Supporting Information

Moon et al. 10.1073/pnas.1109806108 SI Materials and Methods For gp66:I-Ab, LLO-C:I-Ab, OVA-2C:I-Ab, and OVA-3C:I-Ab b Act-2W Transgenic Mice. The design of the Act-2W transgenic tetramers, the peptide sequence for 2W in the I-A β chain ex- mouse was based on the previously generated Act-mOVA pression plasmid was replaced with sequences encoding Lym- transgenic mouse (1). This mouse expressed a chimeric protein phocytic choriomeningitis virus gp protein amino acids 66–77 consisting of the signal peptide from H-2 Kb, the full sequence of (DIYKGVYQFKSV) (7), Listeria monocytogenes listeriolysin O chicken ovalbumin (OVA), and the transmembrane domain of amino acids 190–201 (NEKYAQAYPNVS) (8), and chicken H-2 Db, which results in extracellular membrane expression. ovalbumin OVA-2 and OVA-3, which correspond to amino acids Expression was controlled by the CAGGS vector, which includes 325–335 (QAVHAAHAEIN) and 327–338 (GHAAHAEINEA), the chicken β-actin promoter, the CMV enhancer, and the rabbit respectively (9). For LLO-C:I-Ab, OVA-2C:I-Ab, and OVA-3C: polyadenylation signal (2). To create the Act-2W transgene, the I-Ab tetramers, a disulfide trap strategy was used to prevent coding sequence for 2W peptide (EAWGALANWAVDSA) was peptide movement within the MHCII groove and specifically inserted between the sequences for OVA and the Db trans- lock in the desired peptide binding register (10, 11). In this membrane domain via site-directed mutagenesis (Stratagene) strategy, a cysteine residue was introduced into the linker con- (Fig. S1). The resulting construct was linearized and injected into necting the peptide to the I-Ab beta chain, which then forms B6 oocytes by the University of Minnesota Mouse Genetics a disulfide bond with an introduced cysteine residue at position fi b Laboratory. Founder mice were identi ed by PCR screening of 72 of the I-A alpha chain. For the OVA-3C epitope, residue 327 genomic DNA, and cell surface expression of membrane-bound was changed from V to G to further ensure that the OVA-2C fi fl OVA-2W on blood cells was veri ed by ow cytometry with an epitope would not be presented. antibody to OVA (Abcam). One founder was chosen and bred to Drug-selected stable transfectants were scaled up into 1-L B6 mice to generate a colony of Act-2W transgenic mice that serum-free cultures of Express Five media (Life Technologies) in were subsequently maintained as heterozygotes with respect to either 3-L spinner flasks or 2-L shaker flasks maintained at 125 the transgene. rpm. When densities exceeded 107/mL, expression was induced with the addition of 800 μM CuSO and 2 μg/mL D-biotin Tetramer Construction. Peptide:MHC class II tetramers were 4 (Sigma). After 10–12 d, recombinant biotinylated pMHC was constructed as previously described (3) with some modifications. purified from 0.2-μm filtered supernatants by gravity flow column Drosophila S2 cells were cotransfected via calcium phosphate fi with three separate pRMHa-3 metallothionein promoter-driven chromatography through 4 mL of His Bind nickel af nity resin (EMD Biosciences). Bound material was eluted with 1 M im- expression plasmids and a blasticidin drug-resistance plasmid w fi according to the Drosophila Expression System kit (Life Tech- idazole and 75 kDa protein was further puri ed by size ex- nologies). Two of the pRMHa-3 plasmids encoded extracellular clusion chromatography with Sephacryl S-300 (GE Healthcare domains of the I-Ab alpha and beta chains fused to Fos-Jun Bio-Sciences). acidic and basic leucine zipper motifs, respectively, to improve The molar concentration of biotinylated pMHCII was de- fi stability of the soluble heterodimer (4). The beta chain construct termined by mixing a xed amount with graded amounts of contained the 2W peptide sequence covalently fused to the N streptavidin-phycoerythrin (SA-PE) or streptavidin-allophyco- terminus via a flexible glycine-serine linker, and a 6× His tag at cyanin (SA-APC) (Prozyme), followed by Western blot analysis to the C terminus to enable purification via nickel affinity chro- detect excess biotinylated pMHCII. Tetramers were created by matography. The alpha chain construct contained a C-terminal mixing biotinylated pMHCII with SA-PE or SA-APC at a molar BirA signal sequence to enable site-specific biotinylation by Es- ratio in excess of 4:1 for 30 min at room temperature. Assembled cherichia coli BirA biotin ligase (5), which was encoded by the tetramers were separated from free pMHC by S-300 size exclu- third pRMHa-3 plasmid. Coexpression of these three constructs sion chromatography, concentrated with 100-kDa molecular results in efficient in vivo biotinylation of assembled pMHC weight cutoff spin filters (Millipore), and quantified by absor- heterodimers (6). bance at 566 nm for PE or 650 nm for APC.

1. Ehst BD, Ingulli E, Jenkins MK (2003) Development of a novel transgenic mouse for 7. Oxenius A, et al. (1995) Presentation of endogenous viral proteins in association with the study of interactions between CD4 and CD8 T cells during graft rejection. Am J major histocompatibility complex class II: On the role of intracellular compart- Transplant 3:1355e1362. mentalization, invariant chain and the TAP transporter system. Eur J Immunol 25: 2. Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression 3402e3411. transfectants with a novel eukaryotic vector. Gene 108:193e199. 8. Geginat G, Schenk S, Skoberne M, Goebel W, Hof H (2001) A novel approach of direct 3. Moon JJ, et al. (2007) Naive CD4(+) T cell frequency varies for different epitopes and ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell predicts repertoire diversity and response magnitude. Immunity 27:203e213. epitopes from Listeria monocytogenes. J Immunol 166:1877e1884. 4. Scott CA, Garcia KC, Carbone FR, Wilson IA, Teyton L (1996) Role of chain pairing for 9. Robertson JM, Jensen PE, Evavold BD (2000) DO11.10 and OT-II T cells recognize the production of functional soluble IA major histocompatibility complex class II a C-terminal ovalbumin 323-339 epitope. J Immunol 164:4706e4712. molecules. J Exp Med 183:2087e2095. 10. Stadinski BD, et al. (2010) Diabetogenic T cells recognize insulin bound to IAg7 in an 5. Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin unexpected, weakly binding register. Proc Natl Acad Sci USA 107:10978e10983. holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8:921e929. 11. Chu HH, Moon JJ, Kruse AC, Pepper M, Jenkins MK (2010) Negative selection and 6. Yang J, Jaramillo A, Shi R, Kwok WW, Mohanakumar T (2004) In vivo biotinylation of peptide chemistry determine the size of naive foreign peptide-MHC class II-specific the major histocompatibility complex (MHC) class II/peptide complex by coexpression CD4+ T cell populations. J Immunol 185:4705e4713. of BirA enzyme for the generation of MHC class II/tetramers. Hum Immunol 65: 692e699.

Moon et al. www.pnas.org/cgi/content/short/1109806108 1of2 2W CMV IE β enhancer rabbit -globin polyA

chicken β-actin OVA Db TM promoter Kb leader

Fig. S1. Schematic depiction of Act-2W transgenic construct. The sequence for 2W peptide was embedded within a membrane-bound form of chicken ov- albumin. CMV IE, cytomegalovirus intermediate early; OVA, ovalbumin; TM, transmembrane.

250K A 105 105

200K 4 10 104

150K

3 Lymph Nodes + Spleen 10 103

100K CD8

102 50K 0 0 CD11c + F4/80 CD11c B220 + CD11b + B220 + CD11b

Side Scatter Area Side Scatter 0

0 50K 100K 150K 200K 250K 0102 103 104 105 0103 104 105 Side Scatter Width CD3 CD4

250K B 105 105

200K 104 104

150K

3 Thymus 10 103

100K CD8

102 50K 0

CD11c + F4/80 CD11c 0 B220 + CD11b + B220 + CD11b

Side Scatter Area Side Scatter 0

0 50K 100K 150K 200K 250K 0102 103 104 105 0103 104 105 Side Scatter Width CD3 CD4

Fig. S2. Flow cytometry gating strategy for tetramer-enriched cells. Tetramer-enriched CD4+ and CD8+ T cells from lymph node and spleen (A) or thymus (B) samples were analyzed by flow cytometry using a series of inclusion gates. A side scatter arealow side scatter widthlow gate was set to capture nonaggregated lymphoid cell events. T cells were then gated from these events via positive expression of CD3 and negative expression of a dump panel of non-T-cell lineage markers: B220 for B cells, CD11b for myeloid and NK cells, CD11c for dendritic cells, and F4/80 for macrophages. This gating also allowed for the exclusion of autofluorescent cells, which populate the diagonal region of the plot. T-cell events were then further gated into CD4+ and CD8+ T-cell subsets, which also allowed for a second round of autofluorescent cell exclusion.

Moon et al. www.pnas.org/cgi/content/short/1109806108 2of2