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Effects of STAP-2 on B-cell recovery
Interestingly, pre-B cells were strongly affected by vari- ous inflammatory signals at the early phase after hematopoietic stem cell transplantation (Figure 5). Among these signals, we focused on the TLR4 pathway. B-lineage cells express multiple pathogen recognition receptors, such as TLR, from the earliest stage.7,40 As far as the impact on B progenitors in BM is concerned, two research groups pre- viously showed that signaling through TLR4 inhibits pro-B and pre-B proliferation, and enhances differentiation to IgM+ immature B cells, using different methods.28,29 In our genetically modified mice, STAP-2 displayed synergistic effects on pre-B-cell proliferation with the TLR4 pathway. However, STAP-2 effects did not involve differentiation to IgM+ immature B cells. In macrophages, STAP-2 binds directly to MyD88 and IKKα/β but not to TNFR-associated factor 6 or IL-1R-associated kinase 1.16 As in macrophages, STAP-2 may modulate specific TLR4 functions. We also found that TREM-1 expression is upregulated by STAP-2 overexpression and LPS stimulation. Although it is known that TREM-1 is expressed on B-lineage cells, its potential roles in B lymphopoiesis remain unknown.25-27,41 When the antagonized antibody and neutralized Fc chimera was added into the pre-B-cell cultures, no effects were observed (data not shown). Thus, a gene-modified approach may be required to clarify the function.
Considering the nature of STAP-2 as an adaptor protein, the total protein expression as well as its phosphorylation is essential for the acting process. Previously, we showed that phosphorylated STAP-2 binds directly to several types of inflammatory proteins, including MyD88.16,17,30,31 In this study, we evaluated the transcript under hemato- logic stress. As shown in Figure 7, the expression is sus- tained after LPS treatment. Alternatively, we observed expression changes according to the individual condition after sub-lethal irradiation, indicating that several direct and indirect factors affected by hematologic stress are associated with the expression of STAP-2.
sort LSK or CLP cells via flow cytometry, and, therefore we were unable to identify in which population HSPC expression increased. Based on our in vivo and in vitro results, STAP-2 in CLP may affect B lymphopoiesis under hematologic stress.
In this study, we revealed a detailed mechanism under- lying the impaired effects of STAP-2 on B-cell reconstitu- tion under hematopoietic stress. The target of STAP-2 dur- ing stress hematopoiesis appears to be very specific to B progenitors in BM, and its expression is altered following exposure to hematologic stress, while the expression of STAP-2 is ubiquitous under steady state. Our findings pro- vide insights that may aid in the development of new ther- apeutic approaches, with fewer adverse effects, or increased prognostic value, for patients given severe mye- loablative therapy or allogeneic HSCT.
Disclosures
MI have received speakers bureau from Celgene, Kowa Pharmaceutical, Takeda Pharmaceutical and Novartis outside the submitted work. HS have received honoraria from Jansen, honoraria and research funding from Bristol-Meyer Squibb, Celgene, Fujimoto Pharmaceutical, Mundipharma, Novartis, Ono Pharmaceutical, and Takeda Pharmaceutical outside the submitted work. KO have received speakers bureau from Novartis outside the submitted work. YK have received consul- tancy, honoraria, and research funding from Alexion outside the submitted work. The remaining authors declare no competing financial interests.
Contributions
MI designed research, collected data, analyzed data, and wrote the paper. KO, HShib, and TM analyzed data, and wrote the paper. HShih, JT, HS, DM, and DO collected and analyzed data. YK, RM, JK and KS supplied materials. YK supervised the research and wrote the paper. All authors reviewed and approved the manuscript.
Acknowledgments
The authors would like to thank Dr. Kincade (Oklahoma Medical Research Foundation) for constructive suggestions. We are also grateful for Ms. Habuchi, and Mr. Takashima for tech- nical support.
Funding
This work was supported in part by Japan Society for the Promotion of Science KAKENHI (grant no. 16K09872, 19H03364, 19K08815), Otsuka Pharmaceutical, and Kyowa Kirin Co., Ltd.
Among several candidates, TNFα upregulates the expression of STAP-2 in pre-B cells (Figure 7D). In clinical settings, prolongation of hematologic suppression after chemotherapy is sometimes observed when patients suf- fer from severe complications such as infection. Our find- ings indicate the existence of a complex network induced by hematologic stress under inflammatory milieu. Interestingly, we found Stap-2 expression in the lineage negative HSPC fraction was upregulated following irradi- ation (Figure 7B). Since the intensity of cKit decreases while that of Sca1 increases under hematologic stresses, such as irradiation and LPS administration, it is difficult to
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