Processed platelet HPA1a peptides au naturel

All immunoglobulin G (IgG) immune responses against protein antigens are initiated by activated T helper cells that recognize their cognate antigens on antigen-presenting cells (APCs), such as MHC class II positive dendritic cells (DCs) or macrophages.^2,3  APCs function to internalize protein antigen(s) and proteolytically process them into small peptides of approximately 15 amino acid residues in length so that they can be inserted into the antigen-binding groove of MHC molecules and then re-expressed on the APC surface.^2,3  The MHC-peptide complex is then examined by the T cell's receptor (TCR) as they migrate through the spleen or lymph nodes.^2,3  T cells with sufficient TCR:MHC/peptide affinity together with appropriate APC-dependent costimulatory events (eg, CD28-CD80/86 interaction and cytokine production) will become activated and mediate immune effector functions, such as immunoglobulin class switching from IgM to IgG or initiation of cytotoxic T lymphocyte (CTL) activation.^2,3  Extensive groundbreaking research has led to our current understanding of these processing mechanisms and the identification of immunogenic peptides derived from disease-associated protein antigens, with the goal of potentially modifying the peptides into immunosuppressive or tolerogenic moieties for therapy.

Neonatal alloimmune thrombocytopenia (NAIT) results when maternal IgG alloantibodies arise that are reactive with fetal platelet antigens. These antibodies can cross the placenta during pregnancy, leading to fetal thrombocytopenia and an increased risk of bleeding.⁴  The most common cause of NAIT is an incompatibility in the human platelet antigen (HPA)–1 between the mother and the fetus. The alloantigen system HPA-1 is defined as a leucine/proline polymorphism at residue 33 in the β3 integrin. In homozygous HPA-1b1b (Pro33) women carrying an HPA-1a (Leu33) positive fetus, maternal antibodies against HPA-1a can be formed, and alloimmunization occurs in about 10% of HPA-1a–negative pregnant women. Alloimmunization is strongly associated with HLA-DRB3*0101.^5,6  Severe NAIT occurs at a rate of about 1 in 1100 to 1200 of all pregnancies.⁴  Currently, there is no treatment that can prevent alloimmunization.

Little is known about the underlying cellular immune responses that results in anti–HPA-1a antibody production. Several studies have identified synthetically produced peptides that contain the HPA-1a epitope that either bind to the appropriate HLA molecules or can stimulate T helper cells from HPA-1b1b women,^7,,,,–12  but none to date have confirmed whether these peptides are actually produced within APCs. Identification of naturally processed immunodominant epitopes on the integrin sequence containing the HPA-1a polymorphic site would be an important step toward understanding and controlling the immune response to HPA-1a. These types of studies are technically challenging to perform because of the many complex peptide pools displayed by APCs. However, Anani Sarab et al have solved this problem by using APCs pulsed with the HPA-1a–containing PSI domain and applying sophisticated direct biochemical methods to identity any HPA-1a–containing peptides processed and displayed by HLA molecules. They used an HLA-DRB3*0101 homozygous B-cell line that was pulsed with a recombinant HPA-1a PSI domain as antigen. The HLA-DR class II/peptide complexes were recovered, and the peptides eluted for analysis. Reverse phase–high performance liquid chromatography of the peptide pool revealed a heterogeneous mixture of sequences and many fractions were collected and screened by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. They found that the most abundant peptides generated had molecular masses of 1502 to 1831 Da, reflecting typical lengths of 13 to 15 amino acids. Further characterization using a quadrupole ion trap, equipped with an electrospray ion source, enabled sequence determination of the individual peptides. They identified 4 naturally processed and presented HPA-1a peptides with sequences of up to 17 amino acids in length. All spanned the Leu33 polymorphism and each peptide contained the core residues of the predicted dominant Thcell epitope (Trp25-Leu33). The HPA-1a peptides formed a “nested set” around the core epitope, a feature typical of naturally processed peptides derived from other antigens. These types of peptides arise when class II molecules bind and protect a core amino acid sequence while it is still part of relatively large antigen fragments at an early stage of antigen processing. Subsequent trimming of the fragments is variable, producing a characteristic set of peptides with different lengths spanning the core epitope.

The importance of this study is not only that it confirms that the predominant naturally processed HPA-1a peptide presented by APC are similar to the earlier synthetic peptide studies, but also that the efficiency of processing and display of the helper epitope helps to explain the immunogenicity of HPA-1a. Furthermore, such peptides may provide the basis for novel treatments to tolerize corresponding HPA-1a reactive T helper cells in HPA-1b1b women at risk of NAIT with an HPA-1a–positive fetus.