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. 2021 Feb 25;137(8):1024-1036.
doi: 10.1182/blood.2020006779.

A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner

Ramy Elsaid  1   2   3 , Sylvain Meunier  1   2   3 , Odile Burlen-Defranoux  1   2   3 , Francisca Soares-da-Silva  1   2   4   5 , Thibaut Perchet  1   2   3 , Lorea Iturri  6   7 , Laina Freyer  6 , Paulo Vieira  1   2   3 , Pablo Pereira  1   2   3 , Rachel Golub  1   2   3 , Antonio Bandeira  1   2   3 , Elisa Gomez Perdiguero  6 , Ana Cumano  1   2   3
Affiliations

A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner

Ramy Elsaid et al. Blood. .

Erratum in

Abstract

During embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5+ T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator-expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
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).
Figure 2.
Figure 2.
E13 ETPs have a transcriptional LTi signature and are required for the maturation (CD80+) of mTECs. (A) Single-cell multiplex qPCR analyzed by hierarchical clustering of single E13 and E18 ETPs (80 cells each) for the expression of ILC lineage-specific transcripts (right margin). Each column represents a cell (E13 ETP in salmon, E18 ETP in blue) and each row represents 1 gene (of 41 genes). Highlighted are ILC-associated transcripts (black) and LTi-associated transcripts (in red). Analysis was done by normalizing expression to 2 independent housekeeping genes (Gapdh and Actinb) in R package Phenograph as in Perchet et al and represented in a code color where red represents high expression and blue low expression. Data are pooled from 2 independent experiments. (B) Pregnant female mice were injected at E10.5, E12.5, and E14.5 with 1 mg of anti-IL-7Ra antibody A7R34 (red arrows indicate time points of anti–IL-7Ra injections and purple arrows indicate time points of analysis). Thymic lobes were analyzed at E16.5 for the presence of ETP (Lin−CD117+CD44+CD24low), for Vγ5Vδ1 γδT cells, and for LTi (CD127+CCR6+TcR−CD3− cells) (n = 6), or at P2 for the presence of DN (CD4−CD8−γδ−), DP(CD4+CD8+), CD4+ thymocytes, and of Vγ5Vδ1 γδT cells, for LTi (CD127+CCR6+TcR−CD3− cells), and for CD80+ mTECs (EpCAM+Ly51−UEA.1+CD45−) (n = 24). Plots show numbers of the respective populations per thymus. Data from 2 independent experiments. ****P < .0001.
Figure 3.
Figure 3.
Neonatal thymectomy has no effect on embryonic-derived Vγ5 and Vγ6 γδ T cells. (A) Schematic diagram of neonatal thymectomy experiment. P1 newborn mice were thymectomized and analyzed 6 weeks later. (B) Frequencies of double-positive CD4/CD8 T cells found in the thymus and perithymic tissue isolated from sham and thymectomized mice, respectively. (C) Number of Vγ5+ γδ T cells in the epidermis isolated from 1 ear of sham and thymectomized mice. (D) Number of Vγ6 IL17 γδ T cells, Vγ4 IL17 γδ T cells, and αβ T cells in 1 inguinal lymph node, isolated from sham and thymectomized mice. (E) Number of αβ CD4+CD44− T cells in the spleen. *P < .05, ***P < .001, and ****P < .0001 (Student t test). Data are representative of at least 4 mice in each group from 2 independent experiments. Data are depicted as mean plus or minus standard error of the mean (SEM).
Figure 4.
Figure 4.
Emergence of Il7R⍺+expressing YS progenitors in E9.5 embryos. (A) Schematic representation of the anatomical sites analyzed in mouse embryos: YS, head, FL and the AGM region. (B) Representative flow cytometry plot showing YFP expression on live Lin−CD117+CD41+ E9.5 YS cells from Il7rcreROSA26LSLYFP. (C) Number and percentage of YFP+ per YS (mean plus or minus SEM). Data representative of 14 embryos from 4 independent experiments. (D) Representative flow cytometry plot showing YFP expression on live Lin−CD117+ E9.5 placenta and AGM region cells. (E) Expression of CD115, CD127, CD135, CD16/32, and CD31 within the E9.5 YS Lin−CD117+CD41+ YFP+ and YFP− populations. (F) Experimental strategy for single-cell lineage potential assay of E9.5 YS Lin−CD117+CD41+ YFP+ and YFP− populations. Frequency of wells containing (G) B cells or (H) T cells in cultures of single-sorted E9.5 YS Lin−CD117+CD41+ YFP+ and YFP− cells (180 cells from each population analyzed in 3 independent experiments). (I) Frequency of erythroid and megakaryocytic (E/Mk), myeloid (GM), or mixed E/Mk/GM colonies of single-sorted E9.5 YS Lin−CD117+CD41+ YFP+ and YFP− cells. (J) Frequency of thymic lobes irradiated and reconstituted with CD117+ E9.5 YS YFP+ or YFP−, or with E12.5 LSK or E10.5 YS as controls that contained CD4/CD8 DP cells (left plot) or TCR-Vγ5+ T cells (right plot). FTOCs were analyzed at day 12 after reconstitution.
Figure 5.
Figure 5.
Gene-expression analysis of single E9.5 YS Lin−CD117+ CD41+ YFP+ and YFP−cells. (A) Single-cell multiplex qPCR analyzed by hierarchical clustering of single YFP+ (47 cells) and YFP− (10 cells) E9.5 Lin−CD117+CD41+ cells analyzed for the expression of lymphoid-associated (Il7r, Rag1, Rag2, Flt3), myeloid-associated (Cebpa, Cd32, Csf1r, Csf2r, Csf3r, Irf8, Pu1), B-cell–associated (Pax5, Ebf1), E/Mk-associated (EpoR, Gata1, Mpl, Tal1, Vwf, Klf1), and hematopoiesis-associated (Gata2, Myb, Runx1, Kit, Cd34) genes. (B) Number of Il7r+ and Il7r− cells within E9.5 YS YFP-expressing cells.
Figure 6.
Figure 6.
Thymopoiesis-initiating cells develop exclusively from HSC-derived progenitors. (A) Experimental design of lineage-tracing analysis of Csf1r-expressing cells after OH tamoxifen (OH-TAM) administration at E8.5, E9.5, or E10.5. Arrows indicate pulse and analysis time points. (B) Flow cytometry analysis of E12.5 early thymic progenitors (ETPs). E12.5 thymic lobes of embryos pulsed at indicated time points were analyzed. Left plots show the gating strategy to identify ETP. Right representative histograms show the frequency of Tomato+ or YFP+ cells in ETP. For each experiment, 1 representative analysis is shown. (C) Frequencies of YFP- or Tomato-labeled hematopoietic progenitors and LTi’s in E12.5 FL, fetal blood (FB), and thymi (left plots) and of LSK, B1 B cells, B cells, and Vγ5+ T cells in adult tissues (right plots) of animals pulsed at E8.5, E9.5, or E10.5. Microglia served as controls for labeling efficiency. All data are pooled from minimally 2 independent experiments for embryos analyzed at E12.5 (pulsed at E8.5, n = 8; E9.5, n = 10; E10.5, n = 10); for adult tissues, animals were analyzed between 10 and 12 weeks of age (pulsed at E8.5, n = 6; E9.5, n = 6; E10.5, n = 2). Data are depicted as mean plus or minus SEM except for adult animals pulsed at E10.5 (mean). BM, bone marrow; FT, fetal thymus; PerC, peritoneal cavity.
Figure 7.
Figure 7.
Model depicting the origin and the lineage potential of thymopoiesis-initiating progenitors.
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