Peptides (SIYR, SIYRYYGL; QL9, QLSPFPFDL) were synthesized bystandard F-moc chemistry at the Macromolecular Core Facilityat Pennsylvania State University. Position 5 variants of theQL9 peptide [Y5 (QLSPYPFDL), R5, H5, and M5] were synthesizedat the Protein Sciences Facility at the University of Illinois.QL9 and SIYR were purified by C-18 reverse-phase HPLC. The Hbpeptide (GKKVITAFNEGLK) was synthesized by standard F-moc chemistryand purified by C-18 reverse-phase HPLC at Washington University.
The endogenous peptide transport-deficient human T-lymphoblastoidcell lines T2-Kb and T2-Ld, which have been transfected withthe Kb or Ld heavy chain, respectively, were provided by P.Cresswell (Alexander et al., 1989). The I-Ak, I-Ek –expressingB cell hybridoma, CH27, was used for studies with the classII 3.L2 TCR (Evavold et al., 1992). Cells were propagated inRPMI 1640 media /10% fetal bovine serum, supplemented with L-glutamineand 2-mercaptoethanol at 37°C and 5% CO2. G418 (0.5 mg/ml)was added to T2-Kb and T2-Ld cultures.
scTCR yeast display constructs and libraries
Several mutants of the 2C TCR used here were isolated previously.The 2C-T7 scTCR was engineered for increased yeast surface expression(Kieke et al., 1999) and it has been shown to have an affinity(KD value) for QL9/Ld of 3 µM (unpublished data). The2C-m80 TCR mutant was engineered using yeast display and FACSand the full length TCR has been reported previously to havean affinity for SIYR/Kb of 790 nM (Holler et al., 2003). Morerecent surface plasmon resonance (SPR) measurements of the E.coli expressed, single-chain form of the 2C-m80 TCR have shownit to have an affinity for SIYR/Kb of 150 nM (unpublished data).As the single-chain form was expressed on yeast in the presentstudies, the KD value of 150 nM will be referred to here asthe affinity of the 2C-m80 scTCR. The 2C-m6 TCR was engineeredusing yeast display and FACS and it has been shown to have anaffinity for QL9/Ld of 6 nM (Holler et al., 2000; Holler etal., 2003). The 3.L2 mouse TCR recognizes a peptide from anallelic variant of hemoglobin, presented by the I-Ek Class IIMHC molecule (Evavold et al., 1992). The mutant 3.L2-M15 wasengineered using yeast display to have high affinity for itsligand, hemoglobin peptide Hb/I-Ek (KD = 25 nM) (Weber et al.,2005). Two scTCRs were used as controls that do not bind tothe cognate ligands recognized by the 2C or 3.L2 TCRs. Theseincluded (i) C18-1, a surface-stabilized scTCR mutant (unpublisheddata) of the murine C18 TCR that recognizes a peptide derivedfrom a mutated MAP kinase presented by Kd MHC (Ikeda et al.,1997) and (ii) mWT1-B7, a mouse scTCR raised against Wilms tumorantigen-1 and engineered previously for increased yeast surfaceexpression (unpublished data). The 2C CDR3 library has beendescribed previously (Holler et al., 2000). This yeast librarywas cultured in selective SD-CAA liquid media [2% dextrose (w/v),0.67 % yeast nitrogen base (w/v), and 1% casamino acids (w/v)]with kanamycin (50 µg/ml) at 30°C.
Induction of scTCR expression on the yeast cell surface
scTCR expression on the surface of the Saccharomyces cerevisiaestrain EBY100 was induced as described previously (Boder andWittrup, 1997). Briefly, the yeast display plasmid pCT302, whichencodes a gene fusion linking the scTCR to the yeast cell surfacegene AGA2, controlled by a galactose-driven promoter, was introducedinto EBY100 yeast cells. Expression of the AGA2:scTCR fusiongene was induced by transferring cells growing in SD-CAA togalactose-containing media and shaking at 20°C for at least24 h.
Flow cytometric analysis of scTCR expression and pMHC binding
Yeast that express 2C scTCR mutants (Vß8.2-positive)were detected with the anti-Vß8.2 antibody F23.2.Yeast that express 3.L2 or C18 scTCR mutants (Vß8.3-positive)were detected with the anti-Vß8.3 antibodies KT-8C1(Cedarlane Laboratories) or 1B3.3-PE (BD Pharmingen). Yeastthat express mWT1-B7 scTCR (Vß11-positive) were detectedwith a PE-conjugated anti-Vß11 antibody, RR3-15-PE(BD Pharmingen). F23.2 or KT-8C1 binding was detected by incubatingwith biotinylated goat-anti-mouse IgG (Rockland Inc.), followedby streptavidin:PE (SA:PE) (BD Pharmingen) or by incubationwith PE-conjugated goat F(ab')2 anti-mouse Ig (Southern Biotech).All antibodies and secondary reagents were diluted in phosphate-bufferedsaline containing 0.5% bovine serum albumin (PBS/BSA). Sampleswere analyzed on a Coulter Epics-XL flow cytometer, gating onyeast cells based on light scattering properties. Flow cytometrywas also used to detect binding of yeast-displayed scTCR tosoluble Ld-Ig fusion protein folded with specific peptide (eitherQL9 or Y5). The QL9/Ld-Ig and Y5/Ld-Ig were produced and purifiedas described previously (Chlewicki et al., 2005). To detectpMHC binding by yeast-displayed scTCR, 400 µg/ml QL9/Ld-Igor 200 µg/ml Y5/Ld-Ig was incubated with yeast cells onice for 1 h. Cells were washed with PBS/BSA and resuspendedin PE-labeled goat F(ab'2) anti-mouse Ig.
Peptide loading of cells
APCs (T2-Kb, T2-Ld, or CH27) were pelleted by centrifugationand resuspended in fresh media to a concentration of 106 cells/ml.1 ml aliquots were then dispensed into 1.5 ml microfuge tubes.Peptides were incubated with cells at the following final concentrations:1 µM SIYR, 30 µM QL9, and 10 µM Hb. Singleamino acid variants of QL9 at position 5 were incubated at concentrationspreviously determined to be in excess for maximum stabilizationof Ld on the surface of T2-Ld cells (Schlueter et al., 1996).Peptide/cell mixtures were rocked at room temperature for 60–90min to allow loading of the peptides.
Density differential centrifugation
Induced yeast cells were counted on a hemacytometer and aliquotedinto tubes that contained the peptide-loaded APCs (106 yeastcells per tube). Another aliquot of induced yeast served as‘pre-centrifugation’ control. The yeast/APC mixtureswere allowed to rock at room temperature for 1 h. Followingthe incubation, the yeast/APC mixtures were layered onto threemls of Ficoll-Paque PLUS (Pharmacia) in 15 ml conical tubes.The cells were allowed to settle for 10 min at room temperature,and the tubes were then centrifuged at 1500 r.p.m. (400x g)at 4°C for 30 min. After centrifugation, the visible layerof cells above the Ficoll-Paque (the ‘interface’)was removed from each tube in a 1 ml volume. In some cases,this interface was returned to the rocker for another 30-minincubation, and the procedure was repeated (centrifugation for15 min in this ‘second centrifugation’).
Plasmid rescue and sequencing
Surface display plasmids were rescued from selected yeast clones
using a Zymoprep I Yeast Plasmid Minipreparation Kit (Zymo Research).
Rescued plasmids were introduced into the E. coli
(Invitrogen) by electroporation. Plasmids were purified fromE. coli
using a QIAprep Spin Mini-Prep Kit (Qiagen) and sequenced
at the DNA Core Sequencing Facility at the University of Illinois.