Figure 1.
E13 ETPs retain T/LTi lineage potential in vitro and generate LTi cells in a fetal thymic microenvironment. (A) Experimental strategy for combined T, B, and ILC lineage potential analysis of single ETPs. Single ETPs from either E13 or E18 were sorted onto OP9 stromal cells supplemented with IL-7, Flt3L, KitL, and IL-2. After 36 hours, clones were subdivided in conditions that sustain differentiation of T cells (OP9-DL4 supplemented with IL-7, Flt3L, KitL, and IL-2) or B, myeloid, and ILC/LTi (OP9 supplemented with IL-7, Flt3L, KitL, IL-2, and GM-CSF). (B) Representative flow cytometry plots outlining the gating strategy used to identify ILC1, ILC2, ILC3, and NK cells of E13 and E18 ETP single-cell–derived multi-ILC lineage clone. (C) Pie chart indicating all phenotypic combinations in individual clones detected after in vitro differentiation of E13 (81 clones analyzed of 240 sorted cells; cloning efficiency [CE], 34%) or E18 ETPs (86 clones analyzed of 240 sorted cells; CE, 36%) in 2 independent experiments. Frequency of clones generating only T cells (steel blue); T, NK, ILC1, ILC2, and ILC3 (red) (different in E13 and E18 clones; P < .0001); T and LTi (Rorc+CCR6+) (blue) (different in E13 and E18 clones; P < .0001); T, NK, LCX, 2 other ILC subsets (orange); T and ILC2 (gray); T and 2 other ILC subsets (light green); T, B, and myeloid cells (lime green) (different in E13 and E18 clones; P = .0036). All other combinations were not significantly different in E13 and E18 clones. (D) Flow cytometry plots of Rag2−/−γc−/− E14.5 thymic lobes reconstituted with 500 E13 ETPs or 500 E18 ETPs per lobe and gated CD3− and stained for the expression of CD127 and CCR6 (left plots). Numbers of double-positive (DP) thymocytes per lobe (right plot), thymic LTi per lobe (middle plot) Vγ5Vδ1 γδT cells (left plot). FTOCs were analyzed at day 12 after reconstitution. ****P < .0001. (E) Single E13 ETP were processed as in panel A. After 36 to 48 hours in culture, clones were harvested and colonized single Rag2−/−γc−/− E14.5 thymic lobes. After 24 to 48 hours in hanging drop lobes were transferred into filters and analyzed at day 12 (n = 10 lobes).

E13 ETPs retain T/LTi lineage potential in vitro and generate LTi cells in a fetal thymic microenvironment. (A) Experimental strategy for combined T, B, and ILC lineage potential analysis of single ETPs. Single ETPs from either E13 or E18 were sorted onto OP9 stromal cells supplemented with IL-7, Flt3L, KitL, and IL-2. After 36 hours, clones were subdivided in conditions that sustain differentiation of T cells (OP9-DL4 supplemented with IL-7, Flt3L, KitL, and IL-2) or B, myeloid, and ILC/LTi (OP9 supplemented with IL-7, Flt3L, KitL, IL-2, and GM-CSF). (B) Representative flow cytometry plots outlining the gating strategy used to identify ILC1, ILC2, ILC3, and NK cells of E13 and E18 ETP single-cell–derived multi-ILC lineage clone. (C) Pie chart indicating all phenotypic combinations in individual clones detected after in vitro differentiation of E13 (81 clones analyzed of 240 sorted cells; cloning efficiency [CE], 34%) or E18 ETPs (86 clones analyzed of 240 sorted cells; CE, 36%) in 2 independent experiments. Frequency of clones generating only T cells (steel blue); T, NK, ILC1, ILC2, and ILC3 (red) (different in E13 and E18 clones; P < .0001); T and LTi (Rorc+CCR6+) (blue) (different in E13 and E18 clones; P < .0001); T, NK, LCX, 2 other ILC subsets (orange); T and ILC2 (gray); T and 2 other ILC subsets (light green); T, B, and myeloid cells (lime green) (different in E13 and E18 clones; P = .0036). All other combinations were not significantly different in E13 and E18 clones. (D) Flow cytometry plots of Rag2−/−γc−/− E14.5 thymic lobes reconstituted with 500 E13 ETPs or 500 E18 ETPs per lobe and gated CD3− and stained for the expression of CD127 and CCR6 (left plots). Numbers of double-positive (DP) thymocytes per lobe (right plot), thymic LTi per lobe (middle plot) Vγ5Vδ1 γδT cells (left plot). FTOCs were analyzed at day 12 after reconstitution. ****P < .0001. (E) Single E13 ETP were processed as in panel A. After 36 to 48 hours in culture, clones were harvested and colonized single Rag2−/−γc−/− E14.5 thymic lobes. After 24 to 48 hours in hanging drop lobes were transferred into filters and analyzed at day 12 (n = 10 lobes).

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