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Clonal Hematopoiesis of Indeterminate Potential (CHIP), a phenomenon in which a single hematopoietic stem cell (HSC) disproportionately contributes to the peripheral blood, is associated with an increased likelihood of developing many age-associated diseases. However, the connection to neurodegenerative diseases, such as Alzheimer's Disease (AD) is less clear. Prior studies have established that inflammation plays a central role in AD pathogenesis and mounting evidence suggests systemic inflammatory signals can be transmitted to the CNS, where they may play a direct role in microglia activation and plaque clearance. Here we investigated the role of the two most commonly mutated CHIP-associated genes, Dnmt3a and Tet2, in the pathogenesis of Alzheimer's Disease. We transplanted 5xFAD transgenic mice predisposed to develop familial AD with Dnmt3a-/-, Tet2-/- or wildtype (WT) bone marrow (BM). Mice were then challenged weekly with LPS to mimic systemic chronic inflammation seen in aging. 5xFAD mice transplanted with Dnmt3a-/- BM showed signs of exacerbated AD, including increased cognitive impairment and decreased microglia activation compared to those transplanted with WT BM. Mice transplanted with Dnmt3a-/- BM also had fewer peripheral immune cells infiltrating the brain. In contrast, 5xFAD mice transplanted with Tet2-/- BM showed improved cognitive status, increased amyloid plaque clearance and increased microglia activation, while having a higher percentage of activated infiltrating myeloid cells within the brain compared to WT controls. Our data suggest that Dnmt3a and Tet2 mutations impact peripheral immune cell infiltration leading to changes in microglia activation and AD pathogenesis. Overall, our study of CHIP and AD marks the first report in which Dnmt3a and Tet2, which have opposite roles in DNA methylation, induce opposing effects on disease progression.
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Mean platelet volume (MPV) is an efficient, easily available, and cost-effective marker, which can be used in monitoring glycaemic status in Type 2 Diabetes Mellitus patients. Platelet volume indices (PVI) such as MPV are the indicators of increased platelet activity and can be considered as potential biomarkers for diabetic complications.
Sixty four patients were included to study the link, between blood sugar levels and MPV in conjunction with glycated hemoglobin (HbA1c), in both men and women. Men were notably older with an age of 58.4 years compared to women who had an average age of 50.6 years. Men had a higher MPV of 9.2 fl compared to women 8.4 fl indicating differences in platelet characteristics based on gender within the diabetic population. Additionally, men had HbA1c levels at 8.4% compared to women at 7.9%, which suggests variations in blood sugar control between the genders. Similarly, men showed blood sugar levels at 194.7 mg/dL compared to women's 180 mg/dL. These results highlight the importance of considering gender factors in managing diabetes and stress the need for approaches to enhance patient care and outcomes.
Understanding the relationship between diabetes and thrombosis is crucial for assessing risks and devising management plans. Our research emphasizes the importance of Mean Platelet Volume (MPV) as a marker for evaluating the likelihood of blood clots in individuals, particularly concerning blood sugar control. By acknowledging the significance of MPV in foreseeing clotting events, healthcare providers can improve their ability to pinpoint individuals at risk and tailor treatments accordingly, ultimately enhancing outcomes in diabetes care. Further exploration of MPVs usefulness as a tool in blood clotting issues is necessary to fully exploit its clinical benefits.
Primary myelofibrosis (PMF) has hypercoagulability and venous thromboembolism (SVT). JAK2 V617F mutated patients with lower-risk IPSS have more thrombosis and myeloproliferation vs high-risk IPSS. JAK2 mutated splanchnic endothelium releases von Willebrand factor (VWF), P-selectin, Factor VIII (FVIII), and plasminogen activator inhibitor-1 (PAI-1). We studied VWF, soluble P-selectin (s-P-selectin), P-selectin expression, FVIII, and PAI-1 in 20 patients with PMF according to WHO-criteria. 11/20 were JAK2, 8/20 CALR, and 1/20 MPL mutated. 6/20 were low-, 6/20 intermediate-1-, 8/20 intermediate-2-risk IPSS. The mutations were conducted by ARMS-PCR gel electrophoresis. Hemoglobin and platelets, and VWF, sP-selectin, and PAI-1 were measured by automated analyzer and ELISA, respectively. P-selectin and FVIII were measured by flow cytometry and chromogenic assay, respectively. Patients were 67 years old (23-84 years). Nobody had thrombotic risk factors. 3/20 JAK2 mutated had portal vein thrombosis, 2 with intermediate-2 risk IPSS and 1 with low-risk IPSS, 2/20 JAK2 mutated had mesenteric vein thrombosis and splenic vein thrombosis with intermediate-1 risk IPSS and low-risk IPSS. SVT was diagnosed with computed tomographic scan and abdominal magnetic resonance imaging. JAK2 burden was higher in thrombosis vs without thrombosis (35% vs 10%) as well as hemoglobin (12 g/dl vs 10 g/dl) while platelets were comparable (300x109/L vs 370x109/L9). VWF and s-P-selectin were higher in thrombosis (55±5 ng/mL vs 72±5 ng/mL) vs without thrombosis (30±2 ng/mL vs 35±5 ng/mL) as well as P-selectin (40±5% vs 9±2%). FVIII was higher in thrombosis (330±30%) vs without thrombosis (170±5%). PAI-1 was elevated in thrombosis vs without thrombosis (88±5 ng/ml vs 62±2 ng/ml). These results suggest that PMF-SVT may underly a JAK2 mutated prothrombotic endothelium. Further studies are needed.
Chimeric antigen receptor-engineered T cells and bispecific antibodies have proven to be effective therapies for a range of leukemias, whereas modalities that rely on endogenous T cell responses including immune checkpoint blockade have not proven as beneficial. This has been attributed to a lack of elicited endogenous responses against relatively low mutation burden ALL and AML tumors, particularly in pediatrics. Here we will discuss the identification and characterization of endogenous fusion- and neoantigen-specific T cell responses in pediatric leukemias, as well as attempts to identify such responses arising during CAR T cell therapies via epitope spreading. Novel methods for efficiently detecting these responses will be described, along with the T cell features that characterize successful anti-tumor immunity.
Acute myeloid leukemia (AML) often arises from myelodysplasia (MDS), a pre-leukemic condition characterized by dysplasia and ineffective hematopoiesis. Using mouse models of MDS and AML we identified a global decrease in nucleoporin (NUP) expression that was confirmed in publicly available patient databases. shRNA-mediated downregulation of NUPs in mouse MDS HSPCs and transplantation led to fully penetrant AML with blasts in blood, bone marrow (BM) and spleen. To examine the relevance of NUPs in human disease we downregulated NUPs expression in a model of induced Pluripotent Stem Cell (iPSC)-derived HSPCs harboring the MDS-relevant SRSF2P95L and ASXL1646fs*12 mutations (SA). shNUPS-SA HSPCs overcame exhaustion and loss of CD34 expression, typical of MDS cells. Transformed cells maintained growth for as long as 10 months, acquired phenotypic characteristics of AML blasts and presented a 2.5-fold upregulation of the leukemic biomarker CD123. shNUPS-SA HSPCs engrafted in NSG mice, establishing the transformative potential of NUP downregulation in a humanized in vivo model. RNAseq analysis of HSPCs from mouse and human models of AML versus MDS and patient samples revealed upregulation of genes promoting epithelial-to-mesenchymal transition (EMT). Master regulator analysis between patient-derived MDS and AML HSPCs and their stroma identified the secreted matricellular protein Tenascin X as a candidate regulator of NUPs expression. TNXB levels decreased in BM plasma of AML as compared to MDS patients and in the BM of AML as compared to MDS mice. Mass spectrometry analysis identified the presence of a TNXB protein mainly consisting of the fibrinogen-like domain linked to active TGF-β-mediated activation of EMT. The identification of EMT as a signature of transformation in a non-solid cancer uncovers a novel pathway of AML invasiveness that could be potentially targetable.
Clonal expansion of aberrant hematopoietic stem cells (HSCs) underlies many of the hematological disorders. Cells with somatic mutations can be eliminated through presentation of neoantigens on MHC class I to antigen specific CD8+ cytotoxic T lymphocytes (CTL). However, much remains to be elucidated on how HSCs and CTLs interact with each other. Here, we uncover a unique TNFα-driven interplay between HSCs and CTLs. Co-culture and adoptive transfer experiments using ovalbumin-expressing (OVA+) HSCs (Lin-/c-Kit+/Sca-1+/Flk2-/CD48-/CD150+) and OVA-specific OT-I CTLs demonstrated that OVA+ HSCs efficiently present OVA peptide on MHC class I and directly activated OT-I CTLs, making HSCs one of the most susceptible hematopoietic populations to CTL killing. Indeed, adoptive transfer of OT-I CTLs to mixed chimeric mice that harbor both WT and Jak2V617F OVA+ hematopoietic cells specifically eliminated Jak2V617F OVA+ HSC clones and cured myeloproliferative neoplasm. Intracellular FACS analyses revealed that activated OT-I CTLs produced various cytokines including IFNγ and TNFα and HSCs responded to CTL-derived cytokines to upregulate T cell-attracting chemokines such as CXCL9. Interestingly, the response of HSCs to CTL-derived TNFα was critical for CTLs to induce granzyme B, and TNFα receptor–deficient OVA+ HSCs, but not IFNγ receptor–deficient OVA+ HSCs, escaped from OT-I CTL killing. Of note, such response was unique to HSCs and not observed in granulocyte-monocyte progenitors (GMPs: Lin-/c-Kit+/Sca-1-/CD34+/FcγR+). By contrast, prior exposure to TNFα but not IL-1β or IL-6, rendered OVA+ HSCs, but not OVA+ GMPs, resistant to OT-I CTL killing largely through upregulation of PD-L1 and PD-L2. Taken together, our results reveal the robustness of HSC quality control via MHC class I-dependent interplay with CTLs and highlight TNFα as its critical but context-dependent regulator.
Hematopoietic stem cells (HSCs) are the origin of all blood cells and maintained for throughout life to ensure the homeostasis of the blood system. HSCs exhibit an innate capacity for self-renewal and differentiation, yet their cell fate differs during life-stages. HSCs are predominantly proliferative in the fetal liver yet enter cell cycle dormancy in the adult bone marrow (BM). Systemically and locally produced cytokines specify the cell fate of hematopoietic lineages within the bone marrow (BM). Deficiency in the cytokine thrombopoietin (Thpo) and its receptor Mpl results in an age-progressive decline in hematopoietic stem and progenitor cell (HSPC) and megakaryocytes (MK) number. While HSPC-driven hematopoiesis in developmental and adult-stages differ in proliferative and differentiative nature, no study has addressed whether and how hematopoietic cell sensitivity of Thpo/Mpl signaling varies during these stages. We thus generated a Mpl reporter mouse (Mpl-eGFP and MplCreERT2;CAG-LSL-EGFP) to monitor, induce and trace Mpl expression on hematopoietic cells in the fetal liver (FL)(E14.5) and adult BM. While Mpl was expressed in HSCs, multipotent progenitor (MPP) cells and lineage-specific progenitor cells in the FL, Mpl expression was restricted to HSC and early MPP populations upon short-term (7days after tamoxifen administration) labeling in the adult BM. Furthermore, platelet-biased HSCs but not MK erythroid progenitor (MEP) cells expressed Mpl with short term labeling. We next assessed the effect of inducing deletion of Thpo on hematopoietic cell populations in the BM using Thpofl/fl;CreERT+ (Thpo cKO) mice. Thpo cKO mice exhibited thrombocytopenia and significant decrease in plasma Thpo. BM HSCs, platelet-biased HSCs and MKP numbers sharply and significantly decreased at day7 while other progenitor cell populations retained their number until day 28 of Thpo deletion. Difference in sensitivity to Thpo may shape life-stage dependent functional variation of HSPCs and guide the transition from developmental to adult hematopoiesis.
Immunotherapy leads to long-term survival of cancer patients, yet generalized success has been hampered by insufficient antigen presentation and exclusion of immunogenic cells from the tumor microenvironment. Here, we developed an approach to reprogram tumor cells in vivo by adenoviral delivery of the transcription factors PU.1, IRF8, and BATF3, which enabled them to present antigens as type 1 conventional dendritic cells. Reprogrammed tumor cells remodeled their tumor microenvironment, recruited, and expanded polyclonal cytotoxic T cells, induced complete tumor regressions, and established long-term systemic immunity in different mouse melanoma models. In human tumor spheroids and xenografts, reprogramming to immunogenic dendritic-like cells progressed independently of immunosuppression, which usually limits immunotherapy. Our study paves the way for first-in-human trials and other applications of immune cell reprogramming in vivo.
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