Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E by Scott G. Hansen, Helen L. Wu, Benjamin J. Burwitz, Colette M. Hughes, Katherine B. Hammond, Abigail B. Ventura, Jason S. Reed, Roxanne M. Gilbride, Emily Ainslie, David W. Morrow, Julia C. Ford, Andrea N. Selseth, Reesab Pathak, Daniel Malouli, Alfred W. Legasse, Michael K. Axthelm, Jay A. Nelson, Geraldine M. Gillespie, Lucy C. Walters, Simon Brackenridge, Hannah R. Sharpe, César A. López, Klaus Früh, Bette T. Korber, Andrew J. McMichael, S. Gnanakaran, Jonah B. Sacha, and Louis J. Picker Science Volume 351(6274):714-720 February 12, 2016 Published by AAAS

Fig. 1 MHC restriction of CD8+ T cells elicited by 68-1 RhCMV/SIVgag. MHC restriction of CD8+ T cells elicited by 68-1 RhCMV/SIVgag. (A and B) Peripheral blood mononuclear cells (PBMCs) from a representative RM vaccinated with 68-1 RhCMV/SIVgag (RM Rh22034, one of four similarly analyzed; fig. S3) were stimulated with the indicated epitopic 15-mer peptides pulsed onto the surface of parental MHC-I–negative (neg) cell lines (.221 and K562 cells; negative controls), autologous B lymphoblastoid cell lines (BLCL; positive controls), or the indicated MHC-I transfectants, with CD8+ T cell recognition determined by detection of interferon-γ (IFN-γ) and/or tumor necrosis factor–α (TNF-α) production by flow-cytometric ICS assay (response frequencies of gated CD8+ T cells are shown in each quadrant). The MHC-I molecules tested included both those expressed by Rh22034 (A) and additional RM and human MHC-E molecules not expressed by Rh22034 (B). (C) Mamu-E*02:04, Mamu-E*02:20, and HLA-E*01:03 transfectants were pulsed with the serially diluted concentration of the indicated optimal SIVgag 9-mer epitopic peptides. The transfectants were combined with PBMCs from three to four 68-1 RhCMV/SIVgag–vaccinated RMs for flow-cytometric ICS determination of the frequency of responding CD8+ T cells (IFN-γ– and/or TNF-α–positive). Response frequencies at each peptide dose were normalized to the response observed for the transfectant pulsed with the highest concentration (10 μM) of peptide. Scott G. Hansen et al. Science 2016;351:714-720 Published by AAAS

Fig. 2 MHC-E restriction is limited to CD8+ T cell responses elicited by ΔRh157.5/.4 RhCMV vectors. MHC-E restriction is limited to CD8+ T cell responses elicited by ΔRh157.5/.4 RhCMV vectors. (A) CD8+ T cell responses to SIVgag were epitope-mapped using flow-cytometric ICS to detect recognition of 125 consecutive 15-mer SIVgag peptides [with an overlap of 11 amino acids (a.a.)] in RMs vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIVmac239 (N = 4 to 6 per group shown; see fig. S12 for other studied RMs). Peptides resulting in specific CD8+ T cell responses are indicated by a box, with the color of the box designating MHC restriction as determined by blocking with the pan–MHC-I–blocking mAb W6/32, the MHC-E–blocking peptide VL9, and the MHC-II–blocking peptide CLIP (see the supplementary materials). The minimal number of independent epitopes in these MHC restriction categories is shown at right for each RM. (B) Analysis of SIV-infected CD4+ cell recognition by CD8β+ cells isolated from RMs that were vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIV. The flow profiles at left show IFN-γ and TNF-α production after CD8β+ T cell incubation with autologous SIVmac239-infected CD4+ T cells alone (no block), in the presence of mAb W6/32 plus CLIP, or in the presence of VL9 plus CLIP. All plots are gated on live CD3+ CD8+ cells. The bar graph at right shows the results from all studied RMs. Scott G. Hansen et al. Science 2016;351:714-720 Published by AAAS

Fig. 3 Diversity of MHC-E–restricted epitopes. Diversity of MHC-E–restricted epitopes. (A) Comparison of the total number of distinct MHC-E (green)– versus MHC-Ia (red)–restricted SIVgag epitopes recognized by circulating CD8+ T cells in individual RMs vaccinated with 68-1 RhCMV/SIVgag or conventional viral vectors—the latter including MVA/SIVgag (N = 11), adenovirus type 5/SIVgag (N = 3), and electroporated DNA/gag plus interleukin-12 (N = 4)—or in RMs with controlled SIVmac239 infection (N = 12). The horizontal bars indicate median values (P values are from unpaired, two-tailed Mann-Whitney tests). (B) Comparison of the number of distinct MHC-E–restricted epitopes (per 100 amino acids of protein length) recognized by circulating CD8+ T cells in individual RMs vaccinated with 68-1 RhCMV vectors expressing each of the indicated antigens (RhCMV IE1 responses were evaluated in CMV-naïve RMs that were administered 68-1 RhCMV/SIVgag). The horizontal bars indicate median values for each group. (C) Population-level analysis of the breadth of MHC-E–restricted SIVgag epitope–specific CD8+ T cell responses across 125 consecutive 15-mer gag peptides (with an overlap of 11 amino acids) in 42 RMs vaccinated with the 68-1 RhCMV/SIVgag vector. (D) Sequence logo indicating the frequency of each amino acid in a given position by the height of the letter, based on 11 optimal, MHC-E–restricted SIVgag 9-mer peptide epitopes recognized by CD8+ T cells in RMs vaccinated with the 68-1 RhCMV/SIVgag vector. Blue indicates significant amino acid enrichment in a given position relative to its background frequency in SIVmac239 gag (see supplementary materials). Green highlights the M2 and L9 of the canonical MHC-E–binding motif. (E) The same logo as in (D), colored according to enrichment (blue or green) or underrepresentation (red) among 551 peptides eluted from HLA-E*01:03 in a TAP-deficient setting by Lampen et al. (22) (fig. S15). Amino acids enriched in the second and C-terminal anchor positions among the 551 peptides from (22) were rare among our 11 optimal SIVgag peptides, whereas those that were significantly underrepresented in (22) were enriched in our SIVgag epitopic peptides, highlighted in the actual optimal epitopes listed on the right. The percentage of RMs vaccinated with 68-1 RhCMV/SIVgag (N = 42) that responded to each optimal peptide is noted as the recognition frequency. Scott G. Hansen et al. Science 2016;351:714-720 Published by AAAS

Fig. 4 Structural analysis of MHC-E–peptide binding. Structural analysis of MHC-E–peptide binding. (A) Structural changes in the binding groove of canonical peptide-bound and -unbound HLA-A*02:01 and HLA-E*01:03. Calculations of changes in the volume of the binding groove indicate that, unlike in HLA-A*02:01, the HLA-E*01:03 binding groove does not collapse when the peptide is not bound during the 0.5-µs all-atom molecular dynamics simulations. Error bars indicate the 95% confidence interval, determined using standard error estimates from five independent simulations for each case. The total volume (in cubic angstroms) is calculated from the cavity volumes from the N, F, and C regions, as shown in the three-dimensional structure on the right and described in the supplementary materials. (B) Root-mean-square fluctuations of the backbone atoms of unbound HLA-A*02:01 and HLA-E*01:03 are mapped on the x-ray structure (-ter, terminus). Consistent with (A), the binding groove of HLA-E*01:03 is less flexible compared with that of HLA-A*02:01. The binding groove helices partially unfold in unbound HLA-A*02:01, whereas the unbound HLA-E*01:03 binding groove remains relatively stable. Increasing flexibility is captured by the change in color gradient from blue to white to red. (C) HLA-E*01:03 binding profile obtained from a Rosetta-based docking approach (30) of 11 optimal, MHC-E–restricted, SIVgag epitopic peptides. The backbones of these peptides adopt a similar conformation, as shown by the colors in front of the binding groove cross-section. The bound conformations for these 11 peptides are shown in the insets. Residues buried in HLA-E and exposed are marked in red and white, respectively. (D) Molecular dynamics simulations of docked complexes show that the 11 epitopic peptides are bound in a slightly elevated position relative to VL9 in the MHC-E binding groove and are more solvent-exposed (inset bar graph). (E) Cross sections (viewed from above) of the MHC-E binding groove at two different depths show the differences in the chemical environment recognized by the buried residues of epitopic peptides and VL9. Unlike the hydrophobic environment experienced by the buried residues of the VL9 peptide, epitopic peptides experience a chemically heterogeneous environment at their slightly elevated position in the binding groove (figs. S22 and S23). Scott G. Hansen et al. Science 2016;351:714-720 Published by AAAS