Bootcongres

After organ transplantation, donor alloantigens can be recognized by recipient T cells directly on donor cells, or indirectly as allopeptides presented by MHC on recipient antigen presenting cells (APCs). From experimental animal studies evidence is emerging showing the importance of indirect allorecognition in chronic rejection, donor-specific antibody production and transplant tolerance. However, studies on indirect allorecognition in patients are hampered by the lack of a reliable assay to detect T cells with indirect allospecificity. The aim of this study is to set up a novel technique based on messenger RNA (mRNA) electroporation to measure indirect T cell responses after organ transplantation.

For this purpose, we synthesized mRNA encoding human leukocyte antigen (HLA) A*0201 by in vitro transcription from a DNA template. To exclude the possibility of direct allorecognition, the transmembrane region of the HLA-A*0201 α-chain was deleted from the DNA construct, while the human DC-LAMP gene was included to obtain translocation of the translated protein to the MHC class II compartments. By stimulation with CD154-expressing fibroblasts in the presence of IL-4, peripheral blood B cells of HLA-A*0201^-/HLA-DRB1*0101⁺ donors were expanded and differentiated into antigen presenting B cell blasts (CD40-B). These CD40-B were electroporated with the HLA-A*0201-encoding mRNA and co-cultured with two different CD4⁺ T cell clones recognizing HLA-A*0201 derived peptides presented in HLA-DRB1*0101. After electroporation, HLA-A*0201 was not expressed on the surface of the CD40-B but was detected intracellularly as soon as 2 hours after electroporation. Both CD4⁺ T cell clones produced high amounts of IFN-γ upon co-culture with electroporated CD40-B, similar to the IFN-γ production in response to positive control CD40-B (HLA-A*0201⁺/HLA-DRB1*0101⁺). No IFN-γ was detected in co-cultures of T-cell clones with mock electroporated HLA-A*0201^-/HLA-DRB1*0101⁺ CD40-B. This technique is to be validated on T cells of transplanted patients. 

In conclusion, we set up a novel technique to measure T cells with indirect allospecificity using mRNA electroporation. This technique might be promising to study indirect allorecognition in patients after organ transplantation, and to generate regulatory T cells with indirect allospecificity.