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Effects of STAP-2 on B-cell recovery
hematopoietic stem/progenitor cells (HSPC).5,9,10 Many studies have focused on the pathophysiology of HSC, while few have investigated the role of lineage-committed progenitors, which have great capacity for proliferation.
Treatments for hematologic malignancies such as leukemia and lymphoma have been dramatically improved by recent advances in chemotherapy, immunotherapy and HSC transplantation (HSCT). However, compromising the immune system remains a frequent complication of various types of therapy, and induces the risk of non-relapse mortality. Especially in allogeneic HSCT settings, which is the only curative ther- apy for patients with refractory malignancies and severe BM failure diseases, regeneration of cellular and humoral immunity occurs over one year, while the recovery of innate immune cells, megakaryocytes and erythrocytes is usually observed within one month of HSCT.11 Similar to clinical observations, murine HSCT experiments show rel- atively slow recovery of lymphocytes. Under regenerative conditions, HSC and myeloid-biased multipotent progen- itors (MPP) enter cell-cycle, supporting early recovery of myeloid cells.12,13 However, the mechanisms of lymphoid reconstitution is less well understood.
In 2003, we identified signal-transducing adaptor pro- tein-2 (STAP-2) as a C-FMS/M-CSFR interacting protein.14 STAP-2 contains an N-terminal pleckstrin homology domain, a proline-rich region and an YXXQ motif. Its cen- tral region is distantly related to the Src homology 2-like (SH2) domain. As the adaptor protein structure pre- dicts, we and others identified roles in inflammatory reac- tions, cell survival, migration and cell adhesion in macrophages, T cells or mast cells.15-18 Although interac- tions with inflammatory molecules such as STAT5, MyD88, and IκB kinase (IKK) have been shown in immune cells, the importance of STAP-2 to hematopoiesis in BM remains unknown. Therefore, we investigated STAP-2-mediated regulation of stress hematopoiesis using genetically modified mice.
Methods
Mice
STAP-2 knockout (KO) and transgenic (Tg) mice of the C57BL/6J strain were generated and maintained as described pre- viously.14 For the generation of STAP-2 Tg mice, a cDNA fragment including the full coding region of the human STAP-2 gene was subcloned into a p1026X vector, which consisted of the murine Lck proximal promoter, the Ig intronic H chain enhancer E , and a human growth hormone (hGH) cassette, as previously described.19 Eight to 16-week old mice were used in all experi- ments except for those involving aged mice. Some mice were administered BrdU intraperitoneally (100 mg/kg of body weight) 12 hours prior to BM collection for cell cycle analyses. To examine age-related hematopoiesis, 12-22-month old mice were used. All experimental procedures were conducted under protocols approved by the Institutional Animal Care and Use Committee of Osaka University.
Bone marrow transplantation
Six thousand lineage– Sca1+ cKithigh (LSK) cells sorted from C57BL/6-Ly5.2 (CD45.2) mice were mixed with 6x105 unfraction- ated adult BM cells obtained from wild-type (WT) C57BL/6-Ly5.1 (CD45.1) mice. The mixture of cells was transplanted into C57BL/6-Ly5.1 mice lethally irradiated at a dose of 8.5 Gy.
Flow cytometry
Flow cytometric analysis and sorting were performed using a FACS Aria IIu or FACSCanto (BD Biosciences). The antibodies used for flow cytometric sorting and analysis are listed in Online Supplementary Table S1. Antibodies to CD19, CD45R/B220, CD11b/Mac1, Gr1, Ter119, CD3, and CD8 were used as lineage markers. FITC-Annexin V apoptosis Detection Kit (BD Biosciences) for apoptosis detection, and BrdU Flow Kit (BD Biosciences) for cell proliferation were also used according to the manufacturer’s protocol. FlowJo software (Tree Star) was used for data analysis.
Cultures
The details of culture methods are provided in the Online Supplementary Methods.
RNA-sequencing
Sequencing was performed using an Illumina HiSeq 2500 plat- form in a 75-base single-end mode. The raw data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (GSE127939). The normalized val- ues compared to control were defined to show signal ratios with greater than 2-fold increases or decreases. Bioinformatic analyses were conducted with Ingenuity Pathway Analysis software (Ingenuity Systems).
Real-time quantitative polymerase chain reaction analyses of gene expression
Semi-quantitative and real-time reverse transcriptase poly- merase chain reaction (RT-PCR) was used to assess mRNA expres- sion. Reactions were quantified using fluorescent TaqMan tech- nology.
Statistical analysis
Statistical analysis was carried out using unpaired two-tailed Mann-Whitney tests. The error bars used throughout indicate standard deviation (SD) of the mean.
Results
Loss of STAP-2 accelerates B-cell recovery in bone marrow transplantation
It has been reported that STAP-2 affects immune cell responses by directly binding to a variety of proinflam- matory molecules, including MyD88, IKKα/β, C-FMS, STAT3, and STAT5.14-18 In tumorigenesis, STAP-2 regu- lates cell migration, proliferation and therapy resistance for tyrosine kinase inhibitors.20-23 Hematopoiesis under steady state appears normal in STAP-2 deficient mice;24 we, therefore, hypothesized that STAP-2 may regulate stress hematopoiesis.
Here, we first conducted immunophenotypic character- ization of STAP-2 deficient HSPC with flow cytometry, which were found to be comparable between the WT and knockout (KO) BM under homeostatic conditions (Figure 1A). Next, LSK derived from STAP-2 KO or WT mice (CD45.2+) was intravenously injected into congenic recipients (CD45.1+). One month after HSCT, we found that CD45.2+ donor chimerism in peripheral B cells in KO HSPC transplanted mice was significantly higher than that in controls (WT donor, 51.8±1.0%; STAP-2 KO donor, 72.0±1.5%; P<0.001), while recovery of other lin- eages including Mac1+ or Gr-1+ myeloid and CD3+ T cells was comparable (Figure 1B). The calculation of blood lin-
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