Research ArticleTUMOR IMMUNOLOGY

Generation and molecular recognition of melanoma-associated antigen-specific human γδ T cells

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Science Immunology  14 Dec 2018:
Vol. 3, Issue 30, eaav4036
DOI: 10.1126/sciimmunol.aav4036
  • Fig. 1 Generation of MAA-specific CD8

    + γδ T cells from cord blood HSPCs cultured on OP9-DL4 cells. (A) Flow cytometric analysis of day 55 HSPC/OP9-DL4 cell cocultures for the cell surface expression of CD4 and CD8α (top row) and enrichment by magnetic cell sorting of CD8+ cells is shown (right column). Analyses for CD8α expression and HLA-A*0201/MART-1 [heteroclitic (H)] dextramer staining of cells are shown in both total and CD8α-enriched populations, gated for either DP (middle row) or SP CD8 cells (bottom row). (B) Flow cytometric analysis of day 59 HSPC/OP9-DL4 cell cocultures for cell surface expression of CD4 and CD8α (top row) and enrichment by magnetic cell sorting of CD8+ cells is shown (right) for cells analyzed for αβ TCR, γδ TCR, and CD8β expression. Analyses for HLA-A*0201/MART-1 (heteroclitic) and HLA-A*0201/gp100 dextramer staining of CD8α-enriched populations, as well as for CD8β and γδ TCR expression, are shown. The above results are representative of at least 10 independent cocultures. (C) Cord blood cells were enriched for γδ TCR+ CD4 T cells by magnetic cell sorting from HLA-A2+ samples and stimulated for 18 days in vitro, as described in Material and Methods. Flow cytometric analysis of cultures for expression of Vδ1, Vγ9, γδ TCR, and HLA-A*0201/MART-1 (heteroclitic and WT), as indicated, shows cells gated for the expression of γδ TCR or αβ TCR. These results are representative of at least three independent cultures.

  • Fig. 2 Specificity of CD8

    + γδ T cell clones and their TCRs generated from cord blood HSPCs cultured on OP9-DL4 cells. (A) Flow cytometric analysis of CD8+ clones derived from HSPC/OP9-DL4 cell cocultures for the cell surface expression of HLA-A*0201/MART-1 (heteroclitic), HLA-A*0201/gp100, and/or HLA-A*0201/HIV dextramers, as indicated. HLA-A*0201/MART-1–specific αβ TCR cell clone (IC2) derived from ex vivo cord blood CD8 T cells was used as a positive control (top row). A total of 33 γδ T cell clones were analyzed, and 4 representative clones are shown. (B) Flow cytometric analysis of CD8+ T clones derived from HSPC/OP9-DL4 cell cocultures for the cell surface expression of γδ TCR, αβ TCR, Vδ1, and Vδ2, as indicated. (C) γδ TCR chains obtained from HSPC/OP9-DL4 coculture-generated γδ T cell clones (3C2 and 5F3) were retrovirally transduced into J76 (left column) or J76CD8α (right column). αβ TCR chains obtained from an ex vivo–derived T cell clone (49) were transduced into J76 (left column) or J76CD8αβ (right column). Flow cytometric analysis for HLA-A*0201/MART-1 dextramer staining is shown as histograms for each set of transduced cells, as well as untransduced controls, as indicated. RCN, relative cell number. Shown in untransduced histogram are unstained cells (light gray) and cells stained for HLA-A*0201/MART-1 dextramer (dark gray); for the other histograms, untransduced (light gray) and transduced (dark gray) cells stained for HLA-A*0201/MART-1 dextramer are shown. Frequencies of each populations based on unstained gated cells are indicated. Four γδ TCR clones derived from two independent experiments were analyzed. Each TCR chain was sequenced, and CDR3s are shown in table S2. (D) Specificity of transduced J76 T cells (3C2 or 49) was evaluated using flow cytometry with the indicated HLA dextramers for untransduced cells (top row), γδ TCR (middle row), and αβ TCR (bottom row). Shown for the untransduced panel are unstained cells (light gray) and cells stained as indicated (dark gray); for the other panels, untransduced (light gray) and transduced (dark gray) cells stained for the indicated HLA dextramers are shown. Representative analysis of one of the four transduced γδ TCR chains is shown (n ≥ 3). (E) Cytotoxic T cell assay of sorted activated cord blood PBMCs transduced with 3C2 γδ TCR, as described in fig. S12, showing specific percent lysis at the indicated effector to target ratios of MART-1– or gp100-pulsed T2 cell targets. Representative analysis of γδ TCR–transduced PBMCs is shown (n = 3). MART-1–speficic αβ T cell line, gp100-specific αβ T cell line, and nontransduced PBMCs are also shown.

  • Fig. 3 Three-dimensional complex structure of the 5F3 TCR with HLA-A*02/MART-1.

    (A) Overall crystal structure of 5F3/HLA-A*02/β2M/MART-1 peptide complex (upper left) with the γ and δ chains of the 5F3 TCR colored cyan and magenta, respectively. HLA-A*02, β2M, and MART-1 are colored white, wheat, and yellow, respectively. Shown also is the complex structure of HLA-A*02/β2M/MART-1 with the αβ TCR MEL5 (PDB code: 3HG1; middle) with the α and β chains of the MEL5 TCR colored light green and dark brown. The right panel is the complex structure of CD1d-sulfatide with the γδ TCR DP10.7 (PDB code: 4MNG), where γ and δ chains are colored cyan and magenta, respectively. The lower panels show the positions of the TCR CDR loops on HLA-A*02/MART-1 with the squares indicating the docking orientation. The dashed square in the case of CD1d/DP10.7 denotes that the γ chain does not contact CD1d or the sulfatide ligand. (B) Comparison of the positioning of the CDR loops of the 5F3 γδ TCR and MEL5 αβ TCR on HLA-A*02/MART-1 (left), coloring scheme same as in (A); the numbers 1, 2, and 3 denote CDR loops. Right panel shows comparison of the docking angle of the 5F3 γδ TCR with that of the HLA-A*02/MART-1–reactive αβ TCRs MEL5, DMF4, and DMF5. Spheres represent the position of the conserved intrachain disulfide cysteine residues in the TCR variable domains, and the dashed black lines represent the vectors connecting them. (C) MART-1 peptide recognition by the CDR1γ (cyan) and CDR3δ (magenta) loops. TCR interacting residues are showing as sticks and are labeled. Peptide residues contacted by CDR3δ only are colored yellow, which contacted by both CDR1γ and 3δ loops, and interacting residue is colored orange. Zoomed in view of MART-1 recognition is shown in the inset. 2Fo-Fc electron density maps (contoured at 1σ) for Y32, W98, and D99 are shown as blue mesh.

  • Fig. 4 Footprint mapping of the 5F3 and 3C2 γδ TCRs via alanine-scanning mutagenesis of HLA-A*02 α1 and α2 helices.

    (A) Graph showing the binding results of alanine-scanning mutagenesis. The bars represent the fold change in affinity between the 5F3 TCR (left) and 3C2 TCR (right) with mutant and WT HLA-A*02 (Mut Kd/ WT Kd). Mutations with more than threefold reduction in affinity are colored red, mutations with two- to threefold reduction in affinities are colored orange, mutations with one- to twofold reduction in affinity are colored yellow, mutations with no effect on binding are colored blue, and mutations with less than onefold changes are colored green. Plus sign indicates more than fourfold reduction. Binding analysis was performed twice, and average Kd was used for analysis. (B) The results from (A) are mapped onto a surface representation of the platform domain of HLA-A*02 for the 5F3 TCR (left) and 3C2 TCR (right). HLA-A*02 is colored white, and MART-1 is colored yellow. The mutant residues in α1 and α2 helices are mapped, labeled, and color-coded as shown in (A) on the basis of fold changes in binding due to alanine mutation. (C) Proposed model of the footprints of 5F3 and 3C2 TCRs are shown on the HLA-A*02 as colored ellipses as predicted from the alanine-scanning mutagenesis. Dashed black line indicates the position of linker connecting the MART-1 and β2M in the single-chain construct.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/30/eaav4036/DC1

    Materials and Methods

    Fig. S1. Generation of human CD8 T cells from cord blood OP9-DL4 cell cocultures.

    Fig. S2. Generation of human MART-1+ γδ TCR T cells from cord blood HSPCs cocultured with OP9-DL4 cells.

    Fig. S3. Generation of human MART-1+ γδ TCR cells and clones from cord blood HSPCs cocultured with OP9-DL4 cells.

    Fig. S4. Derivation of human MART-1+ γδ TCR T cells from adult PBMCs.

    Fig. S5. Derivation of human MART-1+ γδ TCR T cells from cord blood naïve PBMCs.

    Fig. S6. Derivation of human MART-1+ γδ TCR T cell line from pooled cord blood naïve PBMCs.

    Fig. S7. Derivation of human MART-1+ γδ TCR T cell clones from individual cord blood PBMCs.

    Fig. S8. Functional characterization of MART-1+ γδ TCR T cell clones generated from cord blood HSPCs cocultured on OP9-DL4 cells and from MART-1+ γδ TCR T cell lines derived from cord blood naïve PBMCs.

    Fig. S9. Specificity of γδ TCR–transduced J76CD8α T cells.

    Fig. S10. Cold target inhibition of γδ TCR staining by HLA/peptide dextramers.

    Fig. S11. Functional characterization of TCR-transduced J76 T cells.

    Fig. S12. Transduction and isolation of PBMCs ectopically expressing 3C2 γδ TCR.

    Fig. S13. Binding analysis of human γδ TCRs to MHCp using BLI.

    Fig. S14. Contribution of germline and recombined residues of the 5F3 γδ TCR in recognition of HLA-A*02/MART-1.

    Table S1. Generation of γδ TCR CD8α+ T cells clones with specificity for HLA-A*0201/MART-1 from HSPC/OP9-DL4 cell cocultures.

    Table S2. Derivation of γδ TCR CD8α+ T cells clones with specificity for HLA-A*0201/MART-1 from naïve PBMCs.

    Table S3. Cloning and sequencing of three in vitro–derived γδ TCR T cell clones.

    Table S4. Data collection and refinement statistics of HLA-A*02-MART-1/5F3 TCR complex.

    Table S5. Contacts residues between γδ TCR and HLA-A*02.

    Table S6. Percentage of BSA contribution of complex components.

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Generation of human CD8 T cells from cord blood OP9-DL4 cell cocultures.
    • Fig. S2. Generation of human MART-1+ γδ TCR T cells from cord blood HSPCs cocultured with OP9-DL4 cells.
    • Fig. S3. Generation of human MART-1+ γδ TCR cells and clones from cord blood HSPCs cocultured with OP9-DL4 cells.
    • Fig. S4. Derivation of human MART-1+ γδ TCR T cells from adult PBMCs.
    • Fig. S5. Derivation of human MART-1+ γδ TCR T cells from cord blood naïve PBMCs.
    • Fig. S6. Derivation of human MART-1+ γδ TCR T cell line from pooled cord blood naïve PBMCs.
    • Fig. S7. Derivation of human MART-1+ γδ TCR T cell clones from individual cord blood PBMCs.
    • Fig. S8. Functional characterization of MART-1+ γδ TCR T cell clones generated from cord blood HSPCs cocultured on OP9-DL4 cells and from MART-1+ γδ TCR T cell lines derived from cord blood naïve PBMCs.
    • Fig. S9. Specificity of γδ TCR–transduced J76CD8α T cells.
    • Fig. S10. Cold target inhibition of γδ TCR staining by HLA/peptide dextramers.
    • Fig. S11. Functional characterization of TCR-transduced J76 T cells.
    • Fig. S12. Transduction and isolation of PBMCs ectopically expressing 3C2 γδ TCR.
    • Fig. S13. Binding analysis of human γδ TCRs to MHCp using BLI.
    • Fig. S14. Contribution of germline and recombined residues of the 5F3 γδ TCR in recognition of HLA-A*02/MART-1.
    • Table S1. Generation of γδ TCR CD8α+ T cells clones with specificity for HLA-A*0201/MART-1 from HSPC/OP9-DL4 cell cocultures.
    • Table S2. Derivation of γδ TCR CD8α+ T cells clones with specificity for HLA-A*0201/MART-1 from naïve PBMCs.
    • Table S3. Cloning and sequencing of three in vitro–derived γδ TCR T cell clones.
    • Table S4. Data collection and refinement statistics of HLA-A*02-MART-1/5F3 TCR complex.
    • Table S5. Contacts residues between γδ TCR and HLA-A*02.
    • Table S6. Percentage of BSA contribution of complex components.

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