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1.
Cell ; 184(22): 5577-5592.e18, 2021 10 28.
Article in English | MEDLINE | ID: mdl-34644529

ABSTRACT

Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover "subTMEs," histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. "Reactive" subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich "deserted" subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.


Subject(s)
Pancreatic Neoplasms/pathology , Tumor Microenvironment , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Cancer-Associated Fibroblasts/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Differentiation , Cell Proliferation , Epithelium/pathology , Extracellular Matrix/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Phenotype , Stromal Cells/pathology , Survival Analysis , Tumor Microenvironment/immunology
2.
Nat Immunol ; 24(12): 2135-2149, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37932456

ABSTRACT

Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cells harbor the T cell receptor (TCR)-derived ζ chain as an intracellular activation domain in addition to costimulatory domains. The functionality in a CAR format of the other chains of the TCR complex, namely CD3δ, CD3ε and CD3γ, instead of ζ, remains unknown. In the present study, we have systematically engineered new CD3 CARs, each containing only one of the CD3 intracellular domains. We found that CARs containing CD3δ, CD3ε or CD3γ cytoplasmic tails outperformed the conventional ζ CAR T cells in vivo. Transcriptomic and proteomic analysis revealed differences in activation potential, metabolism and stimulation-induced T cell dysfunctionality that mechanistically explain the enhanced anti-tumor performance. Furthermore, dimerization of the CARs improved their overall functionality. Using these CARs as minimalistic and synthetic surrogate TCRs, we have identified the phosphatase SHP-1 as a new interaction partner of CD3δ that binds the CD3δ-ITAM on phosphorylation of its C-terminal tyrosine. SHP-1 attenuates and restrains activation signals and might thus prevent exhaustion and dysfunction. These new insights into T cell activation could promote the rational redesign of synthetic antigen receptors to improve cancer immunotherapy.


Subject(s)
Proteomics , Receptors, Antigen, T-Cell , CD3 Complex , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Cell Membrane/metabolism , Lymphocyte Activation , T-Lymphocytes
3.
Nucleic Acids Res ; 52(8): 4215-4233, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38364861

ABSTRACT

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3 and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.


Subject(s)
Chromatin , Gene Regulatory Networks , Heart , Animals , Humans , Mice , Cell Differentiation , Chromatin/metabolism , Chromatin/genetics , Epigenesis, Genetic , Histone Code , Histones/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Regeneration/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Zebrafish/genetics
4.
Am J Hum Genet ; 109(5): 928-943, 2022 05 05.
Article in English | MEDLINE | ID: mdl-35397207

ABSTRACT

Organ fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-ß signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.


Subject(s)
Cardiomyopathy, Hypertrophic , Cilia , Adult , Animals , Cardiomyopathy, Hypertrophic/metabolism , Child , Cilia/genetics , Cilia/metabolism , Fibrosis , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kidney , Liver , Mutation/genetics , Zebrafish/genetics
5.
Blood ; 141(23): 2824-2840, 2023 06 08.
Article in English | MEDLINE | ID: mdl-36696631

ABSTRACT

Peripheral T-cell lymphomas (PTCLs), especially angioimmunoblastic and follicular TCLs, have a dismal prognosis because of the lack of efficient therapies, and patients' symptoms are often dominated by an inflammatory phenotype, including fever, night sweats, weight loss, and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on T-cell follicular helper-type PTCL (TFH-PTCL) disease progression and symptoms. We showed that ITK-SYK-driven murine PTCLs and primary human TFH-PTCL xenografts both induced inflammation in mice, including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving interferon gamma (CD4+ malignant T cells) and interleukin 6 (IL-6; activated granulocytes), ultimately inducing broad JAK activation (JAK1/2/3 and TYK2) in both cell types. Inflammatory granulocyte depletion via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox), or IL-6 deletion within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration, and enhanced mouse survival. Furthermore, unselective JAK inhibitors (ruxolitinib) inhibited both TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions, and an intact JAK/STAT signaling pathway for TFH-PTCL development and also support broad JAK inhibition as an effective treatment strategy in early disease stages.


Subject(s)
Lymphoma, T-Cell, Peripheral , Lymphoma, T-Cell , Humans , Animals , Mice , Lymphoma, T-Cell, Peripheral/pathology , Interleukin-6 , Lymphoma, T-Cell/pathology , Granulocytes/pathology , Inflammation
6.
Mol Ther ; 32(5): 1298-1310, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38459694

ABSTRACT

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.


Subject(s)
CRISPR-Cas Systems , Deoxyribonuclease I , Gene Editing , Keratinocytes , Humans , Gene Editing/methods , Deoxyribonuclease I/metabolism , Deoxyribonuclease I/genetics , Keratinocytes/metabolism , DNA Breaks, Double-Stranded , Chromosome Aberrations , Collagen Type VII/genetics , Collagen Type VII/metabolism , Cells, Cultured
7.
J Allergy Clin Immunol ; 153(1): 243-255.e14, 2024 01.
Article in English | MEDLINE | ID: mdl-37595758

ABSTRACT

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.


Subject(s)
Lymphohistiocytosis, Hemophagocytic , Humans , Mice , Animals , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphohistiocytosis, Hemophagocytic/therapy , Lymphohistiocytosis, Hemophagocytic/diagnosis , T-Lymphocytes , Gene Editing , Mutation , Lymphocytic choriomeningitis virus , Hematopoietic Stem Cells , Membrane Proteins/genetics
8.
J Neurosci ; 43(29): 5290-5304, 2023 07 19.
Article in English | MEDLINE | ID: mdl-37369586

ABSTRACT

The perforant path provides the primary cortical excitatory input to the hippocampus. Because of its important role in information processing and coding, entorhinal projections to the dentate gyrus have been studied in considerable detail. Nevertheless, synaptic transmission between individual connected pairs of entorhinal stellate cells and dentate granule cells remains to be characterized. Here, we have used mouse organotypic entorhino-hippocampal tissue cultures of either sex, in which the entorhinal cortex (EC) to dentate granule cell (GC; EC-GC) projection is present, and EC-GC pairs can be studied using whole-cell patch-clamp recordings. By using cultures of wild-type mice, the properties of EC-GC synapses formed by afferents from the lateral and medial entorhinal cortex were compared, and differences in short-term plasticity were identified. As the perforant path is severely affected in Alzheimer's disease, we used tissue cultures of amyloid precursor protein (APP)-deficient mice to examine the role of APP at this synapse. APP deficiency altered excitatory neurotransmission at medial perforant path synapses, which was accompanied by transcriptomic and ultrastructural changes. Moreover, presynaptic but not postsynaptic APP deletion through the local injection of Cre-expressing adeno-associated viruses in conditional APPflox/flox tissue cultures increased the neurotransmission efficacy at perforant path synapses. In summary, these data suggest a physiological role for presynaptic APP at medial perforant path synapses that may be adversely affected under altered APP processing conditions.SIGNIFICANCE STATEMENT The hippocampus receives input from the entorhinal cortex via the perforant path. These projections to hippocampal dentate granule cells are of utmost importance for learning and memory formation. Although there is detailed knowledge about perforant path projections, the functional synaptic properties at the level of individual connected pairs of neurons are not well understood. In this study, we investigated the role of APP in mediating functional properties and transmission rules in individually connected neurons using paired whole-cell patch-clamp recordings and genetic tools in organotypic tissue cultures. Our results show that presynaptic APP expression limits excitatory neurotransmission via the perforant path, which could be compromised in pathologic conditions such as Alzheimer's disease.


Subject(s)
Alzheimer Disease , Perforant Pathway , Mice , Animals , Perforant Pathway/physiology , Amyloid beta-Protein Precursor/genetics , Alzheimer Disease/pathology , Dentate Gyrus/physiology , Synaptic Transmission/physiology , Synapses/physiology
9.
Kidney Int ; 2024 Aug 30.
Article in English | MEDLINE | ID: mdl-39218392

ABSTRACT

Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease.

10.
Br J Haematol ; 204(2): 595-605, 2024 02.
Article in English | MEDLINE | ID: mdl-37945316

ABSTRACT

Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1.


Subject(s)
Leukemia, Myelomonocytic, Juvenile , Neurofibromatosis 1 , Child , Humans , Leukemia, Myelomonocytic, Juvenile/genetics , Neurofibromatosis 1/genetics , Mutation , Signal Transduction , Genes, Tumor Suppressor
11.
Blood ; 139(7): 1039-1051, 2022 02 17.
Article in English | MEDLINE | ID: mdl-34767620

ABSTRACT

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.


Subject(s)
Bone Marrow Failure Disorders/pathology , Gain of Function Mutation , Heterozygote , Myelodysplastic Syndromes/pathology , Replication Protein A/genetics , Telomere Shortening , Telomere/genetics , Adolescent , Adult , Bone Marrow Failure Disorders/etiology , Bone Marrow Failure Disorders/metabolism , Cell Differentiation , Child , Female , Humans , Infant, Newborn , Male , Middle Aged , Myelodysplastic Syndromes/etiology , Myelodysplastic Syndromes/metabolism , Young Adult
12.
Blood ; 140(10): 1167-1181, 2022 09 08.
Article in English | MEDLINE | ID: mdl-35853161

ABSTRACT

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.


Subject(s)
Leukemia, Myeloid, Acute , Tumor Suppressor Protein p53 , Animals , Apoptosis , Humans , Leukemia, Myeloid, Acute/genetics , Major Histocompatibility Complex , Mice , Proto-Oncogene Proteins c-mdm2/metabolism , Transplantation, Homologous , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Up-Regulation
13.
FASEB J ; 37(1): e22698, 2023 01.
Article in English | MEDLINE | ID: mdl-36520012

ABSTRACT

Folate plays an important role in the modulation of one-carbon metabolism and DNA methylation through a complex biosynthesis pathway. Folate deficiency during pregnancy has been associated with an increased risk for birth defects. This study investigates the extent to which the availability of folate and S-Adenosylmethionine (SAM) affects placental DNA methylation. We hypothesized that maintaining sufficient levels of folate and SAM is particularly important in individuals carrying the MTHFR C677T polymorphism. Maternal- and cord blood was analyzed to genotype the MTHFR rs1801133 SNP. Red blood cell (RBC) folate, vitamin B12, SAM, and S-Adenosylhomocysteine (SAH) were analyzed in cord blood. Epigenome-wide methylation analyses were performed on 90 placenta tissue samples isolated from the fetal side of the placenta; 45 originating from mother-infant dyads homozygous for the MTHFR C677T variant and 45 originating from mother-infant dyads with the homozygous wild type MTHFR677 genotype. Verification of the results was performed using pyrosequencing assays. Genome-wide placental DNA methylation patterns were relatively stable and not significantly affected by levels of one-carbon metabolites. MTHFR genotype was associated with DNA methylation of several loci, including a locus in the MTHFR region. RBC folate and particularly the SAM:SAH ratio did affect overall CpG DNA methylation in some CpG regions when the loci were split according to their CpG island relation. This was most evident in participants carrying the MTHFR C677T variant suggesting a stronger influence of the biosynthesis pathway on the overall placental DNA methylation in MTHFR TT individuals than in MTHFR CC individuals.


Subject(s)
DNA Methylation , Folic Acid , Methylenetetrahydrofolate Reductase (NADPH2) , Placenta , Female , Humans , Infant , Pregnancy , Carbon/metabolism , DNA Methylation/drug effects , DNA Methylation/genetics , Folic Acid/metabolism , Folic Acid/pharmacology , Folic Acid/therapeutic use , Genotype , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Placenta/drug effects , Placenta/metabolism , S-Adenosylmethionine/metabolism , Vitamin B 12/metabolism
14.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Article in English | MEDLINE | ID: mdl-33563755

ABSTRACT

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.


Subject(s)
Antigens, CD20/metabolism , B-Lymphocytes/immunology , Antigens, CD19/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cells, Cultured , Humans , Lymphocyte Activation , Receptors, Antigen, B-Cell/metabolism
15.
Article in German | MEDLINE | ID: mdl-38739266

ABSTRACT

The collaborative project Personalized Medicine for Oncology (PM4Onco) was launched in 2023 as part of the National Decade against Cancer (NKD) and is executed within the Medical Informatics Initiative (MII). Its aim is to establish a sustainable infrastructure for the integration and use of data from clinical and biomedical research and therefore combines the experience and preliminary work of all four consortia of the MII and the leading oncology centers in Germany. The data provided by PM4Onco will be prepared in a suitable form to support decision making in molecular tumor boards. This concept and infrastructure will be extended to 23 participating partner sites and thus improve access to targeted therapies based on clinical information and analysis of molecular genetic alterations in tumors at different stages of the disease. This will help to improve the treatment and prognosis of tumor diseases.Clinical cancer registries are involved in the project to improve data quality through standardized documentation routines. Clinical experts advise on the expansion of the core datasets for personalized medicine (PM). Information on quality of life and treatment outcomes reported by patients in questionnaires, which is rarely collected outside of clinical trials, will make a significant contribution. Patient representatives are involved from the onset to ensure that the important perspective of patients is taken into account in the decision-making process. PM4Onco thus creates an alliance between the MII, oncological centers of excellence, clinical cancer registries, young scientists, patients, and citizens to strengthen and advance PM in cancer therapy.


Subject(s)
Medical Oncology , Neoplasms , Precision Medicine , Humans , Germany , Intersectoral Collaboration , Medical Informatics/organization & administration , Medical Oncology/organization & administration , Models, Organizational , Neoplasms/therapy
16.
Glia ; 71(9): 2117-2136, 2023 09.
Article in English | MEDLINE | ID: mdl-37208965

ABSTRACT

The pro-inflammatory cytokine tumor necrosis factor α (TNFα) tunes the capacity of neurons to express synaptic plasticity. It remains, however, unclear how TNFα mediates synaptic positive (=change) and negative (=stability) feedback mechanisms. We assessed effects of TNFα on microglia activation and synaptic transmission onto CA1 pyramidal neurons of mouse organotypic entorhino-hippocampal tissue cultures. TNFα mediated changes in excitatory and inhibitory neurotransmission in a concentration-dependent manner, where low concentration strengthened glutamatergic neurotransmission via synaptic accumulation of GluA1-only-containing AMPA receptors and higher concentration increased inhibition. The latter induced the synaptic accumulation of GluA1-only-containing AMPA receptors as well. However, activated, pro-inflammatory microglia mediated a homeostatic adjustment of excitatory synapses, that is, an initial increase in excitatory synaptic strength at 3 h returned to baseline within 24 h, while inhibitory neurotransmission increased. In microglia-depleted tissue cultures, synaptic strengthening triggered by high levels of TNFα persisted and the impact of TNFα on inhibitory neurotransmission was still observed and dependent on its concentration. These findings underscore the essential role of microglia in TNFα-mediated synaptic plasticity. They suggest that pro-inflammatory microglia mediate synaptic homeostasis, that is, negative feedback mechanisms, which may affect the ability of neurons to express further plasticity, thereby emphasizing the importance of microglia as gatekeepers of synaptic change and stability.


Subject(s)
Microglia , Tumor Necrosis Factor-alpha , Mice , Animals , Tumor Necrosis Factor-alpha/pharmacology , Receptors, AMPA , Neuronal Plasticity/physiology , Hippocampus , Synaptic Transmission/physiology , Synapses/physiology
17.
Br J Haematol ; 203(2): 264-281, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37539479

ABSTRACT

Acute myeloid leukaemia (AML) relapse after allogeneic haematopoietic cell transplantation (allo-HCT) is often driven by immune-related mechanisms and associated with poor prognosis. Immune checkpoint inhibitors combined with hypomethylating agents (HMA) may restore or enhance the graft-versus-leukaemia effect. Still, data about using this combination regimen after allo-HCT are limited. We conducted a prospective, phase II, open-label, single-arm study in which we treated patients with haematological AML relapse after allo-HCT with HMA plus the anti-PD-1 antibody nivolumab. The response was correlated with DNA-, RNA- and protein-based single-cell technology assessments to identify biomarkers associated with therapeutic efficacy. Sixteen patients received a median number of 2 (range 1-7) nivolumab applications. The overall response rate (CR/PR) at day 42 was 25%, and another 25% of the patients achieved stable disease. The median overall survival was 15.6 months. High-parametric cytometry documented a higher frequency of activated (ICOS+ , HLA-DR+ ), low senescence (KLRG1- , CD57- ) CD8+ effector T cells in responders. We confirmed these findings in a preclinical model. Single-cell transcriptomics revealed a pro-inflammatory rewiring of the expression profile of T and myeloid cells in responders. In summary, the study indicates that the post-allo-HCT HMA/nivolumab combination induces anti-AML immune responses in selected patients and could be considered as a bridging approach to a second allo-HCT. Trial-registration: EudraCT-No. 2017-002194-18.

18.
J Clin Immunol ; 43(2): 371-390, 2023 02.
Article in English | MEDLINE | ID: mdl-36282455

ABSTRACT

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.


Subject(s)
Caliciviridae Infections , Common Variable Immunodeficiency , Norovirus , Humans , Atrophy/complications , Atrophy/pathology , Caliciviridae Infections/immunology , CD8-Positive T-Lymphocytes , Common Variable Immunodeficiency/complications , Common Variable Immunodeficiency/immunology , Immunoglobulin A , Inflammation/complications , Interferons , Norovirus/physiology
19.
Gastroenterology ; 163(4): 965-981.e31, 2022 10.
Article in English | MEDLINE | ID: mdl-35738329

ABSTRACT

BACKGROUND & AIMS: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease. METHODS: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro. RESULTS: Activated CD39+ and CD39+PD-1+ CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39+ and CD39+ PD-1+CD8 T cells, with CD39+ cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course. CONCLUSIONS: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches.


Subject(s)
Apyrase , CD8-Positive T-Lymphocytes , Crohn Disease , Apyrase/blood , Apyrase/genetics , Apyrase/immunology , Biomarkers/blood , CD8-Positive T-Lymphocytes/immunology , Crohn Disease/blood , Crohn Disease/genetics , Crohn Disease/immunology , Cytokines/immunology , Humans , Prognosis , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , T-Lymphocyte Subsets
20.
Eur J Immunol ; 52(9): 1390-1395, 2022 09.
Article in English | MEDLINE | ID: mdl-35099807

ABSTRACT

Recent findings indicate that many immunopathologies are at their roots a consequence of impaired immune responses ("too little" immunity) and not the result of primarily exaggerated immune responses ("too much" immunity). We have summarized this conceptional view as "IMPATH paradox." In this review, we will focus on impaired immune reactions in the context of CD8+ T-cell-mediated immunopathologies. In particular, we will exemplify this concept in two disease models: Virus-triggered primary hemophagocytic lymphohistiocytosis, an inflammatory syndrome caused by genetically impaired cytolytic functions of T cells, and viral hepatitis, where T-cell exhaustion is a major underlying mechanism for impaired effector functions. In both situations, T cells fail to eliminate the source of immune stimulation, which usually serves as an important negative feedback loop curtailing immune reactions. Persistent antigen presentation by APCs and/or infected cells results in continuous stimulation causing chronic inflammation and immunopathology mediated by residual T-cell functions. Hence, immune stimulation or reconstitution rather than immune suppression may be strategies for therapeutic interventions.


Subject(s)
Antigen Presentation , CD8-Positive T-Lymphocytes , Humans , Inflammation/pathology
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