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Figure S1. A. Design of UV-sensitive conditional ligands. A single amino acid within peptides capable of binding the respective HLA molecules was replaced with 3-amino-3-(2-nitro) phenyl-propanoic acid (Anp) at the position indicated by the filled black circle (l). Amino acid position 1 at the N-terminus to position 10 at the C-terminus are indicated by p1 to p10, respectively. These ligands were synthesized by solid phase peptide synthesis and used for refolding reactions. The yield represented by the weight of homogenous HLA monomer per litre of refolding reaction, was listed. When multiple conditional ligands were designed, such as in HLA-A*02:07, -A*11:01, -A*33:03 -B*15:02, -B*40:01, -B*46:01, ligands with the highest yield were selected for further studies (shaded grey). B. Mass spectrometry (MS) analysis with isotopic distribution of conditional ligands by LCMSIT-TOF. The mass to charge ratios (m/z) of peptides were measured after liquid chromatography (LC) separation on a C18 column. As both isomers of the photocleavable 3-amino-3-(2-nitro) phenyl-propanoic acid (Anp) used during solid phase peptide synthesis of the conditional ligand, 2 diasteroisomers could usually be identified. In some cases, however, the two isomers failed to separate during chromatography. The data displayed is for one of the two isomers, mostly for doubly charged molecules (M2+), together with the amino acid sequences of conditional ligands where the letter J represents Anp.

Figure S2. A. Cells were first analyzed for their size and granularity based on their forward scatter area (FSC-A) and side scatter area (SSC-A) profile. Doublets were excluded using side-scatter height (SSC-H) vs side-scatter width (SSC-W) and forward-scatter height (FSC-H) vs forwardscatter width (FSC-W) parameters. Cells present in all three gates were analyzed for their time vs forward scatter area (FSC-A) to exclude cells that were read at the beginning and end of the flow cytometry run. Viable cells were gated based on their negative staining with the Live/Dead® dye (Invitrogen), and these cells were finally analyzed for anti-human CD8 antibodies and HLA-tetramer stainings. All data were analyzed using FlowJo (Tree Star). The example shown is PBMCs stained with HLA-A*2402 tetramers EBV-LMP2419–427 (TYGPVFMSL). B. Direct ex-vivo HLA tetramer staining of PBMCs from various HLA-matched volunteers. PBMCs were isolated and stained with HLA tetramers, followed by flow cytometry analysis. Peptides originating from HCMV-pp65495–503 (NLVPMVATV), HBV-Core18–27 (FLPSDFFPSV), EBV-EBNA4-NP399–408 (AVFDRKSDAK), Influenza A-MP13–21 (SIIPSGPLK), EBV-BRLF1134–142 (ATIGTAMYK), EBV-LMP2419–427 (TYGPVFMSL), HCMV-pp65 341–349 (QYDPVAALF), HCMVpp6591–100 (SVNVHNPTGR), EBV-EBNA3B831–839 (GQGGSPTAM), HCMV-pp65267–275 (HERNGFTVL), HCMV-IE-142–50 (KEVNSQLSL), HCMV-pp65232–240 (CEDVPSGKL), DENVpp2822- 2830 (KPWDIIPMV), Influenza A-NP199–207 (RGINDRNFW), EBV-LMP1156–164 (IALYLQQNW), EBV-EBNA3B279–287 (VSFIEFVGW) were used in the construction of tetramers for HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, -A*24:02, -A*33:03, -B*15:02, -B*40:01, -B*55:02 or –B*58:01 as indicated. For the generation of HLA-A*33:03 tetramers, conditional ligands with the photocleavable moiety at position 8 instead of position 4 was used. C. Culturing of PBMCs to develop antigen-specific T cell lines. PBMCs isolated from healthy volunteers’ blood were stimulated by antigens from EBV-LMP2419–427 (TYGPVFMSL) or HCMVIE-142–50 (KEVNSQLSL) and cultured in 25 U/mL IL-2, 5% AB serum in RPMI for 14 days. HLA tetramer staining for antigen-specific T cells was performed on day 0, 7, 10 and 14 days post stimulation.

Figure S3. A. Design of 9-, 10-, 11-mer peptides derived from the 15-mer antigen. Overlapping 9-, 10-, 11-mer peptides were each tested for their capability to stabilize the corresponding HLA. A total of 18 different peptides were derived from each 15-mer peptide. B. HLA-stability ELISA results for soluble HLA-B*40:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from SARS NP44 15-mer peptide. Non-irradiated HLA with conditional ligand and no peptide added (-UV), irradiated HLA and no peptide added (+UV), or with indicated 9-, 10-, 11-mers peptides. Peptides are labeled as capable (red bars) or incapable (white bars) of stabilizing the corresponding HLA. C. Similar to Part B, HLA-stability ELISA results for soluble HLA-B*40:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from SARS NP65 15-mer peptide. D. Similar to Part B, HLA-stability ELISA results for soluble HLA-B*55:02 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from DENV NS3–108 15-mer peptide. E. Similar to Part B, HLA-stability ELISA results for soluble HLA-B*58:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from SARS 3a2 15-mer peptide. F. Similar to Part B, HLA-stability ELISA results for soluble HLA-B*58:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from HBV Pol33 15-mer peptide. G. Similar to Part B, HLA-stability ELISA results for soluble HLA-B*58:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from HBV Env72 15-mer peptide. H.Similar to Part B, HLA-stability ELISA results for soluble HLA-C*08:01 molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from HBV Env34 15-mer peptide. I. HLA-stability ELISA results for soluble HLA molecules that were ligand-exchanged with 9-, 10-, 11-mers truncated peptides from the indicated15-mer peptide. Non-irradiated HLA with conditional ligand and no peptide added (-UV), irradiated HLA and no peptide added (+UV), or with indicated 9-, 10-, 11-mers peptides.

Table S1. Origin of the parent peptides on which the conditional ligands were based

Table S2. List of known CTL epitopes that are used in this study

Table S3. HLA typing of PBMCs donors.

Table S3. Gene and protein sequences of the pertinent heavy chains.

Table S4.

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