Biology subjects

Mulder, A.

Publications and source records attributed to Mulder, A..

7 recordsLinked to original sources

bioRxiv2025

Verification of emerging genomic mutations in Mycobacterium tuberculosis allows transmission chains to be distinguished in an epidemiological typing cluster extending over thirty years.

Whole genome sequencing (WGS) is able to identify epidemiological links between Mycobacterium tuberculosis isolates. Recent clustering can be ruled out using a pre-defined single nucleotide polymorphism (SNP) threshold. If WGS clusters grow significantly over time limited genetic variability hampers epidemiological investigations. Newly emerging (informative) SNPs in isolates of an extended cluster growing for more than 30 years to >150 cases in the Netherlands were analysed. WGS data was analyzed from 61 sequencing files from 54 patients. Genomic positions that varied within the cluster isolates were carefully screened for minority populations in other isolates from the cluster. A transmission scheme was generated on the basis of WGS data alone then compared to the epidemiological information available. Fifty-two informative SNPs were identified, eight of which were also detected as mixed variants. One emerging SNP in dnaA (1199G>A R400H) has been observed in other transmitted strains and may be under selection. There was high concordance between the transmission chains suggested on basis of the newly emerging SNPs and scenarios identified using classical epidemiological cluster investigations. Analysis of filtered SNPs accumulating in the genome of M. tuberculosis in large clusters contains information on transmission dynamics and can be used to support epidemiological investigations.

microbiology↗

bioRxiv2023

The appearance of sugI mixed loci in three individuals during treatment for MDR-TB, supports the involvement of sugI in Mycobacterium tuberculosis d-cycloserine resistance in vivo.

To study the adaptation of multi-drug resistant Mycobacterium tuberculosis (MDR-TB) during treatment patients diagnosed with MDR-TB were recruited into an observational study. Clinical data and M. tuberculosis DNA at diagnosis and between seven days and two months of MDR-TB treatment were collected. The drugs prescribed were recorded. Interpretable WGS data from 118 isolates from 54 participants was obtained (11 in Belarus and 43 in Moldova) and screened for the presence of unfixed single nucleotide polymorphisms (mixed SNPs / loci). This study was performed shortly after the publication of the 2019 WHO consolidated guidelines on drug-resistant tuberculosis treatment. Existing drug supplies and procurement in one country after the switch to the all oral MDR-TB regimen in addition to patient factors, influenced the selection of and exposure to drugs. Confidently mixed SNPs were identified in samples from multiple participants in only five genes (gyrA, pncA, Rv1129c, Rv1148c, and sugI). All other genes with confidently mixed SNPs were identified in isolates from only a single individual. A significant proportion of the participants (52 of 54 participants) received d-cycloserine as part of their initial treatment, most participants who initially received d-cycloserine did not receive bedaquiline in their initial regimen (all at one site). Three different mixed SNPs were identified in sugI gene from a follow up isolate from three participants (P7A, P7T, and Q6stop). Mutations in sugI have previously been reported in spontaneous in vitro d-cycloserine resistant mutants. Alterations in the sugI gene may indicate a sub optimal d-cycloserine containing regimen and potentially be of clinical significance with respect to adaptation to d-cycloserine. Monitoring the accumulation of low frequency escape mutants may help identify regimens insufficiently powerful to block the accumulation of antimicrobial resistance mutants and identify drug(s) at risk of resistance selection.

microbiology↗

bioRxiv2022

A high-impact COL6A3 mutation alters the response of chondrocytes in neo-cartilage organoids to hyper-physiologic mechanical loading

ObjectivesThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. The pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds chondrocytes and is critical in transducing the biomechanical cues. The objective is to study the functional effects of an OA disease-risk mutation in COL6A3 in interaction with hyper-physiological mechanical cues in a tailored human induced pluripotent stem cells (hiPSCs) derived cartilage organoid model. MethodTo identify pathogenic OA mutations exome sequencing in symptomatic OA patients was performed. To study functional effects, CRISPR-Cas9 genome engineering was used to introduce the mutation in our established human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model in interaction with hyper-physiological mechanical loading conditions. ResultsA high-impact mutation in COL6A3 was identified that resulted in significantly lower binding between the PCM proteins COLVI and fibronectin (FN) and provoked an osteoarthritic chondrocyte state. Moreover, aberrant function of the PCM, secondary to the COL6A3 mutation, abolished the initial stress responses marked particularly by upregulation of PTGS2 encoding cyclooxygenase-2 (COX-2), after hyper-physiological mechanical loading conditions. ConclusionThese findings demonstrate that ablating the characteristic transient COX-2 response after injurious mechanical cues may have a direct negative impact on chondrocyte health. What is already knownO_LIThe etiology of osteoarthritis revolves around the interplay between genetic predisposition and perturbing environmental cues, such as mechanical stress. C_LIO_LIThe pericellular matrix, with its hallmark proteins collagen type VI and fibronectin, surrounds the chondrocytes and is critical in transducing biomechanical cues from the extracellular matrix to chondrocytes henceforth it determines the chondrocyte mechanical environment. C_LIO_LIThe mechanical environment of the chondrocytes is a critical factor that influences chondrocyte health as it determines the balance between synthesis and degradation of the articular cartilage extracellular matrix. C_LI What this study addsO_LIA sustainable human induced pluripotent stem cell-derived in-vitro neo-cartilage organoid model that is tailored to study detailed biologic effects of mechanical cues to chondrocytes. C_LIO_LIAn OA disease-risk mutation in COL6A3 reduces the binding between collagen type VI to fibronectin and provoked an osteoarthritic chondrocyte state. C_LIO_LIUpon hyper-physiological mechanical loading, aberrant function of the pericellular matrix, secondary to the COL6A3 mutation, ablates the initial transient inflammatory response, characterized particularly by PTGS2 encoding cyclooxygenase-2 (COX-2). C_LI How this study might affect research practice or policyO_LIInhibiting COX-2, as an important transient inflammatory response after hyper-physiological mechanical cues, could worsen the loss of structural integrity of the cartilage in osteoarthritis patients. Henceforth, prescription of COX-2 inhibitors as pain treatment for OA patients should be reconsidered. C_LI

molecular biology↗

bioRxiv2021

Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-{zeta} signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

immunology↗

bioRxiv2020

The SPPL3-defined glycosphingolipid repertoire regulates immune responses by improving HLA class I access

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

immunology↗

bioRxiv2020

ERAP2 facilitates a subpeptidome of Birdshot Uveitis-associated HLA-A29

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labelled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.

immunology↗

bioRxiv2020

PAKC: A novel Panel of HLA class I Antigen presentation machinery Knockout Cells from the same genetic origin

With the emergence of immunotherapies, the understanding of functional HLA class I antigen presentation to T cells is more relevant than ever. Current knowledge on antigen presentation is based on decades of research in a wide variety of cell types with varying antigen presentation machinery (APM) expression patterns, proteomes and HLA haplotypes. This diversity complicates the establishment of individual APM contributions to antigen generation, selection and presentation. Therefore, we generated a novel Panel of APM Knockout Cell lines (PAKC) from the same genetic origin. After CRISPR/Cas9 genome-editing of ten individual APM components in a human cell line, we derived clonal cell lines and confirmed their knockout status and phenotype. We then show how PAKC will accelerate research on the functional interplay between APM components and their role in antigen generation and presentation. This will lead to improved understanding of peptide-specific T cell responses in infection, cancer and autoimmunity. Key pointsO_LIWe generated a panel of cell lines to study HLA class I antigen presentation C_LIO_LIWe show how this will spark research in infection, tumor biology and autoimmunity C_LI

immunology↗