Endothelial SIRPα signaling controls thymic progenitor homing for T cell regeneration and antitumor immunity

Thymic homing of hematopoietic progenitor cells (HPCs) is an essential step for the subsequent T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism remains still unknown. Here we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs) and impaired thymic HPC homing. Mechanistically, SIRPα-deficient ECs and CD47-deficient lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in a SHP2- and Src-dependent manner. Furthermore, CD47-signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Functionally, SIRPα signaling is required for T cell regeneration upon sub-lethal total body irradiation (SL-TBI); CD47-SIRPα signaling blockade post SL-TBI diminishes antitumor immunity. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell regeneration and antitumor immunity. Graphic abstract Thymic homing of hematopoietic progenitor cells is fundamental to the T cell-based adaptive immunity, yet the molecular basis of this process is less clear. We discovered that endothelial SIRPα signaling, engaged by migrating cell derived CD47 ligand, regulates thymic homing of hematopoietic progenitor cells for T cell regeneration and antitumor immunity. SIRPα is preferentially expressed on thymic portal endothelial cells. Endothelial SIRPα regulates thymic homing of hematopoietic progenitor cells. CD47-SIRPα downstream signaling induces VE-cadherin endocytosis. CD47-SIRPα signaling blockade impairs thymic T cell regeneration and antitumor immunity.

homing of bone marrow derived hematopoietic progenitor cells (HPCs) is therefore a 72 critical step. It was reported that HPCs enter the thymus via unique blood vessels that 73 are surrounded by perivascular spaces (PVS) (Lind et al., 2001;Mori et al., 2007)  TPECs are enriched with transcripts related to cell adhesion and trafficking, supporting 84 its critical role for thymic homing. However, the molecular basis of TPEC function and 85 its underlying mechanism have not been experimentally determined. 86 Several molecules have been found to play important roles for thymic homing of 87 HPCs. P-selectin and adhesion molecule VCAM-1 and ICAM-1, highly expressed on 88 ECs, as also confirmed in our previous study, have been suggested to mediate adhesion 89 of HPCs on ECs (Lind et al., 2001;Mori et al., 2007;Rossi et al., 2005;Scimone et al., 90 and hematopoietic cell subsets shows 'self' identity and protects them from being 115 cleared by macrophages (Blazar et al., 2001;Jaiswal et al., 2009;Olsson et al., 2005;116 Yamao et al., 2002). Tumor cells express high level of CD47 to escape immune 117 surveillance initiated by macrophages (Chao et al., 2010;Majeti et al., 2009;118 Willingham et al., 2012), which leads to the development of CD47-SIRPα blockade as 119 a promising approach for cancer therapy.

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CD47-SIRPα has also been reported to play important roles in cell adhesion and 121 migration. SIRPα expressed on cDCs, neutrophils, melanoma cells and CHO cells has 122 been reported to promote cell motility (Fukunaga et al., 2004;Liu et al., 2002;Motegi 123 et al., 2003) through SHP2-dependent activation of Rho GTPase and cytoskeleton 124 reorganization (Wollenberg et al., 1996) , (Inagaki et al., 2000). On the other hand, SIRPα 125 expressed on neutrophils and monocytes has been shown to be the ligand for endothelial 126 or epithelial CD47 and promotes junction opening through activating Rho family 127 6 GTPase therefore permitting transmigration of the SIRPα-expressing cells (de Vries et 128 al., 2002;Liu et al., 2002;Stefanidakis et al., 2008). However, the expression and 129 function of SIRPα on ECs remain unknown. 130 In the present study, we uncovered the novel role of TPEC signature molecule SIRPα 131 in thymic homing of progenitor cells, revealed that migrating cell-derived CD47 and 132 EC-SIRPα intracellular signal induce junctional VE-cadherin endocytosis and promote 133 TEM. In addition, we showed that CD47-SIRPα signaling blockade upon thymic injury 134 resulted in impaired T cell regeneration and antitumor immunity.

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Thymic portal endothelial cells preferentially express SIRPα. 138 To explore the molecular mechanisms for thymic homing of the progenitors through 139 TPECs, we started with signature genes of TPECs based on previously published RNA-   Endothelial SIRPα is essential for ETP population maintenance and thymic 155 progenitor homing. 156 ETPs are the very first subset of bone marrow-derived progenitor cells that settle the 157 thymus and are committed to T cell lineage. Studies have suggested ETP population 158 size a direct indicator of thymic HPC homing (Krueger et al., 2010;Shi et al., 2016;159 Zlotoff et al., 2010). Therefore, to test the requirement of SIRPα for thymic progenitor 160 homing, we first analyzed ETP population in Sirpα -/mice. Sirpα -/mice appeared substantial reduction in ETP population was confirmed in Sirpα -/mice (Figure 2A,B). 166 Bone marrow multipotent progenitors including lineage -Sca-1 + c-Kit high (LSK) and 167 common lymphoid progenitors (CLPs) remained unchanged in these mice ( Figure   168 2C,D), suggesting the reduced population size of thymic ETP is unlikely due to 169 impaired generation/homeostasis of bone marrow progenitors.

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Previous studies suggested that thymic entry of the progenitor cells and ETP 171 population maintenance is guided by multiple cues derived from thymic endothelial 172 cells as well as thymic epithelial cells. To specifically test the role of endothelial SIRPα, 173 we generated Sirpα ΔTie2-Cre (Sirpα flox/flox × Tie2-cre) mice. Significant decrease of 174 thymic ETP population was found in Sirpα ΔTie2-Cre mice in a degree similar to that in

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Previous studies have reported SIRPα as a phagocytic checkpoint on tissue 180 macrophages and CD47-null cells are rapidly cleared in congenic wild type (WT) 181 mice (Bian et al., 2016). Possibility exists that phagocytic activity may elevate towards 182 circulating progenitor cells in Sirpα -/or Sirpα ΔTie2-Cre mice before they reach the thymus,  To directly test the role of SIRPα on HPC thymic homing, a short-term thymic 193 homing assay was adopted (Shi et al., 2016), in which massive amount of progenitor-      As CD47-SIRPα blockade is currently a promising therapeutic antitumor strategy, 355 impaired peripheral T cell regeneration after CV1 treatment led us to concern that 356 SIRPα blockade might dampen T cell-mediated antitumor response during CRT. We TPECs interact with progenitors and regulate their thymic entry.

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SIRPα has been well recognized as a "don't eat me" receptor on myeloid cells.

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Interestingly, our current study reveals a previously totally unrecognized function of 373 SIRPα on ECs for TEM regulation. SIRPα-CD47 interaction has been reported to 374 regulate TEM of neutrophils and monocytes (de Vries et al., 2002;Liu et al., 2002;375 Stefanidakis et al., 2008). In these studies, CD47, but not SIRPα, is considered as the  (Figure 4A). The expression level of CD47 on ETPs is also significantly 400 higher than that on TPECs (Figure 4-figure supplement 1E,F). These data suggest  Our study reveals a novel mechanism of adherens junction VE-cadherin endocytosis.

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Both SHP2 and Src kinase have been reported to regulate VE-cadherin endocytosis, via 413 modification of VE-cadherin in different manners (Allingham et al., 2007;Wessel et al., 414 2014). However, how they are activated remains unclear. Here, our data suggest that  Figure 5B). Together with the fact that ITIM motif 420 activates cytosolic SHP2 phosphatase (Motegi et al., 2003;Tsuda et al., 1998), these 421 data suggest SIRPα signal induced VE-cadherin endocytosis is at least in part through 422 ITIM-SHP2 pathway. In addition, we have also found CD47 engagement induces Src 423 activation in ECs (Figure 5G,H). Inhibition of Src kinase via PP2 results in 424 significantly impaired TEM and VE-cadherin endocytosis in WT ECs, but not in Sirpα -

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/-ECs ( Figure 5J and Figure 6E). Therefore, Src kinase is also involved in SIRPα 426 signal induced VE-cadherin endocytosis. How SIRPα coordinates both SHP2 and Src 427 pathways remains to be investigated in future.

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The novel role of SIRPα on TEM regulation might not be limited to TPECs. It is 429 interesting to find that elevated expression level of SIRPα is also found on high       SIRPα-hIg was produced as previously described (Liu et al., 2015). Briefly, pEE12.4- Bone marrow chimeras and short-term homing assay 579 6 weeks old recipient mice received 10 Gy γ radiation once. 5×10 6 donor bone marrow 580 cells were injected intravenously into the recipients on the day or the next day of 581 irradiation. 8 weeks later, mice are ready for subsequent studies. Short-term homing 582 assay: donor bone marrows were taken from femurs and tibias from WT mice, red blood 583 cells were lysed and labeled by 2μM CFSE, and 5×10 7 bone marrow cells were 584 transferred intravenously to the recipients. The recipients were sacrificed 2 days later 585 and detected for donor derived progenitors in the thymus and spleen. Wild type mice were exposed to 5.5 Gy γ radiation once (sub-lethal total body   Statistical analyses were performed using GraphPad 6.0 (Prism). Two-tailed unpaired 681 Student's t-test was used for significance test, unless otherwise specified. All 682 experiments with significant differences were performed independently three times.