Mouse Model Of Transplantation Research Articles

MLL-AF9-Induced Leukaemia Changes during Ontogenic Stages

MLL-AF9 causes acute lymphoid leukaemia (ALL), acute myeloid leukaemia (AML) and mixed phenotype acute leukaemia (MPAL) in infants and children. Infant leukaemia originates in utero, yet this has never been formerly shown for MLL-AF9-driven disease. Multiple mouse models invariably revealed that MLL-AF9 can induce myelo-monocytic AML, however, they failed to recapitulate the ALL and MPAL phenotypes as presented in paediatric patients. To explore the impact of developmental stage for MLL-AF9 leukaemic burden and phenotypes, we established a new inducible (“Tet-off”), reversible, transplantable mouse model in which expression of the fusion is controlled by the 3' stem cell leukaemia (SCL)-enhancer element. We crossed SCL-tTA mice(1) with inducible MLL-AF9 mice(2) to generate double transgenic SCLtTA/iMLL-AF9 mice. To determine the impact of developmental stage, MLL-AF9 was induced from the conceptional stage for prenatal (E0), in neonates for postnatal (P0) and at 20 weeks for adult (≥20 weeks) cohorts. Following induction for 6-8 weeks in vivo MLL-AF9 expression was measured in haematopoietic stem cells (HSCs, (Lin-cKit+Sca-1+CD150+CD48-), lymphoid-myeloid primed progenitors (HPC-1/LMPPs, LSK CD150-CD48+/Flt3+), and granulocyte-monocyte progenitors (GMPs, Lin-cKit+Sca-1CD34+CD16/32hi). We found that MLL-AF9 expression levels were comparable within each population across the age groups but observed higher MLL-AF9 expression in the GMPs compared to HSCs in the postnatal model, concomitant with a moderate expansion in the GMP clonal size. Upon postnatal MLL-AF9 induction, all the mice developed leukaemia with a short latency of 13 weeks and complete penetrance. In contrast, E0 prenatal- or adult-induction resulted in longer latencies of 32 and 24 weeks respectively. Despite sustained MLL-AF9 expression, not all the mice developed leukaemia in prenatal- and adult-induced cohorts. Postnatal induced mice developed myelo-monocytic (AML-M4), megakaryoblastic (AML-M7), or MPAL, whereas prenatal and adult induction of MLL-AF9 developed AML-M4 exclusively. Regardless of leukaemia phenotypes, all diseased mice displayed thrombocytopenia and splenomegaly, and leucocytosis. Independent from the age at which MLL-AF9 was induced, all AML-M4 mice had clonal expansion of GMPs. Adult-induced AML-M4 mice had an increase in HSCs while postnatal-induced mice had an increase in HPC-1/LMPPs. In postnatal-induced MPAL mice, there was expansion of GMPs and HPC-1/LMPP populations, and postnatal-induced AML-M7 mice had clonal expansions of HSC, HPC-1/LMPP, and MEP populations with no expansion in GMPs. Thus, the age when the MLL-AF9 fusion is induced does not only determine the disease latency and penetrance, but also affects lineage determination, and the amount of leukaemic HSPCs. Leukaemic-GMP (L-GMP) and leukaemic-HSC (L-HSC) have been characterized in adult models as leukaemic stem cells (LSC). Transplantation of bulk and L-GMPs from adult-induced AML-M4 cells were able to cause AML with complete penetrance, in line with published data and validating our model. However, prenatal- and postnatal-induced AML-M4 cells were not transplantable. In all age cohorts, bulk AML cells formed serially plateable large and compact colonies and were reversible. Collectively, LSCs exist within a GMP-like population of adult-induced MLL-AF9 AML, they may however reside in different HSPC populations in prenatal- and postnatal-induced disease. In summary, this novel inducible model recapitulates several features of infant, paediatric and adult MLL-AF9 AML demonstrating that ontogenic stage is a determining factor in differences in disease biology.

Cinobufagin Induces Apoptosis of DLBCL Cells By Targeting the Inhibition of G6PD Activity

Background:Globally, there are 150,000 new cases of diffuse large B-cell lymphoma (DLBCL) each year, accounting for approximately 30% of all non-Hodgkin lymphoma cases. Although most patients can be cured with R-CHOP immunochemotherapy, 30-40% of patients progress or relapse after treatment. Therefore, there is an urgent need to find new treatments to improve the survival rate of this group of patients. Natural small molecule drugs have unique advantages as anticancer agents due to their low toxicity and multiple targets. This project aims to explore potentially effective natural compounds as new therapeutic strategies for DLBCL. Methods:Search for suitable natural compounds through GEO database, CMAP server and bioinformatics analysis. The effects of natural compounds on the biological function of DLBCL cells were studied by IC50 assay, apoptosis assay, cell cycle assay and subcutaneous transplanted tumor model. RNA sequencing, metabolomics detection, SEA server and 3D molecular simulation docking analysis were used to explore the drug target. The drug mechanism was verified by G6PD activity assay, EdU incorporation assay, NADPH assay and ROS assay. Results:We used GEO and CMAP databases to identify 20 natural compounds that target DLBCL prognostic genes. Cinobufagin, a natural bufodienol isolated from toad venom, became the main object of this study because of its low IC50 value. Flow cytometry analysis showed that cinobufagin can effectively induce G2 arrest and apoptosis of DLBCL cells. In addition, cinobufagin significantly inhibited the growth of tumor tissue in a mouse model of heterotopic subcutaneous tumor transplantation. The results of transcriptome sequencing suggested that cinobufagin treatment inhibited multiple metabolism-related pathways. Metabolomic analysis showed a decrease in nucleoside metabolite content and inhibition of niacinamide synthesis. Through SEA server and 3D molecular docking simulation analysis, it was found that glucose-6-phosphate dehydrogenase (G6PD) may be a potential target of cinobufagin. G6PD activity assay showed that cinobufagin could inhibit the catalytic ability of G6PD. EdU incorporation experiments showed that cinobufagin inhibited DNA synthesis in DLBCL cells. In addition, cinobufagin also promoted the up-regulation of NADP+/NADPH ratio and the increase of ROS. The addition of ROS scavengers and nucleotides partially reversed the apoptosis-inducing ability of cinobufagin. Conclusion:G6PD is a target of cinobufagin. By inhibiting the enzyme activity of G6PD, cinobufagin can inhibit DNA synthesis and the production of reducing NADPH, thereby inducing apoptosis of DLBCL cells. This suggests that Cinobufagin has potential application as a new drug for the treatment of DLBCL.

Local Oxygen Dictates Hematopoietic Cell Growth and Function

Functional hematopoietic stem (HSC) and progenitor cells (HPC) are responsible for the replenishment of blood/immune cells, are a source of treatment for diseases, and when dysregulated are a source of hematological neoplasia. To identify factors that impact HSC/HPC functional competency, we performed a transcriptomic screen which revealed that human cord blood (CB) HSCs/HPCs capable of early engraftment in an NSG mouse model of transplantation exhibited enriched expression of gene programs that vary with changes in oxygen (O 2) availability compared to non-transplanted input cells. This is physiologically and clinically relevant as HSCs/HPCs and diseased hematopoietic cells such as acute myeloid leukemia (AML) are exposed to O 2 tensions ranging from <1% to 21% as they function in various anatomical sites or are harvested for clinical applications. Thus, there are translational and practical rationales for understanding the growth and functional properties that hematopoietic cells exhibit across the range of O 2 they experience physiologically or artificially, which we examined here. First we found significantly different frequencies of HSCs/HPCs and colony forming units (CFU) in human bone marrow (BM), mobilized peripheral blood (PB), and CB, which have varied O 2 tensions, suggesting O 2 availability may impact hematopoiesis. To directly examine this, human CB CD34+ cells were split and plated in CFU assays or expanded in growth stimulating conditions at 1% O 2, 3% O 2, 5% O 2, 14% O 2 (physiologic tensions), or 21% O 2 (processing tension) . HPCs expanded best and formed more differentiated CFUs in high physiologic O 2 tensions and extra physiologic O 2 tensions, while HSCs expanded better and more potent CFUs were formed in a mid-physiologic O 2 tension (5%). This suggests that O 2 dependent pathways are critical to HSC/HPC growth but there may be a “sweet spot” for early HSCs/HPCs that allows for proliferation while maintaining self-renewal and potency to maintain a pool of primitive cells, possibly mirroring in vivo O 2 responses. In in vivo functional HSC/HPC analyses, NSG mice transplanted with CB units expanded in physiologic O 2 exhibited significantly better early neutrophil recovery and long-term myeloid reconstitution compared to input, suggesting that growth and maintenance of HSC/ myeloid HPC functional competency is better balanced in physiologic O 2 compared to extra physiologic tensions. Platelet recovery and lymphoid reconstitution were significantly better in mice receiving uncultured control CB, suggesting expansion does not affect all HSC/HPC subpopulations equally. Mechanistically, cells grown in low O 2 tensions (1-3%) had higher frequencies of early HSCs/HPCs, significantly lower intracellular ROS levels, a significantly higher percentage of cells in cell cycle G0, and lower levels of phosphorylated γH2AX than those grown in higher O 2. These data suggest that while to some extent HSCs/HPCs require O 2 to proliferate, they also exhibit properties associated with exhaustion when maintained in higher O 2. We next examined the effects varying O 2 tensions have on diseased hematopoietic cells using primary mouse AML cells or oncogene transformed human CB CD34+ cell lines. AML cells proliferated significantly more rapidly and formed more CFUs in higher O 2 tensions, suggesting that O 2 sensing pathways may be a dependency in AML that could affect disease progression as local O 2 tensions become perturbed. AML cells grown in lower O 2 exhibited lower resting levels of phosphorylated γH2AX (measured by flow cytometry) and lower O 2 consumption rates (measured by Seahorse metabolic flux analyzer), suggesting they are less susceptible to cellular stress. In line with this, AML cells maintained in low O 2 exhibited significantly better proliferation and recovery after treatment with the chemotherapeutic cytarabine compared to those in higher O 2 tensions. Thus, diseased cells growing in lower O 2 niches may be less susceptible to treatment and therefore may be a source of chemoresistance or relapse. These data reveal important roles for O 2 in balancing hematopoietic cell growth with normal and diseased cell function. Manipulating O 2 tension or O 2 sensing pathways could be used to improve HSC/HPC expansion for transplantation or to treat disease. Transcriptomic studies of HSCs/HPCs and AML cells are ongoing to determine the O 2 sensing mechanisms that dictate cellular function.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

The Effects of IL-23/IL-18-Polarized Neutrophils on Renal Ischemia-Reperfusion Injury and Allogeneic-Skin-Graft Rejection in Mice.

Neutrophils display heterogeneity and plasticity with different subgroups and immune-regulatory functions under various surrounding conditions. Neutrophils induced by IL-23/IL-18 (referred to N(IL-23+IL-18) neutrophils) have a unique gene-expression profile, with highly expressing IL-17, MHC-II, and costimulatory molecules. The adoptive transfer of N(IL-23+IL-18) neutrophils significantly increased the pathogenesis in a renal ischemia-reperfusion injury mouse model. N(IL-23+IL-18) neutrophils directly and efficiently induced allogeneic T cell proliferation in vitro. N(IL-23+IL-18) neutrophils enhanced the syngeneic T cell response to allogeneic antigens in mixed-lymphocyte reaction assays. The adoptive transfer of the donor or host N(IL-23+IL-18) neutrophils significantly enhanced the antidonor antibody production in an allogeneic-skin-transplanted mouse model, accompanied by increased Tfh cells in the spleens. Therefore, the neutrophil subset induced by IL-23/IL-18 promotes tissue injury and antidonor humoral response in the allogeneic transplantation mouse model.

Targeting PD-1/PD-L1 inhibits rejection in a heterotopic tracheal allograft model of lung transplantation.

Immune checkpoint molecules such as programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) have revolutionized the field of lung cancer treatment. As part of our study, we examined the role of these proteins in acute rejection in a mouse model of heterotopic tracheal transplantation. Recipient mice were untreated (Allo group) or treated with anti-PD-L1 (aPDL1 group) or PD-L1 Fc recombinant protein (PD-L1 Fc group). A further group of C57BL/6 mice received isografts (Iso group). The occlusion rate was significantly higher in the Allo group than in the Iso group (p = 0.0075), and also higher in the aPD-L1 group (p = 0.0066) and lower in the PD-L1 Fc group (p = 0.030) than in the Allo group. PD-L1 Fc recombinant protein treatment significantly decreased interleukin-6 and interferon-γ levels and reduced the CD4+/CD8+ T cell ratio, without increasing PD-1 and T-cell immunoglobulin mucin 3 expression in CD4+ T cells. These data suggest that PD-L1 Fc recombinant protein decreases the levels of inflammatory cytokines and the proportion of CD4+ T cells without exhaustion. The PD-L1-mediated immune checkpoint mechanism was associated with rejection in the murine tracheal transplant model, suggesting a potential novel target for immunotherapy in lung transplantation.

Heterogeneous Intercellular Communication of Hematopoietic Stem Cells in the Mouse Bone Marrow

Introduction.Hematopoietic stem cells (HSCs) are responsible for maintaining the homeostasis of the blood and immune systems. This process must rapidly adapt to bleeding, infection, injury, and disease. HSCs reside in specialized microenvironments in the bone marrow, called “niche”. Several bone marrow cell types have been proposed as the key components of the niche. It is well established that HSCs rely on molecular signals from the niche cells to survive, migrate, proliferate, and differentiate. Recent studies suggest that HSCs differentiate heterogeneously. Additionally, the bone marrow is a heterogeneous environment consisting of more than thirty cell types. This study will test the hypothesis that individual HSCs are engaged in heterogeneous intercellular signaling in the bone marrow. Materials and Methods.We utilized CellChat (Jin et al., 2021), a database for ligand-receptor pairs and associated pathways, to predict cell-cell signaling at the single cell level. We used single cell RNA sequencing data from mouse hematopoietic stem and progenitor cells, blood and immune cells, and non-hematopoietic cells from the bone marrow to determine putative cell-cell interactions. The predicted signaling interactions were then adjusted based on the cell-cell spatial interaction revealed by PhenoCycler measurement (Akoya Biosciences). Mouse bone marrow transplantation experiments were performed to test the functional significance of the identified signaling pathway interactions. Results.We found that individual HSCs send and receive different levels of molecular signals across various signaling pathways. In addition to the expected interactions with the known HSC niche cells, we also found new interactions between HSCs and their progeny cells including myeloid progenitors and immune cells. These cell-cell signaling interactions are consistent with the spatial interaction frequencies of the corresponding cell types as revealed by our imaging data from the PhenoCycler analysis. Further, we found that some signaling pathways are positively or negatively correlated across individual HSCs, indicating complex intercellular signaling networks. The most significant negative correlation was found between HSCs sending MHC-I signals and sending PARs signals (Figure). Moreover, this MHC-I/PARs signaling correlation network also involves signaling from the HSC niche, including those that are known to play roles in HSC homing such as ESAM and VCAM. Therefore, we tested the engraftment efficiency of HSCs that receive PARs and those that do not using the HSC mouse transplantation model. We found that PAR1 + HSCs engraft much less efficiently. Finally, we present the HSC Interaction Data Explorer (HIDE), a database that comprehensively depicts the interactions between HSCs and other bone marrow cells including the involved signaling pathways, ligands and receptors of the pathways, and the pathway correlations. Conclusion.Our study uncovered novel interactions of HSCs with myeloid progenitor cells and immune cells, which were validated by spatial interaction data. These interactions indicate feedback signaling in regulating blood cell regeneration. Our findings further revealed signaling pathway correlation networks across individual HSCs, and we showed their functional significance using transplantation experiments. Finally, we present HIDE as a resource that informs the interactions between HSCs and various bone marrow cells, the relevant signaling pathways, and the pathway correlations. Figure Legend. HSC signaling correlation networks. Each node shows a signaling pathway and direction (e.g., HSCs sending MHC-I). The edges represent the spearman correlation score.

Pgam1 Regulate Adaptive NK Cells in Anti-Viral and Anti-Tumor Response

Natural killer (NK) cells are the innate immune cells that play a role in the anti-viral infections and tumor cells. The adaptive NK cells are characterized with expression of the HLA-E-specific CD94/NKG2C activating receptor. The reconstitution of NKG2C+ adaptive NK cells post HSCT is much faster in NKG2C homozygous(NKG2C wt/wt) donors than NKG2C heterozygous donors (NKG2C wt/del), which resulted in lower incidence of CMV infection. In this study, we aimed to find out the mainly regulators of adaptive NK cells between NKG2C wt/wt and NKG2C wt/del donors. RNA-sequencing analysis were performed. Results shown that NK cells from NKG2C wt/wt donors constant upregulated glycolysis and biosynthesis-related genes, while downregulated in TGF-beta signaling and FOXO signaling pathway(Figure A and B). In order to determine the role of glycolysis in adaptive NK cells, upregulated glycolysis related genes were assessed by q-PCR, and PGAM1 is considered as the most important gene in the expansion of adaptive NK cells(Figure C). PGAM1 is upregulated upon NK cells stimulation via cytokine or target cells, which is related to the CD107a expression. HKB99 is an allosteric inhibitor for PGAM1, which is considered as a potential therapeutic target for many cancers nowadays. NK cells were added with HKB99 in vitro for 12h hours to evaluate the anti-K562 function. After that, Glycolysis is inhibited with the decreasing of ECAR while the OCR is not affected (FigureD). The adding of PGAM1 inhibitor resulted in decreasing of CD107a expression in NK cells, especially in the adaptive NK cells. NK cells activation signaling pathway is also inhibited, including the phosphorylation of mTORC1 protein S6, Syk, zap70, and the PLC-gamma. The PI3K/AKT is inhibited after the PGAM1 inhibition. After that,supplementation of pyruvate is efficient to rescue the inhibition caused by PGAM1 inhibitor. 5mM pyruvate adding can restore the cytokine release of NK cells and the phosphorylation of S6 and syk/zap70(Figure E). PGAM1-NCR-cre mice was constructed in which PGAM1 is knock out in NK cells specifically. In KO group, the ratio of NK cells and the maturation of NK cells are decreased. Upon cytokine stimulation, the activator receptors expression is less in KO mice. Besides, PGAM1 deficiency NK cells exhibited glucose uptake deficiency and disadvantages in proliferation both in vivo and in vitro (Figure F). The differentiation (CD11b+CD27-) and activation marker (DNAM1, Ly49H) are decreased in the competing transplantation mouse model, while the exhaustion markers such as PD-1 and NKG2A is upregulated. Further, MCMV infection models shown that PGAM1 deficiency mice with lower ratio of Ly49H+ adaptive NK cells at the early stage of infection, while Ly49G2 and Ly49C+ cells are not affected(Figure G). Further, Ly49H+ adaptive NK cells shown less expression of Ki67 in PB-NK cells, with the activating markers CD25 is decreased. Higher MCMV load induced inflammation is occurred in PGAM1 deficiency NK cells. Anti-tumor effect was also evaluated. The apoptosis of YAC-1 cells is decrease in coincidence with CD107a expression decreasing. MLL-AF9 leukemia mice model shown in day 18, the Ly49H+ adaptive NK cells in PGAM1 deficiency mice is less than WT mice, and the tumor burden is higher and accompanied with shorter survival under tumor circumstances(Figure H). RNA seq analysis shown compared to WT mice, the downregulated genes in PGAM1 deficiency NK cells are enriched in leukocyte activation and innate immune response also interfered the cytokine-mediated signaling pathway. At last, we monitored expression of PGAM1 and the function of NK cells in patients whose CMV is reactive post HSCT. PGAM1 is elevated post CMV infection, which is correlated with CD107a secretion. ROC curve suggests that PGAM1 may act as a predictor for refractory recurrence CMV infection in patients post HSCT(Figure I). In summary, our studies shown the glycolysis is effectively support the faster adaptive NK cells expansion and CMV response. PGAM1-mediated glycolysis is significant for NK cells anti-tumor and anti-viral function.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

The upregulation of immune checkpoints after photodynamic therapy reducing immune effect for treating breast cancer.

The immune effect induced by photodynamic therapy (PDT) has a limited effect on breast tumor. This study hypothesized that suppressive immune checkpoints on T cells might upregulate after PDT, which may reduce the antitumor effect of PDT for treating breast tumor. This study explored the alteration of immune checkpoint for the first time. A bilateral subcutaneous transplanted breast tumor mice model was established, and right tumors imitated primary tumors, and left tumors imitated distant tumors. Primary tumors were treated with PDT mediated by hematoporphyrin derivatives (HpD-PDT). Costimulatory molecules (ICOS, OX40, and 4-1BB) and immune checkpoints (PD1, LAG-3, CTLA-4, TIM-3, TIGIT) on tumor infiltrating T cells after HpD-PDT were analyzed by flow cytometry. Antitumor and immune effects were also assessed after HpD-PDT combined with anti-PD1 and LAG-3 antibodies. Primary tumors were suppressed, but distant tumors could not be inhibited after HpD-PDT. The number of T cells was increased, but function did not enhance after HpD-PDT. Additionally, costimulatory molecules (ICOS, OX40, and 4-1BB) were not elevated, but the suppressive immune checkpoints on tumor infiltrating T cells were upregulated after HpD-PDT. Notably, PD1+ LAG-3+ CD4+ T and PD1+ LAG-3+ CD8+ T cells were significantly increased. When PD1 and LAG-3 blockade combined with HpD-PDT, both primary and distant tumors were significantly suppressed, and antitumor immune effects were significantly enhanced. HpD-PDT could upregulate the PD1+ LAG-3+ CD4+ T and PD1+ LAG-3+ CD8+ T cells. Dual blockade of PD1 and LAG-3 immune checkpoints can enhance the antitumor effect of HpD-PDT.

CXCR5 + CD8 + T Cell-mediated Suppression of Humoral Alloimmunity and AMR in Mice Is Optimized With mTOR and Impaired With Calcineurin Inhibition.

Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT. We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant. ACT-mediated decrease in germinal center B cells, posttransplant alloantibody titer, and amelioration of AMR in high alloantibody-producing CCR5 knockout kidney transplant recipients were impaired when ACT was combined with CNi and enhanced when combined with mTORi. CNi (but not mTORi) reduced ACT-mediated in vivo cytotoxicity of IgG + B cells and was associated with increased quantity of germinal center B cells. Neither CNi nor mTORi treatment impacted the expression of cytotoxic effector molecules (FasL, Lamp1, perforin, granzyme B) by CD8 + T Ab-supp after ACT. Concurrent treatment with CNi (but not mTORi) reduced in vivo proliferation of CD8 + T Ab-supp after ACT. The increase in quantity of splenic CD44 + CXCR5 + CD8 + T cells that occurs after ACT was reduced by concurrent treatment with CNi but not by concurrent treatment with mTORi (dose-dependent). Impaired efficacy of ACT by CNi is attributed to reduced persistence and/or expansion of CD8 + T Ab-supp cells after ACT. In contrast, concurrent immunosuppression with mTORi preserves CD8 + T Ab-supp cells quantity, in vivo proliferation, and in vivo cytotoxic effector function after ACT and enhances suppression of humoral alloimmunity and AMR.

THU083 Disulfiram Mediated Anti-tumor Effect In Pituitary Neuroendocrine Tumors By Inducing Cuproptosis

Abstract Disclosure: N. Huang: None. Y. Zhang: None. Y. Liu: None. Y. Feng: None. L. Xue: None. Z. Wu: None. Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors. Dopamine agonists and somatostatin analogues are the first-line drugs for PRL and GH-secreting PitNETs clinically. However, there is no effective drug for other subtypes and drug-resistant PitNETs. Thus, it’s impending to discover new therapeutic targets and medicine. Cuproptosis is a copper-dependent form of cell death driven by lipoylated protein aggregation, loss of Fe-S cluster-containing proteins, and induction of HSP70. However, the role of cuproptosis in PitNETs is not clear. In this study, a total of 17 primary samples of PitNETs were collected to perform high-throughput drug screening, an anti-abuse drug disulfiram (DSF) was identified that can induce cell death effectively. We found that disulfiram also has a strong inhibitory effect on pituitary tumor cell lines MMQ, Att20 and GH3 (-87.1%, -62.5%, -92.7%, p<0.001), but the inhibitory effect was eliminated by copper chelator TTM/BCS. This indicated that DSF played the anti-tumor effect requiring the participation of copper ions. To explore the underlying mechanism that DSF induces the cell death of PitNETs, we detected the hallmark of cuproptosis and found that DSF leads to a decrease in the acylation level of proteins related to the tricarboxylic acid cycle, limiting the function of the protein. On the one hand, DSF leads to the downregulation of iron-sulfur cluster-associated proteins, and the oligomerization of dihydrolipoamide S-acetyltransferase (DLAT), which is eventually accompanied by an increase in the protein level of heat shock protein 70 (HSP70) at protein and mRNA level, causing proteotoxic stress leading to cell death. Finally, DSF treatment inhibited the tumor growth in MMQ cell transplanted mouse model and destructed the morphology of the pituitary tumor 3D sphere similarly demonstrating the inhibition effect. In conclusion, we find that the anti-abuse drug disulfiram can induce coproptosis in pituitary tumor cells in a copper-dependent manner and is a potential drug for the treatment of PitNETs. Presentation: Thursday, June 15, 2023

Gut microbiota aggravates neutrophil extracellular traps-induced pancreatic injury in hypertriglyceridemic pancreatitis

Hypertriglyceridemic pancreatitis (HTGP) is featured by higher incidence of complications and poor clinical outcomes. Gut microbiota dysbiosis is associated with pancreatic injury in HTGP and the mechanism remains unclear. Here, we observe lower diversity of gut microbiota and absence of beneficial bacteria in HTGP patients. In a fecal microbiota transplantation mouse model, the colonization of gut microbiota from HTGP patients recruits neutrophils and increases neutrophil extracellular traps (NETs) formation that exacerbates pancreatic injury and systemic inflammation. We find that decreased abundance of Bacteroides uniformis in gut microbiota impairs taurine production and increases IL-17 release in colon that triggers NETs formation. Moreover, Bacteroides uniformis or taurine inhibits the activation of NF-κB and IL-17 signaling pathways in neutrophils which harness NETs and alleviate pancreatic injury. Our findings establish roles of endogenous Bacteroides uniformis-derived metabolic and inflammatory products on suppressing NETs release, which provides potential insights of ameliorating HTGP through gut microbiota modulation.

Initial exploration of a novel fusion protein, IL-4/IL-34/IL-10, which promotes cardiac allograft survival mice through alloregulation.

Immune mediated graft loss still represents a major risk to transplant recipients. Creative approaches to immunosuppression that exploit the recipient's own alloregulatory mechanisms could reduce the need for pharmacologic immunosuppression and potentially induce immune tolerance. In the process of studying recipient derived myeloid derived suppressor cells (MDSCs), we identified key alloregulatory MDSC mechanisms, mediated by isolatable proteins IL-4, IL-34, and IL-10. We sought to purify these proteins and fuse them for subsequent infusion into transplant recipients as a means of inducing an alloregulatory response. In this introductory investigation, we leveraged molecular engineering technology to create a fusion protein (FP) of three cytokine coding sequences of IL-4, IL-34, and IL-10 and demonstrated their expressions by Western Blot analysis. Following purification, we tested whether FP IL-4/IL-34/IL-10 (FP1) can protect heart transplant allografts. Injection of FP1 significantly prolonged allogeneic cardiac graft survival in a dose-dependent fashion and the increase of graft survival time exceeded survival attributable to IL-34 alone. In vitro, MDSCs cells were expanded by FP1 treatment. However, FP1 did not directly inhibit T cell proliferation in vitro. In conclusion, newly developed FP1 improves the graft survival in cardiac transplantation mouse model. Significant additional work to optimize FP1 or include other novel proteins could supplement current treatment options for transplant patients.

Synthesis of mizoribine prodrugs and their in vivo evaluation as immunosuppressive agents

Mizoribine is a well-known immunosuppressive drug, based on a nucleoside scaffold, that targets inosine-monophosphate dehydrogenase (IMPDH). In an effort to increase its in vivo efficacy, three different types of prodrugs (a phosphoramidate prodrug, a lipophilic ester derivative and an amino acid conjugate) were prepared. Screening of these prodrugs in a rapid whole blood assay revealed that the two ester-based mizoribine prodrugs potently inhibited interleukin 2 secretion. Moreover, these prodrugs were able to prolong graft survival, when evaluated in a mouse model of cardiac allograft transplantation. Strikingly, a combination therapy of these mizoribine prodrugs with tacrolimus had a synergistic in vivo effect.

Establishment and evaluation of a modified mouse model of renal subcapsular transplantation of microvolume cells

The renal subcapsular space provides an easily accessible, nutrition-rich pocket that supports engraftment, and as such, is often used as a site for stem and cancer cell transplantation. Renal capsule transplantation requires high technical requirements, the recipient mice have greater surgical damage, the mouse kidney is small and the kidney capsule is fragile, and the operation is easy to fail. The conventional method is not suitable for microvolume cell transplantation to this site in animals with a small kidney, such as mice, due to high risks of cell loss or dislocation or injury to the capsule. In this study, we developed and validated a modified approach for the mouse model of renal subcapsular transplantation of microvolume mouse skeletal stem cells (SSCs). We used a pipette with a refined tip to separate the capsule from the parenchyma. Moreover, we used cells suspended in Matrigel rather than a liquid carrier for transplantation. Using the modified method, we were able to transplant microvolume mouse SSCs as low as 0.2 μL beneath the mouse renal capsule with excellent reproducibility. After 4 weeks of in vivo culture, the implanted mouse SSCs formed grafts on the surface of the parenchyma at the target site of transplantation. Histological staining of the grafts indicated osteogenic, fibrogenic, and lipogenic differentiation. Micro-CT imaging of the grafts revealed bone formation. This modified model could be used to effectively transplant different types of microvolume cells to the renal subcapsular space when the donor cells are difficult to acquire or the recipient mice have a very small size kidney.

Macrophage colony-stimulating factor as a weapon against cytomegalovirus.

Cytomegalovirus (CMV) infection is one of the severe opportunistic infections faced by severely immunocompromised patients. High viral loads cause tissue-invasive disease and expose to death or various indirect effects. Substantial progress was made in monitoring active infection, and antiviral drugs were developed. However, dose-limiting toxicities and genotypic resistance limit therapeutic efficacy and vaccine development is hampered by the complex biology of the virus. In this issue of EMBO Molecular Medicine, Kandalla etal (2023) suggest an innovative strategy using the cytokine macrophage colony-stimulating factor (M-CSF) whose clinical development was left behind two decades ago. By stimulating an endogenous immune defense mechanism, M-CSF promotes viral clearance in a mouse model of hematopoietic stem cell transplantation, without impairing stem cell engraftment. These results reactivate the interest in the potential therapeutic use of this cytokine.

Exploring cardiac impact of oral nicotine exposure in a transplantable Neoplasm Mice Model: Insights from biochemical analysis, morphometry, and molecular docking: Chlorella vulgaris green algae support

Nicotine-induced cardiac tissue damage is a concern for cancer patients, but the exact pathogenesis from nicotine oral exposure is unclear. This study was designed to investigate the impact of nicotine and Chlorella vulgaris (Ch. V) on cardiac glutathione homeostasis, inflammatory response, cardiac damage markers, apoptotic proteins and histopathological findings in an experimentally transplantable neoplasm mouse model (Ehrlich ascites carcinoma; EAC). In the in-vivo experiment, the female Swiss mice were divided into four groups: control, Ch.V (100 mg/kg), Nicotine (100 µg/ml/kg), and a combination group ( Nocotine+ Ch.V) for 40 days. Furthermore, in this study,the effects of C. vulgaris components on caspase-3, TNF-α, and IL-1β activity were explored using Molecular Operating Environment (MOE) docking software to ensure its ability to counteract the toxic effects of nicotine. The results indicated that nicotine has induced significant (P < 0.001) cardiopathic alterations in EAC-bearing mice with changes in cardiac tissue enzymes. C. Vulgaris attenuated the nicotine-induced cardiac glutathione inhibition, suppressed the inflammatory response, exerted antiapoptotic effects, mitigated myocardial injury biomarkers, and repaired cellular and tissue damage. Moreover, the molecular docking results revealed the ability of C. vulgaris to bind with interleukin-1 receptor type 1 (IL1R1) and tumor necrosis factor receptor superfamily member 1 A (TNFRSF1A) in the mice tissues, ameliorating apoptosis and inflammatory processes associated with nicotine-induced cardiotoxicity. This study provides a model for understanding nicotine-induced myocardial injury during experimentally transplantable neoplasm. It highlights C. vulgaris as a beneficial food supplement for cancer patients exposed to nicotine orally.

IMM47, a humanized monoclonal antibody that targets CD24, exhibits exceptional anti-tumor efficacy by blocking the CD24/Siglec-10 interaction and can be used as monotherapy or in combination with anti-PD1 antibodies for cancer immunotherapy.

This study evaluates the anti-tumor mechanism of IMM47, a humanized anti-CD24 mAb. Biolayer interferometry, ELISA and flow cytometry methods were used to measure the IMM47 binding, affinity, ADCC, ADCP, ADCT and CDC activities. In vivo therapeutical efficacy was measured in transplanted mouse models. IMM47 significantly binds granulocytes but not human erythrocytes and blocks CD24's ability to bind to Siglec-10. IMM47 has strong ADCC, ADCT and ADCP activity against REH cells. IMM47's in vivo pharmacodynamics showed that IMM47 has strong anti-tumor effects in human siglec-10 transgenic mouse models with a memory immune response. IMM47 also has powerful synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo and Keytruda, by blocking CD24/Siglec-10 interaction through macrophage antigen presentation with strong ADCC, ADCP, ADCT and CDC activities and with a safe profile. IMM47 binding to CD24 is independent of N-glycosylation modification of the extracellular domain.

Abstract 16 Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood

Abstract Introduction Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.