Broadly targeted CD8⁺ T cell responses restricted by major histocompatibility complex E

Abstract

Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8αβ(+) T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E-restricted CD8(+) T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

Fig. 1. MHC restriction of strain 68-1 RhCMV/SIVgag-elicited CD8+ T cells

(A, B) PBMC from a representative strain 68-1 RhCMV/SIVgag-vaccinated RM (Rh22034, of 4 similarly analyzed, see fig. S3) were stimulated with the indicated epitopic 15mer peptides pulsed onto the surface of parental MHC-I-negative cell lines (.221 and K562; 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 IFN-γ and/or TNF-α production by flow cytometric ICS assay (response frequencies of gated CD8+ T cells 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 9mer epitopic peptides, and combined with PBMC from 3–4 68-1 RhCMV/SIVgag-vaccinated RM for flow cytometric ICS determination of the frequency of responding CD8+ T cells (IFN-γ+ and/or TNF-α+). Response frequencies at each peptide dose were normalized to the response observed with the transfectant pulsed with highest concentration (10μM) of peptide.

Fig. 2. 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 15mer gag peptides (with 11 amino acid overlap) in RM vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIVmac239 (n = 4–6 per group shown; see fig. S12 for other studied RM). 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 anti-pan-MHC-I mAb W6/32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP (see Methods). 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 RM vaccinated with the indicated SIVgag-expressing viral vectors or infected with SIV. The flow profiles at left show IFN-γ and TNF-α production following CD8β+ T cell incubation with autologous SIVmac239-infected CD4+ T cells alone (no block), or in the presence of the pan-MHC-I-blocking mAb W6/32 plus the MHC-II-binding CLIP peptide (W6/32 + CLIP), or MHC-E-binding peptide VL9 plus CLIP (VL9 + CLIP). All plots are gated on live, CD3+, CD8+ cells. The bar graph at right shows the results from all studied RM.

Fig. 3. Diversity of MHC-E-restricted epitopes

(A) Comparison of the total number of distinct MHC E- (green) vs. MHC-Ia (red)-restricted SIVgag epitopes recognized by circulating CD8+ T cells in individual RM vaccinated with strain 68-1 RhCMV/gag vs. conventional viral vectors, the latter including MVA/gag (n = 11), Ad5/gag (n = 3) and electroporated DNA/gag + IL-12 (n = 4), or in RM with controlled SIVmac239 infection (n = 12). The horizontal bars indicate median values (p values from unpaired, two-tailed Mann-Whitney test). (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 RM vaccinated with strain 68-1 RhCMV vectors expressing each of the indicated antigens (note: RhCMV IE1 responses were evaluated in CMV-naïve RM administered strain 68-1 RhCMV/gag). 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 15mer gag peptides (with 11 amino acid overlap) in 42 strain 68-1 RhCMV/gag vector-vaccinated RM. (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 9mer peptide epitopes recognized by CD8+ T cells in strain 68-1 RhCMV vector-vaccinated RM. Blue indicates significant amino acid enrichment in a given position relative to their background frequency in SIVmac239 Gag (see Methods). Green highlights the 2M 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) (see fig. S15). Amino acids enriched in the 2^nd and C-terminal anchor positions among the 551 Lampen et al. peptides were rare among our 11 optimal SIVgag peptides, while those that were significantly underrepresented were enriched, highlighted in the actual peptides on the right. The percentage of strain 68-1 RhCMV/gag-vaccinated RM (n = 42) that responded to each optimal peptide is noted as the “Recognition Frequency”.

Fig. 4. 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 binding groove indicate that, unlike HLA-A*02:01, the HLA-E*01:03 binding groove doesn’t collapse when the peptide is not bound during the 0.5 microsecond all-atom molecular dynamics simulations. Error bar indicates 95% confidence interval using standard error estimates from five independent simulations for each case. Total volume is calculated from the cavity volumes from N, F and C, as shown in the three-dimensional structure and described in the Methods. (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. Consistent with (A), the binding groove of HLA-E*01:03 is less flexible compared to 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 with multiple colors in front of the binding groove cross-section. The bound conformations for these 11 peptides are shown in the colored 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 in the MHC-E binding groove compared to VL9 and are more solvent-exposed (inset bar graph). (E) Cross-section (view from top) 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 VL9 peptide, epitopic peptides experience a chemically heterogeneous environment at their slightly elevated position in the binding groove (see figs. S22 and S23).