Data for: Tumor evolution and immune microenvironment dynamics in primary and relapsed mantle cell lymphoma
Citation and access
Citation and access
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Creator/Principal investigator(s):
Research principal:
Data contains personal data:
Yes
Type of personal data:
Genetic data
Code key exists:
Yes
Sensitive personal data:
Yes
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Method and outcome
Method and outcome
Population:
The population under study consists of patients diagnosed with mantle cell lymphoma. The results of the analysis will refer to this specific group of individuals.
Time method:
Study design:
- Observational study
Sampling procedure:
Description of sampling:
Sample processing for scRNA-seq samples: Mononuclear cells were isolated from bone marrow samples by standard density centrifugation using Lymphoprep (Axis-Shield) following the manufacturer’s instructions. Intestinal, tonsillar, and lymphoid tissues were processed into single-cell suspensions using mechanical dissociation and then filtered through 70 μm containers. Single cells were cryopreserved in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen before use. Cell sorting for scRNAseq samples: The cells used for flow cytometry were thawed in RPMI 1640 cell medium (Gibco). The cells were blocked with human FcR Blocking Reagent (Miltenyi Biotec) for 10 min and subsequently stained with anti-CD19 BV421 and anti-CD235a FITC in FACS buffer (PBS with 2% FBS) for 20 min on ice. Immediately before sorting, the cells were briefly incubated with propidium iodide staining solution. For the MCL samples, live CD235a- cells, CD235a-CD19+ cells, and CD235a-CD19- cells were sorted in FACS buffer using a BD FACSAria Fusion Sorter (BD Biosciences). Live CD235a- cells were used for single-cell sequencing, while live CD235a- CD19+ and CD235a- CD19- cells were used to extract DNA for tumor and germline WGS, respectively. For the control sample, live CD19- cells and live CD19+ cells were sorted separately and then mixed at a 3:7 ratio for single-cell sequencing. scRNA-seq sequencing and data analysis: Using the Chromium Next GEM Single-Cell 5′ Reagent Kit v2 (10x Genomics), the cells were loaded onto separate lanes of the Next GEM Chromium Controller (10x Genomics) for encapsulation (target recovery of 10,000 cells for each sample). Single-cell gene expression (GEX) libraries were constructed following the manufacturer’s protocols. WGS sequencing: For fresh-frozen tissue samples, genomic DNA from tumors and matched blood samples was extracted separately by using a DNeasy Tissue and Blood Kit (Qiagen). For the single-cell suspension MCL samples, FACS (CD19+/CD19-) or an EasySep human B-cell enrichment kit was used to separate B cells and non-B cells to represent the tumor samples and nontumor (germline) controls, respectively. Genomic DNA from the sorted cells was extracted using the DNeasy Tissue and Blood Kit.
Number of individuals/objects:
11
Samples/material - Existing from scientific collection/biobank
Samples/material - Existing from scientific collection/biobank
Name:
Type(s) of sample:
Geographic coverage
Geographic coverage
Geographic location:
Administrative information
Administrative information
Responsible department/unit:
Department of Medical Biochemistry and Biophysics [C2]
Contributor(s):
Ethics Review 1:
Stockholm - 2015/418-31
Ethics Review 2:
Swedish Ethical Review Authority - 2022-02865-02
Funding
Funding
Funding agency:
- Swedish Research Council
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Award number:
2019-01302_VR
Award title:
Regulation of immunoglobulin gene diversification: with relevance for primary immunodeficiency and lymphoid malignancies
Funding information:
The aim of this project is to understand the complex molecular mechanisms involved in the regulation of immunoglobulin (Ig) gene diversification processes, i.e. V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM). We have developed a series of next generation sequencing technology-based strategies to study the in vitro and in vivo V(D)J recombination as well as CSR and SHM patterns in human B-cells. Furthermore, inducible pluripotent stem cells reprogrammed from healthy individuals´ and patients’ fibroblasts have been generated, and will be further differentiated in order to recapture different B cell development stages. Moreover, single cell RNA sequencing and CRISPR-based technologies will be applied to help to identify key factors driving B cell development as well as Ig gene diversification. Combining these approaches, and taking advantage of our unique collection of patient samples with rare primary immunodeficiencies, we hope to delineate the complex molecular mechanism involved in Ig gene diversifications. Finally, we hope to address the question whether illegitimate CSR and SHM events are associated with B cell genome instability and lymphomagenesis. The somatic mutation and translocation patterns will be systematically characterized in B-cell lymphoma in relation to DNA repair defects and chronic viral infection status (exemplified by HBV virus).
Topic and keywords
Topic and keywords
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