Later Stages Of Differentiation Research Articles

Integrative analyses of genetic mechanisms responsible for bone-fat imbalance in osteoporosis.

Osteoporosis manifests through adipocyte accrual and osteoblast diminution within bone marrow. However, the precise mechanisms driving the shift from osteogenesis to adipogenesis in bone marrow mesenchymal stem cells (BMSCs) remain largely undefined. In this study, we harnessed the power of bioinformatic tools to analyze gene expression patterns of BMSCs during adipogenic differentiation and osteoporosis using the data from Gene Expression Omnibus (GEO) repositories (GSE113253 and GSE35956), complemented by in vitro and in vivo experiments to validate the findings. Five distinct expression profiles of differentially expressed genes across the adipogenic timeline were identified. The initial phase is marked by ribosome biogenesis and rRNA processing, which is followed by the metabolism of organic acids and processing of inorganic ions. In contrast, the terminal phase is characterized by lipid transport, accumulation, and metabolism, alongside inorganic cation metabolism, thereby underscoring unique transcriptional signatures during the early and late stages of adipogenic differentiation. In BMSCs derived from osteoporotic samples, there is a notable decline in cellular proliferation and a diminished osteogenic capacity. Critically, the genes common to both adipogenesis and osteoporosis in BMSCs are predominantly involved in the negative regulation of Wnt signaling and cellular proliferation. Key genes including SOCS1, MYC, CEBPB, FYN, AXIN2, and RXRA are identified and show downregulation in BMSCs from aged mice. Subsequent in vitro experiments have validated the regulatory influence of RXRA on both adipogenic and osteogenic differentiations of BMSCs, highlighting its crucial role as a central modulator in bone formation and the pathophysiology of osteoporosis.

A conserved Plasmodium nuclear protein is critical for late liver stage development

Malaria, caused by Plasmodium parasites, imposes a significant health burden and live-attenuated parasites are being pursued as vaccines. Here, we report on the creation of a genetically attenuated parasite by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. In the rodent parasite Plasmodium yoelii, LINUP expression was restricted to liver stage nuclei after the onset of liver stage schizogony. Compared to wildtype P. yoelii, P. yoelii LINUP gene deletion parasites (linup—) exhibited no phenotype in blood stages and mosquito stages but suffered developmental arrest late in liver stage schizogony with a pronounced defect in exo-erythrocytic merozoite formation. This defect caused severe attenuation of the liver stage-to-blood stage transition and immunization of mice with linup— parasites conferred robust protection against infectious sporozoite challenge. LINUP gene deletion in the human parasite Plasmodium falciparum also caused a severe defect in late liver stage differentiation. Importantly, P. falciparum linup— liver stages completely failed to transition from the liver stage to a viable blood stage infection in a humanized mouse model. These results suggest that P. falciparum LINUP is an ideal target for late liver stage attenuation that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine.

METTL16 inhibits differentiation and promotes proliferation and slow myofibers formation in chicken myoblasts

N6-methyladenosine (m6A) plays a crucial regulatory role in muscle growth and development. In our previous studies, we identified a m6A methyltransferase, Methyltransferase like 16 (METTL16), which is associated with chicken muscle development and muscle fiber type conversion. To further understand the regulatory role of METTL16 in chicken muscle function, we analyzed its expression in muscle tissues with different myofiber type compositions and in chicken primary myoblasts (CPMs) at various stages. We also manipulated METTL16 expression in CPMs to examine its effects on cell proliferation, differentiation, muscle fiber type formation, and global m6A RNA methylation status. Our results showed that METTL16 expression increased during myoblast proliferation and gradually decreased in the late differentiation stage. Furthermore, METTL16 exhibited specific expression in slow-twitch muscles. Cell Counting Kit-8 assays, 5-Ethynyl-2'-deoxyuridine staining, RT-qPCR, Western blot, and immunofluorescence analyses showed that METTL16 promotes myoblast proliferation while inhibiting myoblast differentiation. We also observed that METTL16 induces the upregulation of slow-twitch myosin heavy chain (MyHC) and slow-twitch-specific genes in myotubes, while downregulating fast-twitch MyHC and fast-twitch-specific genes. Furthermore, both interference and overexpression of METTL16 led to changes in overall cellular m6A modification levels and Methyltransferase like 3 (METTL3) expression levels. These findings confirm that METTL16 plays a key role in myoblast proliferation, differentiation, and muscle fiber type formation in chickens. Considering the role of myoblasts in chicken muscle growth and meat quality regulation, METTL16 may serve as a key target for molecular selection in chicken meat traits.

Mass Spectrometry Analysis of Neurotransmitter Shifting during Neurogenesis and Neurodegeneration of PC12 Cells.

Parkinson's disease (PD) affects movement; however, most patients with PD also develop nonmotor symptoms, such as hyposmia, sleep disorder, and depression. Dopamine levels in the brain have a critical influence on movement control, but other neurotransmitters are also involved in the progression of PD. This study analyzed the fluctuation of neurotransmitters in PC12 cells during neurogenesis and neurodegeneration by performing mass spectrometry. We found that the dopaminergic metabolism pathway of PC12 cells developed vigorously during the neuron differentiation process and that the neurotransmitters were metabolized into 3-methoxytyramine, which was released from the cells. The regulation of the intracellular and extracellular concentrations of adenosine indicated that adenine nucleotides were actively utilized in neural differentiation. Moreover, we exposed the differentiated PC12 cells to rotenone, which is a suitable material for modeling PD. The cells exposed to rotenone in the early stage of differentiation exhibited stimulated serotoninergic metabolism, and the contents of the serotoninergic neurotransmitters returned to their normal levels in the late stage of differentiation. Interestingly, the nondifferentiated cells can resist the toxicant rotenone and produce normal dopaminergic metabolites. However, when differentiated neuron cells were exposed to rotenone, they were seriously damaged, leading to a failure to produce dopaminergic neurotransmitters. In the low-dosage damage process, the amino acids that functioned as dopaminergic pathway precursors could not be absorbed by the cells, and dopamine and L-dopa were secreted and unable to be reuptaken to trigger the cell damage.

Early markers of induced pluripotent stem cell hematopoietic development

The generation of immune cell populations from induced pluripotent stem cells (iPSCs) is a valuable model to study mechanisms that control hematopoietic development; it also is a promising approach to develop immunotherapeutic strategies to treat various diseases, including hereditary, oncological and infectious ones. To date, it has been demonstrated that iPSCs can differentiate into different immune cells, including macrophages, neutrophils, natural killer cells and T cells. However, the protocols suggested so far are experimental, and they require optimization, standardization and scaling. Solution to these tasks requires methods allowing to predict the efficacy of ongoing differentiation at early differentiation stages. Here, we evaluated whether iPSC hematopoietic/myeloid differentiation can be monitored by means of flow cytometry. Human iPSCs were differentiated into hematopoietic/myeloid cells using two protocols previously suggested for the generation of macrophages from iPSCs. The protocols differed by methods used to induce early and late stages of cell differentiation. At early differentiation stages, the protocols differed by approaches used to induce the generation of mesoderm and hemogenic endothelium, i.e., 2D differentiation in the presence of exogenous factors known to promote mesoderm and hemogenic endothelium development (“factor-dependent” protocol) or 3D differentiation in the absence of exogenous cytokines and growth factors (“spontaneous” protocol). At late differentiation stages, the protocols differed by factors added to the cultures to promote hematopoietic/myeloid specification (i.e., SCF, FGF2, IL-6, IL-3 and M-CSF or only IL-3 and M-CSF). At different stages of differentiation, the expressions of antigens known to be expressed by mesoderm, hemogenic endothelium and hematopoietic cells (i.e., CD309, CD34, CD31, CD43 and CD45) were evaluated. At early differentiation stages, the main phenotypic changes observed in cell cultures were an upregulation of the expression of CD309 (a receptor for vascular endothelial growth factor), the appearance of the expression of sialophorin CD43 and the expression of CD34 antigen. In cells cultured in 2D factor-dependent conditions, these changes appeared earlier and were more pronounced as compared with cells cultured in 3D “spontaneous” conditions. The results suggest that CD309 and/or CD43 are valuable markers for an early prediction of the effectiveness of iPSC differentiation into hematopoietic/ myeloid progeny.

Phenotypic profiling of human induced regulatory T cells at early differentiation: insights into distinct immunosuppressive potential

Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103− counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.

Lamc1 promotes osteogenic differentiation and inhibits adipogenic differentiation of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit multi-lineage differentiation potential and robust proliferative capacity. The late stage of differentiation signifies the functional maturation and characterization of specific cell lineages, which is crucial for studying lineage-specific differentiation mechanisms. However, the molecular processes governing late-stage BMSC differentiation remain poorly understood. This study aimed to elucidate the key biological processes involved in late-stage BMSC differentiation. Publicly available transcriptomic data from human BMSCs were analyzed after approximately 14 days of osteogenic, adipogenic, and chondrogenic differentiation. Thirty-one differentially expressed genes (DEGs) associated with differentiation were identified. Pathway enrichment analysis indicated that the DEGs were involved in extracellular matrix (ECM)-receptor interactions, focal adhesion, and glycolipid biosynthesis, a ganglion series process. Subsequently, the target genes were validated using publicly available single-cell RNA-seq data from mouse BMSCs. Lamc1 exhibited predominant distribution in adipocytes and osteoblasts, primarily during the G2/M phase. Tln2 and Hexb were expressed in chondroblasts, osteoblasts, and adipocytes, while St3gal5 was abundantly distributed in stem cells. Cell communication analysis identified two receptors that interact with LAMCI. q-PCR results confirmed the upregulation of Lamc1, Tln2, Hexb, and St3gal5 during osteogenic differentiation and their downregulation during adipogenic differentiation. Knockdown of Lamc1 inhibited adipogenic and osteogenic differentiation. In conclusion, this study identified four genes, Lamc1, Tln2, Hexb, and St3gal5, that may play important roles in the late-stage differentiation of BMSCs. It elucidated their interactions and the pathways they influence, providing a foundation for further research on BMSC differentiation.

Bioinformatics Identification and Experimental Verification of Disulfidptosis-Related Genes in the Progression of Osteoarthritis.

Osteoarthritis (OA) is a disabling and highly prevalent condition affecting millions worldwide. Recently discovered, disulfidptosis represents a novel form of cell death induced by the excessive accumulation of cystine. Despite its significance, a systematic exploration of disulfidptosis-related genes (DRGs) in OA is lacking. This study utilized three OA-related datasets and DRGs. Differentially expressed (DE)-DRGs were derived by intersecting the differentially expressed genes (DEGs) from GSE114007 with DRGs. Feature genes underwent screening through three machine learning algorithms. High diagnostic value genes were identified using the receiver operating characteristic curve. Hub genes were confirmed through expression validation. These hub genes were then employed to construct a nomogram and conduct enrichment, immune, and correlation analyses. An additional validation of hub genes was performed through in vitro cell experiments. SLC3A2 and PDLIM1 were designated as hub genes, displaying excellent diagnostic performance. PDLIM1 exhibited low expression in early chondrocyte differentiation, rising significantly in the late stage, while SLC3A2 showed high overall expression, declining in the late differentiation stage. Cellular experiments corroborated the correlation of SLC3A2 and PDLIM1 with chondrocyte inflammation. Two hub genes, SLC3A2 and PDLIM1, were identified in relation to disulfidptosis, providing potential directions for diagnosing and treating OA.

Quantification and Profiling of Early and Late Differentiation Stage T Cells in Mantle Cell Lymphoma Reveals Immunotherapeutic Targets in Subsets of Patients.

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue (n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (TH) cells and cytotoxic (TC) cells of early (CD57-) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. TC and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ TC-cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL.

Osteoblast-derived extracellular vesicles exert bone formation effects by WIF1-mediated regulation of mitophagy

BackgroundOsteoporosis arises mainly from an imbalance in bone remodeling, characterized by the impaired ability of osteoblasts (OBs) to form new bone. A significant challenge remains in identifying and validating key genes involved in osteogenic differentiation to develop effective treatments. MethodsThe regulatory role of WIF1 in osteogenic differentiation stages was investigated using Western blot and quantitative polymerase chain reaction assays to measure the expression levels of osteogenic-related genes in MC3T3-E1 cells. Mineralization of OBs was assessed through alizarin red S (ARS) and alkaline phosphatase (ALP) staining assays. To explore the relationship with mitophagy, RNA sequencing was performed to examine the effects of Wif1 overexpression on genes related to osteogenic differentiation and mitophagy processes. Additionally, histological staining and micro-computed tomography were conducted on ovariectomized (OVX) mice treated with Wif1-overexpressing OB-derived extracellular vesicles (EVs) to evaluate their impact on bone loss and osteogenic differentiation in bone marrow stromal cells (BMSCs). ResultsWif1 was identified as a crucial marker for late-stage osteogenic differentiation, playing a role in regulating mitophagy to release functional EVs. In vitro analyses showed that Wif1 overexpression accelerated osteogenic differentiation by activating genes related to late-stage osteogenic differentiation and mitophagy processes. In vivo experiments demonstrated that administering Wif1-overexpressing OB-derived EVs to OVX mice partially reversed bone loss and countered the suppression of osteogenic differentiation in BMSCs. ConclusionsOur findings shed light on the molecular mechanism of Wif1 in osteogenic differentiation and suggested the therapeutic efficacy of Wif1-overexpressing OB-derived EVs in osteoporosis.

P11 A role for keratinocyte arginase in skin wound healing and barrier function

Abstract Introduction and aims Delayed cutaneous wound healing is a major problem that can result in social isolation and significant morbidity including amputation. There are many changes in cellular function in a chronic wound. Notably, the enzyme arginase (ARG)1 is markedly changed in wound healing. We have identified a role for ARG1 in keratinocyte re-epithelialization during wound closure, but the underlying mechanism driving re-epithelialization was not known. We aimed to determine the role of ARG1 in keratinocyte migration, differentiation and barrier formation. Methods Two-dimensional scratch and differentiation assays using immortalized keratinocytes (NTERT cells) treated with the ARG1 inhibitor, nor-NOHA, and short hairpin RNA-based knockdown cells were assessed. Gene and protein expression changes were highlighted. Expression of proteins was further validated using immunohistochemical profiling of human skin equivalents and human wound lesions by spatial transcriptomics. Results ARG1 was strongly expressed both 2–4h postscratching and at late stages of keratinocyte differentiation. Dysregulation of ARG1 expression was spatially highlighted in chronic lesions, further suggesting an involvement in keratinocyte migration and differentiation. ARG1 inhibition led to a significant delay in postscratch keratinocyte migration. Furthermore, upon ARG1 inhibition, a significant decrease was seen in the expression of late differentiation markers involucrin and filaggrin. ARG1 inhibition significantly decreased the expression of host defence genes β-defensin 3, lipocalin 2 and kallikrein 6. ARG1 triggers production of multiple downstream products and we confirmed a role for the pathway involving production of putrescine and urea, as the addition of these metabolites led to the rescue of these phenotypes. Conclusions ARG1 and its downstream metabolites play a major role in keratinocyte migration, differentiation, and barrier formation, which are vital functions for re-epithelialization and wound closure. Therefore, manipulation of the ARG1 pathway may be crucial in the management of chronic lesions.

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Magnesium isotope behavior during titanomagnetite fractionation in basaltic lavas

Knowledge of the behavior of magnesium (Mg) isotopes during magmatic differentiation is a prerequisite for applying Mg isotopes as a tracer of crustal recycling and mantle heterogeneity. Crystal fractionation of mafic silicate minerals leads to limited Mg isotope fractionation; hence, Mg isotopic variations in basalts largely result from contributions of subducted crustal materials into their mantle sources. However, recent studies have revealed that the segregation of iron oxides, such as chromite during early-stage basaltic differentiation and titanomagnetite and ilmenite during late-stage differentiation, can potentially modify Mg isotopic compositions of mantle-derived lavas. Titanomagnetite is much more commonly observed in terrestrial basaltic lavas than chromite and ilmenite, and can also form during early-stage basaltic differentiation. To constrain the effects of titanomagnetite crystallization on Mg isotopic systematics of basaltic magma, we report Mg isotopic data for a suite of well-characterized alkaline basaltic lavas (MgO wt% = 4.99 to 6.52) from Gerba Guracha, western Ethiopian Plateau, which have undergone extensive titanomagnetite fractionation. Their δ26Mg values range from −0.29‰ to −0.17‰ and do not correlate with indicators of titanomagnetite fractionation such as TiO2 and V. These observations suggest that the segregation of titanomagnetite produced limited Mg isotope fractionation, possibly because of its composition-dependent, highly variable Δ26MgTi-Mgt−melt (Δ26MgTi-Mgt−melt = δ26MgTi-Mgt − δ26Mgmelt) values. Considering that most iron oxides occur as complex solid solutions that are controlled by the oxygen fugacity of magmas, the effects of oxide segregation on Mg isotopic systematics of basalts require further evaluation.

Stereoselective Synthesis of Unsymmetrical 1,1-Diborylalkenes

Abstract1,1-Diborylalkene, a class of important diboryl species, serves as the synthetic precursor of multisubstituted olefin, which is a prevalent building block in natural products, functional materials, and pharmaceuticals. Current methods mainly afford symmetrical 1,1-diborylalkenes, and late-stage differentiation of the two identical boryl groups is required to achieve selective difunctionalization. In comparison, stereoselective synthesis of unsymmetrical 1,1-diborylalkenes (UDBA) are less-explored. This Synpacts article provides a brief summary of the achievements in the synthesis of UDBAs. In particularly, we highlight our recent work on the unsymmetrical 1,1-diborylation of alkynes using a neutral sp2–sp3 diboron reagent to access UDBAs and their controllable stepwise derivatization.1 Introduction2 Background: Unsymmetrical 1,1-Diborylalkene Synthesis3 Stereoselective Unsymmetrical 1,1-Diborylation of Alkynes with a Neutral sp2–sp3 Diboron Reagent4 Summary and Outlook

Bone Morphogenetic Protein 2 Enhances Porcine Beige Adipogenesis via AKT/mTOR and MAPK Signaling Pathways.

Bone morphogenetic protein 2 (BMP2) has been reported to regulate adipogenesis, but its role in porcine beige adipocyte formation remains unclear. Our data reveal that BMP2 is significantly induced at the early stages of porcine beige adipocyte differentiation. Additionally, supplementing rhBMP2 during the early stages, but not the late stages of differentiation, significantly enhances porcine SVF adipogenesis, thermogenesis, and proliferation. Furthermore, compared to the empty plasmid-transfected-SVFs, BMP2-overexpressed SVFs had the enhanced lipid accumulation and thermogenesis, while knockdown of BMP2 in SVFs exhibited the opposite effect. The RNA-seq of the above three types of cells revealed the enrichment of the annotation of thermogenesis, brown cell differentiation, etc. In addition, the analysis also highlights the significant enrichment of cell adhesion, the MAPK cascade, and PPARγ signaling. Mechanistically, BMP2 positively regulates the adipogenic and thermogenic capacities of porcine beige adipocytes by activating PPARγ expression through AKT/mTOR and MAPK signaling pathways.

Abstract 7536: CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity

Abstract Background: Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). However, compared to CD8+ T cell exhaustion, the role of CD4+ T cell exhaustion in antitumor immunity remains unclear. Methods and Results: From single-cell sequencing for tumor-infiltrating lymphocytes (TILs) from patients with melanoma and mouse models, we found that CXCL13, which is highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces PD-1+CXCR5+CD4+ follicular helper T (TFH)-like cell infiltration, which could contribute to antitumor immunity. Furthermore, a part of the TFH-like cells in the TME exhibits cytotoxicity and directly attacks MHC-II-expressing tumors. RNA velocity and latent time analysis in single-cell sequencing for melanoma TILs showed that the differentiation of TFH-like cells from the naive CD4+ T cells were different from that of other CD4+ T cells. In addition, the TFH-like cytotoxic CD4+ T cells were at the late stage of differentiation and some non-cytotoxic TFH-like cells might differentiate into TFH-like cytotoxic CD4+ T cells. We additionally found considerable overlapped clonotypes between the cytotoxic and non-cytotoxic clusters. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH-like cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Conclusion: Our findings provide novel insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, which mediates antitumor immunity orchestrally with CD8+ T cells. Citation Format: Joji Nagasaki, Wenhao Zhou, Shusuke Kawashima, Takamasa Ishino, Katsushige Kawase, Youki Ueda, Kazuo Yamashita, Tomofumi Watanabe, Takashi Inozume, Yosuke Togashi. CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7536.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

CAR-T Therapy of Multiple Myeloma, Based on the Congresses ASH-2021 and ASCO-2022

Current treatment of multiple myeloma (ММ) based on proteasome inhibitors, immunomodulating drugs, and monoclonal antibodies has, to a certain extent, reached the limit of its potential. Despite considerable clinical advance, ММ still remains a chronic incurable disease. Tumor-specific T-cell therapy with chimeric antigen receptor (CAR) is a new evolution step towards achieving MM cure. Today, B-cell maturation antigen (BCMA) is regarded as the primary target of CAR-T treatment of MM. This receptor is mainly expressed on the surface of tumor plasma cells in ММ as well as in B-cells of late differentiation stages and normal plasma cells. In 2021–2022, two CAR-T drugs, idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel), were approved for clinical use in the USA and the European Union for patients with relapsed/refractory MM. The studies of these drugs yielded encouraging clinical results. Other antigen (GPRC5D, SLAMF7) cell-based drugs are now in early stages of development. The present review is concerned with latest advances in CAR-T therapy for MM reported at the recent congresses ASH-2021 and ASCO-2022. The review comprehensively discusses the results of the KarMMa (ide-cel, stage II) and CARTITUDE-1 (cilta-cel, stage IB/II) studies. It also provides historical background of CAR-Т cell generation as well as preclinical and on-going clinical trial data on MM. It outlines potential failure causes and prospects of further improvement of the new technology.of its potential. Despite considerable clinical advance, ММstill remains a chronic incurable disease. Tumor-specific T-cell therapy with chimeric antigen receptor (CAR) isa new evolution step towards achieving MM cure. Today,B-cell maturation antigen (BCMA) is regarded as the primary target of CAR-T treatment of MM. This receptor is mainlyexpressed on the surface of tumor plasma cells in ММ aswell as in B-cells of late differentiation stages and normalplasma cells. In 2021–2022, two CAR-T drugs, idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel(cilta-cel), were approved for clinical use in the USA andthe European Union for patients with relapsed/refractoryMM. The studies of these drugs yielded encouraging clinical results. Other antigen (GPRC5D, SLAMF7) cell-baseddrugs are now in early stages of development. The presentreview is concerned with latest advances in CAR-T therapyfor MM reported at the recent congresses ASH-2021 andASCO-2022. The review comprehensively discusses theresults of the KarMMa (ide-cel, stage II) and CARTITUDE-1(cilta-cel, stage IB/II) studies. It also provides historicalbackground of CAR-Т cell generation as well as preclinicaland on-going clinical trial data on MM. It outlines potentialfailure causes and prospects of further improvement of thenew technology.

T cell senescence: a new perspective on immunotherapy in lung cancer.

T cell senescence is an indication of T cell dysfunction. The ability of senescent T cells to respond to cognate antigens is reduced and they are in the late stage of differentiation and proliferation; therefore, they cannot recognize and eliminate tumor cells in a timely and effective manner, leading to the formation of the suppressive tumor microenvironment. Establishing methods to reverse T cell senescence is particularly important for immunotherapy. Aging exacerbates profound changes in the immune system, leading to increased susceptibility to chronic, infectious, and autoimmune diseases. Patients with malignant lung tumors have impaired immune function with a high risk of recurrence, metastasis, and mortality. Immunotherapy based on PD-1, PD-L1, CTLA-4, and other immune checkpoints is promising for treating lung malignancies. However, T cell senescence can lead to low efficacy or unsuccessful treatment results in some immunotherapies. Efficiently blocking and reversing T cell senescence is a key goal of the enhancement of tumor immunotherapy. This study discusses the characteristics, mechanism, and expression of T cell senescence in malignant lung tumors and the treatment strategies.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.