Extended Data Figure 8. Cytotoxicity and the two morphs of morula cells (MC) at point of rejection (POR).

a, Cytotoxicity assays of isolated LGL cells compared to small cells, and to isolated MC. In both cases the LGL cells had significantly higher cytotoxicity compared to the other cell types. The experiment with isolated cells was performed twice with triplicates. Unpaired T-test, two-tailed *P=0.003, **P=0.0013, Mean. b, LGL cells were isolated (upper left) and incubated overnight either in syngeneic (upper right) or in allogeneic challenge (lower left). FSC/ SSC analysis of LGL cell (lower population) and MC (upper population). Small caption of light microscopy pictures of cells after the incubation. (Lower right) Analysis of LGL and morula cells in syngeneic compare to allogeneic challenge. Experiment was performed once with duplicates and validated by light microscopy. Mean. c, An H&E section of B. schlosseri colonies undergoing rejection. In the ampule (AMP) the inactivated form of cytotoxic MC/ large granular lymphocytes like cells (LGL) can be observed (left). On the other hand the activated form with the brown pigmentation of MC can be observed at POR (right). d, Confocal imagery of phagocytosis assays to validate the allogeneic engulfment. In the panels colony labeled with CFSE in green and colony labeled with DiD in red after allogeneic phagocytosis assay. Large phagocytic cells can be seen after engulfment of allogeneic cells or vesicles. Experiment of allogeneic phagocytosis validation by confocal imaging was performed twice. Scale bar 20 μm, e, Example of cytotoxicity assay in different Effector to Target ratios (E:T), where the targets are compatible or rejecting colony cells to the effector colony. In the rejecting colony, specific lysis is significantly higher. The experiment was performed three times with triplicates. ANOVA two-factor with replication, *P=0.0015, Mean, SD.