Search

Recent work identified Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) as clonal progeny of mature B cells. Therefore, it is generally assumed that cHD homogenously represents a B cell lymphoma. In a subset of cHD, however, H/RS cells expressing T cell-associated proteins may be candidates for alternative lineage derivation. Single H/RS cells with cytotoxic T cell phenotype were micromanipulated from three cases of cHD and analyzed by single cell polymerase chain reaction for immunoglobulin heavy (IgH) and light chain (IgL) gene rearrangements, T cell receptor (TCR)-beta gene rearrangements, and germline configuration of the IgH and TCR-beta loci. H/RS cells from two cases of cHD harbored clonal, somatically mutated Ig gene rearrangements, whereas TCR-beta loci were in germline configuration. In contrast, H/RS cells from an additional case harbored clonal TCR-beta variable/diversity/joining (VDJ) and DJ gene rearrangements, whereas the IgH locus was in germline configuration on both alleles. Thus, in two cases of cHD with H/RS cells expressing cytotoxic T cell molecules, the tumor cells are derived from mature B cells that aberrantly express T cell markers. In a third case, however, H/RS cells were derived from a T cell, demonstrating that cHD can also occur as a T cell lymphoma.

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of nivolumab in cHL. However, the mechanism of action of nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells was significantly increased after 5 hours of nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and Hodgkin-Reed-Sternberg cells in cHL after nivolumab administration.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. 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 CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF., (© 2023. The Author(s).)

Introduction: Normal CD30 + B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30 + B cells have been discussed as a potential precursor population for the malignant CD30 + Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30 + B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30 + B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30 + B-cell populations may represent potential precursor lesions of Hodgkin lymphoma., Methods: We microdissected single CD30 + B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes., Results: The CD30 + B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30 + B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity., Discussion: We conclude that in human reactive lymph nodes with relatively many CD30 + B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30 + B-cell populations represent precursor lesions of Hodgkin lymphoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Küppers, Budeus, Hartmann and Hansmann.)

Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (AITL), is characterized by constitutional symptoms, advanced-stage disease, and generalized lymphadenopathy. A genetic hallmark of this lymphoma is the frequent occurrence of the RHOA mutation G17V in neoplastic cells, which is observed in around 60% of patients. Because RHOA is involved in both T-cell receptor downstream signalling and cell migration, we hypothesized that the characteristic presentation of AITL could be the result of enhanced tumor cell migration. Therefore, this study aimed to elucidate the impact of the RHOA variant G17V on the migration of neoplastic T cells. We transfected the T-cell lymphoma cell lines HH and HuT78 to stably express the RHOA-G17V variant. RHOA-G17V-expressing T cells did not exhibit enhanced motility compared to empty-vector-transfected cells in microchannels, a 3D collagen gel, or primary human lymphatic tissue. Cells of the HH cell line expressing RHOA-G17V had an increased number of cells with cleaved collagen compared with the empty-vector-transfected cells. Therefore, we hypothesized that the early spread of AITL tumor cells may be related to remodelling of the extracellular matrix. Accordingly, we observed a significant negative correlation between the relative area of collagen in histological sections from 18 primary AITL and the allele frequency of the RHOA -G17V mutation. In conclusion, our results suggest that the characteristic presentation of AITL with early, widespread dissemination of lymphoma cells is not the result of an enhanced migration capacity due to the RHOA -G17V mutation; instead, this feature may rather be related to extracellular matrix remodelling.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Ustaszewski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The simulation of immune response is a challenging task because quantitative data are scarce. Quantitative theoretical models either focus on specific cell-cell interactions or have to make assumptions about parameters. The broad variation of, e.g., the dimensions and abundance between lymph nodes as well as between individual patients hampers conclusive quantitative modeling. No theoretical model has been established representing a consensus on the set of major cellular processes involved in the immune response. In this paper, we apply the Petri net formalism to construct a semi-quantitative mathematical model of the lymph nodes. The model covers the major cellular processes of immune response and fulfills the formal requirements of Petri net models. The intention is to develop a model taking into account the viewpoints of experienced pathologists and computer scientists in the field of systems biology. In order to verify formal requirements, we discuss invariant properties and apply the asynchronous firing rule of a place/transition net. Twenty-five transition invariants cover the model, and each is assigned to a functional mode of the immune response. In simulations, the Petri net model describes the dynamic modes of the immune response, its adaption to antigens, and its loss of memory.

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αβ sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4 + and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4 + and CD8 + T cells. Degrees of clonal T cell expansion varied between patients (range: 1-18 expanded clones per patient) and was almost exclusively restricted to CD8 + T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells., (© 2022. The Author(s).)

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology., (© 2022. The Author(s).)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) can show variable histological growth patterns and present remarkable overlap with T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL). Previous studies suggest that NLPHL histological variants represent progression forms of NLPHL and THRLBCL transformation in aggressive disease. Since molecular studies of both lymphomas are limited due to the low number of tumor cells, the present study aimed to learn if a better understanding of these lymphomas is possible via detailed measurements of nuclear and cell size features in 2D and 3D sections. Whereas no significant differences were visible in 2D analyses, a slightly increased nuclear volume and a significantly enlarged cell size were noted in 3D measurements of the tumor cells of THRLBCL in comparison to typical NLPHL cases. Interestingly, not only was the size of the tumor cells increased in THRLBCL but also the nuclear volume of concomitant T cells in the reactive infiltrate when compared with typical NLPHL. Particularly CD8 + T cells had frequent contacts to tumor cells of THRLBCL. However, the nuclear volume of B cells was comparable in all cases. These results clearly demonstrate that 3D tissue analyses are superior to conventional 2D analyses of histological sections. Furthermore, the results point to a strong activation of T cells in THRLBCL, representing a cytotoxic response against the tumor cells with unclear effectiveness, resulting in enhanced swelling of the tumor cell bodies and limiting proliferative potential. Further molecular studies combining 3D tissue analyses and molecular data will help to gain profound insight into these ill-defined cellular processes., (© 2022. The Author(s).)

Recently, we have developed novel highly promising gene expression (GE) classifiers discriminating localized nodal (LFL) from systemic follicular lymphoma (SFL) with prognostic impact. However, few data are available in LFL especially concerning hotspot genetic alterations that are associated with the pathogenesis and prognosis of SFL. A total of 144 LFL and 527 SFL, enrolled in prospective clinical trials of the German Low Grade Lymphoma Study Group, were analyzed by fluorescence in situ hybridization to detect deletions in chromosomes 1p, 6q, and 17p as well as BCL2 translocations to determine their impact on clinical outcome of LFL patients. The frequency of chromosomal deletions in 1p and 17p was comparable between LFL and SFL, while 6q deletions and BCL2 translocations more frequently occurred in SFL. A higher proportion of 1p deletions was seen in BCL2 -translocation-positive LFL, compared with BCL2 -translocation-negative LFL. Deletions in chromosomes 1p, 6q, and 17p predicted clinical outcome of patients with SFL in the entire cohort, while only deletions in chromosome 1p retained its negative prognostic impact in R-CHOP-treated SFL. In contrast, no deletions in one of the investigated genetic loci predicted clinical outcome in LFL. Likewise, the presence or absence of BCL2 translocations had no prognostic impact in LFL. Despite representing a genetic portfolio closely resembling SFL, LFL showed some differences in deletion frequencies. BCL2 translocation and 6q deletion frequency differs between LFL and SFL and might contribute to distinct genetic profiles in LFL and SFL., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC -translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum , a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T-cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T-cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T-cell-derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility-associated proteins were regarded, T-cell-derived cHL cell lines showed the highest similarity to ALK - ALCL cell lines. In contrast, T-cell-derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T-cell-derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T-cell-derived cHL. In summary, our cell line-derived data suggest that based on proteomics and migration behaviour, T-cell-derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

T-cell lymphomas are highly heterogeneous and their prognosis is poor under the currently available therapies. Enhancers of zeste homologue 1 and 2 (EZH1/2) are histone H3 lysine-27 trimethyltransferases (H3K27me3). Despite the rapid development of new drugs inhibiting EZH2 and/or EZH1, the molecular interplay of these proteins and the impact on disease progression and prognosis of patients with T-cell lymphomas remains insufficiently understood. In this study, EZH1/2 mutation status was evaluated in 33 monomorphic epitheliotropic intestinal T-cell lymphomas by next generation sequencing and EZH1/2 and H3K27me3 protein expression levels were detected by immunohistochemistry in 46 T-cell lymphomas. Correlations with clinicopathologic features were analyzed and survival curves generated. No EZH1 mutations and one (3%) EZH2 missense mutation were identified. In univariable analysis, high EZH1 expression was associated with an improved overall survival (OS) and progression-free survival (PFS) whereas high EZH2 and H3K27me3 expression were associated with poorer OS and PFS. Multivariable analysis revealed EZH1 (hazard ratio (HR) = 0.183; 95% confidence interval (CI): 0.044-0.767; p = 0.020;) and EZH2 (HR = 8.245; 95% CI: 1.898-35.826; p = 0.005) to be independent, divergent prognostic markers for OS. In conclusion, EZH1/2 protein expression had opposing effects on the prognosis of T-cell lymphoma patients.

Profound knowledge exists about the clinical, morphologic, genomic, and transcriptomic characteristics of most lymphoma entities. However, information is currently lacking on the dynamic behavior of malignant lymphomas. This pilot study aimed to gain insight into the motility of malignant lymphomas and bystander cells in 20 human lymph nodes. Generally, B cells were faster under reactive conditions compared with B cells in malignant lymphomas. In contrast, PD1-positive T cells did not show systematic differences in velocity between reactive and neoplastic conditions in general. However, lymphomas could be divided into two groups: one with fast PD1-positive T cells (e.g., Hodgkin lymphoma and mantle cell lymphoma; means 8.4 and 7.8 µm/min) and another with slower PD1-positive T cells (e.g., mediastinal grey zone lymphoma; mean 3.5 µm/min). Although the number of contacts between lymphoma cells and PD1-positive T cells was similar in different lymphoma types, important differences were observed in the duration of these contacts. Among the lymphomas with fast PD1-positive T cells, contacts were particularly short in mantle cell lymphoma (mean 54 s), whereas nodular lymphocyte-predominant Hodgkin lymphoma presented prolonged contact times (mean 6.1 min). Short contact times in mantle cell lymphoma were associated with the largest spatial displacement of PD1-positive cells (mean 12.3 µm). Although PD1-positive T cells in nodular lymphocyte-predominant Hodgkin lymphoma were fast, they remained in close contact with the lymphoma cells, in line with a dynamic immunological synapse. This pilot study shows for the first time systematic differences in the dynamic behavior of lymphoma and bystander cells between different lymphoma types.

Objective: The NADPH oxidase Nox4 is an important source of H 2 O 2 . Nox4-derived H 2 O 2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model., Methods and Results: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice., Conclusion: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

A hallmark of classical Hodgkin lymphoma (cHL) is the attenuation of B-cell transcription factors leading to global transcriptional reprogramming. The role of miRNAs (microRNAs) involved in this process is poorly studied. Therefore, we performed global miRNA expression profiling using RNA-seq on commonly used cHL cell lines, non-Hodgkin lymphoma cell lines and sorted normal CD77 + germinal centre B-cells as controls and characterized the cHL miRNome (microRNome). Among the 298 miRNAs expressed in cHL, 56 were significantly overexpressed and 23 downregulated ( p < 0.05) compared to the controls. Moreover, we identified five miRNAs (hsa-miR-9-5p, hsa-miR-24-3p, hsa-miR-196a-5p, hsa-miR-21-5p, hsa-miR-155-5p) as especially important in the pathogenesis of this lymphoma. Target genes of the overexpressed miRNAs in cHL were significantly enriched ( p < 0.05) in gene ontologies related to transcription factor activity. Therefore, we further focused on selected interactions with the SPI1 and ELF1 transcription factors attenuated in cHL and the NF-ĸB inhibitor TNFAIP3 . We confirmed the interactions between hsa-miR-27a-5p:SPI1, hsa-miR-330-3p:ELF-1, hsa-miR-450b-5p:ELF-1 and hsa-miR-23a-3p:TNFAIP3, which suggest that overexpression of these miRNAs contributes to silencing of the respective genes. Moreover, by analyzing microdissected HRS cells, we demonstrated that these miRNAs are also overexpressed in primary tumor cells. Therefore, these miRNAs play a role in silencing the B-cell phenotype in cHL.