Biology subjects

Keppler, O. T.

Publications and source records attributed to Keppler, O. T..

9 recordsLinked to original sources

bioRxiv2026

Optimizing CRISPR/Cas9 genome editing in primary human hematopoietic cells to advance studies into HIV biology

Defining how human host factors shape HIV-1 infection in vivo remains essential for the development of genetically engineered cell therapies. Here, we established a non-viral CRISPR/Cas9 ribonucleoprotein-based platform for efficient single and multiplex gene editing in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). Edited HSPCs retained viability, proliferative capacity, primitive immunophenotypes, and multilineage differentiation potential and supported targeted reporter knock-in independently of the cell source, cord blood (CB), bone marrow (BM), and mobilized from peripheral blood (MPB). Following differentiation, SAMHD1 knockout increased HIV-1 susceptibility of HSPC-derived macrophages, MX2 knockout also increased HIV infection in cells pre-stimulated with IFN-2a, and CXCR4 knockout blocked X4-tropic HIV-1 infection of HSPC-derived megakaryocytes. We then transplanted CCR5-, SAMHD1-, or non-targeting control-edited HSPCs into immunodeficient mice and challenged reconstituted animals with R5-tropic HIV-1. CCR5 knockout prevented detectable viral spread, validating the model using a clinically relevant dependency factor. In contrast, SAMHD1 knockout accelerated viral dissemination, with earlier plasma viremia and 3.6-fold higher cumulative viremia, although endpoint viral burden was not significantly different from controls. These findings establish transplantation of CRISPR/Cas9-edited human HSPCs as a modular platform for dissecting HIV-1 host-factor function across hematopoietic lineages ex vivo and in vivo and for evaluating engineered cell-based strategies.

bioengineering↗

bioRxiv2023

ediTONSIL: Activation-Neutral Gene Editing of CD4 T cells in Human ex Vivo Tonsil Cultures

MotivationCD4 T cells are central players of adaptive immunity that rapidly change activation states in response to exogenous T cell receptor (TCR) stimulation. While molecular processes in activated human CD4 T cells from peripheral blood are well studied, resting CD4 T cells are refractory to gene-editing transduction and transfection methods without prior activation. Knowledge on the molecular biology of truly resting CD4 T cells is therefore lacking. We present here the culture and editing workflow ediTONSIL that allows for gene editing of tissue-resident CD4 T cells without compromising their activation state or immunological function. This will enable molecular mechanistic analyses of this cell type in their native/physiological tissue context. SummaryThe molecular and immunological properties of resting CD4 T cell biology are understudied due to the lack of suitable gene editing methods. Here we describe the ex vivo culture and gene editing methodology ediTONSIL for tissue-resident CD4 T cells from human tonsil tissue. CRISPR/Cas9 RNP nucleofection under optimized culture conditions and cytokine concentrations results in knock out efficacies of over 90%, which are e.g. sufficient to prevent HIV-1 infection when targeting the viral entry co-receptor CXCR4. EdiTONSIL does not impair tonsil CD4 T cell viability, activation state or immunocompetence and does not require exogenous activation. Editing can be performed on multiple cell types in bulk cultures or of CD4 T cells previously isolated from tonsils that can be added back to the non-CD4 T cell fraction post gene editing to reassemble into immunocompetent organotypic lymphoid aggregate structures. This highly efficient and versatile workflow for gene editing of tonsillar CD4 T cells enables the dissection of molecular mechanisms in ex vivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/554089v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1ece503org.highwire.dtl.DTLVardef@1af2d0corg.highwire.dtl.DTLVardef@1b67f14org.highwire.dtl.DTLVardef@510d63_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

BackgroundSAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 substrate, in the context of SAMHD1. MethodsCNDAC was tested in 13 acute myeloid leukaemia (AML cell lines), in 26 acute lymphoblastic leukaemia cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. ResultsAlthough the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. ConclusionIntrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

pharmacology and toxicology↗

bioRxiv2021

Impaired function and delayed regeneration of dendritic cells in COVID-19

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DC) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute disease to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage- HLADR+ cells lacking DC markers. Increased frequency of the recently discovered CD163+ CD14+ DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of PD-L1 in conventional DC (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naive CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

immunology↗

bioRxiv2021

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

The immune system of most SARS-CoV-2 infected individuals limits viral spread to the upper airways without pulmonary involvement. This prevents the development of pneumonic COVID-19. However, the protective immunological responses causative of successful viral containment in the upper airways remain unclear. Here, we combine longitudinal single-cell RNA sequencing, proteomic profiling, multidimensional flow cytometry, RNA-Seq of FACS-sorted leukocyte subsets and multiplex plasma interferon profiling to uncover temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients. We compare host responses in a high-risk patient population infected with SARS-CoV-2 but without pulmonary involvement to patients with COVID-19 pneumonia. Our data reveal a distinct immunological signature of successful viral containment, characterized by an early prominent interferon stimulated gene (ISG) upregulation across immune cell subsets. In addition, reduced cytotoxic potential of Natural Killer (NK) and T cells, as well as a monocyte phenotype with immune-modulatory potential are hallmarks of protective immunity. Temporal resolution across disease trajectories highlights ISG upregulation as particularly prominent early in the disease and confirms increased expression also in comparison to healthy controls. We validate this distinct temporal ISG signature by in-depth RNA-seq of FACS-sorted leukocyte subsets in a large prospective ambulatory SARS-CoV-2 infected cohort confirming early and robust ISG upregulation particularly in monocytes and T cells. In conclusion, our data demonstrate a protective ISG phenotype in patients with successful containment of SARS-CoV-2 infection without progression to COVID-19. This early protective interferon response might be exploited as a therapeutic approach and for disease course prediction.

immunology↗

bioRxiv2020

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies. Author summaryHuman hematopoietic stem cells and their derivatives of the myeloid and lymphoid lineages are important targets for gene correction or modifications using the CRISPR-Cas9 technology. Among others, this approach can support site-specific insertion of chimeric antigen receptors (CARs) or T cell receptors (TCRs) into primary T cells. Their subsequent adoptive transfer to patient donors is a promising immunotherapeutic concept that may control chronic infection or certain types of cancer. Human B cells have a similar potential but, in contrast to T cells, they are very sensitive, difficult to handle, and short-lived ex vivo precluding their genetic modification. Here, we provide means to manipulate primary human B cells genetically using in vitro assembled Cas9 ribonucleoprotein complexes and electroporation for their delivery. Our study demonstrates near-to-complete loss of a model target gene and provides examples to evaluate a cellular gene with a critical role during infection with Epstein-Barr virus (EBV).

immunology↗

bioRxiv2020

Micro RNAs are minor constituents of extracellular vesicles and are hardly delivered to target cells

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we analyzed the capacity of EVs to deliver packaged miRNAs into target cells and to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but there was no EV-cell membrane fusion or delivery of cargo. Importantly, the functionality of cells exposed to miRNA-carrying EVs was not affected. These results suggest EV-borne miRNAs do not act as effectors and question their relevancy in paracrine cell-to-cell communication. AUTHOR SUMMARYThe majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication.

cell biology↗

bioRxiv2020

Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

While current combined antiretroviral therapy (cART) allows control of HIV replication in patients and effectively suppresses plasma viral loads, it is unable to target latent reservoirs, which are responsible for virus rebound after discontinuation of therapy. Several histone deacetylase inhibitors (HDACIs) have been shown to target reservoirs and to reactivate latent HIV. While this effect is highly desired, it carries the risk that HIV-1 may be reactivated in tissue compartments were cART concentrations are insufficient and thus leading to de novo infections in this sites. To address this concern, we evaluated the effect of different HDACIs for their ability to reverse HIV latency and to modulate de novo infections. Two of the inhibitors, sodium butyrate and bufexamac, significantly inhibited de novo HIV-1 infection in activated CD4+ T-cells. Transcriptome and proteome analysis indicated global changes of protein abundancies, exhibited reduced proliferation of CD4+ T-cells, and revealed butyrate-based proteasomal degradation of EP300, an important factor for HIV-1 replication. Our results disclose new potential treatment strategies and minimizes the concern of potential reservoir reseeding by HDACIs.

microbiology↗

bioRxiv2018

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analogue drugs important for treating a variety of cancers, including Acute Myeloid Leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We find that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogues tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies.\n\nSignificanceNucleoside analogue drugs are widely used to treat a variety of cancers and viral infections. With an essential role in regulating the nucleotide pool in the cell by degrading cellular nucleotides, SAMHD1 has the potential to decrease the cellular concentration of frequently prescribed nucleotide analogues and thereby decrease their clinical efficacy in cancer therapy. To improve future nucleotide analogue treatments, it is important to understand SAMHD1 interactions with these drugs. Our work thoroughly examines the extent to which nucleotide analogues interact with the catalytic and allosteric sites of SAMHD1. This work contributes to the assessment of SAMHD1 as a potential therapeutic target for cancer therapy and the future design of SAMHD1 modulators that might improve the efficacy of existing therapies.

biochemistry↗