Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice

Abstract

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Figures

Figure 1

Structures and sequences of PR3 and HNE proteins. (A) Native forms of PR3 and HNE are shown as well as versions engineered for expression as fusions to the C-terminal of EGFP in rVV. Amino acid sequences are shown at the termini of the mature proteases as well as within the signal peptide, pro-dipeptide, and propeptide segments cleaved off during cellular processing (reviewed in Korkmaz et al). The positions of the PR1 and PR2 epitopes are indicated in addition to amino acid substitutions introduced to inactivate the enzymatic activity. (B) Alignment of amino acid sequences and consensus of human and murine proteinase 3 and neutrophil elastase ORFs. The alignment uses the unprocessed forms of the polypeptides with N-terminal signal peptides, prodipeptides, and C-terminal propeptides still present. The regions of the PR1 epitope (completely conserved across the 4 proteins) and the PR2 epitopes (nonconserved) are indicated in black boxes.

Figure 2

WB analysis of recombinant PR3 and HNE expression. CV1 cells were infected with rVVs expressing HNE, PR3, and PR3-T fused to EGFP. Cytoplasmic (C) and nuclear (N) fractions were prepared 24 hours after infection with rVVs at an MOI of 1. The blot was probed with an Ab to HNE (A) or to EGFP (B). Apparent molecular weights are indicated in kilodaltons. This image is representative of 3 rVV infection experiments.

Figure 3

Deconvolution of immune responses to PR3. Flow analyses of ICC assays analyzing responses of rVV-EGFP-PR3–immunized HHDII mice to a PR3 peptide library. Splenocytes from immunized mice were expanded for 1 week in culture by stimulation with the whole PR3 library (A) and aliquots of these cultures tested, first by restimulation with peptide pools (D-G), and then in a separate experiment, with single peptides (B-C, H-K) followed by labeling with Abs to CD8 and IFN-γ. The robust response to the PR3 library was shown to be because of 2 peptides (64 and 65) within peptide subpool D (H-I). The minimal cytotoxic epitope common to these 2 peptides was shown to be a 10-mer peptide termed PR2 (K). This deconvolution of PR3 responses to the PR2 epitope was performed in 2 repeat experiments, each using at least 3 mice.

Figure 4

PR1-specific and PR2-specific T cells are detectable in CML patients. (A) Side-by-side plots showing CD8+ T cells specific for the HLA-A2–restricted epitopes PR1 and PR2 as measured by ex vivo ICC on PBMCs from 27 CML patients using peptides as restimulation Ags (Table 1). For each of the 2 Ags, subjects were divided into 2 groups according to whether they had been treated by HSCT. Background responses to an irrelevant HIV-1 gag HLA-A2 epitope peptide were subtracted. The geometric mean percentage of CD8+ T cells recognizing each of the epitopes is indicated, and the error bars indicate the 95% confidence interval on the log scale. The dashed line at 0.01% indicates the detection limit of the assay. P values were calculated using the Mann-Whitney 2-tailed test. (B) Scatter plot of PR1 IFN-γ responses and PR2 IFN-γ responses. The Pearson correlation coefficient (r = 0.66, P < .0002) is calculated using the logarithmic scale. In both panels A and B, the zero values were set to 0.001 for graphical presentation.

Figure 5

Immunodominance of PR2 over PR1 in rVV-EGFP-PR3–immunized mice. (A) Naturally processed PR3 protein was used as Ag for IVS. HHDII mice were immunized with rVV-EGFP-PR3, and splenocytes were expanded for 1 week in culture by stimulation with K562-A2PR3 cells. The cultures were then tested in ICC assays using irrelevant HIV-gag control peptide (i), or with PR1 peptide (ii), or with PR2 peptide (iii) as Ags. The IVS cultures contained PR2-specific CD8+ T cells but not PR1-specific CD8+ T cells. (B) Induction of PR1-specific CD8+ T cells by immunization with vaccinia expressing different proteases. Groups of HHDII mice were immunized with rVV-EGFP-PR3 (i), or with rVV-EGFP-PR3-T (ii), or with rVV-EGFP-HNE (iii). Splenocytes from all 3 groups were expanded by cocultivation with K562-A2 cells loaded with PR1 peptide and after expansion compared in ICC assays using PR1 peptide as Ag. These results are representative of 2 experiments using 3 or 4 mice.

Figure 6

Comparison of PR1 and PR2 peptides for HLA-A2–binding affinity and stability of the MHC-I complexes on the surface of T2 cells. (A) Flow cytometric histogram showing labeling of HLA-A2 on the surface of T2 assays using FITC-conjugated Ab to HLA-A2 following incubation of the cells with 100μM PR1 or PR2 peptides. A known HLA-A2 binding CMV pp65 CTL epitope peptide and a HLA-B7–restricted CMV peptide were used as positive and negative controls. (B) Titration of peptide concentration in the assay of panel A. The binding affinity of the pp65, PR1, and PR2 epitope peptides for HLA-A2 was not significantly different as determined by 1-way ANOVA tests. (C) Time-course experiment to investigate stability of peptide-HLA-A2 complexes following washout of peptide from the T2 cell cultures. Experiments were performed twice each, at peptide concentrations of 3, 6, 12, 25, and 50μM. Panel C shows the experiment using 25μM peptide concentration. The other conditions gave comparable data (data not shown). For further details, see “Peptide-binding assay using T2 cells.”

Figure 7

PR2-specific T cells expanded from PADRE-PR2 fusion peptide immunized mice are functional CTL. PR2-specific murine T cells are cytotoxic to human HLA-A2 LCL infected with rVV expressing PR3 Ag though not HLA-A2 LCL infected with rVV expressing irrelevant EGFP Ag (left panel). PR2 effectors also killed HLA-A2 LCL target cells loaded with PR2 peptide but not an irrelevant peptide (right panel). PR2 effectors were cytotoxic to K562-A2PR3 cells that endogenously express PR3, though not K562-A2 cells (central panel). Each line corresponds to the mean values of 3 individual mice. Error bars indicate the SEM. The specificities of the effector cells for rVV-EGFP-PR3–infected targets versus rVV-EGFP–infected targets, for K562-A2PR3 versus K562-A2 targets, and for PR2 peptide-pulsed targets versus gag peptide-pulsed targets were statistically significant (P < .01) as determined by 1-way ANOVA tests.