Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment.

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

The anti-PD-L1/CTLA-4 bispecific antibody KN046 in combination with nab-paclitaxel in first-line treatment of metastatic triple-negative breast cancer: a multicenter phase II trial.

This multicenter, phase II study (NCT03872791) aims to evaluate the efficacy and safety of the anti-PD-L1/CTLA-4 bispecific antibody KN046 combined with nab-paclitaxel in the first-line treatment of patients with metastatic triple-negative breast cancer (TNBC). The primary endpoints included objective response rate (ORR) and duration of response (DoR), and secondary endpoints included progression-free survival (PFS), overall survival (OS) rate, safety, and the correlation of PD-L1 status with clinical efficacy. This trial met pre-specified endpoints. 27 female patients were enrolled sequentially to receive KN046 in two dose levels (3 mg/kg or 5 mg/kg). Among the 25 evaluable patients, the ORR achieved 44.0% (95% CI, 24.4% - 65.1%), and the median DoR was not mature. The median PFS reached 7.33 months (95%CI, 3.68 - 11.07 months), and the median OS was 30.92 months (95%CI, 14.75 - NE months). In PD-L1 positive patients, PFS was 8.61 months (versus 4.73 months) and the 2-year OS rate was 62.5% (versus 57.1%) compared to PD-L1 negative patients. Patients tolerated well the combination therapy. In general, KN046 combined with nab-paclitaxel showed favorable efficacy and survival benefits with tolerable toxicity in the first-line treatment of metastatic TNBC, especially PD-L1 positive, which is worth further investigation.

Recent advances in enzymatic properties, preparation methods, and functions of glycoside hydrolase from Bifidobacterium: a review.

Bifidobacterium is a major probiotic of intestinal gut flora and exerts many physiological activities, and it is widely applied in the fields of food and medicine. As an important part of Bifidobacterium, glycoside hydrolase plays a role in its physiological activity. With the continuous development and improvement of genetic engineering technology, research on this type of enzyme will play a crucial role in promoting the further development of Bifidobacterium in the field of probiotics. In this review, the preparation methods, enzymatic properties, and functions of glycoside hydrolase extracted from Bifidobacterium are described and summarized. The common method for preparing glycoside hydrolase derived from Bifidobacterium is heterologous expression in Escherichia coli BL21. The optimal pH range for these glycoside hydrolase enzymes is between 4.5 and 7.5; the optimal temperature is between 30 and 50 °C, which is close to the optimal growth condition of Bifidobacterium. Based on substrate specificity, these glycoside hydrolase could hydrolyze synthetic substrates and natural oligosaccharides, including a series of pNP artificial substrates, disaccharide, and trisaccharides, while they have little ability to hydrolyze polysaccharide substrates. This review will be expected to provide a basis for the development of Bifidobacterium as a probiotic element.

Chitosan-based hydrogel wound dressing: From mechanism to applications, a review.

As promising biomaterials, hydrogels are widely used in the medical engineering field, especially in wound repairing. Compared with traditional wound dressings, such as gauze and bandage, hydrogel could absorb and retain more water without dissolving or losing its three-dimensional structure, thus avoiding secondary injury and promoting wound healing. Chitosan and its derivatives have become hot research topics for hydrogel wound dressing production due to their unique molecular structure and diverse biological activities. In this review, the mechanism of wound healing was introduced systematically. The mechanism of action of chitosan in the first three stages of wound repair (hemostasis, antimicrobial properties and progranulation), the effect of chitosan deacetylation and the molecular weight on its performance are analyzed. Additionally, the recent progress in intelligent and drug-loaded chitosan-based hydrogels and the features and advantages of chitosan were discussed. Finally, the challenges and prospects for the future development of chitosan-based hydrogels were discussed.

Identification, quantitation and organoleptic contributions of furan compounds in brandy.

Furan compounds actively contribute to the characteristics of brandy. Herein, we have attempted to identify and quantify the furan compounds present in brandy using three different extraction methods combined with comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry. Threshold determination and omission experiments were carried out to verify their organoleptic contribution. Liquid-liquid extraction using dichloromethane was found to be the optimal extraction method. A total of 21 furan compounds were identified, in which 5 were detected in brandy for the first time. Our quantitative results showed a positive correlation between the furan compound content and the aging time. Among them, ethyl 5-oxotetrahydro-2-furancarboxylate exhibited a very high odor activity value (1.64 < OAV < 179.53) and smoky aroma. Omission tests showed that the three furan compounds with an OAV > 1 made a significant difference to brandy. These findings bring a new perspective to the sensory and chemical characteristics of brandy.

Chemosensory Characteristics of Brandies from Chinese Core Production Area and First Insights into Their Differences from Cognac.

This work aimed to compare the aroma characteristics of representative brandies with different grades from Yantai (one of the Chinese core production areas) and Cognac and to establish relationships between sensory descriptors and chemical composition. Descriptive analysis was performed with a trained panel to obtain the sensory profiles. Forty-three aroma-active compounds were quantified by four different methodologies. A prediction model on the basis of partial least squares analysis was performed to identify candidate compounds that were unique to a certain group of brandies. The result showed that brandies from Yantai could be distinguished from Cognac brandies on the basis of spicy, dried fruit, floral, and fruity-like aromas, which were associated with an aromatic balance between concentrations of a set of compounds such as 5-methylfurfural, γ-nonalactone, and γ-dodecalactone. Meanwhile, brandy with different grades could be distinguished on the basis of compounds derived mostly during the aging process.

Does delayed EBV infection contribute to rising childhood cancers?

Childhood cancer is on the rise in high-income countries. Epidemiological studies suggest that reduced exposure to common infections in early life is to blame. However, no specific infection responsible for protection against cancer has been identified, and the underlying mechanisms remain a matter of speculation. Recent findings that Epstein-Barr virus (EBV) can induce antitumor immunity lead us to hypothesize that the delay in EBV infection in such countries might contribute to the increase in childhood cancers.

Facts and Hopes in the Relationship of EBV with Cancer Immunity and Immunotherapy.

Epstein-Barr virus (EBV), the first identified human tumor virus, infects and takes up residency in almost every human. However, EBV genome-positive tumors arise in only a tiny minority of infected people, presumably when the virus-carrying tumor cells are able to evade immune surveillance. Traditional views regard viral antigens as the principal targets of host immune surveillance against virus-infected cells. However, recent findings indicate that EBV-infected/-transformed B cells elicit both cytotoxic CD8+ and CD4+ T-cell responses against a wide range of overexpressed cellular antigens known to function as tumor-associated antigens (TAA), in addition to various EBV-encoded antigens. This not only broadens the ways by which the immune system controls EBV infection and prevents it from causing cancers, but also potentially extends immune protection toward EBV-unrelated cancers by targeting shared TAAs. The goal of this review is to incorporate these new findings with literature data and discuss future directions for improved understanding of EBV-induced antitumor immunity, as well as the hopes for rational immune strategies for cancer prevention and therapy.

Tender Coconut Water Protects Mice From Ischemia-Reperfusion-Mediated Liver Injury and Secondary Lung Injury.

ABSTRACT: Organ injury by oxidative and inflammatory mediators occurs during ischemia-reperfusion (I/R) of the liver. Remote organ injury secondary to liver I/R increases the systemic insult. Tender coconut water (TCW) has been studied in chemical and fructose-induced liver injury but its ability to decrease tissue injury in clinically relevant injury models is unknown. We evaluated the therapeutic potential of TCW in preventing liver I/R injury and associated remote organ injury. Mice were fed sugar water (SUG; control) or TCW for a week and then subjected to 60 min of liver ischemia followed by reperfusion for 6 h. Plasma alanine transaminase levels, tissue damage, and mRNA levels of Nos2, Tnf, and Il6 were significantly lower in mice fed TCW prior to I/R. Plasma cytokines followed liver cytokine patterns. TCW increased mRNA levels of the anti-oxidant genes Hmox1 and Ptgs2 in the liver of mice subjected to I/R. Remote lung injury from liver I/R was also decreased by TCW feeding as evident by less neutrophil infiltration, decreased pro-inflammatory Il6, and increased anti-inflammatory Il10 mRNA levels in the lung. To examine macrophage activation as a potential mechanism, TCW pretreatment decreased the amount of nitrite produced by RAW264.7 macrophages stimulated with LPS. The levels of Nos2, Il1b, Tnf, and Il6 were decreased while Il10 and Hmox1 mRNA levels were significantly up-regulated upon LPS stimulation of TCW pretreated RAW264.7 macrophages. Collectively, our results indicate that TCW decreased hepatic I/R-mediated damage to liver and lung and suggest that decreased macrophage activation contributes to this effect.

Identification, quantitation and sensorial contribution of lactones in brandies between China and France.

Lactones are important flavor compounds in lots of foodstuffs. They also play an important role in brandy, but have not been studied at large. In this study, solid-phase extraction (SPE) and stir bar sorptive extraction (SBSE) combined with comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GC × GC-TOFMS) were applied to identify and quantify lactones in brandies between China and France. Totally 17 lactones were identified, four of which were detected only in SBSE. Among them, γ-valerolactone, γ-heptalactone, δ-octalactone, γ-undecanolactone and δ-dodecalactone were detected in brandy for the first time. The results of partial least squares-discriminant analysis (PLS-DA) revealed that lactones distinguished regional characteristics among different brandies. The omission test showed that four lactones (OAV > 1) had direct impact on the aroma of brandy, and other seven lactones at sub-threshold (0.1 < OAV < 1) provided peach and apricot aroma characteristics through synergistic effects.

Mechanism of EBV inducing anti-tumour immunity and its therapeutic use.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein-Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein-Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers.

Tender coconut water suppresses hepatic inflammation by activating AKT and JNK signaling pathways in an in vitro model of sepsis.

Tender coconut water (TCW) is a natural plant product rich in phytochemicals and protects against toxic liver injury. However, the mechanism by which TCW inhibits inflammation and tissue damage is unknown. We examined the effect of TCW on primary rat hepatocyte viability, cytokine-induced gene expression and proinflammatory signaling in an in vitro model of sepsis. We observed that TCW improved hepatocyte viability and protected hepatocytes against cytokine-mediated cell death. TCW suppressed IL-1ß-mediated increases in Nos2, Tnf, and Il6 mRNA and increased heme oxygenase 1 (HMOX1) protein. TCW inhibited iNOS expression through activation of AKT and JNK pathways since inhibition of PI3K and JNK signaling reduced TCW's effect on iNOS protein expression and activity. These results demonstrate that TCW reduces proinflammatory gene expression and hepatocyte injury produced by elevated inflammatory cytokines and nitric oxide production.

BCR-dependent lineage plasticity in mature B cells.

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.

Signaling by the Epstein-Barr virus LMP1 protein induces potent cytotoxic CD4+ and CD8+ T cell responses.

The B-lymphotropic Epstein-Barr virus (EBV), pandemic in humans, is rapidly controlled on initial infection by T cell surveillance; thereafter, the virus establishes a lifelong latent infection in the host. If surveillance fails, fatal lymphoproliferation and lymphomagenesis ensue. The initial T cell response consists of predominantly CD8+ cytotoxic T cells and a smaller expansion of CD4+ cells. A major approach to treating EBV-associated lymphomas is adoptive transfer of autologous or allogeneic T cells that are stimulated/expanded on EBV-transformed B cells. Strikingly, the clinical response correlates with the frequency of CD4 cells in the infused T cells. Although in vitro studies suggested that EBV-specific CD4 cells develop cytotoxicity, they have not been comprehensively characterized and the molecular mechanism underlying their formation remains unknown. Our recent work, using a transgenic approach in mice, has revealed a central role for the EBV signaling molecule LMP1 in immune surveillance and transformation of EBV-infected B cells. The mouse model offers a unique tool for uncovering basic features of EBV immunity. Here, we show that LMP1 expression in B cells induces potent cytotoxic CD4 and CD8 T cell responses, by enhancing antigen presentation and costimulation by CD70, OX40 ligand, and 4-1BB ligand. Our data further suggest that cytotoxic CD4 cells hold superior therapeutic value for LMP1 (EBV)-driven lymphomas. These findings provide insights into EBV immunity, demonstrating that LMP1 signaling alone is sufficient to induce a prominent cytotoxic CD4 response, and suggest strategies for immunotherapy in EBV-related and other cancers.

Cell-specific regulation of iNOS by AMP-activated protein kinase in primary rat hepatocytes.

BACKGROUND: AMP-activated protein kinase (AMPK) regulates several metabolic pathways in hepatocytes that are critical to the hepatic response to sepsis and shock. Induction of nitric oxide synthesis is an important response to sepsis, inflammation and shock and many of the stimuli that upregulate inducible nitric oxide synthase (iNOS) also activate AMPK. AMPK inhibits nitric oxide (NO) production in skeletal and cardiac muscle cells, but the role of AMPK in regulating iNOS expression in hepatocytes has not been determined. MATERIALS AND METHODS: Primary cultured rat hepatocytes were preincubated with an AMPK inhibitor, AMPK activators, or transfected with AMPK siRNA before being treated with the proinflammatory cytokines interleukin-1ß (IL-1ß) and interferon-γ (IFNγ). The hepatocyte cell lysate and culture supernatants were collected for Western blot analysis and Griess assay. RESULTS: IL-1ß and IFNγ markedly upregulated iNOS expression and AMPK phosphorylation. IL-1ß + IFNγ-induced NO production and iNOS expression were significantly decreased in hepatocytes treated with the AMPK inhibitor compound C and AMPK knockdown by AMPK siRNA. Cytokine-induced iNOS expression was increased by AMPK activators 1-oxo-2-(2H-pyrrolium-1-yl)-1H-inden-3-olate, AMPK signaling activator III and AICA-riboside. Compound C upregulated Akt and c-Jun N-terminal kinase phosphorylation but decreased IκBα phosphorylation. AICA-riboside exerted opposite effects on these signaling pathways in hepatocytes. CONCLUSIONS: In contrast to other cell types, AMPK increased IL-1ß + IFNγ-induced NO production and iNOS expression through the Akt, c-Jun N-terminal kinase, and NF-κΒ signaling pathways in primary hepatocytes. These data suggest that AMPK-altering medications used clinically may have subsequent effects on iNOS expression and proinflammatory signaling pathways.

Utidelone plus capecitabine versus capecitabine alone for heavily pretreated metastatic breast cancer refractory to anthracyclines and taxanes: a multicentre, open-label, superiority, phase 3, randomised controlled trial.

BACKGROUND: Utidelone, a genetically engineered epothilone analogue, has shown promise as a potential treatment for breast cancer in phase 1 and 2 trials. The aim of this phase 3 trial was to compare the efficacy and safety of utidelone plus capecitabine versus capecitabine alone in patients with metastatic breast cancer. METHODS: We did a multicentre, open-label, superiority, phase 3, randomised controlled trial in 26 hospitals in China. Eligible participants were female patients with metastatic breast cancer refractory to anthracycline and taxane chemotherapy regimens. We randomly assigned participants (2:1) using computer based randomisation and block sizes of 6 to a 21-day cycle of either utidelone (30 mg/m2 intravenously once per day on days 1-5) plus capecitabine (1000 mg/m2 orally twice per day on days 1-14), or capecitabine alone (1250 mg/m2 orally twice per day on days 1-14), until disease progression or unacceptable toxicity occurred. Patients, physicians, and assessors were not masked to treatment allocation; however, an independent radiology review committee used to additionally assess response was masked to allocation. The primary endpoint was centrally assessed (by an independent radiology review committee) progression-free survival, and analysed using the Kaplan-Meier product-limit method in the intention-to-treat population. Safety was assessed in all participants who received at least one dose of study drug. Follow-up is ongoing. This study is registered at ClinicalTrials.gov, number NCT02253459. FINDINGS: Between Aug 8, 2014, and Dec 14, 2015, we enrolled and randomly assigned 270 patients to treatment with utidelone plus capecitabine, and 135 to capecitabine alone. Median follow-up for progression-free survival was 6·77 months (IQR 3·81-10·32) for the utidelone plus capecitabine group and 4·55 months (2·55-9·39) for the capecitabine alone group. Median progression-free survival by central review in the utidelone plus capecitabine group was 8·44 months (95% CI 7·95-9·92) compared with 4·27 months (3·22-5·68) in the capecitabine alone group; hazard ratio 0·46, 95% CI 0·36-0·59; p<0·0001. Peripheral neuropathy was the most common grade 3 adverse event in the utidelone plus capecitabine group (58 [22%] of 267 patients vs 1 [<1%] of 130 patients in the capecitabine alone group). Palmar-plantar erythrodysaesthesia was the most prominent grade 3 adverse event in the capacitabine alone group (in 10 [8%] of 130 patients) and was the next most frequent grade 3 event in the utidelone plus capecitabine group (in 18 [7%] of 267 patients). 16 serious adverse events were reported in the combination therapy group (diarrhoea was the most common, in three [1%] patients) and 14 serious adverse events were reported in the monotherapy group (the most common were diarrhoea, increased blood bilirubin, and anaemia, in two [2%] patients for each event). 155 patients died (99 in the combination therapy arm, 56 in the monotherapy arm). All deaths were related to disease progression except for one in each group (attributed to pericardial effusion in the combination therapy group and dyspnoea in the monotherapy group) that were considered possibly or probably treatment-related. INTERPRETATION: Despite disease progression with previous chemotherapies, utidelone plus capecitabine was more efficacious compared with capecitabine alone for the outcome of progression-free survival, with mild toxicity except for peripheral sensory neuropathy, which was manageable. The findings from this study support the use of utidelone plus capecitabine as an effective option for patients with metastatic breast cancer. FUNDING: Beijing Biostar Technologies, Beijing, China.

Genetic drivers of NF-κB deregulation in diffuse large B-cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of B cell non-Hodgkin lymphoma worldwide and comprises a heterogeneous group of malignancies that originate from the malignant transformation of germinal center (GC) B cells. Over the past decade, significant improvement has been achieved in our understanding of the molecular pathogenesis underlying this disease, thanks in part to the implementation of powerful genomic technologies allowing genome-wide structural and functional analyses. These studies revealed the presence of multiple oncogenic alterations dysregulating signal transduction pathways that are normally required for the normal biology of the cells from which these tumors are derived. Among the pathways identified as recurrent targets of genetic lesions in DLBCL, NF-κB has emerged as a central player in the development and maintenance of this disease, particularly in the less curable, activated B cell (ABC)- like subtype. These lesions reveal vulnerabilities of the lymphoma cells that can be exploited for the design of more rationale therapeutic approaches. The purpose of this review is to summarize recent progresses in understanding the role of NF-κB deregulation in the pathogenesis of DLBCL, with emphasis on the genetic basis underlying its aberrant activation, in relationship to the normal biology of B lymphocytes, and the modelling of these lesions in the mouse.

Resveratrol decreases nitric oxide production by hepatocytes during inflammation.

INTRODUCTION: The production of excessive amounts of nitric oxide (NO) through inducible nitric oxide synthase (iNOS) contributes to organ injury, inflammation, and mortality after shock. Resveratrol (RSV) is a natural polyphenol that decreases shock-induced hepatic injury and inflammation. We hypothesized that RSV would mediate these effects by decreasing hepatocyte iNOS production. METHODS: Rat hepatocytes were isolated, cultured with varying concentrations of RSV, and then stimulated to induce iNOS with interleukin-1 and interferon. Induction of iNOS protein was measured by Western blot, iNOS mRNA by polymerase chain reaction, and NO production was measured by culture supernatant nitrite. Activation of intracellular signaling pathways involving Akt, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) were measured by Western blot using isoform-specific antibodies. RESULTS: RSV decreased the expression of iNOS mRNA, protein, and supernatant nitrite in a dose-dependent manner. Our previous work demonstrated that Akt and JNK both inhibit hepatic iNOS production, whereas NF-κB increases iNOS expression. Analysis of signaling pathways in this study demonstrated that RSV increased JNK phosphorylation but decreased Akt phosphorylation and increased NF-κB activation. CONCLUSION: RSV decreases cytokine-induced hepatocyte iNOS expression, possibly through up-regulation of the JNK signaling pathway. RSV merits further investigation to determine its mechanism as a compound that can decrease inflammation after shock.

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/ß and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models.

An oncogenic role for alternative NF-κB signaling in DLBCL revealed upon deregulated BCL6 expression.

Diffuse large B cell lymphoma (DLBCL) is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-κB activity, a role for oncogenic lesions that activate the alternative NF-κB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-κB pathway, occurs in ∼15% of DLBCLs and that it often coexists with BCL6 translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-κB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-κB pathway in DLBCL development.