Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells

Küppers, Ralf; Schneider, Markus; Hansmann, Martin‐Leo

doi:10.1007/978-1-4939-9151-8_3

Cited by 9 publications

(9 citation statements)

References 28 publications

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“…Fragments of PCNSL cryosections were digested with 2 mL proteinase K (Roche PCR grade) for 4 hours at 55°C, followed by heat inactivation at 95°C for 10 minutes. Seminested polymerase chain reactions (PCRs) for VH, Vk, and Vl genes were performed (30 and 44 cycles, respectively), using the Expand High Fidelity PCR kit (Roche), as described by Küppers et al 14,15 The resulting variable region genes were sequenced and analyzed with IMGT-V-Quest for functionality, V(D)J segment usage, and mutations. Whenever both a functional immunoglobulin (Ig) heavy and light chain variable region gene was obtained, these genes were cloned into a modified pCES vector for the expression of recombinant BCRs as Fab fragments.…”

Section: Ig Variable Region Gene Pcrmentioning

confidence: 99%

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma

Thurner

Preuss

Bewarder

et al. 2018

Blood

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Abstract To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

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Section: Ig Variable Region Gene Pcrmentioning

confidence: 99%

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma

Thurner

Preuss

Bewarder

et al. 2018

Blood

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“…Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and the study was approved by institutional review boards and local Ethics Committees (SHN-06-2018; 15-6184-BO). Pools of 10 HRS along with pools of 10 non-tumor cells and membrane sections without tissue as controls were laser-microdissected as previously described 45 . Following digestion with proteinase K for 3 hours at 55°C and heat inactivation for 10 minutes at 95°C, a semi-nested, two-rounded PCR with exon-spanning primers was performed to amplify exon 3 of IRF4.…”

Section: Methodsmentioning

confidence: 99%

A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma

Schleußner

Cauchy

Franke

et al. 2022

Preprint

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Disease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation. Such mutations can affect TF-interactions with their cognate DNA-binding motifs. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Furthermore, IRF4-C99R thoroughly modifies IRF4 function, by blocking IRF4-dependent plasma cell induction, and up-regulating disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single arginine mutation creates a complex switch of TF specificity and gene regulation. These data open the possibility of designing specific inhibitors to block the neomorphic, disease-causing DNA-binding activities of a mutant transcription factor.

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“…PCR was performed with six framework region 1 subgroup‐specific primers and a JH primer mix (3′ JH mix) for 35 cycles in six separate reactions, using 300 ng of DNA per reaction. Primer sequences have been published before (Kuppers et al , 2013). PCR products were purified from agarose gels and Sanger sequenced with the IGHV primers used for PCR and the BigDye Sequencing Kit (ABI, Heidelberg, Germany) on an Applied Biosystems 3130 Genetic Analyzer (ABI).…”

Section: Methodsmentioning

confidence: 99%

PLK1‐dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV‐infected mice

et al. 2021

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While Epstein–Barr virus (EBV) establishes a life‐long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B‐cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B‐cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo‐like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain‐of‐function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B‐cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

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Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells

Cited by 9 publications

References 28 publications

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma

A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma

PLK1‐dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV‐infected mice

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