T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting

Abstract

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

FIGURE 1.

Combinatorial peptide library scan of the MEL5 CD8⁺ T-cell clone. A, 6 × 10⁴ C1R A2 cells were pulsed in duplicate with each sublibrary from a decamer CPL (100 μg/ml) at 37 °C. After 2 h, 3 × 10⁴ clonal MEL5 CD8⁺ T-cells were added and incubated overnight. Supernatant was harvested and assayed for MIP-1β by ELISA. Histograms show the S.D. of two duplicate assays. Red bars indicate the “index” amino acid at each of the 10 positions. B, summary box plot of CPL analysis. Numbers 1–10 indicate the amino acid position; the sequence at the top shows the industry standard heteroclitic ELAGIGILTV sequence. Combined data from four replicate assays are shown in the box plot; the size and color of the amino acid in single-letter code indicate how well MEL5 CD8⁺ T-cells responded to each sublibrary in accordance with the key shown on the bottom.

FIGURE 2.

Selection of Melan A_26–35 analog peptides that preferentially activate the MEL5 CD8⁺ T-cell clone. 3 × 10⁴ MEL5 or MEL187.c5 clonal CD8⁺ T-cells were incubated overnight with 6 × 10⁴ C1R A2 B-cells prepulsed with various concentrations of the indicated peptides. Supernatants were harvested and assayed for MIP-1β by ELISA. A, recognition of the ELAGIGILTV peptide and the single-substituted variants FLAGIGILTV, ELTGIGILTV, and ELAGIGIITV. B, recognition of the ELAGIGILTV and FLTGIGIITV peptides. C, recognition of the ELAGIGILTV and FATGIGIITV peptides. Error bars represent the S.D. of duplicate assays and, in most cases, are smaller than the plot symbols.

FIGURE 3.

Binding of Melan A_26–35 analog peptides to HLA A*0201. A, SPR stability assay of HLA A*0201-EAAGIGILTV, HLA A*0201-ELAGIGILTV, HLA A*0201-FLTGIGIITV, and HLA A*0201-FATGIGIITV loaded in parallel on the same BIAcore chip. B, T2 cell surface binding assays for each peptide shown in A using 10 μm peptide (S.D. representative of two experiments). Both peptide binding assays were performed as described previously (34). A standard Student's t test with equal variance and equal distribution revealed that the mean fluorescence intensity (MFI) values for all comparisons were significantly different to each other (p < 0.05).

FIGURE 4.

Binding affinity of Melan A_26–35 analog peptides to the MEL5 and MEL187.c5 TCRs. A, SPR equilibrium binding of soluble MEL5 TCR to HLA A*0201-FLTGIGIITV. B, SPR equilibrium binding of soluble MEL187.c5 TCR to HLA A*0201-FLTGIGIITV. C, SPR equilibrium binding of soluble MEL5 TCR to HLA A*0201-FATGIGIITV. D, SPR equilibrium binding of soluble MEL187.c5 TCR to HLA A*0201-FATGIGIITV. The mean affinity for each interaction is shown (n = 2).

FIGURE 5.

Heteroclitic and analog pMHCI tetramer binding and kinetics at the surface of MEL5 and MEL187.c5 CD8⁺ T-cells. A and B, steady state tetramer binding analysis. 5 × 10⁴ MEL5 (A) or MEL187.c5 (B) CD8⁺ T-cells were stained with either PE-conjugated HLA A*0201-ELAGIGILTV tetramer or PE-conjugated HLA A*0201-FATGIGIITV tetramer (10 μg/ml each) for 20 min at 37 °C. C and D, tetramer on-rate analysis for MEL5 (C) and MEL187.c5 (D) CD8⁺ T-cells. MFI, mean fluorescence intensity. E and F, tetramer off-rate analysis for MEL5 (E) and MEL187.c5 (F) CD8⁺ T-cells. C–F, experimental procedures and curve fitting were performed as described previously (18, 49).

FIGURE 6.

Structural analysis of Melan A_26–35 analog peptides in complex with HLA A*0201. A, structural comparison of HLA A*0201-ELAGIGILTV (complexed with MEL5) and HLA A*0201-FATGIGIITV. The FATGIGIITV peptide is shown as green sticks; the ELAGIGILTV peptide is shown as yellow sticks. The MEL5 TCRα chain is shown as a cyan graphic; the MEL5 TCRβ chain is shown as an orange graphic. The HLA A*0201 helices have been omitted for clarity. Importantly, conformational differences at peptide positions 1, 3, and 8 can be observed between the two peptides. These differences result in an ∼5-fold increase in binding affinity for the MEL5 TCR. B, structural comparison of HLA A*0201-FATGIGIITV and HLA A*0201-FLTGIGIITV. The FATGIGIITV peptide is shown as green sticks; the FLTGIGIITV peptide is shown as red sticks. The MEL5 TCRα chain is shown as a cyan graphic; the MEL5 TCRβ chain is shown as an orange graphic. The HLA A*0201 helices have been omitted for clarity. C, interaction of the PheP1 side chain (green sticks) of the FATGIGIITV peptide with Trp-167 (blue sticks) in the MHC α2 domain and Tyr-59 (blue sticks) in the MHC α1 domain. D, interaction of the ThrP3 side chain (green sticks) of the FATGIGIITV peptide with Tyr-99 (blue sticks) in the MHC α2 domain. E, interaction of the IleP8 side chain (green sticks) of the FATGIGIITV peptide with Trp-147 (blue sticks) in the MHC α2 domain. Data collection and refinement statistics are shown in supplemental Table S1.

FIGURE 7.

The optimized analog peptide FATGIGIITV primes large populations of melanoma-reactive CD8⁺ T-cells. A, 6 × 10⁶ peripheral blood mononuclear cells from healthy HLA A*0201⁺ individuals were pulsed with 100 μm peptide (ELAGIGILTV or FATGIGIITV) for 1 h at 37 °C; CD8⁺ T-cell lines were then grown out for 14 days and stained with PE-conjugated HLA A*0201-ELAGIGILTV tetramer (left panels) or PE-conjugated HLA A*0201-EAAGIGILTV tetramer (right panels) as described under “Experimental Procedures.” Representative data are shown from one donor. Data from 10 additional donors are shown in supplemental Table S2. B and C, specific lysis of MEL 526 (B) or MEL 624 (C) melanoma cells exposed to a CD8⁺ T-cell line from one donor. Data from six additional donors are shown in supplemental Fig. S3. All lysis assays were set up using a range of E:T ratios; effectors were enumerated by staining with the HLA A*0201-EAAGIGILTV tetramer. Error bars are S.D. from three experiments.

FIGURE 8.

FATGIGIITV-primed CD8⁺ T-cells are clonotypically distinct from those primed by the heteroclitic peptide ELAGIGILTV. 5 × 10⁴ cells from HLA A*0201-restricted Melan A-specific CD8⁺ T-cell lines were incubated with LIVE/DEAD® fixable aqua amine-reactive fluorescent dye for 15 min at room temperature, washed once, and stained with allophycocyanin-conjugated HLA A*0201-EAAGIGILTV tetramer. Cells were then stained with peridinin chlorophyll protein-conjugated anti-human CD8 and a panel of anti-human Vβ antibodies for 30 min at 4 °C. Corresponding data for HLA A*0201-ELAGIGILTV tetramer⁺ cells from the same CD8⁺ T-cell lines are shown in supplemental Fig. S4.