The nonpolymorphic MHC Qa-1b mediates CD8+ T cell surveillance of antigen-processing defects

Abstract

The nonclassical major histocompatibility complex (MHC) Qa-1b accommodates monomorphic leader peptides and functions as a ligand for germ line receptors CD94/NKG2, which are expressed by natural killer cells and CD8+ T cells. We here describe that the conserved peptides are replaced by a novel peptide repertoire of surprising diversity as a result of impairments in the antigen-processing pathway. This novel peptide repertoire represents immunogenic neoantigens for CD8+ T cells, as we found that these Qa-1b-restricted T cells dominantly participated in the response to tumors with processing deficiencies. A surprisingly wide spectrum of target cells, irrespective of transformation status, MHC background, or type of processing deficiency, was recognized by this T cell subset, complying with the conserved nature of Qa-1b. Target cell recognition depended on T cell receptor and Qa-1b interaction, and immunization with identified peptide epitopes demonstrated in vivo priming of CD8+ T cells. Our data reveal that Qa-1b, and most likely its human homologue human leukocyte antigen-E, is important for the defense against processing-deficient cells by displacing the monomorphic leader peptides, which relieves the inhibition through CD94/NKG2A on lymphocytes, and by presenting a novel repertoire of immunogenic peptides, which recruits a subset of cytotoxic CD8+ T cells.

Figure 1.

Selective recognition of Qa-1^b-expressing target cells. Isolated T cell clones were tested against panels of TAP-deficient lymphoma (EC7.1) and melanoma (B78H1) cells. Both cell lines are also deficient in MHC class I (Howell et al., 2000; Chiang et al., 2003) and were reconstituted with constructs encoding H-2D^b, -K^b, or Qa-1^b. Cytotoxic activity against Qa-1^b–expressing EC7.1 cells (A) and IFN-γ production against Qa-1^b-expressing B78H1 cells (B) by three independent T cell clones. Control Qdm-specific CTL failed to recognize the Qa-1^b expressing targets because of the absence of TAP, unless pulsed with the Qdm peptide (right). Means and standard deviations of triplicate wells are shown for one out of three comparable experiments.

Figure 2.

The T cell receptor, but not NKG2A/C, mediates reactivity of the T cells. (A) Blocking antibody against Qa-1^b demonstrated a direct role of this molecule for T cell recognition. Experiment was performed using EC7.1.Qa-1^b target cells and repeated three times with similar outcome. (B) Blocking CD3 or CD8 with monoclonal antibodies decreased T cell reactivity against TAP-deficient EC7.1.Qa-1^b target cells. Antibodies against NKG2A/C did not alter the T cell response, indicating that only the T cell receptor is critically involved in mediating reactivity. Data are representative of four experiments. (C) Exogenous peptide loading competes with endogenously presented epitopes on EC7.1.Qa-1^b cells and inhibits the recognition of these target cells by Qa-1^b–restricted CTL (left). Qdm (AMAPRTLLL) or Qdm L8K (AMAPRTLKL) peptides were loaded exogenously at the indicated concentrations on EC7.1.Qa-1^b cells and IFN-γ release by CTL was measured. Control peptide was the K^b-binding 8-mer SIINFEKL from OVA. The Qdm L8K mutant peptide–Qa-1^b complexes fail to interact with CD94/NKG2A or CD94/NKG2C (Kraft et al., 2000), indicating that the loaded peptides interfere with T cell receptor–mediated reactivity. Qdm-specific control CTL (right) was activated by the peptides. Means and standard deviations of triplicate wells are shown for one out of three comparable experiments.

Figure 3.

Qa-1^b–restricted CTL are reactive against cells with defined processing deficiencies. (A) LPS-stimulated B cell blasts from wild-type (B6), TAP1^−/−, and β2m^−/− mice were used as targets for Qa-1^b–restricted CTL (left) or control Qdm-specific CTL (right). (B) LPS-stimulated B cell blasts from wild-type (B6), MHC class I knockout, and MHC class I/β2m knockout mice were tested for recognition by Qa-1^b–restricted CTL (left) or control Qdm-specific CTL (right). Graphs display representative experiments out of four performed. Means and standard deviations of triplicates are shown.

Figure 4.

Partial deficiencies in the processing pathway of tumor cells induce the novel Qa-1^b–presented peptide-epitopes. (A) Qa-1^b–restricted CTL respond to Ad5-transformed mouse cells (Ad5MEC) and fibrosarcoma cells (MCA) that are derived from TAP1 knockout mice. Recognition was lost upon gene transfer of mouse TAP1 in these cell lines (+ TAP1). Qdm-specific control CTL displayed opposing specificity, indicating that the Qdm peptide is only presented on TAP-proficient tumor cells (right). (B) Four colon carcinoma cell lines from BALB/c (C26 and CC36) or C57BL/6 (MC38 and CMT93) background were used as targets for both CTL types. Pretreatment with IFN-γ to boost the antigen-processing and presentation machinery resulted in decreased reactivity by the Qa-1^b–restricted CTL, whereas gene transfer of the viral TAP-inhibitor UL49.5 (van Hall et al., 2007) led to strongly increased recognition. Again, Qdm-specific control CTL displayed opposing specificity (right). Means and standard deviations of triplicates are shown from one representative experiment out of four.

Figure 5.

Identification of the TAP-independent peptide repertoire of Qa-1^b. (A) The chimeric Qa-1^b/D^b class I molecule was expressed in EC7.1 cells, which are TAP- and MHC class I–negative. TAP-positive counterparts were generated by introduction of the TAP2 gene. Surface display on EC7.1.Qa-1^b/D^b cells of the chimeric molecule and absence of the endogenous D^b molecule was determined by flow cytometry with Qa-1^b– and D^bα3-specific antibodies and D^bα2-specific antibody, respectively, as indicated in the histogram plots (clone KH95 and also H131-31; not depicted). Staining of TAP-reconstituted cells gave comparable results. Control TAP-proficient RMA cells displayed endogenous Qa-1^b and D^b molecules. (B) TAP expression was analyzed on RMA, RMA-S, EC7.1, and EC7.1.TAP2 cells by Western blot using the antibody TAP2.688 against mouse TAP2. Results confirmed the lack of TAP2 expression on EC7.1 and RMA-S cells. (C) The chimeric construct was recognized by CTL clones: the TAP-negative variant only by Qa-1^b–restricted CTL (black bars) and the TAP-positive variant only by Qdm-specific CTL (gray bars). Means and standard deviations of triplicate wells are shown from one representative experiment out of four. (D) Peptide purification and MS analysis revealed the wide diversity of the TAP-negative peptide repertoire, whereas the TAP-positive peptide repertoire was mainly limited to the Qdm peptide. Data were collected from four independent experiments in the case of TAP-negative repertoire and two independent experiments for TAP-positive repertoire. Number of different peptides with indicated length is depicted of 84 identified peptides that are listed in Table II.

Figure 6.

Immunogenicity of Qa-1^b–presented peptides in vitro and in vivo. (A) Immunogenicity of 59 peptides was determined by compiling the IFN-γ response of 25e T cell cultures, which were induced by immunization with irradiated TAP-negative EC7.1.Qa-1^b.B7 cells. Responses were measured using a matrix-based approach in which each peptide was represented in two independent pools. T cell responses against each pool were scored from zero to three and compiled scores from the two pools were multiplied. (B–D) Percentage of IFN-γ–producing CD8⁺ T cells in the blood of mice that were naive (B) or immunized with the indicated peptide (C). Blood samples were taken and cells were stimulated overnight with medium or specific peptide, stained with antibodies, and analyzed by flow cytometry. Collected data from peptide-specific frequencies of CD8⁺ cells from mice are depicted in (D). Each data point represents one mouse, and data from two independent experiments is shown. (E–G) Cytotoxic reactivity in vivo in the same mice as shown in D. Specific killing was determined in naive (E) or immunized mice (F) by comparing the numbers of CFSE^high targets, which were loaded with relevant peptides, to CFSE^low targets, which were not loaded with peptide. CFSE^intermediate targets were loaded with Qdm and were always comparable to targets without peptide. The means of percentage in vivo killing is depicted with standard deviations (G).