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Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells

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Science Immunology  19 Jul 2019:
Vol. 4, Issue 37, eaau9039
DOI: 10.1126/sciimmunol.aau9039
  • Fig. 1 DS-A2 molecules are peptide receptive and form stable complexes with associated peptides.

    (A) Calculated free energy profiles versus width of the peptide-binding cleft extracted from MD simulations for wt-A2 (gray) and DS-A2 (blue) in the presence (solid line) and in the absence (dotted line) of bound peptide. (B) Process of generating empty MHC-I molecules. (C) Binding of 100 nM NLVPKFITCVATV peptide, measured by fluorescence anisotropy, to purified empty DS-A2 molecules alone (blue) or in the presence of 10 mM dipeptide GM (black). Brown dots represent anisotropy values of nonspecific binding. Data are plotted in duplicates, with the solid line indicating association rate kinetics. Association rates (Kon) = 5.9 ± 0.11 × 105 (DS-A2) and 1.6 ± 0.02 × 105 (DS-A2 plus GM). (D) Thermal stability of purified DS-A2 without peptide (blue dotted line), with 10 mM dipeptide GM (black line), and with 10 μM NLVPMVATV (blue line). The minimum of each curve represents the Tm value (DS-A2, 40°C; DS-A2 + 10 mM GM, 42°C; DS-A2 + 10 μM NLVPMVATV, 60°C). Each sample was analyzed in duplicate or triplicate; representative curves are shown. (E) Crystal structure overlay of DS-A2 (blue) and wt-A2 (gray) associated with NLVPMVATV peptide. The C84-C139 disulfide bond is highlighted in yellow. (F) Overlay of NLVPMVATV peptide in the binding groove of DS-A2 (blue) and wt-A2 (gray). (G) Binding of the C terminus of the NLVPMVATV peptide in the F pocket of the peptide-binding groove of DS-A2 (blue) and wt-A2 (gray). A 2mFoDFc electron density map is shown at contour level 1σ for the engineered disulfide bond and two water molecules that fill the area that is occupied in the wt-A2 by the side chain of tyrosine-84.

  • Fig. 2 Improved and efficient antigen-specific T cell detection using DS-A2 molecules.

    (A) Schematic illustration of pMHC multimer preparation using DS-A2. DS-A2 molecules were first incubated with the desired peptide for 15 min and then multimerized on fluorochrome-labeled streptavidin. (B) Dot plots of flow cytometry analysis for antigen-specific T cell detection across different donor samples. PBMCs were stained with DS-A2 and DS-A24 tetramers (DS) prepared after direct loading of peptides on DS-monomers or with wt-A2 and wt-A24 tetramers (WT) prepared after UV-mediated peptide exchange on wt monomers. (C) Staining index (SI) of DS (blue) and WT (orange) tetramers for plots in (B) (and one additional donor) and (D). Paired t test P = 0.0004 (***). The staining index was calculated as (median of positive − median of negative)/(SD of negative × 2). (D) Dot plots of T cells stained by DS-A2 (DS) and wt-A2 (WT) tetramers for several virus-associated antigens in one donor’s PBMCs. As controls, an HIV-derived A2 epitope and a nonbinding peptide were used. (E) Representative dot plots of FLU MP (GILGFVFTL)–specific T cell detections in donor PBMCs comparing DS-A2 and wt-A2 tetramers assembled from pMHC monomers produced by classical folding, UV-mediated peptide exchange or using empty DS-A2. (F) Staining index comparison for tetramers prepared using DS- and wt-A2 molecules across several viral specificities and donor PBMCs. Paired t test was used for statistical analysis; P = 0.015 [*] (DS-UV versus WT-UV) and P = 0.013 [*] (WT-UV versus empty DS). ns, not significant. (G) TCR recognition profile of DS-A2 and wt-A2 molecules. Shannon logos display the amino acid requirements for pMHC-TCR interaction at each position of the peptide. TCR recognition profiles were generated using a DNA barcode–labeled library of 192 peptide-A2 multimers with amino acid substitutions of the original KLLEIAPNC peptide. TCR recognition for each of these peptide variations was measured with pMHC dextramers prepared from DS-A2 molecules or from wt-A2 molecules for two T cell clones that recognized the Merkel cell polyomavirus–derived epitope A2-KLLEIAPNC. Numbers on dot plots represent the percentage of pMHC multimer–positive cells out of total CD8+ T cells.

  • Fig. 3 DS-A2 molecules used in combinatorial color encoding effectively detect virus- and melanoma-associated antigen-specific T cells.

    (A) Flow cytometry plot of antigen-specific CD8+ T cells detected using combinatorial color encoding. Dot plots represent detected antigen-specific CD8+ T cells; gray dots represent pMHC multimer–negative or single color–positive cells, and colored dots represent dual-color multimer-positive cells. Staining was conducted using DS-A2 (DS) and wt-A2 (WT) in parallel (wt-A2 tetramers were generated after UV-mediated peptide exchange). (B) Fold change of staining index, comparing DS-A2 over wt-A2 tetramers for each color label used for the given T cell population detected in (A). (C) Melanoma-associated antigen-specific T cells detected using DS-A2 and wt-A2 (UV exchange) tetramers from expanded TILs of a patient with melanoma. A library of 21 melanoma-associated antigenic peptides was screened using the combinatorial color encoding strategy. Dot plots of three identified T cell populations. (D) Fold change in staining index, similar to (B), for melanoma-associated antigen-specific T cells shown in (C). (E) Correlation of antigen-specific T cell frequencies identified using DS-A2 tetramers and wt-A2 tetramers from donor PBMCs and in the patient with melanoma [combined from Figs. 2 (B, D, and E) and 3 (A and C) and fig. S6]. (F) Comparative staining index and frequency of OT1 splenocytes detected by DS and wt tetramers of murine MHC H-2Kb. SIINFEKL peptide and six variant peptides, highlighted in red, with different affinities to the OT1-TCR (plotted high to low on the x axis) were used to compare the detection efficiency of DS with wt–H-2Kb tetramers. Dot plots are shown in fig. S7.

  • Fig. 4 Empty-loadable MHC-I multimers for efficient detection of antigen-specific T cells.

    (A) Schematic illustration of empty-loadable MHC-I multimers assembled using DS–MHC-I molecules. Empty-loadable DS-A2 multimers were stored at −20°C and loaded with peptide on demand. (B) Conditions for peptide loading to empty-loadable multimers. Detection of GILGFVFTL-specific T cells after addition of FLU MP GILGFVFTL peptide is shown as the frequency of multimer-positive CD8+ T cells at varying peptide concentration and time. (C) Detection of antigen-specific CD8+ T cells using DS-ELTs for A2, A24, and H-2Kb MHC alleles compared with wt and DS tetramers. Wt tetramers were prepared after UV-mediated peptide exchange. (D) Stability of empty-loadable multimers. DS-A2 ELTs conjugated with APC were stored for up to 1 week at 4°, 20°, and 37°C and then loaded with CMV pp65 (NLVPMVATV)–specific peptide to detect T cells from donor PBMCs. The frequency (left) and MFI (right) of detected antigen-specific T cells are shown for varying conditions. Dot plots are shown in fig. S8. (E) Peptide dissociation rate measured as FLU MP GILGFVFTL–specific T cell detection efficiency by wt-A2 (orange) or empty-loadable DS-A2 multimers (blue). pMHC multimers (without excess peptide) were incubated at 4°, 20°, and 37°C up to 1 week and used for T cell detection from donor PBMCs. The frequency (left) and MFI (right) of detected antigen-specific T cells are shown for varying conditions. Dot plots are shown in fig. S9. (F) Dot plots showing T cell detection efficiency of DS-A2 ELTs stored for 6 months. DS-A2 ELTs conjugated with different fluorophores stored at −20°C for 6 months were incubated with FLU MP peptide and used for T cell detection. Compare with freshly prepared DS-A2 ELTs in (C).

  • Fig. 5 Detection of neoantigen-specific T cell recognition in cancer.

    (A) Antigen-specific T cells in melanoma TILs identified by the combinatorial color encoding strategy using empty-loadable DS-A2 multimers. pMHC multimers for 54 A2-restricted peptides (43 neoantigens, 5 melanoma-associated cancer antigens, and 6 virus-derived antigens) were generated, each in a two-color combination. Frequency of pMHC multimer–positive cells out of total CD8+ T cells is shown as colored dots across three categories. Multimer-negative cells are plotted in gray for each pMHC combination (zero values are converted to 0.0001 to plot on log scale). (B) Dot plots of the antigen-specific T cell populations from (A), each is shown with indicated fluorochromes and peptide sequences. The mutated amino acid position of the neoantigens is highlighted in red.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/37/eaau9039/DC1

    Fig. S1. MD simulation of DS-A2.

    Fig. S2. Dipeptide-mediated folding efficiency of DS-MHC molecules.

    Fig. S3. Peptide binding measurements and comparative purification profiles.

    Fig. S4. Gating strategies for flow cytometry analysis.

    Fig. S5. Purity and biotinylation efficiency.

    Fig. S6. Detection of virus-derived antigen-specific T cells from donor PBMCs in a combinatorial encoding strategy.

    Fig. S7. Detection of low-avidity pMHC-TCR interactions using DS–H-2Kb multimers.

    Fig. S8. T cell detection plots for the stability analysis of DS-A2 empty-loadable tetramers.

    Fig. S9. T cell detection plots of peptide dissociation analysis.

    Table S1. Crystal structure data collection and refinement statistics.

    Table S2. Protein yield after in vitro folding and purification of wt and DS-A2 molecules.

    Table S3. Raw data (Excel file).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. MD simulation of DS-A2.
    • Fig. S2. Dipeptide-mediated folding efficiency of DS-MHC molecules.
    • Fig. S3. Peptide binding measurements and comparative purification profiles.
    • Fig. S4. Gating strategies for flow cytometry analysis.
    • Fig. S5. Purity and biotinylation efficiency.
    • Fig. S6. Detection of virus-derived antigen-specific T cells from donor PBMCs in a combinatorial encoding strategy.
    • Fig. S7. Detection of low-avidity pMHC-TCR interactions using DS–H-2Kb multimers.
    • Fig. S8. T cell detection plots for the stability analysis of DS-A2 empty-loadable tetramers.
    • Fig. S9. T cell detection plots of peptide dissociation analysis.
    • Table S1. Crystal structure data collection and refinement statistics.
    • Table S2. Protein yield after in vitro folding and purification of wt and DS-A2 molecules.

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