Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen

Reviewer #1 (Public Review):

Summary:
This study investigated the role of CD47 and TSP1 in extramedullary erythropoiesis by utilization of both global CD47-/- mice and TSP1-/- mice.

Strengths:
Flow cytometry combined with spleen bulk and single-cell transcriptomics were employed. The authors found that stress-induced erythropoiesis markers were increased in CD47-/- spleen cells, particularly genes that are required for terminal erythroid differentiation. Moreover, CD47 dependent erythroid precursors population was identified by spleen scRNA sequencing. In contrast, the same cells were not detected in TSP1-/- spleen. These findings provide strong evidence to support the conclusion that the differential role of CD47 and TSP1 in extramedullary erythropoiesis in mouse spleen.

Weaknesses:
Methods and data analysis are appropriate. However, some clarifications are required. The discussion section needs to be expanded.
1). The sex of mice that were used in the study is unknown.
2). In the method of Single-cell RNA sequencing (page 10), it mentioned that single cell suspensions from mouse spleens were depleted of all mature hematopoietic cell lineages by passing through CD8a microbeads and CD8a+ T cell isolation Kit. As described, it is confusing what cell types are obtained for performing scRNAseq. More information is required for clarity.
3). The constitutive CD47 knockout mouse model is utilized in this study. The observed accumulation of erythroid precursors in the spleens of CD47-/- mice suggests a chronic effect of CD47 on spleen function. Can the current findings be extrapolated to acute scenarios involving CD47 knockdown or loss, as this may have more direct relevance to the potential side effects associated with anti-CD47-mediated cancer therapy? Please expand on this topic in the discussion section.

Reviewer #2 (Public Review):

Summary:
The authors used existing mouse models to compare the effects of ablating the CD47 receptor and its signaling ligand Thrombospondin. The CD47-KO model used in this study was generated by Kim et al, 2018, where hemolytic anemia and splenomegaly was reported. This study analyzes the cell composition of the spleens from CD47-KO and Thsp-KO, focusing on early hematopoietic and erythroid populations. The data broadly shows that splenomegaly in the CD47-KO is largely due to an increase in committed erythroid progenitors as seen by Flow Cytometry and single-cell sequencing, whereas the Thsp-KO shows a slight depletion of committed erythroid progenitors but is otherwise similar to WT in splenic cell composition.

Strengths: The techniques used are appropriate for the study and the data support the main conclusions of the study. This study provides novel insights into a putative role of Thsp-CD47 signaling in triggering definitive erythropoiesis in the mouse spleen in response to anemic stress and constitutes a good resource for researchers seeking to understand extramedullary erythropoiesis.

Weaknesses: The Flow cytometry data alone supports the authors' main conclusion and single-cell sequencing confirms them but does not add further information, other than those already observed in the Flow data. The single-cell sequencing analysis and presentation could be improved by using alternate clustering methods as well as separating the data by genotype and displaying them in order for readers to fully grasp the nuanced differences in marker expression between the genotypes. Further, it is not clear from the authors' description of their results whether the increased splenic erythropoiesis is a direct consequence of CD47-KO or a response to the anemic stress in this mouse model. The enrichment of cKit+ Ter119+ Sca1- cells in CD47-KO indicates that these are likely stress erythroid progenitors. Another CD47-KO mouse model (Lindberg et al 1996) has no reported erythroid defects and was also not examined in this study.

Author Response

Reviewer 1:

1. The missing mouse gender information will be incorporated into the revised manuscript. For flow cytometry, two male and two female mice of each genotype were used. For single cell RNA sequencing, two female and one male mouse of each genotype were used. For the bulk RNA sequencing four male cd47−/− mice and four male wildtype mice were used.

2. The bulk RNA sequencing analysis identified elevated expression of erythropoietic genes in CD8+ spleen cells from cd47−/− versus wildtype mice that were obtained using magnetic bead depletion of all other lineages. Therefore, we used the same Miltenyi negative selection kit as the first step to prepare the cells for single cell RNA sequencing. These untouched cells were then depleted of most mature CD8 T cells using a Miltenyi CD8a(Ly2) antibody positive selection kit. An important consideration underlying this approach was recognizing that the commercial magnetic bead depletion kits used for preparing specific immune cell types are optimized to give relatively pure populations of the intended immune cells using wildtype mice. Our previous experience studying NK cell development in the cd47−/− mice taught us that NK precursors, which are rare in wildtype mouse spleens, accumulate in cd47−/− spleens and were not removed by the antibody cocktail optimized for wildtype spleen cells (Nath et al Front Immunol 2018). The present data indicate that erythroid precursors behave similarly.

3. Anemia is a prevalent side effect of several CD47 therapeutic antibodies being developed for cancer therapy. Anemia would be expected to induce erythropoiesis in bone marrow and possibly at extramedullary sites. Human spleen cells are not accessible to directly evaluate extramedullary erythropoiesis in cancer patients, but analysis of circulating erythroid precursors or liquid biopsy methods could be useful to detect induction of extramedullary erythropoiesis by these therapeutics. We are currently investigating the ability of CD47 antibodies to directly induce erythropoiesis using a human in vitro model.

Reviewer 2:

1. The reviewer asked, “whether the increased splenic erythropoiesis is a direct consequence of CD47-KO or a response to the anemic stress in this mouse model.” Our data supports both a direct role for CD47 and an indirect role resulting from the response to anemic stress. We cited our previous publications describing increased Sox2+ stem cells in spleens of Cd47 and Thbs1 knockout mice, but we neglected to emphasize another study where we found that bone marrow from cd47−/− mice subjected to the stress of ionizing radiation exhibited more colony forming units for erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) progenitors compared to bone marrow from irradiated wildtype mice (Maxhimer Sci Transl Med 2009). Taken together, our published data demonstrates that loss of CD47 results in an intrinsic protection of hematopoietic stem cells from genotoxic stress. This function of CD47 is thrombospondin-1-dependent and is consistent with the up-regulation of early erythroid precursors in the spleens of both knockout mice but cannot explain why the Thbs1−/− mice have fewer committed erythroid precursors than wildtype. We cited studies that documented increased red cell turnover in cd47−/− mice but less red cell turnover in Thbs1−/− mice compared to wildtype mice. Increased red cell clearance in cd47−/− mice is mediated by loss of the “don’t eat me” function of CD47 on red cells. In wildtype mice, clearance is augmented by thrombospondin-1 binding to the clustered CD47 on aging red cells (Wang, Aging Cell 2020). Thus, anemic stress in the mouse strains studied here decreases in the order cd47−/− > WT > Thbs−/−. This is consistent with the increased committed erythroid progenitors reported here in cd47−/− spleens and decreased committed progenitors in the Thbs1−/− spleens.

2. The cd47−/− mice used for the current study are the same strain as those reported by Lindberg et al in 1996, with additional backcrossing onto a C57BL/6 background.