Wild-type Recipients Research Articles

Regulation of cardiac allograft immune responses by microRNA-155

IntroductionA better understanding of the immune mechanisms involved in allograft rejection after transplantation is urgently needed to improve patient outcomes. As microRNA-155 (miR155) plays a critical role in inflammation, we postulated that a deficiency of miR155 will improve cardiac allograft survival and enhance tolerance induction after heart transplantation. MethodsWe developed an acute rejection mouse model through heterotopic BALB/c cardiac transplantation to C57BL/6 (wild-type) and C57BL/6 miR155 knock-out (miR155KO) mice. Further, we induced tolerance in both groups through a costimulatory blockade with CTLA4-Ig (200 μg; post-transplant day 2) and MRI antibodies (250 μg; post-transplant day 0), targeting CD28/B7 and CD40/CD154 signals, respectively. Finally, we examined the effects of injecting 100 μg of small extracellular vesicles (sEVs) isolated from wild-type mice undergoing rejection into tolerant miR155KO mice. ResultsMean survival time (MST) of the cardiac allografts in wild-type and miR155KO mice was 7 and 15 days, respectively (p < 0.0001). Costimulatory blockade increased MST to 65 days and > 100 days in the wild-type and miR155KO recipients, respectively (p < 0.001). Injection of sEVs isolated from wild-type mice undergoing rejection into tolerant miR155KO mice decreased the allograft survival to 9 days, significantly lower than the tolerant miR155KO mice without injection of sEVs (>100 days; p < 0.0001). ConclusionmiR155KO mice have improved cardiac allograft survival and enhanced induction of tolerance after heterotopic cardiac transplantation. Injection of sEVs from wild-type mice undergoing rejection into the miR155KO mice reversed these benefits.

Abstract Or117: JNK2-mediated gut-heart crosstalk in aging-associated AF pathogenesis.

Introduction: Aging is an independent risk factor for atrial fibrillation (AF). We recently revealed a critical role of activated cardiac stress kinase JNK2 in AF risk. Yet, the mechanisms underlying JNK2 activation in the aged heart remain unclear. Emerging evidence suggests that organ crosstalk critically contributes to the development of heart diseases. Interestingly, increased AF episodes were clinically observed in patients with active gastrointestinal (GI) diseases (with a hallmark of enhanced intestinal epithelial barrier permeability (‘leaky gut’), but reduced AF during remission. While both GI dysfunction and enhanced AF risk are seen in aged humans and animal models, whether aging-associated leaky gut leads to AF pathogenesis through activated JNK2 remains unknown. Methods: To assess the leaky gut and AF link, young (Yg, 2 mo) wildtype (WT) and JNK2dn mice (cardiac-specific overexpression of inactive dominant negative (dn) JNK2) were used for aged fecal matter transplant (FMT), while aged (Ag, 24 mo) mice were transplanted with Yg fecal matter to rescue aging-associated AF. Atrial tachycardia/fibrillation (AT/AF) incidence was measured using our well-established protocol, and GI permeability was measured using a serum dextran (4-kDa) FITC fluorescence assay 7 days post-FMT. Results: Aged WT mice had an elevated FITC level (4.9±1.5 µg/ml vs 0.98±0.04 µg/ml; n=11,8; p&lt;0.01) with increased AT/AF inducibility compared to Yg mice (n=7,10; p&lt;0.05). Interestingly, Yg WT recipients subjected to aged FMT (Ag-to-Yg) exhibited an ~3-fold increase in GI permeability (n=14; p&lt;0.01) and significantly enhanced AT/AF inducibility relative to Yg sham controls (8 of 13 vs 0 of 10; p&lt;0.05), while Yg-to-Yg FMT did not alter GI permeability and AT/AF inducibility in Yg recipients (0 of 5, p=NS). Strikingly, cardiac-specific JNK2 inhibition prevented aged-FMT-caused AT/AF in Yg JNK2dn recipient mice (0 of 10; p&lt;0.01), suggesting that activated cardiac JNK2 underlies leaky gut-evoked AF in aging. Further, Yg-to-Ag FMT eliminated aged-associated AT/AF inducibility in aged recipients (0 of 6). Conclusion: Aging-associated GI dysfunction enhances cardiac JNK2 activation, which in turn drives AF risk. Targeting cardiac JNK2 could be a novel anti-AF therapeutic strategy.

Chronic TNF in the aging microenvironment exacerbates Tet2 loss-of-function myeloid expansion

AbstractSomatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cells (HSCs) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing tumor necrosis factor (TNF) favors TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2 mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF–/– genotypes reconstituted with WT CD45.1+ and Tet2–/– CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2–/– cells in old WT recipient mice, with strong skewing toward the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF–/– recipient mice, suggesting that TNF signaling is essential for the expansion Tet2-mutant myeloid clones. Examination of human patients with rheumatoid arthritis with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

Improved Preservation of Rat Small Intestine Transplantation Graft by Introduction of Mesenchymal Stem Cell-Secreted Fractions.

Segmental grafts from living donors have advantages over grafts from deceased donors when used for small intestine transplantation. However, storage time for small intestine grafts can be extremely short and optimal graft preservation conditions for short-term storage remain undetermined. Secreted factors from mesenchymal stem cells (MSCs) that allow direct activation of preserved small intestine grafts. Freshly excised Luc-Tg LEW rat tissues were incubated in preservation solutions containing MSC-conditioned medium (MSC-CM). Preserved Luc-Tg rat-derived grafts were then transplanted to wild-type recipients, after which survival, injury score, and tight junction protein expression were examined. Luminance for each graft was determined using in vivo imaging. The findings indicated that 30-100 and 3-10kDa fractions of MSC-CM have superior activating effects for small intestine preservation. Expression of the tight-junction proteins claudin-3, and zonula occludens-1 preserved for 24h in University of Wisconsin (UW) solution containing MSC-CM with 50-100kDa, as shown by immunostaining, also indicated effectiveness. Reflecting the improved graft preservation, MSC-CM preloading of grafts increased survival rate from 0% to 87%. This is the first report of successful transplantation of small intestine grafts preserved for more than 24h using a rodent model to evaluate graft preservation conditions that mimic clinical conditions.

Early-life infection depletes preleukemic cells in a mouse model of hyperdiploid B-cell acute lymphoblastic leukemia

AbstractEpidemiological studies report opposing influences of infection on childhood B-cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus (CMV) infection as a model early-life infection, we show that virus exposure within 1 week of birth induces profound depletion of transplanted E2A-PBX1 and hyperdiploid B-ALL cells in wild-type recipients and in situ–generated PLC in Eμ-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of interleukin (IL)-12p40–driven interferon (IFN)-γ production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for potential inhibitory effects of early-life infection on B-ALL progression and could inform novel therapeutic or preventive strategies.

T cell responsiveness to IL-10 defines the immunomodulatory effect of costimulation blockade via anti-CD154 and impacts transplant survival.

Costimulation blockade (CoB)-based immunotherapy is a promising alternative to immunosuppression for transplant recipients; however, the current limited understanding of the factors that impact its efficacy restrains its clinical applicability. In this context, pro- and anti-inflammatory cytokines are being recognized as having an impact on T cell activation beyond effector differentiation. This study aims at elucidating the impact of direct IL-10 signaling in T cells on CoB outcomes. We used a full-mismatch skin transplantation model where recipients had a T cell-restricted expression of a dominant negative IL-10 receptor (10R-DN), alongside anti-CD154 as CoB therapy. Unlike wild-type recipients, 10R-DN mice failed to benefit from CoB. This accelerated graft rejection correlated with increased accumulation of T cells producing TNF-α, IFN-γ, and IL-17. In vitro experiments indicated that while lack of IL-10 signaling did not change the ability of anti-CD154 to modulate alloreactive T cell proliferation, the absence of this pathway heightened TH1 effector cell differentiation. Furthermore, deficiency of IL-10 signaling in T cells impaired Treg induction, a hallmark of anti-CD154 therapy. Overall, these findings unveil an important and novel role of IL-10 signaling in T cells that defines the success of CoB therapies and identifies a target pathway for obtaining robust immunoregulation.

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 2962: Chemotherapy induced senescence drives peripheral neuropathy

Abstract Chemotherapy is a mainstay of cancer therapy. Unfortunately, while chemotherapy can profoundly impact disease free survival, it is often accompanied with devastating side effects including hair loss, bone loss, cognitive impairment (referred to as "brain fog"), and peripheral neuropathies. While many of these side effects may subside after the completion of chemotherapy, some persist for a lifetime, substantially compromising the quality of life for survivors. Particularly insidious is chemotherapy-induced peripheral neuropathy (CIPN), which can impair a patient's ability to care for themselves. Shockingly, in over 30% of patients, CIPN persists even after the cessation of chemotherapy, subjecting them to a lifetime of numbness and/or pain. Paclitaxel (PTX), a common treatment for breast cancer, induces CIPN in 61-92% of patients depending on the administered dose. Unfortunately, there are currently no preventative or curative measures for CIPN resulting in side effects that persist for months or even years after the cessation of chemotherapy. Considering chemotherapy can trigger cellular senescence, leading to the release of a diverse array of factors collectively known as the senescence-associated secretory phenotype (SASP) that are capable of negatively influencing neighboring cells. To determine if senescence played a role in CIPN we treated with PTX and found that animals displayed reduced axon innervation in the paws and diminished paw withdrawal responses during the von Frey assay, characteristic symptoms of CIPN. To determine if senescent drove PTX-induced CIPN, we utilized the INKATTAC (INK) mouse, a model in which a p16INK4a promoter drives an inducible suicide transgene to eliminate p16+ senescent cells following treatment with AP20187 (AP). Employing the INK mouse, we successfully prevented CIPN by eliminating PTX-induced senescent cells. Furthermore, through bone marrow (BM) transplant studies using BM from INK mice into wildtype recipients, we failed to prevent CIPN. This result establishes that senescent immune cells, do not contribute to CIPN. In an independent approach, we found that senolytic drugs (kill senescent cells), such as dasatinib and quercetin (DQ) or ABT737 (ABT), effectively prevented and reversed CIPN in mice. Additionally, since the p38MAPK pathway has been shown to regulate SASP mRNA stability, we treated with p38 and MK2 inhibitors and found that inhibition prevented CIPN in mice. To address the mechanism behind CIPN we have carried out single cell RNA sequencing (scRNA-seq) to identify the cell population senescing in response to chemotherapy. These analyses will allow us to understand the mechanisms that drive CIPN and may lead to new treatments for patients suffering from CIPN. Citation Format: Taylor Malachowski, Ganesh Kumar Raut, Xianmin Luo, Satarupa Mullick Bagchi, Sheila Stewart. Chemotherapy induced senescence drives peripheral neuropathy [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 2962.

Abstract 139: Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models

Abstract Osteosarcoma (OS) manifests during puberty, coinciding with rapid bone growth, a feature requiring exploration. We selectively deleted p53 or both p53 and Rb1 alleles in growth-dependent chondrocyte-origin models, utilizing Col10a1-Cre or Agc-CreERT2, incorporating with ROSA-Tomato for tracing and isolating the mutant cell collection. Col10p53flfl(tm) and Col10p53flflRbfl+(tm) mutants exhibited 100% penetrance in osteoblastic OS, with the latter displaying shortened latencies (mean: 335 vs 265 days) and increased metastases. Tamoxifen-induced AgcCreERT2 models highlighted growth stage-dependent OS development, correlating penetrance inversely and latencies positively with mouse age at tamoxifen treatment. Tumors exclusively occurred in endochondral bones, near growth plates. Whole-genome profiling of tm+ tumor cells revealed genetic variations, including SNPs, Indels, CNVs, and SVs. CNVs involved genes altered in human OS, emphasizing losses in Dmd and gains in Myc, Vegfa, Foxm1, Akt1, Aurkb, and Ccen1. A common CNV (losses, Chr. 10: 34,158,531-34,443,999, Log ratio ←0.05) occurred in all sequenced primary and metastatic samples, with male specimens sharing a loss in the Y chr. p region (approximately 725,200-799,409). Orthotopic transplantation of tm+ p53− stromal cells from 4-month-old Col10p53flfltm mice gave rise to bone tumors and lung migration in wild-type recipients, explicitly establishing the transplanted cells as the direct origin of the cancer precursors within early tm+ p53− stromal populations. Whole-genome sequencing unveiled a discernible progression of genomic alterations from 1 to 4-month-old tm+ p53− stromal cells, culminating in donor-derived tumors. Notably, only these specific donor-derived tumors, and not the early tm+ p53− stromal cells, exhibited the Chr. 10 CNV observed in autochthonous malignancies. Micro-metastases, identified by fluorescence imaging, were prevalent, alongside macro-metastases (~10% in Col10p53flflRbfl+tm and ~25% in AgcCreERT2p53flflRbfl+tm cohorts). Notably, amputation of primary OS sites in mice surviving longer revealed larger and more abundant tm+ cell clusters. Some micro-metastases not only expressed signature genes typical of macro-lung metastases but also shared genomic variations with corresponding primaries, including the prevalent loss in Chr. 10. Enhanced immune cell infiltration was evident in lungs hosting micro-metastases, suggesting a potential immune response triggered by pre-tumor early seeding cells. Murine OS models faithfully replicate human OS features, underscoring chondrocytes as significant contributors to OS cell sources. The inclusion of a tomato reporter enables precise tracking, rendering these models invaluable for studying OS etiology, particularly in unraveling pre-tumor molecular events in both primary and metastatic settings. Citation Format: Xin Zhou, Hannah Beird, Yanhua Yi, Zhaohui Xu, Zhongting Zhang, Harjeet Singh, Wendong Zhang, Yifei Wang, Sylvester Jusu, Michael Roth, Jonatha Gill, Richard Gorlick. Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models [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 139.

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [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 1540.

Abstract 1484: 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia

Abstract Myelodysplastic syndromes (MDS) are a diverse group of blood cancers characterized by peripheral blood cytopenias and dysplastic blood cell morphology. Up to 40% of patients with high risk MDS will transform to acute myeloid leukemia (AML), an aggressive blood cancer with significant morbidity and an overall 5-year survival of less than 35%. Both MDS and AML have been linked to hypermethylation of cytosine residues. Hypermethylation in the 5’ regulatory region can result in epigenetic downregulation of gene expression, and it has been speculated that hypermethylation of tumor suppressor genes can contribute to malignant transformation. DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5’-cytosine methylation, with the aim of inducing the re-expression of tumor suppressor genes resulting in a halt of tumor progression. 5-Aza-4’-thio-2’-deoxycytidine (Aza-TdCyd or ATC) is a cytidine analog that incorporates an aza modification (nitrogen in place of carbon) of the cytosine base and a thioether modification of the deoxyribose sugar. ATC is a promising new DNMT1i that has demonstrated efficacy in preclinical studies of solid tumors. Based on these prior studies, we sought to characterize the potential therapeutic effect of ATC in MDS.Mice that express a NUP98::HOXD13 (NHD13) transgene develop an MDS that closely recapitulates all of the key features of human MDS, including pancytopenia, dysplastic morphology, and transformation to AML. We transplanted NHD13 and wild-type (WT) hematopoietic stem and progenitor cells into WT recipients, and used this model of MDS to investigate the therapeutic potential of ATC. We found that ATC treatment led to both T and B lineage lymphoid leukemia (ALL) originating from the NHD13 cells. Surprisingly, ATC treatment also led to T and B lineage ALL in the WT cells, and further experiments demonstrated that non-transplanted, healthy WT mice developed T and B cell ALL after treatment with ATC. In sum, all mice that received at least 8 cycles of ATC therapy at the 1.0 mg/kg dose developed leukemia. Whole exome sequencing of tumors revealed thousands of acquired mutations, almost all of which were C&amp;gt;G transversions in a previously unrecognized, specific 5’-NCG-3’ context. These mutations involved a number of genes well-known to be involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Treatment of the human U937 and CEM cell lines in vitro showed thousands of acquired C&amp;gt;G transversions in a similar context. Taken together, these findings demonstrate that ATC can be a potent mutagen in human and mouse cells, both in vitro and in vivo. Citation Format: Ryan Matthew Bertoli, Yang Jo Chung, Michael Difilippantonio, Anthony Wokasch, Madison R. Marasco, Haley Klimaszewski, Susannah Garber, Yuelin Jack Zhu, Robert L. Walker, Dengchao Cao, James Doroshow, Paul Meltzer, Peter D. Aplan. 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia [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 1484.

Autophagy differentially regulates tissue tolerance of distinct target organs in graft-versus-host disease models.

Tissue-intrinsic mechanisms that regulate severity of systemic pathogenic immune-mediated diseases, such as acute graft-versus-host disease (GVHD), remain poorly understood. Following allogeneic hematopoietic stem cell transplantation, autophagy, a cellular stress protective response, is induced in host nonhematopoietic cells. To systematically address the role of autophagy in various host nonhematopoietic tissues, both specific classical target organs of acute GVHD (intestines, liver, and skin) and organs conventionally not known to be targets of GVHD (kidneys and heart), we generated mice with organ-specific knockout of autophagy related 5 (ATG5) to specifically and exclusively inhibit autophagy in the specific organs. When compared with wild-type recipients, animals that lacked ATG5 in the gastrointestinal tract or liver showed significantly greater tissue injury and mortality, while autophagy deficiency in the skin, kidneys, or heart did not affect mortality. Treatment with the systemic autophagy inducer sirolimus only partially mitigated GVHD mortality in intestine-specific autophagy-deficient hosts. Deficiency of autophagy increased MHC class I on the target intestinal epithelial cells, resulting in greater susceptibility to damage by alloreactive T cells. Thus, autophagy is a critical cell-intrinsic protective response that promotes tissue tolerance and regulates GVHD severity.

Toll-like receptor 2 deficiency promotes the generation of alloreactive γδT17 cells after cardiac transplantation in mice

Homograft rejection is the main cause of heart transplantation failure. The role of TLR2, a major member of the toll-like receptor (TLR) family, in transplantation rejection is has yet to be elucidated. In this study, we used a mouse model of acute cardiac transplantation rejection to investigate whether the TLR2 signalling pathway can regulate cardiac transplantation rejection by regulating alloreactive IL-17+γδT (γδT17) cells. We found that the expression of TLR2 on the surface of dendritic cells (DCs) and macrophages increased during acute transplantation rejection. In addition, our investigation revealed that γδT17 cells exert a significant influence on acute cardiac transplantation rejection. TLR2 gene knockout resulted in an increase in alloreactive γδT17 cells in the spleen and heart grafts of recipient mice compared with wild-type recipient mice and an increase in the mRNA expression of IL-17, IL-1β, CCR6, and CCL20 in the heart grafts. In an in vitro experiment, a mixed lymphocyte reaction was conducted to assess the impact of TLR2 deficiency on the generation of γδT17 cells, which further substantiated a significant increase compared to that in wild-type controls. Furthermore, the mixed lymphocyte reaction showed that TLR2 regulated the production of γδT17 cells by regulating the ability of DCs to secrete IL-1β. These results suggest that TLR2 signalling is important for regulating the generation of γδT17 cells after cardiac allograft transplantation.

Haematopoietic innate interleukin 17A production drives immunopathology in female mouse genital Chlamydia muridarum infection.

Chlamydia trachomatis infection is the leading cause of bacterial urogenital infection and has been demonstrated to drive inflammation and scarring of the reproductive tract. Recent studies have identified key triggers of proinflammatory adaptive immune responses driven by innate leukocytes and epithelia driving immunopathology. Utilizing chimeric mouse models, we investigated the definitive source and role of IL17 and IL17 signalling receptors during early Chlamydia muridarum infection of the female urogenital tract. Bone marrow transplants from wild-type (WT) and IL17A-/- mice to recipients demonstrated equivocal infection kinetics in the reproductive tract, but interestingly, adoptive transfer of IL17A-/- immune cells to WT recipients resulted in no infertility, suggesting a haematopoietic (as opposed to tissue) source of IL17 driving immunopathology. To further delineate the role of IL17 in immunopathology, we infected WT and IL17 receptor A (IL17RA)-/- female mice and observed a significant reduction in immunopathology in IL17RA-/- mice. WT bone marrow transplants to IL17RA-/- recipient mice prevented hydrosalpinx, suggesting signalling through IL17RA drives immunopathology. Furthermore, early chemical inhibition of IL17 signalling significantly reduced hydrosalpinx, suggesting IL17 acts as an innate driver of disease. Early during the infection, IL17 was produced by γδ T cells in the cervico-vagina, but more importantly, by neutrophils at the site of infertility in the oviducts. Taken together, these data suggest innate production of IL17 by haematopoietic leukocytes drives immunopathology in the epithelia during early C. muridarum infection of the female reproductive tract.

Hypercholesterolemia impairs collateral artery enlargement by ten-eleven translocation 1-dependent hematopoietic stem cell autonomous mechanism in a murine model of limb ischemia

ObjectiveThe extent of collateral artery enlargement determines the risk of limb loss due to peripheral arterial disease. Hypercholesterolemia impairs collateral artery enlargement, but the underlying mechanism remains poorly characterized. This study tests the hypothesis that hypercholesterolemia impairs collateral artery enlargement through a ten-eleven translocation 1 (Tet1)-dependent hematopoietic stem cell (HSC)-autonomous mechanism that increases their differentiation into proinflammatory Ly6Chi monocytes and restricts their conversion into proangiogenic Ly6Clow monocytes. MethodsTo test our hypothesis, we induced limb ischemia and generated chimeric mouse models by transplanting HSCs from either wild-type (WT) mice or hypercholesterolemic mice into lethally irradiated WT recipient mice. ResultsWe found that the lethally irradiated WT recipient mice reconstituted with HSCs from hypercholesterolemic mice displayed lower blood flow recovery and collateral artery enlargement that was nearly identical to that observed in hypercholesterolemic mice, despite the absence of hypercholesterolemia and consistent with an HSC-autonomous mechanism. We showed that hypercholesterolemia impairs collateral artery enlargement by a Tet1-dependent mechanism that increases HSC differentiation toward proinflammatory Ly6Chi monocytes and restricts the conversion of Ly6Chi monocytes into proangiogenic Ly6Clow monocytes. Moreover, Tet1 epigenetically reprograms monocyte gene expression within the HSCs. Restoration of Tet1 expression in HSCs of hypercholesterolemic mice restores WT collateral artery enlargement and blood flow recovery after induction of hindlimb ischemia. ConclusionsThese results show that hypercholesterolemia impairs collateral artery enlargement by a novel Tet1-dependent HSC-autonomous mechanism that epigenetically reprograms monocyte gene expression within the HSCs.

Wnt/β-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Absence of MHC Class I Expression on Target Cells Exacerbates Iron Dependent CD4+ T Cell Mediated Killing

Introduction: Little is known about the target-cell intrinsic features that affect tissue susceptibility to T-cell mediated cell death. The major histocompatibility complex (MHC) Class I presents antigens to CD8+ T cells, while MHC class II presents antigens to CD4+ T cells, and this process is critical for infectious, tumor, auto- and allo -immunity. However, MHC Class I has been suggested to have a non-canonical role iron metabolism, but whether it plays a role in tissue tolerance to CD4+ T cell mediated damage is not known. Herein, we investigated the role of MHC Class I on target cells in CD4+ T-cell mediated damage to intestinal epithelial cells (IECs) utilizing clinically relevant models of T-cell mediated gastrointestinal damage from Graft-versus-Host Disease (GVHD), a major complication of allogeneic stem cell transplantation. Methods: Because MHC class I is expressed on all nucleated cells, to specifically assess the role of MHC Class I on IECs, we generated and utilized intestine-specific MHC Class I knockout mice (B2m∆IEC) as recipients for MHC Class II-disparate bm12–&amp;gt;B2m∆IEC allogeneic stem cell transplant (allo-SCT). We utilized flow cytometry, immunological, molecular and biochemical assays to assess cell death pathways and tissue damage in IECs. We performed mechanistic studies to assess the role of iron and utilized clinically relevant iron chelation treatment to assess survival after treatment. Results: Here, we demonstrate that absence of MHC Class I on target cells exacerbates CD4+ T cell mediated killing of IECs leading to greater mortality in the KO animals than WT recipients (P&amp;lt;0.01) (Fig.1). Compared with wild-type (WT) mice, B2m∆IEC mice, despite an increase in mortality following MHC II disparate allo-SCT, did not demonstrate significant changes in T cell activation, or antigen presentation. In vitro co-culture cell killing assays demonstrate that B2m knockout cells have increased cell death compared with WT cells when co-cultured with activated bm12 CD4+ T cells. Because B2m knockout mice have increased liver iron deposition, we hypothesized that the increase in GI damage might be a consequence of iron related cell death in the IECs in B2m∆IEC mice. We demonstrate that absence of B2m on the intestine is sufficient to drive an increase in tissue iron levels in the liver and intestine. Furthermore, B2m∆IEC allo-recipient mice demonstrated increased intracellular labile iron in IECs. Given the increased mortality in B2m∆IEC allo-recipient mice and the increase in intracellular iron in IECs, we investigated the role of iron-dependent cell death, ferroptosis. We show that IECs from B2m∆IEC allo-recipient mice have increased ferroptosis compared with WT animals (P&amp;lt;0.05). Finally, chelation of iron with administration of clinically utilized iron chelator (Deferasirox) ameliorated excess mortality from GVHD in the B2m∆IEC mice. Conclusion: Absence of MHC Class I expression on target cells exacerbates CD4+ T cell mediated killing by enhancing iron dependent ferroptosis. These data demonstrate that in contrast to immunological paradigm, class I regulates CD4+ T cell mediated cytotoxicity even as it is essential for CD8+ T cell immunity.

Trisomy 8 Remodels Chromatin and Activates Transcription of Runx1-Target Genes in Hematopoietic Stem Cell

Myelodysplastic syndrome (MDS) is a poor prognosis cancer that predominantly affects the elderly, arising from hematopoietic stem cell (HSC), resulting in bone marrow failure and predisposition to acute myelogenous leukemia (AML). It has long been known that numerical chromosome anomalies such as trisomy 8 are critical for the development of MDS. Trisomy 8 is known to be associated with relatively poor prognosis, and is a criterion for the diagnosis of MDS in patients. Systemic inflammation has been implicated in the selective advantage for clonal hematopoietic stem cells and the progression of MDS, such as trisomy 8 MDS associated with Behcet's disease. Next generation sequencing studies revealed that trisomy 8 MDS cells highly mutated RUNX1 transcriptional factor (TF) and ASXL1, an epigenetic modifier, both of which regulate HSC function and the differentiation, relative to MDS cells without trisomy 8, suggesting that those mutations may help trisomy 8 to drive the development of MDS. However, previous clinical studies failed to identify genes on the chromosome 8 commonly amplified among trisomy 8 MDS patients, which may account for the pathogenesis of MDS, the molecular mechanism of development of trisomy 8 MDS remains unclear. Thus, we hypothesized that trisomy 8 initiates the transformation by multiple malfunctions of epigenetic and transcriptional regulators in HSC other than a gene dosage effect due to the extra chromosome 8. In order to determine how trisomy 8 affects hematopoiesis, we established a new trisomy 8 model by introducing a human chromosome 8 to mimic trisomy 8 in mouse ES cells. We succeeded in generating trisomy 8 chimeric mice and confirmed the mice showing emergence of hematopoiesis in fetal liver but significantly decreased the chimerism of trisomy 8 cells than the control mice. Wecompetitively transplanted trisomy 8 fetal liver cells at E14.5 into lethally-irradiated wild-type primary recipient mice, and moreover serial transplanted hematopoietic stem and progenitor cells (HSPCs) which isolated from the primary recipient mice. We found that trisomy 8 reduced the self-renewal capacity of HSPC and impeded the differentiation, but it was not sufficient to develop myeloid malignancies in mice. To elucidate the molecular mechanism underlying the impaired hematopoiesis by trisomy 8, we performed RNA sequencing in HSCs isolated from the primary transplanted mice. Gene set enrichment analysis revealed that in comparison with the control HSCs, trisomy 8 HSCs had significantly positive enrichments in type I and type II interferon response genes, but also the canonical target genes of Runx1 and PU.1 TFs were positively enriched in trisomy 8 HSCs. In addition, ATAC-seq analysis revealed that trisomy 8 HSCs changed chromatin accessibility enriched with the binding motif of Runx TF, compared to control HSCs. Given the changes in chromatin accessibility in trisomy 8 HSC, we performed chromatin conformation capture sequencing (Hi-C) in HSPCs and found that trisomy 8 induced changes in chromatin structures such as topologically associating domain (TAD) and compartment A/B in the other chromosomes, which were partly associated with changes in transcription of genes. Since the Runx1 gene is critical for HSC integrity and differentiation, and loss-of-function mutations of RUNX1 were often found in human myeloid malignancies with trisomy 8, we attempted to determine whether Runx1 deletion cancel the impaired self-renewal capacity of trisomy 8 HSC. We found that Runx1 deletion partially canceled the impaired repopulating capacity of trisomy 8 cells in recipient mice, suggesting the selective advantage of loss of function mutation of the RUNX1 gene in trisomy 8 mosaic people and patients with trisomy 8 MDS. Furthermore, as Janus kinase (JAK) and TANK-binding kinase 1 (TBK1) are critical for interferon signalling and activation of their downstream target genes, we revealed that the inhibition of either JAK or TBK function ameliorated trisomy 8-induced chromatin remodelling in HSCs. Overall, trisomy 8 changes chromatin structure and transcription of genes on other chromosomes in HSC accompanied with the malfunction of Runx1 TF, at least in part, due to the activation of interferon signaling, and results in the impaired hematopoiesis. We demonstrated that trisomy 8 creates the basis for transformation of MDS via remodeling chromatin structures in HSC.

CYP26 Activity Maintains the Bone Marrow Niche and Stem Cell Homeostasis

Vitamin A and its active compound, retinoic acid (RA), manifest pleomorphic activities in hematopoiesis. Vitamin A homeostasis is maintained systemically through hepatic CYP26. Emerging data suggests that CYP26 mediated RA metabolism also plays a role in controlling hematopoietic stem and progenitor cells (HSPCs) behavior (Ghiaur G et al. PNAS 2013, Schonberger K et al. Cell Stem Cell 2022). We generated Cyp26a1/ Cyp26b1 inducible knockout (CYP26KO) using ROSA26CreERT Cyp26a1/ Cyp26b1 conditional mice to study the impact on hematopoiesis and the bone marrow microenvironment (BME). Tamoxifen induces complete CYP26KO in these mice and subsequently higher retinoid levels and systemic inflammation (Snyder JM et al. FASEB J 2020). Here, we characterize the hematopoietic, bone and bone marrow (BM) phenotype of these mice. Compared to the CYP26 wild type controls (CTR), we observed leukocytosis and mild anemia as well as signs of extramedullary hematopoiesis in CYP26KO mice (Table). Spleen histology and flow cytometry showed an increased number of all mature elements, including megakaryocytes as well as increased absolute numbers of all hematopoietic progenitor populations (HPCs): LSK, CMP, GMP and MEP (Table). BM analysis showed decreased cellularity and myeloid biased in CYP26KO mice (Table). There was an increased frequency of colony forming unit cell (CFU-C) in the BM of CYP26KO mice but overall preserved absolute numbers when accounted for cellularity (Table). We observed increased frequency of LSK (Table) but decreased numbers of CMP, GMP and MEP in the BM of CYP26KO mice. Both CFU assay and flow cytometry showed myeloid biased HPCs found in the BM of CYP26KO mice (Table). When analyzing the stem cell compartment, short term stem cells and multipotent progenitor cells number were decreased in CYP26KO BM (p&amp;lt;0.01), but the long-term HSCs (LT-HSC) were relatively preserved (0.04 ± 0.01 vs. 0.17 ± 0.04 x10 6/femur, CTR vs CYP26KO, p&amp;lt;0.05). Both flow cytometry and CFU assay confirmed the presence of circulating HPCs in CYP26KO mice (Table). Thus, hematopoiesis of CYP26KO mice migrated away from the BM and into extramedullary tissues. The hematopoietic phenotype was most likely due to the cell extrinsic role of CYP26 since bone marrow transplantation experiments confirmed that a CYP26KO recipient (regardless of the genotype of the hematopoietic cells) and not CYP26 wild type recipient recapitulates the observed hematopoietic findings. This observation, together with necropsy findings of thin, frail bones in CYP26KO mice, prompted a closer look at the BM matrix/microenvironment of these mice. CYP26KO mice had smaller BM cavities, decreased trabecular bone and an increased fat/bone marrow volume ratio (Table). Consistent with these findings, CYP26KO mesenchymal stroma cells (MSC) had no osteoblastic differentiation potential in vitro but showed preserved adipogenic potential. The number of CFU-fibroblasts was decreased in the BM of CYP26KO mice but were found circulating in the PB and were dramatically increased in the spleen of these mice (Table). Single cell RNAseq showed quantitative and qualitative alterations in the BME populations with relative reduction of Cxcl12 abundant MSC2, which explained the qRT-PCR observation of lower levels of Cxcl12 mRNA in MSC isolated from CYP26KO mice. More so, Spp1 mRNA was downregulated in various MSC populations of CYP26KO bone marrow, consistent with their osteopenia and with potential implications for the observed HSPC phenotype. Receptor ligand analysis revealed that various components of the BME have impaired interactions with the extracellular matrix. Thus, the leading hypothesis is that absence of CYP26 leads to a dysregulated BME and to migration of a HSCs-niche unit out of the BM and into secondary hematopoietic organs. Interestingly, scRNAseq experiments uncovered upregulation of S100a8/9 in the CYP26KO neutrophil progenitors, which may be in response to systemic stress. The observed bone alterations (osteopenia and increased BM fat) coupled with noted changes in hematopoiesis (i.e. myeloid bias and inflammatory stress) may represent early signs of aging of the bone and bone marrow and suggest a central role of retinoid homeostasis in this process.

Novel Mouse Model That Recapitulates the Hematologic Phenotype of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that presents during early childhood and is characterized by macrocytic anemia, congenital malformations, and predisposition to cancer. More than 90% of DBA patients are diagnosed during their first year of life (median age 12 weeks). The pathogenesis of DBA is linked to loss-of-function mutations in genes encoding ribosomal proteins (RP), although mutations in 3 non-RP genes ( GATA1, TSR2, and HEATR3) have been detected. DBA patients present with a high degree of clinical heterogeneity with varying severity and responses to steroid therapy. DBA provides a unique disease model to study how RP deficiency impacts the phenotype and pathogenesis of ribosomopathies. However, one challenge is the lack of animal models that faithfully recapitulate the clinical features of DBA in terms of disease severity and onset age. Heterozygous loss-of-function mutations in RPL11 are found in 5-20% of DBA patients, but previous mouse models carrying Rpl11 haploinsufficiency have only a mild anemia. Here, we report a novel mouse model of DBA with inducible haploinsufficient expression of Rpl11 which results in a severe macrocytic anemia in juvenile mice. Mx1-CreRpl11+/flox experimental mice were generated by cross-breeding homozygous Rpl11 flox/flox and Mx1-cre breeders. Haploinsufficiency of Rpl11 ( Rpl11+/Δ) was induced by intraperitoneal injection of polyinosinic-polycytidylic acidto mice with Mx1-CreRpl11 +/flox on postnatal day 8 and 10. Diseased mice with Rpl11+/Δ showed macrocytic anemia at 2 weeks (wks) post-induction with hemoglobin (Hb) &amp;lt; 10 g/dL, eventually dying of severe anemia (Hb&amp;lt; 2 g/mL) by 30 wks (median survival 27 wks, p&amp;lt;0.001, n=7 )( Figure 1). We observed a 50% decreased expression of Rpl11 in blood neucleated cells from Rpl11+/Δ mice and several characteristic clinical features of DBA. Erythrocyte adenosine deaminase was significantly elevated in the blood of all Rpl11+/Δ mice when compared to wild type (WT) littermates (median 5 vs 1 EU/g Hg, p&amp;lt;0.001, n=6). Similarly, EPO concentrations were significantly elevated in Rpl11+/Δ mice compared to WT mice (median 38752 vs 23 pg/mL, p&amp;lt;0.01, n=6). On postmortem examination, the DBA mice displayed splenomegaly. FACS analysis data demonstrated that the differentiation of erythropoiesis is significantly blocked in peripheral blood, bone marrow (BM), and spleen of Rpl11+/Δ mice compared to WT mice (p&amp;lt;0.05, n = 5). In addition, methylcellulose colony assays revealed that CFU-E colonies were significantly decreased in BM from Rpl11+/Δ mice in comparison to WT mice (median 23.5 vs 40.5 per 0.2 million BM cells in the presence of EPO at a concentration of 1 unit/mL, p&amp;lt;0.05, n=4). To determine whether BM hematopoietic stem cells/progenitor cells from Rpl11+/Δ mice can transfer the DBA phenotype, we transplanted bone marrow cells of Rpl11+/ Δ mice (CD45.2+) into sublethally irradiated WT mice (CD45.1+). Our data demonstrate that the BM cells from Rpl11+/Δ donor mice can induce lethal macrocytic anemia in WT recipient mice. Studies on the molecular mechanisms underlying the dysregulated erythropoiesis in Rpl11+/Δ mice are ongoing. In conclusion, we have successfully generated the first DBA mouse model that recapitulates the hematologic features of DBA patients. This model provides an ideal tool to study the pathogenesis of DBA and other ribosomopathies.