Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

Deletion of master regulators of the B cell lineage reprograms B cells into T cells. Here we found that the transcription factor Hoxb5, which is expressed in uncommitted hematopoietic progenitor cells but is not present in cells committed to the B cell or T cell lineage, was able to reprogram pro-pre-B cells into functional early T cell lineage progenitors. This reprogramming started in the bone marrow and was completed in the thymus and gave rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immunological functions that closely resembled those of their natural counterparts. Hoxb5 repressed B cell 'master genes', activated regulators of T cells and regulated crucial chromatin modifiers in pro-pre-B cells and ultimately drove the B cell fate-to-T cell fate conversion. Our results provide a de novo paradigm for the generation of functional T cells through reprogramming in vivo.

Publisher Correction: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

In the version of this article initially published, some identification of the supplementary information was incorrect. The items originally called Supplementary Tables 1, 2, 3, 4 and 5 should be Source Data Figures 1, 2, 4, 5 and 7, respectively; those originally called Supplementary Tables 6, 7 and 8 should be Supplementary Tables 1, 2 and 3, respectively; and those originally called Source Data Figures 1, 2, 4, 5 and 7 should be Supplementary Tables 4, 5, 6, 7 and 8, respectively. The errors have been corrected in the HTML version of the article.

Effects of Smoking on Major Adverse Cardiovascular Events in Patients With Coronary Artery Spasm: A Systematic Review and Meta-Analysis.

BACKGROUND: Smoking is an established independent risk factor for coronary artery spasm (CAS), but its effects on major adverse cardiovascular events (MACE) in patients with CAS have not been systematically assessed. METHODS: This systematic review and meta-analysis of studies published from January 2000 to July 2023 was conducted to examine the relationship between smoking and MACE in patients with CAS. Data on MACE were obtained from both smoking and non-smoking CAS patient groups. The effects of smoking on MACE in patients with CAS were assessed through meta-analysis, utilising Stata 17.0 software for all statistical analyses. RESULTS: Nine studies, encompassing 9,376 patients, from Japan (5 studies), Korea (4 studies) and Spain (1 study) were included in the final analysis. Meta-analysis revealed that smoking significantly impacted MACE in patients with CAS (RR 1.965; 95% CI 1.348-2.865), a finding further validated by sensitivity analyses. Subgroup analyses identified a stronger correlation between smoking and increased MACE endpoints in Japanese patients and in those with >3 years of follow-up. CONCLUSIONS: This meta-analysis strongly indicates that smoking escalates the risk of MACE in patients with CAS, with a more pronounced association observed in Japanese patients and those with extended follow-up periods.

Transcription factor Hoxb5 reveals the unidirectional hierarchy of hematopoietic stem cell pool.

Hoxb5 exhibits preferential expression in hematopoietic stem cells (HSCs) and uniquely marks the long-term HSCs (LT-HSCs). Previous studies have demonstrated the remarkable capability of Hoxb5 to alter cell fates when enforced expression in blood progenitors, such as B cell progenitors and multipotent progenitors. Additionally, Hoxb5 deficiency does not hinder the generation of LT-HSCs. However, the specific impact of Hoxb5 deletion on LT-HSCs has remained unexplored. To address this, we developed a conditional Hoxb5 knockout-reporter mouse model, wherein Hoxb5 was knock out by the Vav-cre recombinase, and the endogenous Hoxb5 promoter drove the expression of the blue fluorescent protein (BFP). Our findings revealed that the primary recipients, who transplanted with HSCs indicating Hoxb5 deficiency by the presence of BFP (BFP-positive HSCs), exhibited comparable levels of donor chimerism and lineage chimerism to recipients transplanted with HSCs that spontaneously did not express Hoxb5 and thus lacked BFP expression (BFP-negative HSCs). However, during the secondary transplantation, recipients receiving total bone marrow (BM) from the primary recipients with BFP-positive HSCs showed significantly higher levels of donor chimerism and more robust multi-lineage chimerism compared to those receiving total BM from the primary recipients with BFP-negative HSCs. Our results indicate that deleting Hoxb5 in LT-HSCs transiently influences their lineage differentiation bias without compromising their long-term self-renewal capacity. These findings highlight the primary role of Hoxb5 in regulating lineage commitment decisions in LT-HSCs, while emphasizing that its presence is not indispensable for the maintenance of long-term self-renewal capacity.

Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR-T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour-killing efficacy and persistence. Results showed all constructs enhanced tumour-killing and prolonged survival in tumour-bearing mice. In particular, CAR1 (CD8 TMD-CD3ζ SD)-NK cells showed superior efficacy in treating tumour-bearing animals and exhibited enhanced persistence when combined with OX40 co-stimulatory domain. Of note, CAR1-NK cells were most effective at lower effector-to-target ratios, while CAR4 (CD8 TMD-OX40 CD- FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1-, CAR2 (CD8 TMD- FcεRIγ SD)-, CAR3 (CD8 TMD-OX40 CD- CD3ζ SD)- and CAR4-NK cells over those treated with CAR5 (CD28 TMD- FcεRIγ SD)-, CAR6 (CD8 TMD-4-1BB CD-CD3ζ 1-ITAM SD)- and CAR7 (CD8 TMD-OX40 CD-CD3ζ 1-ITAM SD)-NK cells, with CAR5-NK cells showing the weakest anti-tumour activity. Increased expression of exhaustion markers, especially in CAR7-NK cells, suggests that combining CAR-NK cells with immune checkpoint inhibitors might improve anti-tumour outcomes. These findings provide crucial insights for developing CAR-NK cell products for clinical applications.

Prolonged generation of multi-lineage blood cells in wild-type animals from pluripotent stem cells.

Regenerating prolonged multi-lineage hematopoiesis from pluripotent stem cells (PSCs), an unlimited cell source, is a crucial aim of regenerative hematology. In this study, we used a gene-edited PSC line and revealed that simultaneous expression of three transcription factors, Runx1, Hoxa9, and Hoxa10, drove the robust emergence of induced hematopoietic progenitor cells (iHPCs). The iHPCs engrafted successfully in wild-type animals and repopulated abundant and complete myeloid-, B-, and T-lineage mature cells. The generative multi-lineage hematopoiesis distributed normally in multiple organs, persisted over 6 months, and eventually declined over time with no leukemogenesis. Transcriptome characterization of generative myeloid, B, and T cells at the single-cell resolution further projected their identities to natural cell counterparts. Thus, we provide evidence that co-expression of exogenous Runx1, Hoxa9, and Hoxa10 simultaneously leads to long-term reconstitution of myeloid, B, and T lineages using PSC-derived iHPCs as the cell source.

Hoxb5 reprogrammes murine multipotent blood progenitors into haematopoietic stem cell-like cells.

OBJECTIVES: The expression of transcription factor Hoxb5 specifically marks the functional haematopoietic stem cells (HSC) in mice. However, our recent work demonstrated that ectopic expression of Hoxb5 exerted little effect on HSC but could convert B-cell progenitors into functional T cells in vivo. Thus, cell type- and development stage-specific roles of Hoxb5 in haematopoietic hierarchy await more extensive exploration. In this study, we aim to investigate the effect of Hoxb5 expression in multipotent blood progenitor cells. MATERIALS AND METHODS: A Mx1cre/RosaLSL-Hoxb5-EGFP/+ mouse model was used to evaluate the effect of Hoxb5 expression in blood multipotent progenitor cells (MPP). Golden standard serial transplantation experiments were used to test the long-term haematopoiesis potential of Hoxb5-expressing MPP. Single-cell RNA-seq analysis was used to characterize the gained molecular features of Hoxb5-expressing MPP and to compare with the global transcriptome features of natural adult HSC and fetal liver HSC (FL HSC). RESULTS: Here, with a mouse strain engineered with conditional expression of Hoxb5, we unveiled that induced expression of Hoxb5 in MPP led to the generation of a de novo Sca1+ c-kit+ CD11b+ CD48+ (CD11b+ CD48+ SK) cell type, which can repopulate long-term multilineage haematopoiesis in serial transplantations. RNA-seq analysis showed that CD11b+ CD48+ SK cells exhibited acquired features of DNA replication and cell division. CONCLUSIONS: Our current study uncovers that Hoxb5 can empower MPP with self-renewal ability and indicates an alternative approach for generating HSC-like cells in vivo from blood lineage cells.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors.

Regeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-induced NK (iNK) cells are a source of off-the-shelf cell products for universal immune therapy. Conventional methods for iNK cell regeneration from hPSCs include embryoid body (EB) formation and feeder-based expansion steps, which are time-consuming and cause instability and high costs of manufacturing. Here, we develop an EB-free, organoid aggregate method for NK cell regeneration from hPSCs. In a short time-window of 27-day induction, millions of hPSC input can output over billions of iNK cells without the necessity of NK cell expansion feeders. The iNK cells highly express classical toxic granule proteins, apoptosis-inducing ligands, as well as abundant activating and inhibitory receptors. Functionally, the iNK cells eradicate human tumor cells via mechanisms of direct cytotoxicity, apoptosis, and antibody-dependent cellular cytotoxicity. This study provides a reliable scale-up method for regenerating human NK cells from hPSCs, which promotes the universal availability of NK cell products for immune therapy.

Tribulus terrestris L. protects glomerular endothelial cells via the miR155-H2AC6 interaction network in hypertensive renal injury.

BACKGROUND: Hypertensive renal injury is one of the most lethal complications of hypertension. At present, renin-angiotensin-aldosterone system (RAAS) blockers are considered the best drugs for the treatment of renal injury in hypertension because of their nephroprotective effect of reducing proteinuria, but there are no specific drugs for this purpose, however, clinical trials proved that Chinese medicine has a protective effect on target organs in the treatment of hypertension. Tribulus terrestris L. (TrT), a traditional Chinese medicine (TCM), has potential applications due to its reno-protective and immunomodulatory effects. METHODS: We investigated the underlying reno-protective mechanism of TrT on Angiotensin II (AngII)-induced hypertensive renal injury in glomerular endothelial cells by integrating the differential expression profiles of micro RNA (miRNA) and messenger RNA (mRNA) to construct a miRNA-mRNA interaction network associated with hypertensive kidney injury, followed by quantitative real-time polymerase chain reaction (qRT-PCR) for validation. RESULTS: Seventy-six differentially expressed mRNAs (DEmRNAs) and 1 differentially expressed miRNAs (DEmiRNAs) were identified in the control group and the AngII-induced hypertensive renal injury group, respectively. 110 DEmRNAs and 27 DEmiRNAs were identified in the TrT treatment group and the AngII-induced group, respectively. The core component of the miRNA-mRNA network was miR-155-5p. Our study showed that miR-155-5p expression levels were more decreased in the AngII-induced hypertensive renal injury group than the control group. TrT treatment also significantly upregulated miR-155-5p. Additionally, we found that miR-155-5p expression levels were negatively correlated with H2A clustered histone 6 (H2AC6). CONCLUSIONS: The results of this study indicate that TrT has a reno-protective effect on AngII-induced hypertensive renal injury by miR-155-5p, which negatively regulates the expression of H2AC6. Our findings offer a new therapeutic strategy and have identified an effective candidate target for the treatment of hypertensive renal injury in clinical settings.

Identification of lncRNA-mRNA regulatory network associated with isolated systolic hypertension and atherosclerotic cerebral infarction.

BACKGROUND: Increasingly, evidence has shown that long non-coding RNAs (lncRNAs) play an important role in isolated systolic hypertension (ISH). However, a systematic lncRNA-messenger RNA (mRNA) regulatory network is still absent in isolated systolic hypertension and atherosclerotic cerebral infarction patients (ISH & ACI). This research aimed to establish a lncRNA-mRNA co-expression network in patients with ISH & ACI, to probe into the potential functions of lncRNA in such patients. METHODS: Expression profiles of lncRNA and mRNAs were collected and compared, from 8 patients with ISH and 8 patients with ISH & ACI by RNA-seq data. Differentially expressed lncRNAs and mRNAs were screened out via high-throughput sequencing in the plasma of ISH/ACI patients and control ISH patients. Then, a lncRNA-mRNA interaction network was built using the Pearson correlation coefficient by Cytoscape software. The expression levels of the hub genes and lncRNAs were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in another 10 ISH/ACI patients and 10 control patients. This study was approved by the responsible institutional review board (IRB) and informed consent was provided by participants. RESULTS: A total of 2,768 differentially expressed lncRNAs and 747 differentially expressed mRNAs were identified. We identified two hub genes (CD226 and PARVB) and 11 lncRNAs in the lncRNA-mRNA interaction network. The results of qRT-PCR and cell assay verified that lncRNAs ENST00000590604 and CD226 are highly expressed in patients of ISH & ACI. Further, CD226 was associated with vascular endothelial cells growth and stability through the platelet activation and focal adhesion pathway. CONCLUSIONS: We established a novel mRNA-lncRNA interaction network. The lncRNAs ENST00000590604 and CD226 might be the potential biomarkers of ISH & ACI.

Two-step protocol for regeneration of immunocompetent T cells from mouse pluripotent stem cells.

Numerous efforts have been attempted to regenerate T cells in culture dish from pluripotent stem cells (PSCs). However, in vitro generated T cells exhibited extremely low activity and compromised immunocompetency in vivo. Here, we describe a two-step protocol for regenerating functional T cells using an inducible Runx1-Hoxa9-PSC (iR9-PSCs) line. The procedure mainly includes generation of induced hematopoietic progenitor cells (iHPCs) in vitro, transplantation, and development of functional induced T cells (iT) in vivo via transplantation. The entire induction process in vitro requires 21 days before iHPCs transplantation. The development of mature T cells in vivo takes 4 to 6 weeks post-transplantation. We provide a simple and reproducible approach for functional T cell regeneration from iR9-PSCs for research purpose.

Hematopoietic lineage-converted T cells carrying tumor-associated antigen-recognizing TCRs effectively kill tumor cells.

Tumor-associated antigen (TAA) T-cell receptor (TCR) gene-engineered T cells exhibit great potential in antitumor immunotherapy. Considering the high costs and low availability of patient-derived peripheral blood T cells, substantial efforts have been made to explore alternatives to natural T cells. We previously reported that enforced expression of Hoxb5 converted B cells into induced T (iT) cells in vivo Here, we successfully regenerated naive OT1 (major histocompatibility complex I restricted ovalbumin antigen) iT cells (OT1-iT) in vivo by expressing Hoxb5 in pro-pre-B cells in the OT1 transgenic mouse. The OT1-iT cells can be activated and expanded in vitro in the presence of tumor cells. Particularly, these regenerated OT1-iT cells effectively eradicated tumor cells expressing the TAA (ovalbumin) both in vitro and in vivo This study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.

Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.

Achievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-to-hematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαß repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition.

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

A protocol for generating induced T cells by reprogramming B cells in vivo.

Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells in vivo. This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research.

Overexpression of Short Variant Form of New Kelch Family Protein Leads to Erythroid and Megakaryocyte Dysplasia by Targeting Megakaryocyte-Erythroid Progenitors.

Nd1-S is the nuclear-localizing short variant form of Nd1 (Ivns1abp) encoding a Kelch family transcription factor. While the function of Nd1 has been investigated in the context of metastasis and doxorubicin-induced cardiotoxicity, little is known about its role in hematopoiesis. In this study, we investigated the function of Nd1-S in hematopoiesis by transplanting the Nd1-S-overexpressing murine hematopoietic stem and progenitor cells (HSPCs) into recipient mice (Nd1-S mice). Enforced expression of Nd1-S led to erythroid and megakaryocyte dysplasia, demonstrated by dramatically decreased red blood cells and platelets, and megakaryocytes in the peripheral blood and bone marrow of the Nd1-S mice. Moreover, phenotypic megakaryocyte-erythroid progenitors (MEPs) accumulated in these Nd1-S mice with aberrant morphology and defective colony-forming capability. Furthermore, these phenotypic MEPs showed impaired pathways regulating erythroid differentiation and megakaryocyte development. Therefore, our study provides de novo evidence that overexpression of Nd1-S in HSPCs leads to erythroid and megakaryocyte dysplasia in vivo by targeting MEPs.

Pyrimidoindole derivative UM171 enhances derivation of hematopoietic progenitor cells from human pluripotent stem cells.

In the field of hematopoietic regeneration, deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells, remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines, UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here, using a serum-free, feeder-free, and chemically defined induction protocol, we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34+CD45+ derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol, the derived hematopoietic progenitors failed to engraft in NOG mice, indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols, UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro.