Convergent evolution and B-cell recirculation in germinal centers in a human lymph node.

Germinal centers (GCs) play a central role in generating an effective immune response against infectious pathogens, and failures in their regulating mechanisms can lead to the development of autoimmune diseases and cancer. Although previous works study experimental systems of the immune response with mouse models that are immunized with specific antigens, our study focused on a real-life situation, with an ongoing GC response in a human lymph node (LN) involving multiple asynchronized GCs reacting simultaneously to unknown antigens. We combined laser capture microdissection of individual GCs from human LN with next-generation repertoire sequencing to characterize individual GCs as distinct evolutionary spaces. In line with well-characterized GC responses in mice, elicited by immunization with model antigens, we observe a heterogeneous clonal diversity across individual GCs from the same human LN. Still, we identify shared clones in several individual GCs, and phylogenetic tree analysis combined with paratope modeling suggest the re-engagement and rediversification of B-cell clones across GCs and expanded clones exhibiting shared antigen responses across distinct GCs, indicating convergent evolution of the GCs.

MALT1-dependent cleavage of CYLD promotes NF-κB signaling and growth of aggressive B-cell receptor-dependent lymphomas.

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.

Lymphoma-associated mutations in autoreactive memory B cells of patients with Sjögren's syndrome.

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with SjÓ§gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

AKT supports the metabolic fitness of multiple myeloma cells by restricting FOXO activity.

Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival.

AKT signaling restrains tumor suppressive functions of FOXO transcription factors and GSK3 kinase in multiple myeloma.

The phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM.

Repertoire Analysis of B-Cells Located in Striated Ducts of Salivary Glands of Patients With Sjögren's Syndrome.

A major complication of primary Sjögren's syndrome (pSS) is development of mucosa associated lymphoid tissue (MALT) B-cell lymphoma, particularly in salivary glands. These lymphomas express FcRL4 and are characteristically associated with lymphoepithelial lesions. Neoplastic B-cells may be derived from non-neoplastic glandular intraductal B-cells, also virtually all expressing FcRL4. A characteristic feature of MALT lymphomas is the production of rheumatoid factors (RFs), which are largely encoded by stereotypic immunoglobulin variable heavy chain (IGHV) sequences. The aim of this study was to examine whether there is a relationship between the intraductal and periductal B-cells and whether the intraductal B-cells are selected for RF. RNA was extracted from laser-microdissected infiltrated ductal areas and periductal infiltrates from frozen parotid gland tissue sections of 5 pSS patients. PCR amplified IGHV transcripts were cloned into pCR™4-TOPO vector and subsequently sequenced. Microdissected ducts yielded 96 unique IGHV sequences derived from intraductal B-cells, while 119 unique IGHV sequences were obtained from periductal infiltrates. No major difference in VH-gene usage was observed between intraductal and periductal B-cells. Nearly all (>90%) IGHV sequences derived from both intraductal and periductal B-cells were mutated. Clonal expansions as defined by shared VDJ rearrangements were also present among both intraductal and periductal B-cells: in total 32 clones were found, from which 12 were located within ducts, 15 in periductal areas, and five clones shared members in both areas. We observed 12 IGHV rearrangements encoding for RF sequences from which two were derived from intraductal B-cells and 10 from periductal B-cells. Nine RF sequences were part of a clone. Together these findings indicate that intraductal and periductal B-cells are closely related to each other. Intraductal B-cells are most likely derived from periductal B-cells. We did not obtain evidence that RF-specific B-cells are enriched within the striated ducts. We speculate that in principle any activated B-cell can enter the striated ducts from the periductal infiltrate, irrespective of its antigenic specificity. Within the ducts, these B-cells may receive additional activation and proliferation signals, to further expand at these sites and by acquisition of driver-mutations develop toward lymphoma.

Salivary Gland Mucosa-Associated Lymphoid Tissue-Type Lymphoma From Sjögren's Syndrome Patients in the Majority Express Rheumatoid Factors Affinity-Selected for IgG.

OBJECTIVE: Patients with SjÓ§gren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)-type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity. METHODS: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30-encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively. RESULTS: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15-encoded RFs, a recurrent replacement mutation in the IGKV3-15-third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc. CONCLUSION: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas.

The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors.

The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients.

Acquisition of N-Glycosylation Sites in Immunoglobulin Heavy Chain Genes During Local Expansion in Parotid Salivary Glands of Primary Sjögren Patients.

Previous studies revealed high incidence of acquired N-glycosylation sites acquired N-glycosylation sites in RNA transcripts encoding immunoglobulin heavy variable region (IGHV) 3 genes from parotid glands of primary Sjögren's syndrome (pSS) patients. In this study, next generation sequencing was used to study the extent of ac-Nglycs among clonally expanded cells from all IGVH families in the salivary glands of pSS patients. RNA was isolated from parotid gland biopsies of five pSS patients and five non-pSS sicca controls. IGHV sequences covering all functional IGHV genes were amplified, sequenced, and analyzed. Each biopsy recovered 1,800-4,000 unique IGHV sequences. No difference in IGHV gene usage was observed between pSS and non-pSS sequences. Clonally related sequences with more than 0.3% of the total number of sequences per patient were referred to as dominant clone. Overall, 70 dominant clones were found in pSS biopsies, compared to 15 in non-pSS. No difference in percentage mutation in dominant clone-derived IGHV sequences was seen between pSS and non-pSS. In pSS, no evidence for antigen-driven selection in dominant clones was found. We observed a significantly higher amount of ac-Nglycs among pSS dominant clone-derived sequences compared to non-pSS. Ac-Nglycs were, however, not restricted to dominant clones or IGHV gene. Most ac-Nglycs were detected in the framework 3 region. No stereotypic rheumatoid factor rearrangements were found in dominant clones. Lineage tree analysis showed in four pSS patients, but not in non-pSS, the presence of the germline sequence from a dominant clone. Presence of germline sequence and mutated IGHV sequences in the same dominant clone provide evidence that this clone originated from a naïve B-cell recruited into the parotid gland to expand and differentiate locally into plasma cells. The increased presence of ac-Nglycs in IGHV sequences, due to somatic hypermutation, might provide B-cells an escape mechanism to survive during immune response. We speculate that glycosylation of the B-cell receptor makes the cell sensitive to environmental lectin signals to contribute to aberrant B-cell selection in pSS parotid glands.

B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains.

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.

The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre-B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre-B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity.

In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

Stereotypic rheumatoid factors that are frequently expressed in mucosa-associated lymphoid tissue-type lymphomas are rare in the labial salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: Among autoimmune diseases, Sjögren's syndrome (SS) is most strongly associated with the development of malignant B cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT)-type lymphoma. Previously, we have shown that in ∼40% of cases of salivary gland MALT lymphoma, high-affinity stereotypic rheumatoid factor (RF) B cell receptors, specific for IgG-Fc, are expressed. This study was undertaken to investigate whether in the inflamed salivary glands of patients with SS, a similar RF-biased Ig repertoire is present. METHODS: Extensive analyses of the B cell Ig VH region repertoire were performed on microdissected tissue samples from the labial salivary glands of 4 patients with SS. RESULTS: All SS labial salivary glands harbored expanded B cell clones, of which 1 or 2 were highly expanded and detected in >50% of the microdissected samples. However, among the identified 464 distinct Ig clonotypes, only 3 stereotypic RF-expressing clones were detected. In 2 patients with SS, an RF-expressing clone was detected at low frequency in 1 of the microdissected samples, whereas 1 patient with SS harbored a highly expanded RF-expressing clone that was detected in all microdissected samples and also detected in the peripheral blood. Two years after analysis of this sample, the latter patient developed a diffuse large B cell lymphoma originating from the same RF clone. CONCLUSION: Inflamed labial salivary glands in patients with SS generally harbor 1 or 2 highly expanded B cell clones. The repertoire strongly biased toward stereotypic RFs in salivary gland MALT lymphomas is not a reflection of a similar repertoire in the inflamed salivary glands of patients with SS; rather, in the latter, the repertoire is based on a strong selection advantage of incidental stereotypic RF-expressing B cells.

A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi.

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5(+)CD19(+) B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for ß-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. ß-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for ß-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to ß-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.

Activation-induced cytidine deaminase splice variants are defective because of the lack of structural support for the catalytic site.

Recently, conflicting results were reported on the hypermutation activity of activation-induced cytidine deaminase (AID) splice variants. With the generation of single point mutations, we studied the structure-function relationship of the amino acids that are commonly absent from all described splice variants. The results from this analysis pointed to several amino acids that are required for class switch recombination (CSR), without perturbing cellular localization or nucleocytoplasmic shuttling. A defect in deaminase activity was found to underlie this CSR deficiency. Interestingly, the most debilitating mutations concentrated on hydrophobic amino acids, suggesting a structural role for this part of the protein. Indeed, by generating homologous amino acid replacements, CSR activity could be restored. These results are in agreement with recent reports on the protein structure of the AID homolog APOBEC3G, suggesting a similar protein composition. In addition, the findings underscore that AID splice variants are unlikely to have preservation of catalytic activity.

CD20 deficiency in humans results in impaired T cell-independent antibody responses.

CD20 was the first B cell differentiation antigen identified, and CD20-specific mAbs are commonly used for the treatment of B cell malignancies and autoantibody-mediated autoimmune diseases. Despite this the role of CD20 in human B cell physiology has remained elusive. We describe here a juvenile patient with CD20 deficiency due to a homozygous mutation in a splice junction of the CD20 gene (also known as MS4A1) that results in "cryptic" splicing and nonfunctional mRNA species. Analysis of this patient has led us to conclude that CD20 has a central role in the generation of T cell-independent (TI) antibody responses. Key evidence to support this conclusion was provided by the observation that although antigen-independent B cells developed normally in the absence of CD20 expression, antibody formation, particularly after vaccination with TI antigens, was strongly impaired in the patient. Consistent with this, TI antipolysaccharide B cell responses were severely impeded in CD20-deficient mice. Our study therefore identifies what we believe to be a novel type of humoral immunodeficiency caused by CD20 deficiency and characterized by normal development of antigen-independent B cells, along with a reduced capacity to mount proper antibody responses.