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.

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.

The Immunopeptidome from a Genomic Perspective: Establishing the Noncanonical Landscape of MHC Class I-Associated Peptides.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

Antigen-specific TCR-T cells from Rag2 gene-deleted pluripotent stem cells impede solid tumour growth in a mouse model.

The technology of adoptive transfer of T-cell receptor (TCR) engineered T cells is wildly investigated as it has the potential to treat solid cancers. However, the therapeutic application of TCR-T cells is hampered by the poor quality derived mainly from patients' peripheral blood, as well as heterogeneous TCRs caused by the mismatch between transgenic and endogenous TCRs. To improve the homogeneity, antigen-specificity and reduce possible autoreactivity, here we developed a technique to generate antigen-specific T cells from Rag2 gene-deleted pluripotent stem cells (PSCs) and further measured their anti-tumour efficacy. PSCs were first targeted with OT1 TCR into the Rag2 locus to prevent TCR rearrangement during T-cell development. The engineered PSCs were then differentiated through a two-step strategy, in vitro generation of haematopoietic progenitor cells, and in vivo development and maturation of TCR-T cells. Finally, the response to tumour cells was assessed in vitro and in vivo. The regenerated OT1-iT displayed monoclonal antigen-specific TCR expression, and phonotypic normalities in the spleen and lymph node tissues. Importantly, the OT1-iT cells eliminated tumour cells while releasing specific cytokines in vitro. Furthermore, adoptive transfer of OT1-iT cells suppresses solid tumour growth in tumour-bearing animals. Our study presents a novel and straightforward strategy for producing antigen-specific TCR-T cells in vivo from PSCs, allowing for allogeneic transplantation and therapy of solid tumours.

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.

Loss of Nupr1 promotes engraftment by tuning the quiescence threshold of hematopoietic stem cell repository via regulating p53-checkpoint pathway.

Hematopoietic stem cells (HSCs) are dominantly quiescent under homeostasis, which is a key mechanism of maintaining the HSC pool for life-long hematopoiesis. Dormant HSCs poise to be immediately activated on urgent conditions and can return to quiescence after regaining homeostasis. To date, the molecular networks of regulating the threshold of HSC dormancy, if exist, remain largely unknown. Here, we unveiled that deletion of Nupr1, a gene preferentially expressed in HSCs, activated the quiescence HSCs under homeostatic status, which conferred engraftment competitive advantage on HSCs without compromising their stemness and multi-lineage differentiation abilities in serial transplantation settings. Following an expansion protocol, the Nupr1-/- HSCs proliferate more robustly than their wild type counterparts in vitro. Nupr1 inhibits the expression of p53 and the rescue of which offsets the engraftment advantage. Our data unveil the de novo role of Nupr1 as an HSC quiescence-regulator, which provides insights into accelerating the engraftment efficacy of HSC transplantation by targeting the HSC quiescence-controlling network.

Grade repetition and bullying victimization in adolescents: A global cross-sectional study of the Program for International Student Assessment (PISA) data from 2018.

BACKGROUND: Grade repetition is practiced worldwide and varies considerably across the globe. Globally, around 32.2 million students repeated a grade at the primary education level in 2010. Although a large body of research has documented grade repetition's academic and non-academic effects, the limited evidence on associations between grade repetition and school bullying is inconsistent and ambiguous. This study aimed to investigate the global association of grade repetition with bullying victimization in a large-scale school-based cross-sectional study. METHODS AND FINDINGS: We used the latest global data from the Program for International Student Assessment (PISA) 2018. PISA 2018 was conducted between March and August 2018 in 80 countries and economies among students aged 15-16 years attending secondary education. The students reported their experiences of repeating a grade at any time point before the survey and of being bullied in the past 12 months. The outcome measures were 6 types of bullying victimization. We accounted for the complex survey design and used multivariate logistic regression models to estimate the odds ratios (ORs) with 95% confidence intervals (CIs) of grade repetition with bullying victimization after adjusting for potential confounders (sex; age group; migrant status; school type; economic, social, and cultural status; and parental emotional support). This study included 465,146 students (234,218 girls and 230,928 boys) with complete data on grade repetition and bullying victimization in 74 countries and economies. The lifetime prevalence of grade repetition was 12.26%, and 30.32% of students experienced bullying at least a few times a month during the past 12 months. Grade repetition was statistically significantly associated with each type of bullying victimization. The OR (95% CI) of overall bullying victimization for grade repeaters compared with their promoted peers was 1.42 (95% CI 1.32-1.52, p < 0.001). The sex-specific analysis produced similar results in both boys and girls. Furthermore, girls who repeated a grade had higher risks of being made fun of, being threatened, having possessions taken away, and being pushed around than boys. The major limitation is that this study only included students attending schools and therefore may be subject to possible selection bias. In addition, the cross-sectional design hinders us from establishing causality between grade repetition and bullying victimization. CONCLUSIONS: In this study, we observed that, globally, both boys and girls who repeat a grade are at increased risk of being bullied compared with promoted peers, but girls may experience higher risks than boys of specific types of bullying associated with repeating a grade. These findings provide evidence for the association of grade repetition with bullying victimization. Sex differences in risk of experiencing some types of bullying suggest that tailored interventions for girls who repeat a grade may be warranted.

MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

OBJECTIVES: Exploring approaches of extending the haematopoiesis time window of MPPs and lineage-committed progenitors might produce promising therapeutic effects. NUP98-HOXA10hd (NA) fusion protein can expand long-term haematopoietic stem cells (HSCs) and promote engraftment competitiveness without causing obvious oncogenesis. Our objectives were to investigate the roles of NA fusion protein in MPP and downstream lineage-committed progenitor context. MATERIAL AND METHODS: 300 sorted MPPs (Lin- CD48- c-kit+ Sca1+ CD135+ CD150- ) were mixed with 5 × 105 total BM helper/competitor cells and injected into irradiated recipients. For secondary transplantation, 5 × 106 total BM cells from primary recipient mice were injected into lethally irradiated recipients. NA-MPP recipient mice were sacrified for flow cytometric analysis of bone marrow progenitors at indicated time points. Sorted MPPs and myeloid progenitors were used for RNA-seq library preparation. RESULTS: We showed that NA-expressing MPPs achieved significantly longer multi-lineage haematopoiesis (>44-week) than natural MPPs (20-week). NA upregulated essential genes regulating long-term haematopoiesis, cell cycle, epigenetic regulation and responses to stress in MPPs. These molecular traits are associated with the earlier appearance of a Sca1- c-kit+ myeloid progenitor population, and more abundant cellularity of lineage-committed progenitor as well as bone marrow nucleated cells. Further, the NA-derived primary bone marrow cells, which lack NA-LSK cells, successfully repopulated secondary multi-lineage haematopoiesis over 20 weeks. CONCLUSIONS: This study unveiled that NA fusion protein promotes MPP and lineage-committed progenitor engraftment via extending long-term multi-lineage haematopoiesis.

Mesenchymal stem cells suppress leukemia via macrophage-mediated functional restoration of bone marrow microenvironment.

Bone marrow (BM) mesenchymal stem cells (MSCs) are critical components of the BM microenvironment and play an essential role in supporting hematopoiesis. Dysfunction of MSCs is associated with the impaired BM microenvironment that promotes leukemia development. However, whether and how restoration of the impaired BM microenvironment can inhibit leukemia development remain unknown. Using an established leukemia model and the RNA-Seq analysis, we discovered functional degeneration of MSCs during leukemia progression. Importantly, intra-BM instead of systemic transfusion of donor healthy MSCs restored the BM microenvironment, demonstrated by functional recovery of host MSCs, improvement of thrombopoiesis, and rebalance of myelopoiesis. Consequently, intra-BM MSC treatment reduced tumor burden and prolonged survival of the leukemia-bearing mice. Mechanistically, donor MSC treatment restored the function of host MSCs and reprogrammed host macrophages into arginase 1 positive phenotype with tissue-repair features. Transfusion of MSC-reprogrammed macrophages largely recapitulated the therapeutic effects of MSCs. Taken together, our study reveals that donor MSCs reprogram host macrophages to restore the BM microenvironment and inhibit leukemia development.

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.

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.

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.

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.

Trim27 confers myeloid hematopoiesis competitiveness by up-regulating myeloid master genes.

Trim27 (Zinc finger protein RFP) is a potential regulator of hematopoietic stem cells (HSC), yet its role in hematopoiesis remains elusive. Here, we investigated the roles of Trim27 in hematopoiesis by enforcing its expression in mouse and human hematopoietic stem and progenitor cells (HSPC). Ectopic expression of Trim27 in mouse fetal liver (FL) HSPC confers repopulating advantage with myeloid dominance. However, the number of HSC from Trim27 group was comparable with empty vector control group, indicating that overexpression of Trim27 unlikely expanded HSC. Transcriptome analysis of Trim27-overexpressing myeloid progenitor cells (MP) indicated that Trim27 up-regulated essential regulators of myelopoiesis, including Spi1 and Cebpg, up-regulated myeloid proliferation-related signaling genes Nras, Runx1, and Cbfb, up-regulated JAK/STAT signaling inhibitors Socs2, Socs3, and Cish, and up-regulated myeloid maturation-related genes Adam8 and Dek. Moreover, the myeloproliferative advantage caused by overexpressing Trim27/TRIM27 is conserved between mouse and human hematopoiesis. To our knowledge, this is the first study showing that Trim27 confers competitive hematopoiesis by promoting myeloid bias differentiation of HSPC, but not by expanding HSC.

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.