Fig 2: Rat thymus formation using rat ESCs and Foxn1Cas9; Rosa26_ms embryos by blastocyst complementation. (A) Scheme of generation of rat thymus using the Cas9-sgRNAms system. (B) Thymus in Foxn1Cas9; Rosa26_ms2 mouse host with rat ESC chimera at day 5 post birth (right). EGFP signals indicate the rat-derived cells. Rosa26_ms2 mouse littermate was used as controls (left). BF means bright field. (C) Thymus sections in interspecies chimeras. Anti-K5 antibody was used to detected mouse and rat medullary thymic epithelial cells and anti-K8 antibody for mouse cortical thymic epithelial cells but not rat cortical thymic epithelial cells. The EGFP signal indicates rat thymic epithelial cells in the interspecies chimera. Hoechst indicates the nucleus. The bars show 2.5 mm in (B) and 400 µm in (C). (D) FCM analysis of splenocytes in Foxn1Cas9; Rosa26_ms2 mouse host with rESC chimera at day 5 post birth. (E) Percentages of the mouse CD3-positive cells in the mouse CD45-positive splenocytes. (F) The ratio of mouse CD4 and mouse CD8 in the splenocyte. Wild type mouse (mWT, n = 5), Foxn1Cas9; Rosa26_ms2 mouse (mCas; ms2, n = 5), wild type mouse host with rat ES chimera (mWT-rat, n = 3), Foxn1Cas9; Rosa26_ms2 mouse host with rat ESC chimera (mCas; ms2-rat, n = 4) were analyzed in E and F. The other chimeras’ FCM data are shown in Figs. S10 and S11. One-way ANOVA, Tukey honestly significant difference test was used in (E) and (F). **p < 0.01; ***p < 0.001.

Fig 3: PD‐L1+ hESC‐RPE evades killing when cocultured with allogeneic hPBMCs in vitro. (A) The expression of PD‐L1 in hESC‐RPE transduced with PD‐L1 overexpression or empty vector lentiviruses, as measured by qPCR, n = 5 independent experiments. One‐way ANOVA followed by Tukey's post hoc test. Data presented as mean ± SEM; **p < 0.01. (B) Immunofluorescence showing PD‐L1 expression in control (CTL) or PD‐L1+ RPE. Scale bar, 20 μm. (C) The bar graph showing the percentage of hPBMCs cytotoxicity against CTL and PD‐L1+ RPE cells at the cocultured ratio of 10:1 (PBMC: RPE), n = 5 donors. Unpaired Student's t‐test. Data presented as mean ± SEM; ***p < 0.001. (D) The morphology and survival of CTL and PD‐L1+ RPE after coculturing with hPBMCs at the ratio of 10:1 (PBMC: RPE). Scale bar, 100 μm. The bar graph represents the number of adherent hESC‐RPE cells. n = 5 donors. Two‐way ANOVA followed by Sidak's post hoc test. Data presented as mean ± SEM; ****p < 0.0001. (E) Flow cytometry histogram of the proliferation in CD4 and CD8 T cell populations after cocultured with CTL or PD‐L1+ RPE (left); the statistical graph of the mean fluorescence intensity (MFI) of Violet (right). hPBMCs cultured alone were used as the negative control. hPBMCs were pre‐stimulated with anti‐CD3/CD28 antibodies. n = 5 donors. One‐way ANOVA followed by Tukey's post hoc test. Data presented as mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001. (F) The percentage of CD69+ cells in CD4 (up) and CD8 (down) T cell populations when cocultured with CTL or PD‐L1+ RPE. hPBMCs cultured alone were used as the negative control. hPBMCs stimulated with CD3/CD28 were used as the positive control. n = 5 donors. One‐way ANOVA followed by Tukey's post hoc test. Data presented as mean ± SEM; **p < 0.01, ***p < 0.001.

Fig 4: Flow cytometry gating strategy for identification of immune cell populations in the spinal cord. Representative flow cytometry plots illustrating the gating strategy used to identify myeloid cells (CD45highCD11b+ myeloid cells and CD45lowCD11b+ microglia), neutrophils (CD45highCD11b+RP1+), CD4+ and CD8+ T cells (CD45highCD3+CD4+ and CD45highCD3+CD8+) and B cells (CD45highCD45R+) in the spinal cord.

Fig 5: Comparison of CD4+ and CD8+ levels in whole blood of rats in each group. (A) The CD4+ and CD8+ levels in rats detected by Flow cytometry. (B) Comparison of CD4+ level in whole blood of rats in each group. (C) Comparison of CD8+ level in whole blood of rats in each group. *p < 0.05.