T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a–specific, HLA-DRB3*0101–restricted CD4+ T cells

Author:

Ahlen Maria Therese12,Husebekk Anne12,Killie Mette Kjær1,Skogen Bjørn12,Stuge Tor B.12

Affiliation:

1. Department of Immunology and Transfusion Medicine, University Hospital of North Norway, Tromsø; and

2. Department of Immunology, University of Tromsø, Tromsø, Norway

Abstract

Abstract T-cell responses have been implicated in the development of HPA-1a–induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a–specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a–specific T cells could be isolated from HPA-1a–immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a–alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3+CD4+ T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a–specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.

Publisher

American Society of Hematology

Subject

Cell Biology,Hematology,Immunology,Biochemistry

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