Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

Cancer metastasis accounts for approximately 90% of all cancer-related deaths; however, the underlying mechanisms remain largely unknown. It has been known proteins that control F-actin dynamics are crucial for cancer metastasis. In this study, we revealed how an F-actin binding protein, Switch-associated protein 70 (SWAP-70), contributes to breast cancer metastasis. Moreover, immunotherapy is a promising approach to treat metastatic cancer cells by enhancing the function of the host immune system against cancer. Our lab has conducted extensive studies on how SWAP-70 regulates the function of several immune cell types, including dendritic cells (DCs), B cells, and mast cells. These cells have been reported to contribute to tumor immunity. Thus, we hypothesized that SWAP-70 plays a role in tumor immunity. To characterize the function of SWAP-70 in metastasis, we generated 4T1, mouse breast cancer, SWAP-70 knockout (KO) cells using Crispr/Cas9 technology. A syngeneic orthotopic model was used to recapitulate clinical disease progression, and the results showed that SWAP-70 led to significant metastasis to the lungs and bones in immunocompetent mice. Several functional assays have revealed that SWAP-70 promotes anchorage-independent growth, cell migration, invasion, and adhesion in 4T1 cells. Biophysical measurements showed that SWAP-70 contributes to cellular mechanics. To investigate how SWAP-70 in host cells affects tumor immunity, SWAP-70 deficient mice were injected with E0771 mouse breast cancer cells to study tumorigenicity. SWAP-70 deficient mice showed delayed primary tumor growth and less distant metastasis. Isolated SWAP-70−/− DCs were impaired in generating CD8 T cell responses pulsed with soluble OVA protein, but not with OVA peptide, suggesting that the antigen uptake, processing, and presentation process in SWAP-70−/− DCs may be diminished. Taken together, our findings describe the potential mechanisms by which the loss of SWAP-70 hinders cancer metastasis and provide several insights into how targeting SWAP-70 could be a potential therapeutic approach to target cancer.

In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 employ diverse mechanisms to suppress innate and adaptive immunity, which is critical for maintaining immune homeostasis and self-tolerance. Here, we propose Swap-70 as a novel member of the Foxp3-dependent canonical Treg cell signature. We show that Swap-70-/- mice have increased numbers of Foxp3+ Treg cells with an effector/memory-like phenotype that exhibit impaired suppressor function in vitro, but maintain overall immune homeostasis in vivo. Upon formation of an immunological synapse with antigen presenting cells in vitro, cytosolic SWAP-70 protein is selectively recruited to the interface in Treg cells. In this context, Swap-70-/- Treg cells fail to downregulate CD80/CD86 on osteoclast precursor cells by trans-endocytosis and to efficiently suppress osteoclastogenesis and osteoclast function. These data provide first evidence for a crucial role of SWAP-70 in Treg cell biology and further highlight the important non-immune function of Foxp3+ Treg cells in bone homeostasis mediated through direct SWAP-70-dependent mechanisms.