Extended Data Fig. 8: Mixed lymphocyte reaction (MLR) results at post-transplant time points and evidence for recipient thymopoiesis in the explanted thymokidney.

(A) Percentages of donor-reactive CD4⁺ and CD8⁺ T cells in FCM CFSE dye dilution MLR assays performed at POD28 and POD49. Pig-specific responses declined between POD28 and 49 (mean of frequency ± SD is shown for two independent experiments) and was lower than responses to third party allogeneic human stimulators at the same time points. (B) FCM plots showing CFSE dilution of responding recipient T cells at POD49 vs donor pig. CD4⁺ T cells were identified as CD8^— and γδ^— T cells. (C) Anti human CD45 antibody specificity control using pig thymocytes. (D) FCM plots for human CD45 FMO and (E) CD45-stained tube (n = 499,000 cells). (F) Cell suspension from explanted thymokidney tissue stained for various markers. In Tube 1, thymocytes were gated on (i) live cells and (ii) human CD45⁺ cells. From (iii), which comprised 0.48% (n = 33 cells), we analyzed CD4 vs CD8 (iv), revealing 18.2% (n = 6 cells) double-negative (DN), 30.3% (n = 10 cells) double-positive (DP), 33.3% (n = 11 cells) single-positive CD4 (SPCD4), and 18.2% (n = 6 cells) single-positive CD8 (SPCD8) cells. The DN population was further gated and expression of CD34 (0%) (v), CD38 (83.3%, n = 5 cells) (vi) and CD1a (0%) (vii) is shown. The presence of these markers is consistent with the presence of early DN thymocyte populations and the presence of DP cells is consistent with active thymopoiesis. In Tube 2, we gated on thymocytes (i), live cells (ii), and human CD45⁺ and pig CD3^- cells (0.21%, n = 298 cells). (G) FCM analysis of pre-transplant decedent PBMCs to demonstrate percentages of CD4⁺ and CD8⁺ cells, which comprised 74% and 20.0% of CD3⁺ cells, respectively. Gates used for adjacent plots are shown in red polygons.