*Purpose: Swine thymus transplantation is a promising approach to clinical tolerance for pig-to-human xenotransplantation. In humanized mice, a swine thymus graft supports de novo selection of a diverse, pig-tolerant human TCR repertoire that can mount a vaccination response. A transplanted pig thymus must support a TCR repertoire with robust human-restricted immune function. We hypothesized that human TCRs that are positively selected on swine rather than human MHC may have reduced ability to respond to antigens presented by human antigen-presenting cells. We assessed the impact of positive selection in a pig thymus on a human-restricted TCR.

*Methods: We used a TCR transgenic humanized mouse system to study whether Mart1, a human HLA-A2-restricted TCR that does not crossreact to pig cells in vitro¸ can be positively selected in a pig thymus.

*Results: The Mart1 TCR was positively selected with reduced efficiency in a human HLA-A2- or a pig thymus compared to a human HLA-A2+ thymus. While similar proportions of total thymocytes expressed a GFP expression marker (A2+: 15.0±6.46%; A2-: 19.2±5.85%; Sw: 11.2±6.73%; no significance by ANOVA) in all conditions, Mart1+ cells were enriched in A2+ compared to A2- or pig thymi (A2+: 3.51±0.553%; A2-: 1.25±0.58%; Sw: 0.63±0.48%; A2+ vs. A2-, p = 0.049; A2+ vs. Sw, p = 0.010 by ANOVA). Mart1+ CD8SP thymocytes were enriched in A2+ thymi, indicating selection to the CD8SP fate as expected, but were reduced in A2- or Sw thymi (A2+ 2.28±0.59; A2-: 0.26±0.06; Sw: 0.34±0.24, A2+ vs. A2-, p = 0.034; A2+ vs. Sw, p = 0.029 by ANOVA). However, Mart1+ cells were detected in the periphery at similar frequencies among GFP+ cells in all conditions (CD4: A2+: 10.3±6.25%; A2-: 6.69±2.49%; Sw: 4.53±1.69%; CD8: A2+: 41.5±11.6%; A2-: 45.5±15.6%; Sw: 12.1±7.22%; no significance by ANOVA). The cells were functional and proliferated ex vivo when pulsed with Mart1 peptide.

*Conclusions: Our results suggest that while positive selection of some human-restricted T cells in a transplanted pig thymus may be inefficient, some human-restricted TCRs that are selected, even inefficiently, may be able to repopulate and function normally in the periphery of future thymus-transplanted patients.

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