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During persistent antigen stimulation, PD-1 + CD8 T cells are maintained by progenitor exhausted PD-1 + TCF-1 + CD8 T cells (Tpex). Tpex respond to PD-1 blockade, and regulation of Tpex differentiation into more functional Tex is of major interest for cancer immunotherapies. Tpex express high levels of Inducible Costimulator (ICOS), but the role of ICOS for PD-1 + CD8 T cell responses has not been addressed. In chronic infection, ICOS-deficiency increased both number and quality of virus-specific CD8 T cells, with accumulation of effector-like Tex due to enhanced survival. Mechanistically, loss of ICOS signaling potentiated FoxO1 activity and memory-like features of Tpex. In mice with established chronic infection, ICOS-Ligand blockade resulted in expansion of effector-like Tex and reduction in viral load. In a mouse model of hepatocellular carcinoma, ICOS inhibition improved cytokine production by tumor-specific PD-1 + CD8 T cells and delayed tumor growth. Overall, we show that ICOS limits CD8 T cell responses during chronic antigen exposure.

In this issue of JEM, Yada et al. (https://doi.org/10.1084/jem.20222178) demonstrate that effective antibody affinity selection in germinal centers relies on the store-operated calcium entry (SOCE) component of the B cell receptor (BCR) signaling network. Therefore, active BCR signaling is as relevant to positive selection as the function of BCRs as endocytic receptors, answering a question that had puzzled experts for a while. These findings transform our understanding of the mechanisms supporting adaptive immune responses (to vaccines, for example) and have important implications for interpreting the genomics and pathogenesis of germinal center-derived B cell lymphomas., (© 2023 Dominguez-Sola.)

T cell-independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer's patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.

Cell cycle progression, or its arrest upon checkpoint activation, is directed by a complex array of cellular processes dependent on the diffusion of chemical signals. These signals regulate the onset of each cell cycle phase and prevent undesired phase transitions. Functional complementation is a robust strategy to identify such signals, by which mutant phenotypes are rescued through complementation with candidate factors. Here we describe a method that reclaims a five-decade old mammalian cell-cell fusion strategy of functional complementation to study the molecular control of cell cycle progression. The generation of cell-cell fusions (heterokaryons) allows for the analysis, via immunofluorescence, of cell cycle regulator dynamics and evaluating the effective rescue of cell cycle progression in specific genetic settings.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.

Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests.

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Germinal centers are short-lived microanatomical compartments with essential roles in adaptive immunity. These lymphoid structures can be identified in secondary lymphoid organs using both flow cytometry and immunohistological analyses, but only the latter provides useful architectural and spatial information. Here we describe how to use immunofluorescence and immunohistochemistry with specific antibodies to precisely highlight the cellular and architectural features of germinal centers, both in human and mouse secondary lymphoid organs, and to study their normal development and disturbance in disease.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)-mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor-mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice., (© 2016 by The American Society of Hematology.)

The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations in the gene encoding the KMT2D (or MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of these mutations and their role in lymphomagenesis are unknown. Here we show that FL- and DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B cells and enhances B cell proliferation in mice. Moreover, genetic ablation of Kmt2d in mice overexpressing Bcl2 increases the incidence of GC-derived lymphomas resembling human tumors. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may thus represent a rational therapeutic approach for targeting early tumorigenic events.

The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC's diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

Purpose: According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases., Experimental Design: We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry., Results: We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification., Conclusion: We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment., (©2013 AACR)

MEF2B encodes a transcriptional activator and is mutated in ∼11% of diffuse large B cell lymphomas (DLBCLs) and ∼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.

c-Myc oncogenic activity is thought to be mediated in part by its ability to generate DNA replication stress and subsequent genomic instability when deregulated. Previous studies have demonstrated a nontranscriptional role for c-Myc in regulating DNA replication. Here, we analyze the mechanisms by which c-Myc deregulation generates DNA replication stress. We find that overexpression of c-Myc alters the spatiotemporal program of replication initiation by increasing the density of early-replicating origins. We further show that c-Myc deregulation results in elevated replication-fork stalling or collapse and subsequent DNA damage. Notably, these phenotypes are independent of RNA transcription. Finally, we demonstrate that overexpression of Cdc45 recapitulates all c-Myc-induced replication and damage phenotypes and that Cdc45 and GINS function downstream of Myc., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

Germinal centers (GCs) are sites of B-cell clonal expansion, hypermutation, and selection. GCs are polarized into dark (DZ) and light zones (LZ), a distinction that is of key importance to GC selection. However, the difference between the B cells in each of these zones in humans remains unclear. We show that, as in mice, CXCR4 and CD83 can be used to distinguish human LZ and DZ cells. Using these markers, we show that LZ and DZ cells in mice and humans differ only in the expression of characteristic "activation" and "proliferation" programs, suggesting that these populations represent alternating states of a single-cell type rather than distinct differentiation stages. In addition, LZ/DZ transcriptional profiling shows that, with the exception of "molecular" Burkitt lymphomas, nearly all human B-cell malignancies closely resemble LZ cells, which has important implications for our understanding of the molecular programs of lymphomagenesis.

Chromosomal translocations causing deregulated c-MYC expression are detectable in most Burkitt lymphoma cases. However, little is known about the additional lesions necessary for lymphomagenesis. Now, two independent studies, one of which was performed by Sander et al. in this issue of Cancer Cell, identify constitutive PI3K signaling and CyclinD3 mutations as cooperating lesions in both mice and humans. The results have directly actionable therapeutic implications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We report that diffuse large B cell lymphoma (DLBCL) commonly fails to express cell-surface molecules necessary for the recognition of tumor cells by immune-effector cells. In 29% of cases, mutations and deletions inactivate the β2-Microglobulin gene, thus preventing the cell-surface expression of the HLA class-I (HLA-I) complex that is necessary for recognition by CD8(+) cytotoxic T cells. In 21% of cases, analogous lesions involve the CD58 gene, which encodes a molecule involved in T and natural killer cell-mediated responses. In addition to gene inactivation, alternative mechanisms lead to aberrant expression of HLA-I and CD58 in >60% of DLBCL. These two events are significantly associated in this disease, suggesting that they are coselected during lymphomagenesis for their combined role in escape from immune-surveillance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. Here we show that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions.

Myc is a well known proto-oncogene encoding for a transcription factor whose activity is tightly regulated in the cellular context. Myc was the first oncogene recognized to activate the ARF tumor suppressor gene which suppresses cell proliferation partly through stabilization of the p53 tumor suppressor protein but which also has p53-independent growth-suppressive functions. Recent studies have indicated that mouse p19ARF negatively regulates Myc's transcriptional activity. We here show that the human p14ARF directly associates with Myc and relocates Myc from the nucleoplasm to the nucleolus. We found that p14ARF interacts with the Myc-Max complex and the binding of p14ARF does not interfere with Myc-Max interaction in vitro. Protein interaction assays define the Myc BoxII as a critical domain required for interaction with p14ARF. Moreover, we identify 30 amino acids encompassing Myc BoxII domain required for p14ARF interaction and colocalization in vivo. Finally, we show that p14ARF down regulates Myc activated transcription and that this activity cannot be addressed to an intrinsic p14ARF repressor domain.