Project description:While immune signaling has emerged as a defining feature of the glioma microenvironment, local selection of responding T cells and their anti-tumor potential as a population are difficult to measure directly in patients. High-throughput sequencing of T cell receptor repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. Here, we define new immunophenotypes in glioma based on TCRseq and RNA-Seq of tumor tissue, non-neoplastic brain tissue, and peripheral blood from patients. Using information theory, we characterize antigen-driven selection in glioma and its relationship with the expression of distinct immune-functional pathways in the tumor microenvironment. Finally, we identify a strong relationship between usage of certain TCR in peripheral blood and the divergence of the infiltrating T cell population from the peripheral repertoire. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to anti-glioma vaccines and immunotherapy. We characterized the T cell receptor (TCR) repertoires of 11 high-grade glioma patients, three low-grade glioma patients, and thee non-glioma patients by TCRseq of brain-infiltrating T cells and matching peripheral blood. In addition, we obtained gene expression profiles from brain tissue of each patient by RNA-Seq. We additionally measured the TCR repertoires exclusively from peripheral blood of one additional non-glioma patient.
Project description:We dissect HCV imprinting of B cell repertoires in patients with chronic disease. The persistent character of the oncogenic B cell repertoire generated by HCV may point to a chronically elevated lymphoma risk in these patients even years after HCV cure.
Project description:Here, by using mass spectrometry-based methods IgG1 and IgA1 clonal repertoires were monitored quantitatively and longitudinally in more than 50 individual serum samples obtained from 17 COVID-19 patients admitted to intensive care units because of acute respiratory distress syndrome. These serological clonal profiles were used to examine how each patient reacted to a severe SARS-CoV-2 infection. All 17 donors revealed unique polyclonal repertoires and changes after infection. Substantial changes over time in the IgG1 and_or IgA1 clonal repertoires were observed in individual patients, with several new clones appearing following the infection, in a few cases leading to a few very high abundant IgG1 and_or IgA1 clones dominating the repertoire. Several of these clones were de novo sequenced through combinations of top-down, middle-down and bottom-up proteomics approaches. This revealed several sequence features in line with sequences deposited in the SARS-CoV-specific database of antibodies. In other patients, the serological Ig profiles revealed the treatment with tocilizumab, as after treatment, this IgG1-mAb dominated the serological IgG1 repertoire. Tocilizumab clearance could be monitored and a half-life of approximately 6 days was established in these patients. Overall, our longitudinal monitoring of IgG1 and IgA1 repertoires of individual donors reveals that antibody responses are highly personalized traits of each patient, affected by the disease and the chosen clinical treatment. The impact of these observations argues for a more personalized and longitudinal approach in patients' diagnostics, both in serum proteomics as well as in monitoring immune responses.
Project description:Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 33 HA20 patients, we show that heterozygous TNFAIP3 loss skews immune repertoires towards lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas.
Project description:Measurable residual disease (MRD) in acute myeloid leukemia (AML) is monitored through detection of leukemia associated phenotypic protein markers (LAPMs) in bone marrow aspirates, hindering disease real-time monitoring. We explored peripheral blood (PB) extracellular vesicles (EVs)-based methods for MRD monitoring. To confirm that LAPMs are present in AML-derived EVs, EVs were isolated from OCI-AML3 cells by dif-ferential centrifugation, and characterized according to their size (nanoparticle tracking analysis), morphology (transmission electron microscopy) and protein cargo (proteomic analysis and Western blot). CD14 and CD33 were detected in OCI-AML3 cells and their released EVs. To select a method to isolate EVs from the PB of AML patients, three tech-niques were tested: size exclusion chromatography followed by ultrafiltration (SEC-UF), Total Exosome Isolation Kit (Invitrogen) and Exo-spin™ Exosome Purification Kit (Cell Guidance Systems). SEC-UF allowed EVs isolation with higher purity and less aggregates than the other techniques. LAPMs were detected in those EVs but their presence depended on the isolation method. Finally, EVs from seven AML patients’ plasma were isolated by SEC-UF. LAPMs were identified in paired samples at diagnosis and remission, with dif-ferential expression throughout disease evolution. This work highlights the possibility of real-time MRD monitoring through LAPMs’ analysis in AML patient’s circulating EVs.
Project description:To study longitudinal dynamics of IGH BCR repertoires and clonal lineages evolution of memory B-cells, plasmablasts and plasma cells from peripheral blood of healthy donors, which were sampled three times within a year
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
Project description:Circulating tumour DNA (ctDNA) methylation profiling is a promising non-invasive tool for disease monitoring, but longitudinal interpretation is limited by variable sequencing coverage and the loss of clinically relevant signals. We developed REMODEL (Recovery-based Epigenetic Monitoring of Dynamic methylation for Longitudinal analysis), an integrated framework combining coverage-aware binning, count-based longitudinal statistics, and a novel Epigenetic Recovery Score (ERS) to quantify post-treatment methylation recovery. Applied to 17 serial plasma samples from two T-cell lymphoma patients (2,025 genomic bins), REMODEL distinguished durable remission, characterized by coordinated demethylation (86.4% of bins) and sustained positive ERS, from relapse, marked by widespread hypermethylation (64.9% of bins) and persistently negative ERS despite clinical remission. By integrating dynamic methylation detection with recovery modelling, REMODEL provides an interpretable framework for longitudinal ctDNA methylation analysis and minimal residual disease monitoring.