Friday October 11, 2019 from 16:45 to 17:45
Human T cell repertoire selection in porcine thymus grafts
Mohsen Khosravi Maharlooei1, Aleksandar Obradovic1, Haowei Li1, Markus Holzl1, Nichole Danzl1, Grace Nauman1, Christopher A. Parks1, Kazuhiko Yamada1, Megan Sykes1.
1Columbia Center for Translational Immunology, Columbia University, New York, NY, United States
Introduction: T cells selected on porcine (SW) MHC in pig thymus may not optimally recognize antigens presented by human MHC (HLA) in the periphery. Our group has previously shown defects in Tregs that develop in a xenogeneic thymus. Also, as the there are no human medullary thymic epithelial cells (mTECs) in the transplanted pig thymus, we hypothesized that there would be lack of negative selection of human tissue restricted antigen (TRA)-specific T cells and also a lack of Tregs specific for human TRAs. To address these, we used humanized mice generated with human hematopoietic stem cells (HSCs) and swine (SW) thymus grafts (SW/HU mice) and compared them to HU mice prepared with autologous human fetal thymus (HU/HU mice). We performed high throughput TCRβ CDR3 sequencing on thymic and peripheral T cell subsets of these mice to compare TCR repertoires.
Methods: We generated HU/HU vs SW/HU mice with the same human HSCs in each group. The native mouse thymus was removed. In preliminary studies, we investigated human T-cell repertoire formation using high throughput TCRβ CDR3 sequencing in HU/HU mice with autologous vs allogeneic and porcine thymi.
Results: While similar proportions of SP-CD4 and SP-CD8 cells were present in SW and HU thymus grafts, SW/HU mice had lower proportions of CD8 cells among T cells and of Tregs among CD4 cells in the periphery. Increased responsiveness to human TRAs was observed among human T cells of SW/HU compared to HU/HU mice. Replicate HU/HU mice generated diverse and highly divergent TCRβ repertoires. Repertoire narrowing and increased CDR3β sharing between mice and T cell subsets was observed during thymocyte selection. While hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo-crossreactive and autoreactive CDR3βs. Preliminary CDR3β TCR sequencing to compare HU/HU vs SW/HU mice revealed diverse and highly divergent repertoires in both groups. Diversity was reduced in peripheral T cell subsets compared to thymocytes ones in both groups, indicating that post-thymic selection occurred. Sharing was similar between mice with replicate autologous vs xenogeneic thymus.
Conclusion: Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. Initial studies suggest that SW thymi have a similar trend for positive selection of cross-reactive TCRs. Defects in post-thymic interactions and tolerance to human TRAs among T cells selected in SW thymus might be overcome by the addition of human TECs to porcine thymus grafts.