Acute graft-versus-host disease (GVHD) remains a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with limited treatment options for steroid-refractory cases. Current therapies broadly suppress immune responses, increasing infection risk and impairing graft function. We hypothesized that selective depletion of donor immune cells through HLA allele-specific targeting could offer a safer and more precise alternative. To test this, we developed a monoclonal antibody (AN7) against HLA-A2 by immunizing HLA-A24 transgenic mice with recombinant HLA-A2 tetramers, followed by hybridoma screening. AN7 specifically recognized HLA-A2 and HLA-A68, and was reformatted into murine IgG2a and human chimeric IgG1 recombinant antibodies. These retained binding specificity and mediated robust complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) against HLA-A2+ target cells. To evaluate therapeutic potential, we established a fully MHC-mismatched allo-HSCT model using HLA-A2 transgenic donor bone marrow in lethally irradiated BALB/c recipients. A single ultra-low dose of AN7 (1-100 ng) administered post-transplant significantly improved survival and reduced GVHD while preserving donor hematopoiesis. These findings highlight the potential of allele-specific antibody-mediated clearance as a mechanistically distinct approach to donor immune modulation in GVHD.

IntroductionCD19-directed chimeric antigen receptor (CD19-CAR) T-cell therapy has markedly improved outcomes in relapsed and refractory B-cell malignancies, but its efficacy remains limited by insufficient in vivo persistence and functional exhaustion. We have generated functionally rejuvenated T-cells (rejTs) by reprogramming antigen-specific cytotoxic T lymphocytes (CTLs) into induced pluripotent stem cells (iPSCs) and redifferentiating them into CTLs with restored proliferative capacity. In this study, we explored a vaccine synergy strategy to enhance the persistence of CAR-rejuvenated CTLs (CARrejTs) through T-cell receptor (TCR) restimulation.MethodsSARS-CoV-2 spike protein-specific rejTs (COVID19-rejTs) were established from iPSCs derived from spike protein-specific CTLs. A CD19-CAR was introduced into these iPSCs to generate dual-antigen recognition CARrejTs targeting CD19 and COVID-19 spike protein (1919-CARrejTs). Subsequently, 1919-CARrejTs were assessed for cytotoxicity, proliferative capacity, and exhaustion phenotype using 51Cr release assays, sequential rechallenge assays, and CFSE-based proliferation analysis with CAR- or TCR-dependent stimulation.Results1919-CARrejTs uniformly expressed both CD19-CAR and spike protein-specific TCRs, retained antigen-specific cytotoxicity, and exhibited a rejuvenated phenotype with higher expression of granzyme B and perforin and lower expression of exhaustion markers compared with conventional CD19-CAR-T cells. Dual-antigen recognition enhanced cytotoxicity under matched antigen presentation, and 1919-CARrejTs maintained durable tumor control in sequential rechallenge assays. CFSE dilution analysis revealed that TCR-mediated stimulation by spike protein-specific peptide provided strong proliferative capacity of 1919-CARrejTs in an HLA-dependent manner.ConclusionThe combination of iPSC-mediated rejuvenation and dual-antigen recognition via CAR and native TCR confers superior cytotoxicity, persistence, and proliferative potential compared to conventional CD19-CAR-T cells. These findings provide a proof-of-concept for a vaccine-synergy strategy in which in vivo TCR restimulation supports selective expansion and sustained antitumor effect of dual-antigen recognition T-cells that can be a promising treatment approach for B-cell malignancies.

Plots of top 100 DMRs (by area statistic & FWER) between DNMT3AR882H/WT re-mutated and DNMT3AWT/WT samples for patient SU575

Supplementary Figures S1-S11, Additional information for Supplementary Tables S1-S8 and Supplementary Data S1-S6. Fig. S1: Gene editing of primary human patient specimens. Fig. S2: DNMT3A activity assay validates restoration of DNMT3A activity upon correction of DNMT3AR882 missense mutations. Fig. S3: Isolation and functional validation of pre-leukemic HSCs from primary AML specimens. Fig. S4: Genotyping of gene-edited pre-leukemic HSCs. Fig. S5: DNMT3A genotyping in single-cell RNA-Seq data. Fig. S6: Engraftment and genotyping of gene-edited leukemic blasts. Fig. S7: Generation and features of AML-derived iPSC line iSU444. Fig. S8: In vivo correction of DNMT3AR882H. Fig. S9: Whole genome bisulfite sequencing of DNMT3A corrected leukemic cells. Fig. S10: Genotyping of secondary transplant recipients. Fig. S11: Representative loci showing stable methylation and late hypermethylation.

Aims:Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and Results:Isogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. Conclusions:Our study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM.

The persistent shortage of human organs for transplantation has intensified efforts to develop alternative sources, specifically xenotransplantation and exogenesis. Xenotransplantation uses genetically engineered pigs to provide organs, tissues, and cells for clinical use. Significant progress has occurred in developing multigene-modified pigs that lack glycan xenoantigens while expressing human complement and coagulation regulators. These modifications have successfully mitigated hyperacute, antibody-mediated, and cellular rejection in preclinical nonhuman primate models. Recent compassionate-use cases in humans have demonstrated the feasibility of heart, kidney, and liver xenotransplantation, although achieving long-term survival remains a challenge. Complementing this approach, exogenesis aims to generate human-compatible organs within animal hosts through interspecies chimerism. Although advances in establishing organ niches and overcoming xeno-barriers have yielded preliminary success in heart, pancreas, and muscle development, formidable immune and developmental hurdles remain. Together, these approaches offer promising strategies to expand the donor organ pool and address the growing global demand for transplantation. Further advances in genetic engineering, immune modulation, and developmental biology, supported by rigorous preclinical and clinical evaluation, will be critical for widespread translation. This review outlines the current progress, major challenges, and future directions in xenogeneic and exogenic organ generation.

Organ shortage remains a major challenge in transplantation medicine. Interspecies blastocyst complementation offers a promising strategy to generate human organs in livestock. However, efficient xenogeneic donor cell engraftment remains challenging. Here, we identify an innate immune barrier wherein host macrophages selectively eliminate viable xenogeneic donor cells, a process we term xenophagocytosis. Mechanistically, xenogeneic cells display elevated phosphatidylserine, an "eat-me" signal recognized by host macrophages through phagocytic receptor Axl. We demonstrate three orthogonal strategies for xenophagocytosis blockade: genetic ablation of macrophages or the Axl receptor in the host embryo or overexpression of the "don't-eat-me" signal CD47 or the phosphatidylserine-regulating flippase ATP11C in donor cells. Xenophagocytosis blockade enhances rat and human donor chimerism in mouse embryos and improves interspecies pancreas complementation efficiency. These findings reveal a previously unrecognized innate immune barrier that safeguards species integrity during early embryogenesis and provide mechanistic insights to enhance xenogeneic chimerism for generating human organs in livestock.

Hematopoietic stem cells (HSCs) are blood-forming stem cells that can reconstitute the entire blood system after transplantation, which provides curative options for both blood and non-blood diseases. Despite their known utility, broader applications of human HSCs are hampered by the lack of a stable culture protocol. Previously, we developed a long-term mouse HSC expansion protocol using a synthetic polymer to replace serum albumin, thus mitigating inflammatory responses that trigger HSC differentiation. Although this approach, with ongoing improvements, has been applied to human HSC cultures, achieving long-term expansion of human HSCs remains challenging. To address this, we optimized a previously published cytokine-free culture for human HSCs, which utilizes butyzamide, a thrombopoietin (TPO) receptor agonist (TPO-RA), and 740 Y-P, a PI3K activator, as replacements for TPO and stem cell factor (SCF), respectively. In a short-term (7-day) culture, we found that supplementing SCF in addition to 740 Y-P, or substituting butyzamide with lusutrombopag, an FDA-approved TPO-RA, enhanced the ex vivo expansion of phenotypic hematopoietic stem and progenitor cells (lineage⁻CD41⁻CD34⁺) from CD34+ umbilical cord blood (UCB) cells. In xenograft transplantation model using immunodeficient NSG mice, a mid-term (14-day) physioxic (5% O₂) culture supplemented with lusutrombopag and SCF, named “5LS culture”, resulted in robust expansion of engraftable human HSCs (mean human chimerism: 22.9% [14-day expanded] vs. 0.96% [unmanipulated], with multilineage reconstitution of CD33⁺ myeloid and CD19⁺ B cells; N = 7 mice, 16 weeks post-transplantation). Molecularly, SCF consistently upregulated phosphorylation of AKT, STAT5, and p38 in phenotypic multipotent progenitors/HSCs (pMPPs/pHSCs: lineage⁻CD41⁻CD34⁺EPCR⁺) across different samples, whereas 740 Y-P did not, suggesting a mechanistic insight why SCF addition improves human HSC expansion. We next extended the culture duration to 28 days. Interestingly, despite successful 14-day expansion in the 5LS culture, CD34⁺ UCB cells cultured for 28 days predominantly reconstituted CD33⁺ myeloid cells but lost their lymphoid reconstitution potential in NSG mice. Further optimization identified that FLT3 ligand (FLT3-L) supplementation in a 5LS culture (resulting in the “5LSF culture”) improved both the expansion and the maintenance of phenotypic HSCs (pHSCs: lineage⁻CD41⁻CD34⁺EPCR⁺CD90⁺ITGA3⁺) over 28-day culture of CD34+ UCB cells. Similar results were observed using CD34⁺ mobilized peripheral blood cells. Single-cell RNA sequencing with UMAP visualization and clustering analyses revealed that while HSC/MPP clusters from 5LS cultures were skewed toward granulocyte-monocyte progenitors (GMPs), this skewing was absent in the 5LSF condition. Notably, xenotransplantation demonstrated partial restoration of CD19⁺ B cell reconstitution from 28-day expanded CD34⁺ UCB cells in the 5LSF culture (mean B cell contribution among human cells: 0% [5LS culture] vs. 32.1% [5LSF culture], N = 7 mice, 16 weeks post-transplantation). These results highlight that FLT3-L is critical for maintaining multipotency of HSCs in long-term culture.To assess whether the 5LSF culture supports genetic modification, we performed lentiviral transduction and CRISPR-Cas9/AAV6-mediated targeted gene editing in CD34⁺ UCB cells. Lentiviral vectors successfully transduced the pHSC population. Targeted editing of a reporter cassette in a HLF (a transcription factor specifically expressed in HSCs) locus confirmed efficient editing in HLF⁺ HSCs, underscoring the utility of 5LSF culture for HSC genetic engineering. Finally, a limiting dilution assay using unmanipulated, 14-day-expanded, and 28-day-expanded CD34+ UCB cells enabled estimation of functional HSC frequencies and fold expansion over a 28-day culture. The 5LSF culture exhibited the highest fold expansion of functional HSCs (109.0-fold expansion) after 28 days. In contrast, the 5LS culture demonstrated greater fold expansion of functional HSCs after 14 days (60.6-fold expansion) compared to the 5LSF culture (23.1-fold expansion), which is consistent with the results from the phenotypic characterization. These findings provide evidence that human functional HSC can stably expands long-term in the 5LSF culture. In conclusion, we have developed a long-term expansion protocol for human HSCs that facilitates both clinical and research applications of these functional stem cells.

Intercellular transmission of messenger RNA (mRNA) is being explored in mammalian species using immortal cell lines. Here, we uncover an intercellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells under the condition impermissible for primed hPSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis shows the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 d of coculture with mouse embryonic stem cells, surface marker analysis and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, interspecies mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes, which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and interspecies cellular communications.

A long-standing question in biology is the extent to which cells function autonomously as opposed to requiring interactions with other cells or environmental factors. Here, we develop a framework to use interspecies chimeras to precisely decompose evolutionary divergence in any cellular trait into cell-intrinsic and cell-extrinsic components. Applying this framework to thousands of gene expression levels in reciprocal rat-mouse chimeras, we found that most divergence is cell intrinsic, though extrinsic factors also play an integral role. For example, cell-extrinsic regulation of a transcription factor can propagate to its target genes, leading to cell-type-specific extrinsic regulation of both their mRNA and their protein levels. We also show that imprinted genes are dramatically misexpressed in chimeras, suggesting a mismatch between rapidly evolving intrinsic and extrinsic imprinting mechanisms. Overall, our conceptual framework opens up new avenues to investigate the mechanistic basis of the evolution, development, and regulation of myriad cellular traits in any multicellular organism.

Naive pluripotent stem cells (PSCs) are counterparts of early epiblast in the mammalian embryo. Mouse and human naive PSCs differ in self-renewal requirements and extraembryonic lineage potency. Here, we investigated the generation of chimpanzee naive PSCs. Colonies generated by resetting or reprogramming failed to propagate. We discovered that self-renewal is enabled by inhibition of Polycomb repressive complex 2 (PRC2). Expanded cells show global transcriptome proximity to human naive PSCs and embryo pre-implantation epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee naive PSCs can transition to multilineage competence or can differentiate into trophectoderm and hypoblast, forming tri-lineage blastoids. They thus provide a higher primate comparative model for studying pluripotency and early embryogenesis. Genetic deletions confirm that PRC2 mediates growth arrest. Further, inhibition of PRC2 overcomes a roadblock to feeder-free propagation of human naive PSCs. Therefore, excess deposition of chromatin modification H3K27me3 is an unexpected barrier to naive PSC self-renewal.

In vivo gene therapy targeting hematopoietic stem cells (HSCs) holds significant therapeutic potential for treating hematological diseases. This study uses adeno-associated virus serotype 6 (AAV6) vectors and Cre recombination to systematically optimize the parameters for effective in vivo HSC transduction. We evaluated various genetic architectures and delivery methods of AAV6, establishing an optimized protocol that achieved functional recombination in more than two-thirds of immunophenotypic HSCs. Our findings highlight that second-strand synthesis is a critical limiting factor for transgene expression in HSCs, leading to significant under-detection of HSC transduction with single-stranded AAV6 vectors. We also demonstrate that HSCs in the bone marrow (BM) are readily accessible to transduction, with neither localized injection nor mobilization of HSCs into the bloodstream, enhancing transduction efficacy. Additionally, we observed a surprising preference for HSC transduction over other BM cells, regardless of the AAV6 delivery route. Together, these findings not only underscore the potential of AAV vectors for in vivo HSC gene therapy but also lay a foundation that can inform the development of both in vivo AAV-based HSC gene therapies and potentially in vivo HSC gene therapies that employ alternative delivery modalities.

The tumor microenvironment (TME) is deeply involved in cancer progression and treatment resistance. Although humanized mouse models have been developed by transplanting human cells into immunodeficient mice, they fail to fully reconstitute the TME. Blastocyst complementation using Flk-1 (Vegfr2, Kdr) knockout hosts offers a potential solution. However, the generation of interspecies human-mouse chimeras using blastocyst complementation has not yet been successful. As a foundational step, this study aims to demonstrate that donor-derived TME can be constructed using this method in intraspecies chimeric mice. We generated chimeric mice by injecting Azami-Green (AG)-positive C57BL/6 (B6) mouse-derived embryonic stem cells (ESCs) into ICR Flk-1 knockout embryos. We observed that vascular endothelial cells (VECs), hematopoietic cells, and tissue-resident macrophages were derived from the injected AG-positive ESCs. We engrafted B6-derived tumor cells into the chimeras and identified tumor-infiltrating lymphocytes, tumor-associated macrophages, and VECs derived from donor cells. Moreover, tumor-infiltrating CD8+ T cells in these chimeric mice showed cytotoxic activity comparable to that in wild-type mice. We anticipate that this intraspecies chimeric mouse model can serve as a valuable tool for basic research. Furthermore, future humanized tumor models generated via blastocyst complementation have the potential to significantly advance anticancer drug development in the preclinical phase.

Stem cell-based human embryo models offer a unique opportunity for functional studies of the human-specific features of development. Here we genetically and epigenetically manipulate human blastoids, a 3D embryo model of the blastocyst1, to investigate the functional effect of HERVK LTR5Hs, a hominoid-specific endogenous retrovirus, on pre-implantation development. We uncover a pervasive cis-regulatory contribution of LTR5Hs elements to the hominoid-specific diversification of the epiblast transcriptome in blastoids. Many of the LTR5Hs genomic insertions in the human genome are unique to our own species. We show that at least one such human-specific LTR5Hs element is essential for the blastoid-forming potential via enhancing expression of the primate-specific ZNF729 gene, encoding a KRAB zinc-finger protein. ZNF729 binds to GC-rich sequences, abundant at gene promoters associated with basic cellular functions, such as cell proliferation and metabolism. Despite mediating recruitment of TRIM28, at many of these promoters ZNF729 acts as a transcriptional activator. Together, our results illustrate how recently emerged transposable elements and genes can confer developmentally essential functions in humans.

Diamond-Blackfan anemia (DBA) is a rare hematological disorder characterized by red blood cell aplasia. Advances in genomic studies have identified mutations in ribosomal protein genes (such as RPS19, RPL11, RPS26) and non-ribosomal genes (GATA1, EPO, ADA2, TSR2) as the underlying causes of DBA. In addition to anemia, DBA patients often present with bone development-associated defects, including short stature, thumb or craniofacial anomalies, and an increased risk of developing osteosarcoma. These findings suggest a dysregulated non-hematopoietic bone marrow microenvironment (BME) in DBA patients, which is not well understood. Mesenchymal stem cells (MSCs) are a crucial component of the BME and can differentiate into osteoblasts, adipocytes, and chondrocytes. In this study, we determined the molecular defects of DBA MSCs using both mouse and human RPL11 haploinsufficient MSCs. Mouse DBA MSCs were derived from RPL11+/fl carrying Cre-ERt2 and Mx1-Cre, in which the Rpl11 gene is deleted upon injecting tamoxifen or polyinosinic:polycytidylic acid (poly(I:C)), respectively. Additionally, we used cell-permeable Cre in vitro to delete the gene and observe the immediate effect of gene deletion. For human MSCs, we either knocked out the RPL11 gene using the CRISPR/Cas9 system or used DBA patient-derived induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells. Using these models, we identified a decrease in the proliferation of MSCs from RPL11 mutant mice and a DBA patient compared to healthy MSCs (7.04x104±1.01 and 3.62x104±1.00 in WT and DBA, respectively). The reduced proliferation observed in DBA MSCs is due to G2/M phase cell cycle arrest. Interestingly, DBA cells did not show elevated p53 compared to WT MSCs, which is often observed in DBA patients. DBA cells showed a significant reduction of G2/M phase-associated mRNA and proteins such as cyclin A, cyclin B, and CDK1. DBA MSCs showed an increased accumulation of binucleated cells (27.01%±7.501) compared to healthy MSCs (4.995%±1.596), suggesting cytokinesis failure as the underlying cause for G2/M phase arrest. Cytokinesis failure is known to activate the Hippo pathway in cells. When the Hippo pathway is activated, a core kinase called LATS1/2 (large tumor suppressor kinases 1/2) phosphorylates a downstream target called YAP (yes-associated protein), a transcription co-activator that mediates cell proliferation, survival, cytoskeleton arrangement, and osteogenic differentiation in MSCs. Phosphorylation of YAP inhibits the nuclear translocation of YAP through cytoplasmic retention or protein degradation. Western blot analysis and fluorescence microscopy showed a 30% increase in cytoplasmic YAP and a reduction in nuclear YAP in DBA MSCs compared to normal MSCs. As a result of YAP inactivation, actin remodeling and osteogenic differentiation potential were significantly reduced in DBA MSCs. Both mouse and human DBA MSCs showed increased cortical actin accumulation and loss of lamellipodia and stellate morphology. Furthermore, DBA MSCs expressed low RUNX2 (Runt-related transcription factor 2), which is an essential transcription factor for osteoblast differentiation. As expected, both mouse and human DBA MSCs show reduced osteogenic differentiation potential compared to healthy MSCs. Based on these results, we hypothesize that reactivating YAP nuclear translocation increases proliferation in DBA MSCs. To test this hypothesis, we treated mouse DBA and healthy MSCs with a LATS inhibitor at 10nM, 100nM, 1µM, 5 µM, and 10 µM. After 7 days of treatment, DBA MSCs partially restored proliferation compared to control MSCs, with statistical significance starting from 1 µM (p-value < 0.0001 compared to the DMSO control). Among other drugs tested, such as corticosteroids and L-leucine, the LATS inhibitor was the only effective drug that restored proliferation. In addition, the LATS inhibitor restored the lamellipodia formation and stellate morphology of DBA MSCs in a dose-dependent manner.In conclusion, we characterized and identified the impaired YAP signaling pathway of bone marrow mesenchymal stem cells in DBA. Results from this study will lay the groundwork for understanding the bone marrow microenvironment of DBA and identifying potential targets for more effective treatment in DBA.

T cells play a critical role in immune defense by eliminating virus-infected and malignant cells. Their antigen specificity is determined by T cell receptors (TCRs), heterodimers composed of α and β chains, whose diversity is generated through V(D)J recombination and junctional nucleotide insertions. It is well known that, unlike the β chain which follows the principle of allelic exclusion and is expressed from only one allele, the α chain can undergo rearrangement at both alleles and may be expressed from both loci. Identifying the TCRαβ sequences of cytotoxic T lymphocyte (CTL) clones with robust cytotoxicity is essential for advancing TCR-engineered T cell therapies. Although the TCR repertoire is highly diverse, dominant clones occasionally emerge that possess strong reactivity against specific antigens. Analyzing these dominant clones offers opportunities to identify antigen-specific TCRs with therapeutic potential and to understand the principles of immunological memory. We have established a platform to generate rejuvenated antigen-specific CTLs (rejTs) by reprogramming T cells into induced pluripotent stem cells (T-iPSCs) and redifferentiating them into CTLs. These rejTs proliferate vigorously and exhibit strong antigen-specific cytotoxicity, allowing efficient functional evaluation and potential application in adoptive T cell therapies. In our attempt to generate Epstein-Barr virus (EBV) LMP2 (419–427, TYGPVFMSL)-specific rejTs, we obtained CTL clones with identical TCRαβ sequences in two independent experiments. RejTs derived from these clones demonstrated potent cytotoxicity, suggesting that they represent dominant clones. Interestingly, these clones expressed two functionally rearranged TCRα chains (α₁ and α₂), both paired with the same TCRβ chain. To determine which TCRα chain mediated LMP2 recognition, we knocked out endogenous TCRs in HLA-matched donor T cells using CRISPR/Cas9 and transduced them with either TCRα₁β or TCRα₂β. Only TCRα₁β-transduced T cells bound the LMP2/HLA-A24 tetramer and exhibited specific cytotoxicity against LMP2-expressing, HLA-matched lymphoblastoid cell lines (80% vs 0.5% lysis at an E:T ratio of 40:1), indicating that TCRα₁β mediates the observed anti-EBV activity. To explore whether TCRα₂β also recognizes a different epitope of , we applied three deep learning–based TCR specificity prediction tools (MixTCRpred, TCRex, and ERGO-II), which identified candidate EBV epitopes. MHC binding predictions (NetMHC v4.0) confirmed strong binding to the donor's HLA alleles. We are currently validating the predicted epitopes to assess the contribution of TCRα₂β to the cytotoxicity observed in this dominant clone.

Hematopoietic stem cells (HSCs) are crucial for maintaining hematopoietic homeostasis and are localized within distinct bone marrow (BM) niches. While BM niches are often considered similar across different skeletal sites, we discovered that the alveolar BM (al-BM) in the mandible harbors the highest frequency of immunophenotypic HSCs in nine different skeletal sites. Transplantation assays revealed significantly increased engraftment from al-BM compared to femur, tibia, or pelvis BM, likely due to a higher proportion of alveolar HSCs. Moreover, hematopoietic progenitor cells (c-Kit+ Sca-1+ Lin-) in al-BM exhibited increased quiescence and reduced apoptosis, indicating superior maintenance and survival characteristics. We also observed an enrichment of mesenchymal stromal cells and skeletal stem cells in al-BM, suggesting a more supportive microenvironment. These findings indicate that al-BM provides a unique microenvironment conducive to higher frequency of HSCs, offering new insights into site-specific hematopoiesis.

Background To overcome organ shortage during transplantation, interspecies organ generation via blastocyst complementation has been proposed, although not yet in evolutionarily distant species. To establish high levels of chimerism, low chimerism is required early in development, followed by high chimerism, to effectively complement the organ niche. Very few human cells are expected to contribute to chimerism in heterologous animals. Previous studies had demonstrated increased donor chimerism in both intra- and interspecies chimeras in rodents, using insulin-like growth factor 1 receptor (Igf1r) knockout (KO) mice; deletion of the Igf1r gene in the mouse host embryo created a cell-competitive niche. The current study aimed to generate IGF1R–KO pigs and evaluate whether they have the same phenotype as Igf1r-KO mice. Methods To generate IGF1R–KO pigs, genome-editing molecules were injected into the cytoplasm of pig zygotes. The fetuses were evaluated at 104 days of gestation. Results IGF1R–KO pigs were generated successfully. Their phenotypes were almost identical to those of Igf1r-KO mice, including small lungs and enlarged endodermal organs in fetuses, and they were highly reproducible. Conclusions Pigs may allow the generation of organs using blastocyst complementation with developmentally-compatible xenogeneic pluripotent stem cells over a large evolutionary distance.