CD8+ T cell targeting of tumor antigens presented by HLA-E

Abstract

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8⁺ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8⁺ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E⁺ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8⁺ T cell-based immunotherapies.

Fig. 1.. HLA-E expression in PCa.

IHC analysis of PCa or adjacent normal tissue for expression of HLA-E, PAP, and PIN4. TMAs were prepared and processed as described in Materials and Methods. Representative IHC of PCa tumor tissue and adjacent normal tissue are shown for Gleason 7, 8, and 9 tumors stained for HLA-E using a polyclonal antiserum. Pathological tumor (pT) stages of PCa are indicated with pT2c representing localized bilateral PCa, whereas pT3b represents locally advanced PCa. Staining for PAP and PIN4 is additionally shown for the tumor tissue. The results for all TMAs are summarized in data file S1.

Fig. 2.. RhCMV elicits robust and durable T cell responses to TAAs.

(A) Six male RMs were inoculated with 68-1 RhCMV/RhPAP and with 68-1 RhCMV/SIVgag. RMs 1 to 3 were additionally coinoculated with RhCMV/WT-1, whereas RMs 4 to 6 were coinoculated with 68-1 RhCMV/MSLN. At day 140, RMs 1 to 3 received 68-1 RhCMV/MSLN, whereas RMs 4 to 6 received 68-1 RhCMV/WT-1. (B) CD4⁺ and CD8⁺ T cell responses were measured in peripheral blood mononuclear cells (PBMCs) using overlapping peptide pools for each of the antigens by ICS for CD69 and IFNγ and/or TNFα at each of the indicated time points. The background subtracted mean response frequencies are shown (+SEM). Days of inoculation are indicated by arrowheads. Individual response frequencies are shown in data file S3.

Fig. 3.. Epitope specificity and MHC restriction of TAA-targeting CD8⁺ T cells.

CD8⁺ T cell responses in PBMC of RMs 1 to 6 (Fig. 2) were measured by ICS for IFNγ and TNFα to individual overlapping consecutive 15–amino acid oligomer peptides that span the indicated proteins. Assay limit of detection was determined as described previously (51), with 0.05% after background subtraction being the minimum threshold. Above background responses (IFNγ and/or TNFα) are shown by a square along the length of each protein with the peptide numbers shown below. Results of blocking experiments are indicated by the color of the square: Peptides restricted by MHC-II (blue) were blocked with class II-associated invariant chain peptide (CLIP) and anti–human leukocyte antigen-DR (HLA-DR) antibody, whereas MHC-E–restricted peptides (green) were blocked with VL9 peptide and pan–MHC-I antibody. Indeterminate responses are shown in purple. Supertopes, i.e., epitopes recognized in all RMs (including Fig. 5), are boxed in. Overall analysis of 15–amino acid oligomer peptide responses is shown in data file S3. a.a., amino acid.

Fig. 4.. The 68-1.2 RhCMV vectors elicit MHC-Ia–restricted CD8⁺ T cells to RhPAP.

(A) Three male RMs were inoculated with 68-1.2 RhCMV/RhPAP. CD8⁺ and CD4⁺ T cell responses were determined by ICS in PBMC using an overlapping peptide mix for RhPAP at the indicated time points. (B) Memory differentiation phenotypes of RhPAP-specific T cells responding to peptide pools in each RM were determined on the basis of CD28 and CCR7 expression, delineating central memory (T_CM, CD28⁺, CCR7⁺), transitional effector-memory (T_TrEM, CD28⁺, CCR7⁻), and effector-memory (T_EM, CD28⁻, CCR7⁻). (C) CD8⁺ T cell responses and MHC restriction to individual 15–amino acid oligomer peptides from the RhPAP mix were determined as in Fig. 3. All peptide-specific responses were MHC-Ia restricted (shown in red color), i.e., they were blocked by pan–MHC-I antibody but not by VL9 peptide or anti–MHC-II antibodies. Results for each RM in (A) and (B) and overall analysis of 15–amino acid oligomer peptide responses in (C) are listed in data file S3.

Fig. 5.. Rh189-deleted 68-1 RhCMV elicits canonical MHC-Ia–restricted T cells to SIV Gag but not to TAAs.

Six RMs were coinoculated with 68-1 RhCMVΔRh189 expressing the indicated antigens as shown in fig. S3. MHC restriction of individual 15–amino acid oligomer peptides from each antigen for CD8⁺ T cells from four RMs of this cohort was determined as described in Fig. 3. Above background responses are shown as squares at the position along the antigen with the colors indicating blocking by reagents specific for MHC-II (blue), MHC-E (green), or MHC-Ia (red). Supertope peptides are shown by boxes. Indeterminate responses are shown in purple. Overall analysis of 15–amino acid oligomer peptide responses is shown in data file S3.

Fig. 6.. Stimulation of MHC-E–restricted, PAP-specific CD8⁺ T cells by PAP-expressing K562 cells.

(A) Immunoblot of cell lysates of indicated K562-derived cell lines. Expression of endogenous and transfected HLA-E in K562 cells was demonstrated with two HLA-E–specific antibodies MEM-E/02 and 3D12 (5, 90, 91). Note that nontransfected K562 cells express low amounts of HLA-E (88). V5 epitope–tagged RhPAP was detected with PAP-specific (MAB6240, R&D Systems) or V5-specific mouse monoclonal antibodies. A GAPDH-specific mAb was used as loading control. (B) ICS for IFNγ and TNFα of CD8⁺ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cells in the presence or absence of VL9 peptide. (C) Results from five independent experiments showing the average relative frequency (background-subtracted and normalized to K562-E/PAP) of CD69 and IFNγ and/or TNFα-positive CD8⁺ T cells from 68-1 RhCMV/RhPAP-immunized RM responding to the indicated cell lines in the presence or absence of VL9. Individual results are shown in data file S3. Statistical significance was determined by paired t test (not significant, P > 0.05). (D) Immunoblot for HLA-E and FLAG-tagged acid phosphatases. Lysates of stable cell lines of AA7 cells (= K562 cells deleted for HLA-E; fig. S7) transfected with HLA-E alone or together with human PAP, ACP2, or ACPT were electrophoretically separated and probed with the indicated antibodies by immunoblot. (E) ICS for IFNγ and TNFα of CD8⁺ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines. (F) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8⁺ T cells responding to the indicated cell lines were background subtracted and normalized to AA7-E/PAP. Individual results are shown in data file S3. Statistical significance was determined using paired t test.

Fig. 7.. Recognition of PCa cell lines by MHC-E–restricted, PAP-specific CD8⁺ T cells.

(A) Immunoblot of cell lysates of the indicated cell lines for HLA-E, PAP, HLA-Ia, and GAPDH. Antibodies used were MEM-E/02 for HLA-E (90), HC10 for HLA-Ia heavy chains (92), and MA5-15738 (Invitrogen) for GAPDH. The anti-PAP antibody (MAB6240 R&D Systems) detected a lower molecular weight band in DU145 cells that is likely nonspecific since DU145 cells are known to be PAP negative (93, 94). (B) ICS for IFNγ and TNFα production of CD8⁺ T cells from a 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines in the presence or absence of VL9 peptide. In addition, we inhibited potential MHC-II presentation by adding anti-DR and CLIP peptide to all stimulations. (C) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8⁺ T cells from 68-1 RhCMV/RhPAP-immunized RM after background subtraction. The number of repeat experiments is indicated. Individual results are shown in data file S3. Statistical significance was calculated using paired Mann-Whitney U test (not significant, P > 0.05).

Fig. 8.. Recognition of primary PCa cells by MHC-E–restricted, PAP-specific CD8⁺ T cells.

Left: ICS for IFNγ and TNFα production by CD8⁺ T cells from two 68-1 RhCMV/RhPAP-immunized RM after coincubation with PCa cell suspensions in the presence or absence of VL9 peptide. In addition, we inhibited MHC-II presentation by adding anti-DR antibody and CLIP peptide in all stimulations. Right: Summary of results from the indicated number of primary PCa samples showing the average frequency of CD69 and IFNγ and/or TNFα-positive CD8⁺ T cells from 68-1 RhCMV/RhPAP-immunized RM in the absence or presence of VL9 peptide after background subtraction. Individual results are shown in data file S3. Statistical significance was calculated using paired t test.