Mechanism Of Transcription Factor PBX1 Regulates Early Development Of NK Cells

Natural killer(NK)cells exert anti-tumor immune surveillance and resist pathogenic microbial infection by cytotoxicity and secretion of cytokines.NK cells also act as regulatory cells in autoimmune disorders and promote fetal implantation and development at the maternal-fetal interface.With the development of cellular immunotherapy,NK cells are expected to be used in the treatment of cancer,infections,autoimmune diseases and even reproductive diseases.However,in vitro expansion of NK cells remains challenging due to limited understanding of the programmed development of NK cells.NK cells in adult mice develop from hematopoietic stem cells(HSCs).Common lymphoid progenitors(CLPs)are derived from HSCs and give rise to NK-committed progenitor(pre-NKP)cells.Pre-NKP cells change to rNKP cells by expression of CD 122.Then,rNKP cells give rise to immature NK cells(iNKs)while acquiring NK1.1.How CLPs change to pre-NKP cells is particularly important because this stage determines commitment to the NK lineage.NFIL3 determines NK-cell commitment from CLPs,which illustrates further that NFIL3 is an inherent factor that regulates early development of NK cells.With respect to the downstream signals of NFIL3,it has been reported that NFIL3 promotes NK-cell development by directly regulating expression of the transcription factors Tox,Eomes and Id2.However,the factor that can upregulate NFIL3 expression directly is not known.PBX1 is a homeodomain transcription factor which has critical roles in embryonic processes.Conditional knockout of PBX1 in the hematopoietic compartment of mice shows that PBX1 is important for self-renewal of long-term HSCs.The number of CLPs,B cells and NK cells is reduced significantly in the absence of PBX1 in hematopoietic cells while numbers of T cells remain unchanged.Therefore,in addition to PBX1 affecting self-renewal of HSCs,there should be another reason why development of NK cells from CLPs is impaired in the absence of PBX1.Also,the essential target of PBX1 during lymphocyte development has not been identified.Here,we focused on the upstream transcriptional regulation mechanism of NFIL3.We investigated if the transcription factor PBX1 can regulate NFIL3 directly and the detailed molecular mechanism.We obtained following results.1.Impaired development of NK-cell precursors in the absence of PBX1To investigate the role of PBX1 in NK-cell development,we generated Pbx1f/f;Vav1Cre mice,in which PBX1 is knocked out specifically in hematopoietic cells.Flow-cytometry staining showed that deficiency of PBX1 resulted in a significant reduction of pre-NKP cells,rNKP cells and NK cells.However,T cells were not affected.So PBX1 could regulate NK-cell development by acting downstream of CLPs.2.NFIL3 expression decreased in the absence of PBX1PBX1 deficiency results in a reduced number of NK-cell precursors,which is consistent with Nfil3 deficiency.To investigate whether PBX1 could contribute to NFIL3 expression,we measured NFIL3 expression in NK cells of Pbx1f/f;Vav1Cre mice by intracellular flow-cytometry staining.Expression of NFIL3 and the downstream factor EOMES in NK cells was decreased significantly in the absence of PBX1.Therefore PBX1 deficiency could result in reduction of NFIL3 expression.3.PBX1 can bind to the promoter of Nfil3To investigate if PBX 1 affects NIFL3 directly,we analyzed the promoter of Nfil3 and found a potential PBX1 binding site.To ascertain if PBX1 can bind to the Nfil3 promoter,we undertook a ChIP assay with mice bone-marrow NK cells.PBX1 was bound to the promoter region of the Nfil3 locus,which included the sequence we predicted.We demonstrate that PBX1 can binds to the Nfil3 promoter directly in vitro through luciferase reporter assay and DNA-pulldown assay.4.Impaired early development of NK cells in Nfil3proPBX1bs-/-miceGiven that other effects of PBX 1 deficiency led to reduction in the number of CLPs(which is inconsistent with NFIL3 deficiency),we generated Nfil3proPBX1bs-/-mice with a knocked out region(-534 to-425)that included the binding site of PBX 1 in the Nfil3 promoter using CRISPR-Cas9 technology.The percentage of pre-NKP cells,rNKP cells and NK cells in Nfil3proPBX1bs-/-mice decreased,whereas that of CLPs,B cells and T cells was unchanged.Consistently,early development of NK cells was impaired after deleting the binding site of PBX 1 in the Nfil3 promoter.These findings demonstrated that PBX1 regulated early NK-cell development by binding to the Nfil3 promoter.5.PBX1 binding to the promoter region of Nfil3 is dependent upon N286Because PBX1 was shown to bind with the promoter of Nfil3,next we investigated which amino acids of PBX 1 mediated such binding.We generated a fusion of PBX 1 deletion mutants or substitution mutants with a green fluorescent protein(GFP)reporter and performed luciferase reporter assay and DNA-pulldown assay.We found PBX1 binding to the Nfil3 promoter was dependent upon asparagine N286 in the homeodomain.In summary,we demonstrated that the transcription factor PBX1 could regulate NFIL3 transcription positively via asparagine N286 in the homeodomain and,thus,regulate early development of NK cells.Our study facilitates related research on PBX1 and aids understanding of NK-cell development.It also lays a theoretical foundation for in vitro expansion of NK cells for cellular immunotherapy.Our research highlights are:(1)we identified(for the first time)a transcription factor that can bind to the Nfil3 promoter to promote NK-cell development;(2)we discovered that PBX1 affects NK-cell development during the stage CLP give rise to NK-committed progenitor;(3)we revealed that PBX1 binds to the Nfil3 promoter via asparagine N286 in the homeodomain.