Transplantation In Mice Research Articles

Rictor/mTORC2 signalling contributes to renal vascular endothelial-to-mesenchymal transition and renal allograft interstitial fibrosis by regulating BNIP3-mediated mitophagy.

Renal allograft interstitial fibrosis/tubular atrophy (IF/TA) constitutes the principal histopathological characteristic of chronic allograft dysfunction (CAD) in kidney-transplanted patients. While renal vascular endothelial-mesenchymal transition (EndMT) has been verified as an important contributing factor to IF/TA in CAD patients, its underlying mechanisms remain obscure. Through single-cell transcriptomic analysis, we identified Rictor as a potential pivotal mediator for EndMT. This investigation sought to elucidate the role of Rictor/mTORC2 signalling in the pathogenesis of renal allograft interstitial fibrosis and the associated mechanisms. The influence of the Rictor/mTOR2 pathway on renal vascular EndMT and renal allograft fibrosis was investigated by cell experiments and Rictor depletion in renal allogeneic transplantation mice models. Subsequently, a series of assays were conducted to explore the underlying mechanisms of the enhanced mitophagy and the ameliorated EndMT resulting from Rictor knockout. Our findings revealed a significant activation of the Rictor/mTORC2 signalling in CAD patients and allogeneic kidney transplanted mice. The suppression of Rictor/mTORC2 signalling alleviated TNFα-induced EndMT in HUVECs. Moreover, Rictor knockout in endothelial cells remarkably ameliorated renal vascular EndMT and allograft interstitial fibrosis in allogeneic kidney transplanted mice. Mechanistically, Rictor knockout resulted in an augmented BNIP3-mediated mitophagy in endothelial cells. Furthermore, Rictor/mTORC2 facilitated the MARCH5-mediated degradation of BNIP3 at the K130 site through K48-linked ubiquitination, thereby regulating mitophagy activity. Subsequent experiments also demonstrated that BNIP3 knockdown nearly reversed the enhanced mitophagy and mitigated EndMT and allograft interstitial fibrosis induced by Rictor knockout. Consequently, our study underscores Rictor/mTORC2 signalling as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis progression, exerting its impact through regulating BNIP3-mediated mitophagy. This insight unveils a potential therapeutic target for mitigating renal allograft interstitial fibrosis.

Abstract 1156: Akkermansia (Strain BAA835) And The Microbial ADP-heptose Pathway Are Associated With Reduced Neointimal Hyperplasia After Arterial Injury

Introduction: Despite advances in surgical therapy for atherosclerosis, up to 50% of procedures such as balloon angioplasty, stenting, and bypass fail due to restenosis from neointimal hyperplasia, a cell proliferative process potentiated by inflammation. We hypothesized that neointimal hyperplasia is associated with a distinct gut microbiome profile and can be modulated by fecal transplantation. Methods: We performed fecal transplantation (FT) of C57BL6 mice using donors with differential microbiome profiles and susceptibility to neointimal hyperplasia (Sprague-Dawley [SD] and Lewis [LE] rats, high and low susceptibility, respectively). In separate experiments, germ-free mice underwent FT using C57BL6 donors with and without Akkermansia muciniphila BAA835 ( Amuc ) prior to arterial injury. Microbial communities were characterized using 16S rRNA gene amplicon and shotgun metagenome sequencing. Arterial and immune effects were assessed using frozen arterial sections, multi-parameter flow cytometry of mesenteric lymph nodes, or multiplex cytokine assays after ex vivo stimulation of whole blood with lipopolysaccharide. Results: Paradoxically, neointimal hyperplasia after injury was 67.7% greater in FT recipients of low- compared to high-susceptibility stools (P=0.01). In fecal communities of both FT recipient groups, relative abundance of A. muciniphila (r=-0.66, P<0.001) and of the ADP-heptose biosynthesis pathway (r=-0.7, P<0.001) were negatively associated with neointimal hyperplasia. A. muciniphila was differentially elevated (P<0.001) in SD compared to LE FT recipients and was the main contributing taxa for the enrichment (P<0.001) of the ADP-heptose biosynthesis pathway in the SD recipient group. In mice colonized with communities that differed only by the presence of Amuc , neointimal hyperplasia was 57.8% greater in the Amuc - group compared to Amuc+ (P=0.01). Amuc+ recipients had lower levels of IL-12, MIP-1beta, and MCP-1 in unstimulated blood and higher IP-10 and MCP-1 levels in stimulated blood. Conclusion: A. muciniphila and microbial ADP-heptose biosynthesis pathway are inversely associated with neointimal hyperplasia and are potential targets for novel microbe-based strategies to mitigate restenosis risk.

A minor tweak in transplant surgery protocols alters the cellular landscape of the arterial wall during transplant vasculopathy.

Transplant vasculopathy (TV) is a major complication after solid organ transplantation, distinguished by an arterial intimal thickening that obstructs the vascular lumen and leads to organ rejection. To date, TV remains largely untreatable, mainly because the processes involved in its development remain unclear. Aortic transplantation in mice, used to mimic TV, relies on highly variable experimental protocols, particularly regarding the type of anastomosis used to connect the donor aorta to the recipient. While the amount of trauma undergone by a vessel can dramatically affect the resulting pathology, the impact of the type of anastomosis on TV in mice has not been investigated in detail. In this study, we compare the cellular composition of aortic grafts from BALB/C donor mice transplanted into C57BL/6J recipient mice using two different anastomosis strategies: sleeve and cuff. While both models recapitulated some aspects of human TV, there were striking differences in the cellular composition of the grafts. Indeed, aortic grafts from the cuff group displayed a larger coverage of the neointimal area by vascular smooth muscle cells compared to the sleeve group. Aortic grafts from the sleeve group contained higher amounts of T cells, while the cuff group displayed larger B-cell infiltrates. Together, these data indicate that a seemingly minor technical difference in transplant surgery protocols can largely impact the cellular composition of the graft, and thus the mechanisms underlying TV after aortic transplantation in mice.

Targeted depletion of PD-1-expressing cells induces immune tolerance through peripheral clonal deletion.

Thymic negative selection of the T cell receptor (TCR) repertoire is essential for establishing self-tolerance and acquired allograft tolerance following organ transplantation. However, it is unclear whether and how peripheral clonal deletion of alloreactive T cells induces transplantation tolerance. Here, we establish that programmed cell death protein 1 (PD-1) is a hallmark of alloreactive T cells and is associated with clonal expansion after alloantigen encounter. Moreover, we found that diphtheria toxin receptor (DTR)-mediated ablation of PD-1+ cells reshaped the TCR repertoire through peripheral clonal deletion of alloreactive T cells and promoted tolerance in mouse transplantation models. In addition, by using PD-1-specific depleting antibodies, we found that antibody-mediated depletion of PD-1+ cells prevented heart transplant rejection and the development of experimental autoimmune encephalomyelitis (EAE) in humanized PD-1 mice. Thus, these data suggest that PD-1 is an attractive target for peripheral clonal deletion and induction of immune tolerance.

Bazedoxifene attenuates dextran sodium sulfate-induced colitis in mice through gut microbiota modulation and inhibition of STAT3 and NF-κB pathways

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract for which treatment options remain limited. In this study, we used a dual-luciferase-based screening of an FDA-approved drug library, identifying Bazedoxifene (BZA) as an inhibitor of the NF-κB pathway. We further investigated its therapeutic effects in a dextran sodium sulfate (DSS)-induced colitis model and explored its impact on gut microbiota regulation and the underlying molecular mechanisms. Our results showed that BZA significantly reduced DSS-induced colitis symptoms in mice, evidenced by decreased colon length shortening, lower histological scores, and increased expression of intestinal mucosal barrier-associated proteins, such as Claudin 1, Occludin, Zo-1, Mucin 2 (Muc2), and E-cadherin. Used independently, BZA showed therapeutic effects comparable to those of infliximab (IFX). In addition, BZA modulated the abundance of gut microbiota especially Bifidobacterium pseudolongum, and influenced microbial metabolite production. Crucially, BZA's alleviation of DSS-induced colitis in mice was linked to change in gut microbiota composition, as evidenced by in vivo gut microbiota depletion and fecal microbiota transplantation (FMT) mice model. Molecularly, BZA inhibited STAT3 and NF-κB activation in DSS-induced colitis in mice. In general, BZA significantly reduced DSS-induced colitis in mice through modulating the gut microbiota and inhibiting STAT3 and NF-κB activation, and its independent use demonstrated a therapeutic potential comparable to IFX. This study highlights gut microbiota's role in IBD drug development, offering insights for BZA's future development and its clinical applications.

Discovery of Novel Dual Inhibitors Targeting Mutant IDH1 and NAMPT for the Treatment of Glioma with IDH1Mutation.

The targeting of cancer cell intrinsic metabolism has emerged as a promising strategy for antitumor intervention. In the study, we identified the first-in-class small molecules that effectively inhibit both mutant isocitrate dehydrogenase 1 (mIDH1) and nicotinamide phosphoribosyltransferase (NAMPT), two crucial targets in cancer metabolism, through structure-based drug design. Notably, compound 23h exhibits excellent and balanced inhibitory activities against both mIDH1 (IC50 = 14.93 nM) and NAMPT (IC50 = 12.56 nM), leading to significant suppression of IDH1-mutated glioma cell (U87 MG-IDH1R132H) proliferation. Significantly, compound 23h has the ability to cross the blood-brain barrier (B/P ratio, 0.76) and demonstrates remarkable in vivo antitumor efficacy (20 mg/kg) in the U87 MG-IDH1R132H orthotopic transplantation mouse models without any notable toxicity. This proof-of-concept investigation substantiates the viability of discovering small molecules that concurrently target mIDH1 and NAMPT, providing valuable leads for the treatment of glioma and an efficient approach for the discovery of multitarget antitumor drugs.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice

BackgroundTransplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. MethodsWe transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. ResultsThe human immune cells were engrafted for more than three months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+, T cell responses in vitro. hiPS-CM sheets disappeared approximately 17–24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. ConclusionhiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism

AbstractAntibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

Astragaloside IV-PESV inhibits prostate cancer tumor growth by restoring gut microbiota and microbial metabolic homeostasis via the AGE-RAGE pathway.

Prostate cancer (PCa) is becoming the most common malignancy in men worldwide. We investigated the effect of astragaloside IV combined with PESV on the gut microbiota and metabolite of PCa mice and the process of treating PCa. Nude mice were genetically modified to develop tumors characteristic of PCa. The treatment of PCa mice involved the administration of a combination of astragaloside IV and peptides derived from scorpion venom (PESV). Feces were collected for both 16S rDNA and metabolic analysis. Fecal supernatant was extracted and used for fecal transplantation in PCa mice. Tumor development was observed in both PCa mice and nude mice. Tumor histopathology was examined, and the expression of inflammatory factors and the AGE-RAGE axis in PCa tissues were analyzed. PCa mice treated with Astragaloside IV in combination with PESV showed a significant reduction in tumor volume and weight, and stabilization of gut microbiota and metabolites. At the Genus level, significant differences were observed in Porphyromonas, Corynebacterium, Arthromitus and Blautia, and the differential metabolites were PA16_016_0, Astragaloside+, Vitamin A acid, Nardosinone, a-Nortestoster, D-Pantethine, Hypoxanthine, Pregnenolone, cinnamic acid, Pyridoxa, Cirtruline and Xanthurenate. There was a correlation between gut microbiota and metabolites. After the fecal transplantation, tumor growth was effectively suppressed in the PCa mice. Notably, both the mRNA and protein levels of the receptor for advanced glycation end products (RAGE) were significantly decreased. Furthermore, the expression of inflammatory factors, namely NF-κB, TNF-α, and IL-6, in the tumor tissues was significantly attenuated. Conversely, upregulation of RAGE led to increased inflammation and reversed tumor growth in the mice. Astragaloside IV combined with PESV could treat PCa by intervening in gut microbiota composition and metabolite by targeting RAGE.

Characterization and validation of fatty acid metabolism-related genes predicting prognosis, immune infiltration, and drug sensitivity in endometrial cancer.

Endometrial cancer is considered to be the second most common tumor of the female reproductive system, and patients diagnosed with advanced endometrial cancer have a poor prognosis. The influence of fatty acid metabolism in the prognosis of patients with endometrial cancer remains unclear. We constructed a prognostic risk model using transcriptome sequencing data of endometrial cancer and clinical information of patients from The Cancer Genome Atlas (TCGA) database via least absolute shrinkage and selection operator regression analysis. The tumor immune microenvironment was analyzed using the CIBERSORT algorithm, followed by functional analysis and immunotherapy efficacy prediction by gene set variation analysis. The role of model genes in regulating endometrial cancer in vitro was verified by CCK-8, colony formation, wound healing, and transabdominal invasion assays, and verified in vivo by subcutaneous tumor transplantation in nude mice. A prognostic model containing 14 genes was constructed and validated in 3 cohorts and clinical samples. The results showed differences in the infiltration of immune cells between the high-risk and low-risk groups, and that the high-risk group may respond better to immunotherapy. Experiments in vitro confirmed that knockdown of epoxide hydrolase 2 (EPHX2) and acyl-CoA oxidase like (ACOXL) had an inhibitory effect on EC cells, as did overexpression of hematopoietic prostaglandin D synthase (HPGDS). The same results were obtained in experiments in vivo. Prognostic models related to fatty acid metabolism can be used for the risk assessment of endometrial cancer patients. Experiments in vitro and in vivo confirmed that the key genes HPGDS, EPHX2, and ACOXL in the prognostic model may affect the development of endometrial cancer.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Abstract LB316: Endocrine pancreas alteration in pancreatic ductal adenocarcinoma

Abstract Background: The pancreas is central to energy homeostasis, playing essential roles in digestion and regulation of blood glucose. Exocrine pancreas contains large amounts of of hydrolytic enzyme which is responsible for the digestion. Acinar cells, as well as ductal cells, represent the prime components of exocrine pancreas. They are both supposed to be the origin of pancreatic ductal adenocarcinoma(PDAC) and the researches focused on these exocrine cells, as well as other components in pancreatic cancer, are numerous. Islet cells are crucial for glucose homeostasis and their identity are proved to be altered significantly during diabetes by single-cell RNA-sequencing(scRNA-seq). However, the presence or extent of variation of islet cells in PDAC is not established yet. Moreover, due to the low proportion of endocrine cells in pancreas, it is hard to analyze the data of endocrine cells by using published scRNA-seq data of PDAC. As a consequence of this underrepresentation, the function of endocrine cells in PDAC remains unclear. Methods: We used scRNA-seq, multiplex immunohistochemistry (mIHC), high-throughput spatial transcriptomic sequencing (Stereo-seq) to detect the alteration of endocrine pancreas in PDAC of human and mouse model. KPC mice (KrasLSL-G12Dp53LoxPPdx1-CreER) and pancreatic orthotopic transplantation mice were applied to verify the islet cell alteration in multiple stages of PDAC. We also performed islet isolation and purification to enhance the proportion of endocrine cells when preparing single-cell suspension. Cell co-culture experiments were used to determine the interaction between exocrine-endocrine compartments. Results: Through mIHC of islet cells in PDAC tissues and normal pancreas tissues of mice (KPC vs. C57BL/6J) as well as human (PDAC patients vs. healthy donors), we found the decreased proportions of inulin positive (INS+) cells and increased proportions of glucagon positive (GCG+) cells in PDAC tissues. Furthermore, the proportion of pancreatic polypeptide positive (PPY+) cells was significantly elevated in PDAC tissue in both human and murine. Then, we quantified the double-positive cells and found the proportions of INS+GCG+, INS+PPY+ and GCG+PPY+ were markedly increased in PDAC tissue. Additionally, through the experiments in mouse model, we found the islet cells altered gradually within the multiple stage of PDAC(normal-PanIN-PDAC). Finally, it was found that PDAC cells could induce the transdifferentiation of pancreatic α and β cells into GCG+PPY+ and INS+PPY+ double-positive cells, which further promoted PDAC proliferation in a paracrine-dependent manner and formed a reciprocal interaction. Conclusions: Our study systematically maps the alteration of pancreatic endocrine cells in PDAC and elucidates the potential interaction mechanisms between endocrine and exocrine cells during PDAC carcinogenesis. Meanwhile, it is suggested that pancreatic endocrine cells should be considered as an important component of the PDAC microenvironment. Citation Format: Yuan Chen, Xinpeng Yin, Ruiyuan Xu, Rexiati Ruze, Jianlu Song, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Endocrine pancreas alteration in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB316.

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Antibody-Suppressor CXCR5+CD8+ T Cells Are More Potent Regulators of Humoral Alloimmunity after Kidney Transplant in Mice Compared to CD4+ Regulatory T Cells.

Adoptive cell therapy (ACT), especially with CD4+ regulatory T cells (CD4+ Tregs), is an emerging therapeutic strategy to minimize immunosuppression and promote long-term allograft acceptance, although much research remains to realize its potential. In this study, we investigated the potency of novel Ab-suppressor CXCR5+CD8+ T cells (CD8+ TAb-supp) in comparison with conventional CD25highFoxp3+CD4+ Tregs for suppression of humoral alloimmunity in a murine kidney transplant (KTx) model of Ab-mediated rejection (AMR). We examined quantity of peripheral blood, splenic and graft-infiltrating CD8+ TAb-supp, and CD4+ Tregs in KTx recipients and found that high alloantibody-producing CCR5 knockout KTx recipients have significantly fewer post-transplant peripheral blood and splenic CD8+ TAb-supp, as well as fewer splenic and graft-infiltrating CD4+ Tregs compared with wild-type KTx recipients. ACT with alloprimed CXCR5+CD8+ T cells reduced alloantibody titer, splenic alloprimed germinal center (GC) B cell quantity, and improved AMR histology in CCR5 knockout KTx recipients. ACT with alloprimed CD4+ Treg cells improved AMR histology without significantly inhibiting alloantibody production or the quantity of splenic alloprimed GC B cells. Studies with TCR transgenic mice confirmed Ag specificity of CD8+ TAb-supp-mediated effector function. In wild-type recipients, CD8 depletion significantly increased alloantibody titer, GC B cells, and severity of AMR pathology compared with isotype-treated controls. Anti-CD25 mAb treatment also resulted in increased but less pronounced effect on alloantibody titer, quantity of GC B cells, and AMR pathology than CD8 depletion. To our knowledge, this is the first report that CD8+ TAb-supp cells are more potent regulators of humoral alloimmunity than CD4+ Treg cells.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma [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 2367.

Abstract 2190: Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach

Abstract Obesity is a highly prevalent metabolic disease and an established risk factor for colon cancer. However, critical biological pathways underpinning this relationship remain undetermined. Herein, we integrate human and murine colon tumor transcriptomic data to identify obesity-responsive pathways conserved between species. Human colon cancer samples (n=155) were collected as part of the clinical ColoCare Study, a prospective cohort study of patients newly diagnosed with colorectal cancer. Enrolled patients were diagnosed with stage I-III colon cancer and treatment naïve at time of tissue collection. Patients were classified as having a BMI &amp;lt;30 kg/m2 (i.e., non-obese) or BMI ≥30 kg/m2 (i.e., obese) at baseline. Age, sex, tumor stage, and collection site were adjusted for in all analyses. Patients were 63.3 ± 13.7 years, 51% male, and 34% had a BMI ≥30. C57BL/6J adult female mice were fed a low-fat control diet (Research Diets D12450J, n= 8) or a high-fat diet-induced obesity regimen (D12492, n=7) for ~23 weeks, at which time 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were orthotopically transplanted into the colonic submucosa via colonoscopy-guided injection. Obese mice weighed more than controls (controls 26.4 ± 3.7 g vs. DIO 45.9 ± 9.7 g) and had more mesenteric fat surrounding the colon (controls 201 ± 143 mg vs. 1176 ± 589 mg). Tumors were excised after 4 weeks of growth. RNA was extracted from bulk human tumor samples and from the epithelial-enriched EpCAM+ cell population in murine tumor samples for RNA sequencing. Genes differentially expressed between the reference and obese groups were identified using DESeq2. For gene set enrichment analysis, genes were preranked by log2 fold change for both human and mouse datasets. There were 76 and 132 differentially expressed genes (DEGs; FDRq&amp;lt;0.10) identified in the human and mouse datasets, respectively. None of these 208 DEGs were common to both datasets. Thirty-five Hallmark gene sets in the human dataset and 25 Hallmark gene sets in the mouse dataset were significantly enriched in the obese group; 18 of these gene sets were common to both mouse and human datasets. These overlapping pathways were largely related to inflammation (e.g., inflammatory response, IL6 JAK STAT3 signaling, TNFɑ signaling) and metabolism (e.g., cholesterol homeostasis, glycolysis, and fatty acid metabolism). Additional analysis revealed 204 genes common between the leading edges of both transcriptomic datasets. These transdisciplinary data suggest that obesity induces a set of metabolic and inflammatory processes and/or pathways in colon tumors which is conserved between mice and humans. Future studies will mechanistically evaluate the role of candidate genes and pathways in obesity-associated colorectal carcinogenesis in our AKPS orthotopic organoid transplant mouse model. Citation Format: Elaine M. Glenny, Tengda Lin, Victoria M. Bandera, Biljana Gigic, Aik Choon Tan, Babak Mirminachi, Saratchandra S. Khumukcham, Alessandro Carpanese, Christy A. Warby, Olena Aksonova, Caroline Himbert, Jennifer Ose, Chris Stubben, David Nix, Kenneth Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Alexander Brobeil, Martin A. Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Jatin Roper, Cornelia M. Ulrich, Stephen D. Hursting. Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach [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 2190.

Bahcc1 is critical for the aberrant epigenetic program in a mouse model of MLL-ENL–mediated leukemia

AbstractIn leukemogenesis, genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) drives individual context-dependent programs of malignant transformation. In light of the various differentiation stages of HSPCs based on a recently revised definition using CD150/CD48, our analyses showed that a subpopulation of long-term repopulating HSCs was most susceptible to MLL-ENL–mediated transformation. An analysis of the molecular mechanism identified Bromo-adjacent homology domain and coiled-coil containing 1 (Bahcc1), which encodes a reader molecule of trimethylated histone H3 lysine 27 (H3K27me3), as a candidate gene involved in distinct susceptibility to leukemic transformation. Interestingly, Bahcc1 was previously reported to be highly expressed in acute myeloid leukemia (AML) with an unfavorable prognosis, including some cases of MLL-rearranged AML. We found that MLL-ENL upregulated Bahcc1 through binding to its promoter, and that Bahcc1 was involved in MLL-ENL–mediated immortalization at least partly through repression of H3K27me3-marked Cdkn1c. Analyses using bone marrow transplantation in mice showed that depletion of Bahcc1 suppressed the leukemogenic activity of MLL-ENL. In a public database, high BAHCC1 expression was found to be associated with a poor prognosis in pediatric AML, in which BAHCC1 expression was significantly lower in MLL-AF9-AML than in other MLL-fusion-AML. These findings shed light on the distinct immortalization potential of HSPCs and suggest a novel MLL-fusion-Bahcc1 axis, which may lead to development of molecular targeted therapy against MLL-fusion–mediated leukemia.

Therapeutic efficacy and anti-inflammatory mechanism of baicalein on endometriosis progression in patient-derived cell line and mouse model

BackgroundBaicalein is a flavonoid extracted from the roots of Scutellaria baicalensis G. that has anti-inflammatory and antitumor effects. However, therapeutic mechanisms of baicalein in patients with endometriosis in vivo have yet to be elucidated. As a chronic inflammatory gynecological disease, endometriosis causes pain and infertility, and has no complete treatment to date. Current treatment strategies cause several side effects and have high recurrence rates. PurposeThis study aimed to identify the in vivo therapeutic effects of baicalein on endometriosis and verify the action mechanisms of baicalein, focusing on regulating inflammation. MethodsIn this study, an autologous transplant mouse model and patient-derived immortalized human ovarian endometriotic stromal cells (ihOESCs) were used to investigate the therapeutic activities of baicalein. The mouse model was administered with 40 mg/kg baicalein by oral gavage for 4 weeks, and the treatment outcomes of baicalein-treated mice were compared with vehicle- and dienogest-treated groups. ihOESCs were treated with 0–5 μg/ml baicalein for in vitro studies. ResultsBaicalein significantly alleviated the progression of endometriosis in mouse models. Baicalein reduced the expression of proinflammatory cytokines in endometriotic lesions and ihOESCs, and cytokine expression and T cell proportions in mouse spleen. in vitro results showed that baicalein increased mitochondrial calcium flux and induced mitochondrial depolarization and ROS generation in ihOESCs. Ultimately, baicalein inactivated the MAPK/PI3K signaling and induced cell death in ihOESCs. ConclusionIn conclusion, baicalein effectively attenuated the progression of endometriosis through its anti-inflammatory activities. Baicalein can be an alternative or supplemental treatment for endometriosis to ameliorate the side effects of hormonal therapy.

Biomimetic Hydrogels Promote Pseudoislet Formation to Improve Glycemic Control in Diabetic Mice.

Islet or β-cell transplantation is currently considered to be the ideal treatment for diabetes, and three-dimensional (3D) bioprinting of a bionic pancreas with physiological stiffness is considered to be promising for the encapsulation and transplantation of β-cells. In this study, a 5%GelMA/2%AlgMA hybrid hydrogel with pancreatic physiological stiffness was constructed and used for β-cell encapsulation, 3D bioprinting, and in vivo transplantation to evaluate glycemic control in diabetic mice. The hybrid hydrogel had good cytocompatibility and could induce insulin-producing cells (IPCs) to form pseudoislet structures and improve insulin secretion. Furthermore, we validated the importance of betacellulin (BTC) in IPCs differentiation and confirmed that IPCs self-regulation was achieved by altering the nuclear and cytoplasmic distributions of BTC expression. In vivo transplantation of diabetic mice quickly restored blood glucose levels. In the future, 3D bioprinting of β-cells using biomimetic hydrogels will provide a promising platform for clinical islet transplantation for the treatment of diabetes.

Human amniotic mesenchymal stem cells inhibit immune rejection injury from allogeneic mouse heart transplantation: A preliminary study on the microRNA expression

BackgroundMesenchymal stem cell therapy is a new treatment for immune rejection in heart transplantation. The aim of this paper is to investigate the effect of human amniotic mesenchymal stem cells (hAMSCs) on alleviating immune rejection of allogeneic heart transplantation in mice and its possible underlying mechanism. MethodsWe injected hAMSCs into cervical ectopic heart transplantation model mice via tail vein to observe the survival time, the pathological changes of donor myocardium, and the fluorescent distribution of hAMSCs after the transplantation. MicroRNAs (miRs) with significantly differential expression were obtained by RNA sequencing and bioinformatic analysis, and a dual luciferase reporter gene assay together with real-time quantitative PCR (qRT-PCR) was performed to verify the relationship between miRs and their targeting genes. ResultsThe intervention of hAMSCs prolonged the graft survival time and alleviated the pathological damage of the donor heart. The injected hAMSCs were distributed mainly in the liver, spleen, and kidney, only a very small portion in the donor and recipient hearts. In the allogeneic transplantation models, the expression of miR-34b-5p significantly increased after hAMSC treatment. MiR-34b-5p showed a knockdown effect on gene Fc gamma receptor 2B (FCGR2B). ConclusionshAMSCs can reduce the immune rejection injury after allogeneic heart transplantation. This effect may be associated with the upregulation of miR-34b-5p expression to knock down its targeting gene FCGR2B.