*P < 0·05; **P < 0·01; ***P < 0·001. Fig. S3. Thymocyte populations from non-obese diabetic (NOD)-scid IL2rγnull- bone marrow, liver, thymus (NSG–BLT) not irradiated and mice from each group were then implanted with 1 mm3 fragments of human fetal thymus

and liver in the renal subcapsular space. All mice were then injected intravenously with 1 × 105 to 5 × 105 CD34+ haematopoietic stem cells derived from the autologous human CD3-depleted fetal liver. At 12 weeks post-implant, thymic tissues were recovered and the total number of CD45+ cells (a) and the proportion of CD4 and CD8 single-positive and double-positive cells (b) were determined using flow cytometry. **P < 0·001. Fig. S4. Irradiation does not alter the activation status of human T cells in haematopoietic stem cells-engrafted non-obese Acalabrutinib in vitro diabetic (NOD)-scid IL2rγnull (NSG) mice implanted with human thymic tissues. NSG mice were irradiated RXDX-106 order with 200 cGy or not irradiated (0 cGy) and mice from each group were then implanted with 1 mm3 fragments of human fetal thymus and liver in the renal subcapsular space (thymic implant) or left unmanipulated (no thymic implant). All mice were then injected intravenously with 1 × 105 to 5 × 105 CD34+ haematopoietic stem cells derived from the autologous

human CD3-depleted fetal liver. Human CD4+ T cells (a,b,c) and CD8+ T cells (d,e,f) were examined for the expression of CD45RA in the peripheral blood at 12 (a,d) and 16 (b,e) weeks and in the spleen at 16 weeks (c,f). The values shown represent the percentages of human CD4+ or CD8+ T cells expressing CD45RA. Data from NSG mice injected with human HSC in the absence of irradiation is not shown due to the very low levels of T cell development.

Representative flow cytometry histograms for expression of CD45RA and CD62L on CD4+ (g,h) and CD8+ (i,j) T cells is shown for mice implanted with human fetal thymus and liver tissues. *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0·0001. Fig. S5. Human CD4 and CD8 T cells from non-obese diabetic (NOD)-scid IL2rγnull-bone marrow, Epothilone B (EPO906, Patupilone) liver, thymus (NSG–BLT) mice produce cytokines following in-vitro stimulation. NSG mice were either irradiated with 200 cGy or not irradiated and mice from each group were then implanted with 1 mm3 fragments of human fetal thymus and liver in the renal subcapsular space. All mice were then injected intravenously with 1 × 105 to 5 × 105 CD34+ haematopoietic stem cells derived from the autologous human CD3-depleted fetal liver. The ability of human CD4 T cells (a,c,e,g) and human CD8 T cells (b,d,f,h) from the spleens of mice from each group to produce interferon (IFN)-γ (a,b), interleukin (IL)-2 (c,d), IL-17A (e,f) and IL-22 (g,h) was determined at 12 weeks after tissue implant. Splenocytes were stimulated ex vivo with phorbol myristate acetate (PMA) and ionomycin for 5 h in a standard intracellular cytokine assay, as described in Materials and methods. *P < 0·05; ***P < 0·001. Fig. S6.