Non-natural and photo-reactive amino acids as biochemical probes of immune function

Abstract

Wilms tumor protein (WT1) is a transcription factor selectively overexpressed in leukemias and cancers; clinical trials are underway that use altered WT1 peptide sequences as vaccines. Here we report a strategy to study peptide-MHC interactions by incorporating non-natural and photo-reactive amino acids into the sequence of WT1 peptides. Thirteen WT1 peptides sequences were synthesized with chemically modified amino acids (via fluorination and photo-reactive group additions) at MHC and T cell receptor binding positions. Certain new non-natural peptide analogs could stabilize MHC class I molecules better than the native sequences and were also able to elicit specific T-cell responses and sometimes cytotoxicity to leukemia cells. Two photo-reactive peptides, also modified with a biotin handle for pull-down studies, formed covalent interactions with MHC molecules on live cells and provided kinetic data showing the rapid clearance of the peptide-MHC complex. Despite "infinite affinity" provided by the covalent peptide bonding to the MHC, immunogenicity was not enhanced by these peptides because the peptide presentation on the surface was dominated by catabolism of the complex and only a small percentage of peptide molecules covalently bound to the MHC molecules. This study shows that non-natural amino acids can be successfully incorporated into T cell epitopes to provide novel immunological, biochemical and kinetic information.

Figure 1. Chemical structures of the non-natural amino acids used in this study.

Figure 2. HLA western blot following biotin-peptide pull-down.

Panel a. T2 or control cells were treated with reagents indicated below overnight at 37°C and then were exposed or were not exposed to UV light. Lysates were then resolved on SDS gels and blotted for the presence of HLA-A. Lane 1: WT1B-S1F_A in control HLA-A0201-negative cells (KG-1); Lane 2: T2 cells without peptide; Lane 3: T2 cells and WT1B-S1F_A without UV irradiation; Lane 4: T2 cells and WT1B-S1F_A plus 1 min short wavelength UV irradiation; Lane 5: Positive control cells whole lysate with antibody. Panel b. Following photo-activation of peptide, T2 cells were incubated for the times indicated on the gel, prior to lysis and western blot analysis. Lane 1: Positive control cells with whole lysate and antibody; Lane 2: T2 cells without peptide. Lanes 3–5: T2 cells and WT1B-S1F_A plus 1 min short wavelength UV incubated for 0, 18, and 24 hr before analysis. Panel c. Conducted as in panel ‘b’ but using the WT1B-S1F_bz peptide. Lane 1: T2 cells without peptide; Lane 2: WT1B-S1F_bz with no UVirradiation; Lanes 3–5: T2 cells and WT1B- S1F_bz plus 30 min long wavelength UV followed by incubation for 0, 24, and 48 hr before analysis.

Figure 3. CD8+ IFN-γ ELISPOT from an HLA-A0201 donor.

The assay was conducted as described in Materials and Methods. T cells were stimulated in vitro with the WT1J peptides shown in the X axis and then challenged with the peptides shown in the legend. The Y axis represents the number of spots per 1×10⁵ CD8+ T cells.

Figure 4. Cytotoxicity assay with CD8 T cells from an HLA-A0201 donor after three stimulations in vitro.

The assay was conducted as described in Materials and Methods. The X axis shows different ratios between T cells to target cells. The targets and the peptides used to pulse them are shown in the legend. The Y axis shows percentage of cytotoxicity.