Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo

Abstract

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8⁺ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo.

Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay.

Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC₅₀ value of 21 μm or lower. The peptides elicited an interferon-gamma response in vivo.

Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

Keywords: Graft-versus-host disease; Hematopoietic stem cell transplantation; Human leukocyte antigen-DPB1; Predicted Indirectly ReCognizable HLA Epitopes-I; T-cell epitopes.

Fig. 1.

Overview of HLA types used as inputs for the PIRCHE algorithm. a HLA-DPB1 allele frequencies in the DKMS cohort (20). DPB1 alleles with an allele frequency <0.02 (15.65%) include the following HLA-DPB1 alleles with an allele frequency higher than 0.1%: DPB1*05:01, DPB1*13:01, DPB1*17:01, DPB1*10:01, DPB1*14:01, DPB1*11:01, DPB1*06:01, DPB1*19:01, DPB1*09:01, DPB1*15:01, DPB1*23:01, DPB1*16:01, DPB1*20:01, and DPB1*02:02. b Example of the input for the PIRCHE algorithm. Depicted is the first patient genotype, which included the HLA-DPB1*04:01 allele, combined with 19 different donors with the same HLA-A, -B, -C, -DRB1, and -DQB1 alleles, but differing in their HLA-DPB1 allele. The other 18 patient genotypes consisted of the same HLA-A, -B, -C, -DRB1, and -DQB1 typing, but differed in their HLA-DPB1 allele (not shown).

Fig. 2.

Log dose-inhibition curves with indicated IC₅₀ values from the inhibition data of all test peptides (b–f) and negative and positive control peptides (g, h) for the HLA-A*02:01 allele. IC₅₀ values for binding to HLA-A*02:01 were obtained by performing nonlinear regression analysis on the inhibition data of all peptides. Error bars indicate standard deviations. a Overview of the log dose-inhibition curves of all tested peptides including negative and positive control peptides shows variation in binding affinities between the five tested HLA-DPB1-derived peptides. b–f Log-inhibition curves for HLA-A*02:01 binding of RMCRHNYEL (b), YIYNREEFV (c), YIYNREELV (d), YIYNREEYA (e), and YIYNRQEYA (f) peptides having IC₅₀ values of 21.0 μm, 4.9 μm, 3.9 μm, 10.6 μm, and 19.9 μm, respectively. g, h Log-inhibition curves for HLA-A*02:01 binding of negative (KVNVSPSKK) (g) and positive (VLHDDLLEA) (h) control peptides. The negative control peptide did not show any binding to the HLA-A*02:01 allele and the positive control peptide showed binding to HLA-A*02:01 with an IC₅₀ value of 3.9 μm.

Fig. 3.

HLA-DPB1-derived peptides RMCRHNYEL, YIYNREEFV, YIYNREELV, and YIYNRQEYA loaded on T2A2 cells elicited a T-cell response in HLA-A*02:01 transgenic mice. T-cell alloreactivity against immunized HLA-DPB1-derived peptides in HLA-A*02:01 mice was examined using an IFN-γ ELISpot assay with peptide-loaded T2A2 cells. Spot-forming cells for the immunized peptides were corrected for the mean signal from a nonimmunized control peptide (*p < 0.05; **p < 0.01; ***p < 0.001).