Endothelial SIRPα signaling controls VE-cadherin endocytosis for thymic homing of progenitor cells
- The Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, China
- College of Life Sciences, University of the Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
Signal regulatory protein alpha (SIRPα) is preferentially expressed on thymic portal endothelial cells (TPECs).
(A) Expression profile of top 20 signature genes of Ly6C-Selp+ ECs (TPECs), which have absolute FC > 2 and p < 0.01 in TPECs versus either Ly6C+Selp- or Ly6C+Selp+ thymic EC subsets and are in GO …
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Figure 1—source data 1
Source data file for Figure 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Signal regulatory protein alpha (SIRPα) expression on thymic portal endothelial cells (TPECs).
(A) Representative imaging of CD31+Ly6C- TPECs with or without the structure of perivascular space (PVS). The left panel showed a complete cross section of a blood vessel with all fluorescence …
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Figure 1—figure supplement 1—source data 1
Source data file for Figure 1-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 2 supplements
Signal regulatory protein alpha (SIRPα) is essential for early T cell progenitor (ETP) population maintenance, thymic progenitor homing, and proper T cell development.
(A) Representative flow cytometric analysis of ETPs (lineage-CD25-CD44+cKit+) in the thymus of Sirpa-/- and control mice. (B) Proportion of ETP population of total thymocytes and corresponding cell …
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Figure 2—source data 1
Source data file for Figure 2.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Signal regulatory protein alpha (SIRPα) deficiency has minor effect on T cell development at steady state.
(A–C) Cellularity of the thymus (A), proportion (B), and total cell number (C) of major thymocyte subsets (DN, double negative, DP, double positive, CD4 SP, CD4 single positive, and CD8 SP, CD8 …
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Figure 2—figure supplement 1—source data 1
Source data file for Figure 2-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Endothelial signal regulatory protein alpha (SIRPα) is essential for thymic progenitor homing.
(A) Representative dot plot of thymic epithelial cells (TECs, EpCAM+CD45-), TEC subsets (mTECs, UEA-1+Ly51- and cTECs, UEA-1-Ly51+), thymic endothelial cells (ECs, CD31+CD45-), and EC subsets …
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Figure 2—figure supplement 2—source data 1
Source data file for Figure 2-figure supplement 2.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
Endothelial cell (EC)-signal regulatory protein alpha (SIRPα) controls bone marrow progenitor cell transendothelial migration (TEM).
(A) Representative flow cytometric analysis of thymic EC composition in Sirpa-/- and control mice. (B,C) Proportion of thymic EC subsets (B) and corresponding cell numbers (C) in the thymus, n = 4 …
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Figure 3—source data 1
Source data file for Figure 3.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Signal regulatory protein alpha (SIRPα) does not control endothelial cell (EC) development and growth.
(A) Representative flow cytometric analysis of thymic ECs (CD31+CD45-) in the thymus of Sirpa-/- and control mice. (B,C) Proportion (B) and corresponding cell number (C) of ECs in total thymocytes, …
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Figure 3—figure supplement 1—source data 1
Source data file for Figure 3-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Migrating cell-derived CD47 guides their transendothelial migration (TEM).
(A) CD47 expression measured by flow cytometry in various subsets of T cell lineage. BM-LSK: Lineage-Sca1+cKit+ lymphoid progenitor cells in bone marrows, n = 3 in each group, data are …
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Figure 4—source data 1
Source data file for Figure 4.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Migrating cell-derived CD47 guides their transendothelial migration (TEM).
(A) CD47 expression in various subsets of T cell lineage as described in Figure 4A. Shallow lines are the isotype control of the subset of corresponding colors. Data are representative of three …
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Figure 4—figure supplement 1—source data 1
Source data file for Figure 4-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
Signal regulatory protein alpha (SIRPα) intracellular signal controls transendothelial migration (TEM) via SHP2 and Src.
(A) Relative TEM efficiency of lymphocytes through wild type (WT), intracellular-truncated Sirpa-ΔICD, and Sirpa-/- MS1 endothelial monolayer, n = 3 in each group, data are representative of three …
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Figure 5—source data 1
Source data file for Figure 5.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Construction of signal regulatory protein alpha (SIRPα) mutant MS1 cell lines.
(A) Schematic view of Cas9-mediated truncation of Sirpa intracellular region. sgRNA targeting exon 6 of Sirpa, the first exon encoding for the cytoplasmic region, was designed to create frameshift …
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Figure 5—figure supplement 1—source data 1
Source data file for Figure 5-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig5-figsupp1-data1-v1.zip
Figure 6 with 1 supplement
CD47-signal regulatory protein alpha (SIRPα) signaling promotes VE-cadherin endocytosis.
(A) Representative imaging of endocytosed VE-cadherin in the presence of lymphocytes, scale bars represent 20 μm. (B) Statistical analysis of VE-cadherin endocytosis in MS1 endothelial cells. …
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Figure 6—source data 1
Source data file for Figure 6.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
CD47-signal regulatory protein alpha (SIRPα) signaling promotes VE-cadherin endocytosis.
(A) Confocal microscopy of distribution of VE-cadherin on wild type (WT) and Sirpa-/- MS1 monolayers, bar represents 20 μm. (B) VE-cadherin signal per cell calculated by total VE-cadherin signal …
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Figure 6—figure supplement 1—source data 1
Source data file for Figure 6-figure supplement 1.
- https://cdn.elifesciences.org/articles/69219/elife-69219-fig6-figsupp1-data1-v1.xlsx
Figure 7
Schematic diagram of signal regulatory protein alpha (SIRPα) signaling in thymic portal endothelial cells (TPECs).
SIRPα on TPECs is engaged by CD47 on progenitor cells to initiate an inward signaling which induces VE-cadherin endocytosis and transendothelial migration for thymic homing of the progenitors.
Author response image 1
Reconstitution of mixed WT and Cd47-/- bone marrow chimeras.
(A) Schematic view of mixed bone marrow chimera. (B) FACS analysis of chimerism of T cells in the peripheral blood in the host 6 weeks after bone marrow adoptive transfer. (C,D) Statistics of WT and …
Author response image 2
Development of WT and Cd47-/- Lin- progenitors in non-irradiated hosts.
(A) Schematic view of the experiment. (B) Proportion of donor cells in the mixture before transfer. (C) Proportion of donor-derived thymocytes 2 weeks later. (D,E) Statistics of proportion of WT and …
Author response image 3
Reconstitution of bone marrow-derived progenitors in Sirpα-/- mice.
(A) Schematic view of the experiment. (B,C) Statistics of proportion (B) and cell number (C) of donor derived cells in the thymus 4 weeks after SL-TBI and adoptive transfer. n=6 in each group, …
Author response image 4
Analysis of lymphoid progenitors in Sirpα mice.
Gating strategy for common lymphoid progenitors (CLPs) and lymphoid myeloid progenitors (LMPPs). (B,C) Statistics of cell number (B) and proportion (C) of LMPPs in the bone marrow, n=3 in Sirpα+/- …
Author response image 5
Thymic EC composition upon SIRPα blockade.
(A) Cell number of thymic endothelial cells in the thymus after indicated treatment. (B,C) Cell number (B) and proportion (C) of total thymic portal endothelia cells (TPECs) of a thymus. n=3 in each …
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69219/elife-69219-transrepform1-v1.docx
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Supplementary file 1
Source data file for GSE_83114 RNA-seq analysis.
- https://cdn.elifesciences.org/articles/69219/elife-69219-supp1-v1.xlsx
