Research ArticleHIV

CD4+ T cell–mediated HLA class II cross-restriction in HIV controllers

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Science Immunology  08 Jun 2018:
Vol. 3, Issue 24, eaat0687
DOI: 10.1126/sciimmunol.aat0687
  • Fig. 1 Immunological synapse formation by public TCR-transduced cells.

    (A) Bar graphs summarizing the conjugate formation of the F24-transduced (red), F25-transduced (blue), and F5-transduced (green) J76 cells with Gag293-pulsed B-EBV cells (n = 3). (B) Correlation between TCR affinity (Table 1) and conjugate formation capacity (n = 3). r, Pearson coefficient. (C) Representative images obtained by imaging flow cytometry comparing mIS formation. CD3 recruitment to the T cell–APC contact site is indicated by a white arrow. DAPI, 4′,6-diamidino-2-phenylindole. (D) Bar graph comparing the % mIS formed between J76 cells expressing F24 (red), F25 (blue), or F5 (green), and DR11-expressing B-EBV cells, with means ± SD reported (n = 4 except for F5, n = 3). (E) Bars depict the actin mean pixel intensity (MPI) values represented as the percentage of the maximal response (F24 + DR11–B-EBV), with data points reported as black symbols (n = 3 except for F5, n = 2). Statistical differences are computed using unpaired Student’s t test. Background was subtracted for all samples. *P < 0.05, **P < 0.01, ***P < 0.005.

  • Fig. 2 TCR-transduced T cells express cytotoxic markers upon Gag293 stimulation.

    (A to D) Fluorescence-activated cell sorting plots depicting the expression of CD107a and GrB (A and C) or CD107a and perforin (B and D) by F24-transduced (left), F25-transduced (middle), or F5-transduced (right) primary CD4+ T cells. The % of double positive cells is highlighted in red. (E to H) Bar graphs summarizing the proportion of cytotoxic CD107a+ GrB+ (E and G) or CD107a+ perforin+ (F and H) CD4+ T cells (E and F) or CD8+ T cells (G and H) transduced with F24 (red), F25 (blue), or F5 TCR (green). Data are depicted as % of the maximal response obtained with F24-transduced T cells, with means ± SD reported for CD107+ GrB+ (n = 3) and means reported for CD107+ perforin+ (n = 2), with data points reported as black symbols. Statistical differences are computed using the unpaired Student’s t test. Background was subtracted from all data points. **P < 0.01, ***P < 0.005.

  • Fig. 3 Viral suppression by TCR-transduced CD4+ and CD8+ T cells.

    (A) Schematic representation of the experimental system. Viral inhibition was measured by the % decrease of green fluorescent protein–positive (GFP+) infected DCs (iDC). (B) Representative histograms displaying the viral inhibition effect mediated by F24-transduced CD4+ T cells (right) compared with nontransduced CD4+ T cells (left). The % infected iDC is highlighted in red. (C to E) Viral inhibition activity of TCR-transduced CD4+ T cells from healthy donors expressing HLA-DR1 (C), HLA-DR11 (D), or HLA-DR15/DRB5 (E). E:T ratios: 5:1 (blue), 1:1 (green), and 0.5:1 (orange). Bars depict the means obtained from two (C) or three (D and E) independent experiments. Background was subtracted from all data points. (F to H) Viral inhibition activity of TCR-transduced CD8+ T cells from HLA-DR1 (F), HLA-DR11 (G), or HLA-DR15/DRB5 (H) healthy donors. Bars in (F) to (H) depict the means of two independent experiments. The mean % of viral inhibition obtained by TCR-transduced CD4+ T cells (I) or CD8+ T cells (K) coculture at a 5:1 E:T ratio is plotted in function of TCR affinities. (J) The mean % of viral inhibition obtained by TCR-transduced CD4+ T cells is plotted in function of the mean % conjugates formed (reported in Fig. 1A). Data points are reported as black symbols.

  • Fig. 4 Mapping of the core Gag293 epitope and peptide presentation.

    (A) F24-transduced J76 cells were cocultured with APC pulsed with serial dilutions of either full-length Gag293 or peptide truncations. TCR activation was measured by CD69 induction, with background subtracted from all samples. Means ± SD are reported (n = 3). (B to D) J76 cells transduced with either F24 (red), F25 (blue), or F5 (green) were cocultured with HLA-DR11–expressing (B), HLA-DRB5–expressing (C), or HLA-DR1–expressing (D) L cells in the presence of the core-epitope RQ13 or alanine mutants. Means ± SD are reported (n = 3). (E to J) Structures of the peptide-HLA complexes with the Gag293 peptide (blue stick) or the RQ13 peptide (pink stick) (F to H). The β chain of HLA-DR11 is colored green (E, F, I, and J), HLA-DR15 is purple (G and I), HLA-DR1 is orange (H and J), and the HLA-DR α chain is pale gray. Structure overlay of HLA-DR11–RQ13 (green) with HLA-DR15–RQ13 (purple) (I) and HLA-DR15–RQ13 (orange) (J), with the peptide colored similarly to the HLA-DR β chain. The HLA-DR β chain residues 63, 67, and 73 Cα atoms are shown as spheres.

  • Fig. 5 F24 TCR cross-recognition of the RQ13 epitope presented by multiple HLA-DR alleles.

    Top: The F24 TCR (α chain in pale pink, and β chain in pale blue) recognizing RQ13 (black sticks) presented by HLA-DR11 (A), HLA-DR15 (B), and HLA-DR1 (C). The HLA-DR α chain is colored pale gray, whereas the β chains of HLA-DR11, HLA-DR15, and HLA-DR1 are shown in green, purple, and orange, respectively. The CDR1α, CDR2α, and CDR3α loops are shown in teal, green, and purple, whereas the CDR1β, CDR2β, and CDR3β loops are shown in red, orange, and yellow, respectively. Pie charts represent the contribution of each F24 TCR segments toward the peptide-HLA complex (left) or toward the peptide only (right) on HLA-DR11 (A), HLA-DR15 (B), and HLA-DR1 (C) presenting the RQ13 epitope. Bottom panels show the footprint of the F24 TCR on the surface of each HLA-DR–RQ13 complex. The colors correspond to each TCR segment involved in the contact according to the top panels; the magenta and blue spheres represent the center of mass for Vα and Vβ, respectively.

  • Fig. 6 The F24 TCR recognition is primarily focused on the HIV epitope.

    F24 TCR interactions with the HLA-DR11 chain (α chain in gray and β chain in green cartoon) (A) via the CDR2β loop (orange) and the F24 TCR β-chain framework (pale blue), (B) CDR3β residues (yellow), and (C) the CDR1α (teal) and CDR3α (purple) loops. Interactions of the RQ13 peptide (black sticks) (D) with F24 TCR CDR1α (teal), (E) the two CDR3 loops (α, purple; β, yellow), (F) CDR1β (red), CDR2β (orange), and CDR3β residues (yellow). The structures are shown in cartoon representation, interacting residues are depicted in sticks, and hydrogen bonds and van der Waals interactions are shown in blue dashed lines.

  • Fig. 7 Antigen-binding cleft conformational changes, and energetic footprints of the public TCRs on the RQ13 peptide, in the context of multiple HLA-DR alleles.

    Overlay of HLA-DR molecules in their free conformation in green, orange, and purple for HLA-DR11 (A), HLA-DR1 (B), and HLA-DR15 (C), respectively, with their F24 TCR-bound structure in pink, yellow, and blue, respectively. The RQ13 epitope is colored according to the bound HLA-DR molecule and represented as loop. The black circle highlights the hinge of the β-chain helix that changes conformation upon F24 TCR binding in HLA-DR1 (B) and HLA-DR15 (C) molecules. (D to F) Energetic footprint of F24 (D), F5 (E), and F25 (F) TCRs on the HLA-DR11–RQ13 complex. Energetic contribution determined by SPR, and the impact of each mutation was classified as no effect (<threefold affinity decrease, colored black), moderate (three- to fivefold affinity reduction, orange), critical (>fivefold affinity decrease, red), or improving the interaction (>threefold increase in affinity, blue). (G to I) The affinity of the TCR F24 mutants was determined by SPR. The RQ13 peptide is represented as black loop. The effect of each mutation is represented on the surface and colored as per the HLA-DR11 mutant in (D).

  • Table 1 Affinity measurements.

    Kdeq is in μM; kon is in M−1 s−1 × 104; koff is in s−1; t1/2 is in s. ND, not determined. The error is representative of the SD of the experiment performed in duplicate from at least two independent experiments (n ≥ 2).

    KdeqDR1-Gag293*DR1-RQ13DR11-Gag293*DR11-RQ13DR15-Gag293DR15-RQ13DRB5-Gag293*DRB5-RQ13
    F246.97 ± 0.2210.56 ± 2.620.86 ± 0.151.16 ± 0.485.09 ± 0.456.90 ± 0.622.58 ± 0.192.49 ± 0.80
    F2551.50 ± 3.00153.4 ± 10.653.58 ± 0.255.36 ± 1.41152.0 ± 15.56159.6 ± 29.6916.00 ± 0.6037.28 ± 2.02
    F5>100173.0 ± 9.9011.06 ± 1.7314.33 ± 3.18122.0 ± 14.1474.48 ± 13.3474.45 ± 6.2575.6 ± 4.53
    konDR1-Gag293DR1-RQ13DR11-Gag293DR11-RQ13DR15-Gag293DR15-RQ13DRB5-Gag293DRB5-RQ13
    F241.15 ± 0.541.28 ± 0.2410.00 ± 0. 3010.41 ± 0.794.95 ± 0.034.50 ± 0.055.29 ± 0.544.54 ± 0.09
    F25NDND9.14 ± 1.365.42 ± 0.07NDNDND0.21 ± 0.02
    F5NDND1.51 ± 0.070.89 ± 0.03NDNDNDND
    koffDR1-Gag293DR1-RQ13DR11-Gag293DR11-RQ13DR15-Gag293DR15-RQ13DRB5-Gag293DRB5-RQ13
    F240.418 ± 0.0130.172 ± 0.0160.062 ± 0.0020.050 ± 0.0010.142 ± 0.0060.153 ± 0.0080.179 ± 0.0090.167 ± 0.003
    F25NDND0.253 ± 0.0050.113 ± 0.004NDNDND0.498 ± 0.076
    F5NDND0.160 ± 0.0450.107 ± 0.001NDNDNDND
    t1/2DR1-Gag293DR1-RQ13DR11-Gag293DR11-RQ13DR15-Gag293DR15-RQ13DRB5-Gag293DRB5-RQ13
    F241.64.011.111.84.84.53.84.1
    F25NDND2.76.1NDNDND1.4
    F5NDND4.36.5NDNDNDND

    *Kdeq values previously published (10).

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/3/24/eaat0687/DC1

      Materials and Methods

      Fig. S1. HLA-DR polymorphism.

      Fig. S2. Representative TCR binding curves determined by SPR across different HLA-DR molecules.

      Fig. S3. Gating strategies and masks used for the identification of cellular conjugates and immunological synapses.

      Fig. S4. Representative example of TCR transduction efficiency in human peripheral blood mononuclear cells from a healthy donor.

      Fig. S5. Gating strategy used for the single-cycle viral inhibition assay.

      Fig. S6. Correlation between cytotoxic phenotype and viral inhibition.

      Fig. S7. TCR-mediated elimination of HIV-infected DCs.

      Fig. S8. Omit maps and refined maps of the peptide–HLA-DR complexes.

      Fig. S9. Impact of HLA-DR polymorphism on epitope presentation and antigen-binding cleft conformation.

      Fig. S10. The F24 and F5 TCRs engage with the HLA-DR11–RQ13 in the same fashion.

      Fig. S11. Structural changes in the TCRs are limited to the CDR3β loop.

      Fig. S12. Representative binding curves determined by SPR for HLA-DR11 mutants.

      Table S1. List of peptides used in the study.

      Table S2. Data collection and refinement statistics of peptide–HLA-DR structures.

      Table S3. Data collection and refinement statistics of TCR peptide–HLA-DR structures.

      Table S4. Contact table of F24 TCR–HLA-DR11–RQ13.

      Table S5. Contact table of F24 TCR–HLA-DR15–RQ13.

      Table S6. Contact table of F24 TCR–HLA-DR1–RQ13.

      Table S7. Data collection and refinement statistics of F24 TCR structure.

      Table S8. Energetic contribution of the HLA-DR11 residues as measured by SPR.

      Table S9. Affinity measurement of the F24 TCR mutants.

      References (3032)

    • Supplementary Materials

      Supplementary Material for:

      CD4+ T cell–mediated HLA class II cross-restriction in HIV controllers

      Moran Galperin, Carine Farenc, Madhura Mukhopadhyay, Dhilshan Jayasinghe, Amandine Decroos, Daniela Benati, Li Lynn Tan, Lisa Ciacchi, Hugh H. Reid, Jamie Rossjohn*, Lisa A. Chakrabarti*, Stephanie Gras*

      *Corresponding authors. Email: jamie.rossjohn{at}monash.edu (J.R.); chakra{at}pasteur.fr (L.A.C.); stephanie.gras{at}monash.edu (S.G.)

      Published 8 June 2018, Sci. Immunol. 3, eaat0687 (2018)
      DOI: 10.1126/sciimmunol.aat0687

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. HLA-DR polymorphism.
      • Fig. S2. Representative TCR binding curves determined by SPR across different HLA-DR molecules.
      • Fig. S3. Gating strategies and masks used for the identification of cellular conjugates and immunological synapses.
      • Fig. S4. Representative example of TCR transduction efficiency in human peripheral blood mononuclear cells from a healthy donor.
      • Fig. S5. Gating strategy used for the single-cycle viral inhibition assay.
      • Fig. S6. Correlation between cytotoxic phenotype and viral inhibition.
      • Fig. S7. TCR-mediated elimination of HIV-infected DCs.
      • Fig. S8. Omit maps and refined maps of the peptide–HLA-DR complexes.
      • Fig. S9. Impact of HLA-DR polymorphism on epitope presentation and antigenbinding cleft conformation.
      • Fig. S10. The F24 and F5 TCRs engage with the HLA-DR11–RQ13 in the same fashion.
      • Fig. S11. Structural changes in the TCRs are limited to the CDR3β loop.
      • Fig. S12. Representative binding curves determined by SPR for HLA-DR11 mutants.
      • Table S1. List of peptides used in the study.
      • Table S2. Data collection and refinement statistics of peptide–HLA-DR structures.
      • Table S3. Data collection and refinement statistics of TCR peptide–HLA-DR structures.
      • Table S4. Contact table of F24 TCR–HLA-DR11–RQ13.
      • Table S5. Contact table of F24 TCR–HLA-DR15–RQ13.
      • Table S6. Contact table of F24 TCR–HLA-DR1–RQ13.
      • Table S7. Data collection and refinement statistics of F24 TCR structure.
      • Table S8. Energetic contribution of the HLA-DR11 residues as measured by SPR.
      • Table S9. Affinity measurement of the F24 TCR mutants.
      • References (30–32)

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