T cell responses to human platelet antigen–1a involve a unique form of indirect allorecognition
Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor Brynjar Stuge
Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor Brynjar Stuge
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Research Article Immunology

T cell responses to human platelet antigen–1a involve a unique form of indirect allorecognition

Abstract

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloantigen on fetal platelets. In most cases the alloantigen is formed by a single amino acid, integrin β3 Leu33, referred to as human platelet antigen–1a (HPA-1a). Production of anti–HPA-1a antibodies likely depends on CD4+ T cells that recognize the same alloantigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of antigen recognition by HPA-1a–specific T cells, we assayed native and synthetic variants of the integrin β3 peptide antigen for binding to DRA/DRB3*01:01-positive antigen-presenting cells and for T cell activation. We found that HPA-1a–specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.

Authors

Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor Brynjar Stuge

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Figure 2

HPA-1a–specific T cells do not exclusively recognize the Leu33 residue in the HPA-1a peptide.

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HPA-1a–specific T cells do not exclusively recognize the Leu33 residue i...
(A) CFSE-labeled HPA-1a–specific T cell clones were stimulated with DRA/DRB3*01:01-positive APCs (D4BL4) pulsed with a panel of designed peptides in different concentrations, without MLE. T cell proliferation was determined after 7 days, illustrated by 1 of 4 experiments (D7T4). (B) T cell activation was also measured by intracellular cytokine staining (IFN-γ) by flow cytometry. T cells were gated by light scatter and CFSE staining. One representative of 3 experiments with 10 μM peptide pulsing of D4BL4 cells, and (C) the effect of peptide pulsing concentrations on T cell activation. (D) Binding of peptides to D4BL4; same cell line used for T cell activation. Cells were pulsed with biotinylated peptides (10 μM) with or without AdEtOH, stained with Streptavidin–R-PE, and analyzed in flow cytometry. The discrimination of peptide-binding efficiency is enhanced when using AdEtOH as an MLE. Substitutions of Leu33 to Arg/Glu reduced binding, while efficient binding was seen with substitutions with small, aliphatic residues Leu33 to Val/Ile. Comparative binding experiments were conducted twice with 2 replicates; representative histograms are shown. Twelve-mer peptides were used in all experiments. HPA, human platelet antigen; MLE, MHC-loading enhancer; AdEtOH, adamantane ethanol.