human CD8A/B genes, Cignal Lenti AP-1 Luciferase Reporter (Qiagen—SAbiosciences, #CLS-011 L) and NY-ESO-1 specific TCRs derived either from immunization of VelociTTM mice (WO 2016/164492) or from published comparator NY-ESO-1 TCRs (1G4 and clone 113: Li, Y. et al. Nat Biotechnol 2005; 23:349-354). ..

    Selection:

    Article Title: Compositions and methods related to aptamer-based reversible cell selection
    Article Snippet: .. J.RT3-T3.5 and Jurkat (Clone E6-1) cell lines used for counter selection and nucleofection, respectively, were purchased from ATCC. .. The Epstein-Barr virus transformed lymphoblastoid cell line (TM-LCL) used in the rapid expansion protocol (REP) of T cells was made from mononuclear cells as previously described.43 CD19+ and OKT3+ K562 cells used for functional assays were generated by lentivirally transducing parental K562 parental cells (ATCC) with CD19- or OKT3-expressing constructs.

    Transduction:

    Article Title: Compositions and methods for rapid cloning of t-cell receptors
    Article Snippet: .. Transduction of Jurkat (E6-1; ATCC) or J.RT3-T3.5 (ATCC) was performed similarly but without activating reagents and cytokines and using reduced volumes of retroviral supernatant. ..

    Retroviral:

    Article Title: Compositions and methods for rapid cloning of t-cell receptors
    Article Snippet: .. Transduction of Jurkat (E6-1; ATCC) or J.RT3-T3.5 (ATCC) was performed similarly but without activating reagents and cytokines and using reduced volumes of retroviral supernatant. ..



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