Bhlhe40 Promotes CD4+ T Helper 1 Cell and Suppresses T Follicular Helper Cell Differentiation during Viral Infection.

In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.

Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Modulation of PKM2 inhibits follicular helper T cell differentiation and ameliorates inflammation in lupus-prone mice.

OBJECTIVES: Expansion of follicular helper T (Tfh) cells and abnormal glucose metabolism are present in patients with systemic lupus erythematosus (SLE). Pyruvate kinase M2 (PKM2) is one of the key glycolytic enzymes, and the underlying mechanism of PKM2-mediated Tfh cell glycolysis in SLE pathogenesis remains elusive. METHODS: We analyzed the percentage of Tfh cells and glycolysis in CD4+ T cells from SLE patients and healthy donors and performed RNA sequencing analysis of peripheral blood CD4+ T cells and differentiated Tfh cells from SLE patients. Following Tfh cell development in vitro and following treatment with PKM2 activator TEPP-46, PKM2 expression, glycolysis, and signaling pathway proteins were analyzed. Finally, diseased MRL/lpr mice were treated with TEPP-46 and assessed for treatment effects. RESULTS: We found that Tfh cell percentage and glycolysis levels were increased in SLE patients and MRL/lpr mice. TEPP-46 induced PKM2 tetramerization, thereby inhibiting Tfh cell glycolysis levels. On the one hand, TEPP-46 reduced the dimeric PKM2 entering the nucleus and reduced binding to the transcription factor BCL6. On the other hand, TEPP-46 inhibited the AKT/GSK-3ß pathway and glycolysis during Tfh cell differentiation. Finally, we confirmed that TEPP-46 effectively alleviated inflammatory damage in lupus-prone mice and reduced the expansion of Tfh cells in vivo. CONCLUSIONS: Our results demonstrate the involvement of PKM2-mediated glycolysis in Tfh cell differentiation and SLE pathogenesis, and PKM2 could be a key therapeutic target for the treatment of SLE.

Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

Mutational, immune microenvironment, and clinicopathological profiles of diffuse large B-cell lymphoma and follicular lymphoma with BCL6 rearrangement.

BCL6-rearrangement (BCL6-R) is associated with a favorable prognosis of follicular lymphoma (FL), but the mechanism is unknown. We analyzed the clinicopathological, immune microenvironment (immune checkpoint, immuno-oncology markers), and mutational profiles of 10 BCL6-R-positive FL, and 19 BCL6-R-positive diffuse large B-cell lymphoma (DLBCL) cases (both BCL2-R and MYC-R negative). A custom-made panel included 168 genes related to aggressive B-cell lymphomas and FL. FL cases were nodal, histological grade 3A in 70%, low Ki67; and had a favorable overall and progression-free survival. DLBCL cases were extranodal in 60%, IPI high in 63%, non-GCB in 60%, EBER-negative; and had a progression-free survival comparable to that of DLBCL NOS. The microenvironment had variable infiltration of M2-like tumor-associated macrophages (TAMs) that were CD163, CSF1R, LAIR1, PD-L1, and CD85A (LILRB3) positive; but had low IL10 and PTX3 expression. In comparison to FL, DLBCL had higher TAMs, IL10, and PTX3 expression. Both lymphoma subtypes shared a common mutational profile with mutations in relevant pathogenic genes such as KMT2D, OSBPL10, CREBBP, and HLA-B (related to chromatin remodeling, metabolism, epigenetic modification, and antigen presentation). FL cases were characterized by a higher frequency of mutations of ARID1B, ATM, CD36, RHOA, PLOD2, and PRPRD (p < 0.05). DLBCL cases were characterized by mutations of BTG2, and PIM1; and mutations of HIST1H1E and MFHAS1 to disease progression (p < 0.05). Interestingly, mutations of genes usually associated with poor prognosis, such as NOTCH1/2 and CDKN2A, were infrequent in both lymphoma subtypes. Some high-confidence variant calls were likely oncogenic, loss-of-function. MYD88 L265P gain-of-function was found in 32% of DLBCL. In conclusion, both BCL6-R-positive FL and BCL6-R-positive DLBCL had a common mutational profile; but also, differences. DLBCL cases had a higher density of microenvironment markers.

Epidermal loss of Bcl6 exacerbates MC903-induced atopic dermatitis-like skin inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease where Th2-type immune responses are dominant. In the lesional skin of AD, keratinocytes show differentiation defects and secrete proinflammatory cytokines and chemokines, amplifying Th2-type responses in AD. We previously reported that inducible loss of B-cell lymphoma 6 (Bcl6), a transcription repressor and a master transcriptional regulator of follicular helper T cells and germinal center B cells, in the whole body results in upregulation of Th2-related cytokines in mouse skin. However, the role of Bcl6 in keratinocytes remains to be clarified. Here, we observed that BCL6 positively regulates the expression of keratinocyte differentiation markers and plasma membrane localization of adherence junctional proteins in keratinocyte cell culture. Although keratinocyte-specific loss of Bcl6 alone did not induce AD-like skin inflammation, it aggravates MC903-induced AD-like skin inflammation in mice. In addition, Bcl6 expression is decreased in the epidermis of lesional skin from MC903-induced AD-like skin inflammation in mice. These results strongly suggest that Bcl6 downregulation in keratinocytes contributes to the development and aggravation of AD-like skin inflammation in mice.

BCL6 attenuates hyperoxia-induced lung injury by inhibiting NLRP3-mediated inflammation in fetal mouse.

BACKGROUND: The transcriptional repressor B-cell lymphoma 6 (BCL6) has been reported to inhibit inflammation. So far, experimental evidence for the role of BCL6 in bronchopulmonary dysplasia (BPD) is lacking. Our study investigated the roles of BCL6 in the progression of BPD and its downstream mechanisms. METHODS: Hyperoxia or lipopolysaccharide (LPS) was used to mimic the BPD mouse model. To investigate the effects of BCL6 on BPD, recombination adeno-associated virus serotype 9 expressing BCL6 (rAAV9-BCL6) and BCL6 inhibitor FX1 were administered in mice. The pulmonary pathological changes, inflammatory chemokines and NLRP3-related protein were observed. Meanwhile, BCL6 overexpression plasmid was used in human pulmonary microvascular endothelial cells (HPMECs). Cell proliferation, apoptosis, and NLRP3-related protein were detected. RESULTS: Either hyperoxia or LPS suppressed pulmonary BCL6 mRNA expression. rAAV9-BCL6 administration significantly inhibited hyperoxia-induced NLRP3 upregulation and inflammation, attenuated alveolar simplification and dysregulated angiogenesis in BPD mice, which were characterized by decreased mean linear intercept, increased radical alveolar count and alveoli numbers, and the upregulated CD31 expression. Meanwhile, BCL6 overexpression promoted proliferation and angiogenesis, inhibited apoptosis and inflammation in hyperoxia-stimulated HPMECs. Moreover, administration of BCL6 inhibitor FX1 arrested growth and development. FX1-treated BPD mice exhibited exacerbation of alveolar pathological changes and pulmonary vessel permeability, with upregulated mRNA levels of pro-inflammatory cytokines and pro-fibrogenic factors. Furthermore, both rAAV9-BCL6 and FX1 administration exerted a long-lasting effect on hyperoxia-induced lung injury (≥4 wk). CONCLUSIONS: BCL6 inhibits NLRP3-mediated inflammation, attenuates alveolar simplification and dysregulated pulmonary vessel development in hyperoxia-induced BPD mice. Hence, BCL6 may be a target in treating BPD and neonatal diseases.

BCL6 is required for the thymic development of TCRαß+CD8αα+ intraepithelial lymphocyte lineage.

TCRαß+CD8αα+ intraepithelial lymphocytes (CD8αα+ αß IELs) are a specialized subset of T cells in the gut epithelium that develop from thymic agonist selected IEL precursors (IELps). The molecular mechanisms underlying the selection and differentiation of this T cell type in the thymus are largely unknown. Here, we found that Bcl6 deficiency in αß T cells resulted in the near absence of CD8αα+ αß IELs. BCL6 was expressed by approximately 50% of CD8αα+ αß IELs and by the majority of thymic PD1+ IELps after agonist selection. Bcl6 deficiency blocked early IELp generation in the thymus, and its expression in IELps was induced by thymic TCR signaling in an ERK-dependent manner. As a result of Bcl6 deficiency, the precursors of IELps among CD4+CD8+ double-positive thymocytes exhibited increased apoptosis during agonist selection and impaired IELp differentiation and maturation. Together, our results elucidate BCL6 as a crucial transcription factor during the thymic development of CD8αα+ αß IELs.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Selectively targeting BCL6 using a small molecule inhibitor is a potential therapeutic strategy for ovarian cancer.

Ovarian cancer is one of the tumors with the highest fatality rate among gynecological tumors. The current 5-year survival rate of ovarian cancer is <35%. Therefore, more novel alternative strategies and drugs are needed to treat ovarian cancer. The transcription factor B-cell lymphoma 6 (BCL6) is critically associated with poor prognosis and cisplatin resistance in ovarian cancer treatment. Therefore, BCL6 may be an attractive therapeutic target for ovarian cancer. However, the role of targeting BCL6 in ovarian cancer remains elusive. Here, we developed a novel BCL6 small molecule inhibitor, WK369, which exhibits excellent anti-ovarian cancer bioactivity, induces cell cycle arrest and causes apoptosis. WK369 effectively inhibits the growth and metastasis of ovarian cancer without obvious toxicity in vitro and in vivo. meanwhile, WK369 can prolong the survival of ovarian cancer-bearing mice. It is worth noting that WK369 also has significant anti-tumor effects on cisplatin-resistant ovarian cancer cell lines. Mechanistic studies have shown that WK369 can directly bind to the BCL6-BTB domain and block the interaction between BCL6 and SMRT, leading to the reactivation of p53, ATR and CDKN1A. BCL6-AKT, BCL6-MEK/ERK crosstalk is suppressed. As a first attempt, our study demonstrates that targeting BCL6 may be an effective approach to treat ovarian cancer and that WK369 has the potential to be used as a candidate therapeutic agent for ovarian cancer.

Non-IG::MYC in diffuse large B-cell lymphoma confers variable genomic configurations and MYC transactivation potential.

MYC translocation occurs in 8-14% of diffuse large B-cell lymphoma (DLBCL), and may concur with BCL2 and/or BCL6 translocation, known as double-hit (DH) or triple-hit (TH). DLBCL-MYC/BCL2-DH/TH are largely germinal centre B-cell like subtype, but show variable clinical outcome, with IG::MYC fusion significantly associated with inferior survival. While DLBCL-MYC/BCL6-DH are variable in their cell-of-origin subtypes and clinical outcome. Intriguingly, only 40-50% of DLBCL with MYC translocation show high MYC protein expression (>70%). We studied 186 DLBCLs with MYC translocation including 32 MYC/BCL2/BCL6-TH, 75 MYC/BCL2-DH and 26 MYC/BCL6-DH. FISH revealed a MYC/BCL6 fusion in 59% of DLBCL-MYC/BCL2/BCL6-TH and 27% of DLBCL-MYC/BCL6-DH. Targeted NGS showed a similar mutation profile and LymphGen genetic subtype between DLBCL-MYC/BCL2/BCL6-TH and DLBCL-MYC/BCL2-DH, but variable LymphGen subtypes among DLBCL-MYC/BCL6-DH. MYC protein expression is uniformly high in DLBCL with IG::MYC, but variable in those with non-IG::MYC including MYC/BCL6-fusion. Translocation breakpoint analyses of 8 cases by TLC-based NGS showed no obvious genomic configuration that enables MYC transactivation in 3 of the 4 cases with non-IG::MYC, while a typical promoter substitution or IGH super enhancer juxtaposition in the remaining cases. The findings potentially explain variable MYC expression in DLBCL with MYC translocation, and also bear practical implications in its routine assessment.

A MYC-rearrangement is a negative prognostic factor in stage II, but not in stage I diffuse large B-cell lymphoma.

MYC oncogene rearrangements (MYC-R) negatively affect survival in patients with Ann Arbor stage III-IV diffuse large B-cell lymphoma (DLBCL), but their impact in limited stage (LS) I-II is unclear. Therefore, we assessed the impact of MYC-R on progression-free survival (PFS) and overall survival (OS) in LS DLBCL patients at the population level. We identified 1,434 LS DLBCL patients with known MYC-R status diagnosed between 2014 and 2020, who received R-CHOP(-like) regimens using the Netherlands Cancer Registry, with survival follow-up until February 2022. Stage I patients with (n = 83, 11%) and without (n = 650, 89%) a MYC-R had similar 2-years PFS (89% and 93%, p = 0.63) and OS (both 95%, p = 0.22). Conversely, stage II DLBCL patients with a MYC-R (n = 90, 13%) had inferior survival outcomes compared to stage II patients without a MYC-R (n = 611, 87%) (PFS 70% vs. 89%, p = 0.001; OS 79% vs. 94%, p < 0.0001). Both single MYC-R (single hit, n = 36) and concurrent BCL2 and/or BCL6 rearrangements (double/triple hit, n = 39) were associated with increased mortality and relapse risk. In conclusion, in stage II DLBCL a MYC-R is negatively associated with survival. In stage I DLBCL, however, survival outcomes are excellent irrespective of MYC-R status. This challenges the diagnostic assessment of MYC-R in stage I DLBCL patients.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway.

Hypoxia is an important characteristic of Tibetan plateau environment. It can lead to apoptosis, but the mechanism of apoptosis caused by hypoxic stress needs further clarification. Here, cattle kidney cell MDBK were used as cell model. The effect of hypoxic stress on apoptosis and its molecular mechanism were explored. MDBK cells were treated with hypoxic stress, apoptosis and mitochondrial apoptotic pathway were significantly increased, and the expression of B-cell lymphoma 6 (BCL6) was significantly decreased. Overexpressing or inhibiting BCL6 demonstrated that BCL6 inhibited the apoptosis. And the increase of apoptosis controlled by hypoxic stress was blocked by BCL6 overexpressing. MDBK cells were treated with hypoxic stress, the expression and the nuclear localization of p53 were significantly increased. Overexpressing or inhibiting p53 demonstrated that hypoxic stress suppressed the expression of BCL6 through p53. Together, these results indicated that hypoxic stress induced the apoptosis of MDBK cells, and BCL6 was an important negative factor for this regulation process. In MDBK cells, hypoxic stress suppressed the expression of BCL6 through p53/BCL6-mitochondrial apoptotic pathway. This study enhanced current understanding of the molecular mechanisms underlying the regulation of apoptosis by hypoxic stress in MDBK cells.

IRF4 induces M1 macrophage polarization and aggravates ulcerative colitis progression by the Bcl6-dependent STAT3 pathway.

Ulcerative colitis (UC) is an idiopathic chronic intestinal inflammation. An increasing body of evidence shows that macrophages play an important role in the pathogenesis of UC. Interferon regulatory factor 4 (IRF4) is crucial for the development of autoimmune diseases via regulating immune cells. This research was designed to explore the function of IRF4 in UC and its association with macrophage polarization. The in vitro model of UC was established by stimulating colonic epithelial cells with tumor necrosis factor α (TNF-α). A mouse model of UC was constructed by injecting C57BL/6 mice with dextran sulfate sodium salt. Flow cytometry was used to assess percentage of CD11b+ CD86+ and CD11b+ CD206+ cells in bone marrow macrophages. Occult blood tests were used to detect hematochezia. Hematoxylin and eosin staining assay was used to assess colon pathological changes. Enzyme-linked immunosorbent assay (ELISA) was used to detect concentrations of inflammatory cytokines. The interaction of IRF4 and B-cell lymphoma 6 (Bcl6) was confirmed using GST pull-down and coimmunoprecipitation assays. Our findings revealed that IRF4 promoted cell apoptosis and stimulated M1 macrophage polarization in vitro. Furthermore, IRF4 aggravated symptoms of the mouse model of UC and aggravated M1 macrophage polarization in vivo. IRF4 negatively regulated Bcl6 expression. Downregulation of Bcl6 promoted apoptosis and M1 macrophage polarization in the presence of IRF4 in vitro and in vivo. Moreover, Bcl6 positively mediated the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In conclusion, IRF4 aggravated UC progression through promoting M1 macrophage polarization via Bcl6/JAK2/STAT3 pathway. These findings suggested that IRF4 might be a good target to competitively inhibit or to treat with UC.

Prognostic significance of copy number gains of MYC detected by fluorescence in situ hybridization in large B-cell lymphoma.

The MYC protooncogene plays a critical role in many cellular processes. MYC translocations are recurrent in large B-cell lymphomas (LBCLs) where they exhibit a negative effect on survival. Gain of MYC copies is also frequently identified; however, there is no consensus on the frequency and prognostic significance of MYC copy gains. We collected FISH data for MYC with reflex testing for BCL2 and BCL6 and IHC results at diagnosis for a cohort of 396 de novo and transformed LBCL cases and compared progression-free (PFS) and overall survival (OS) to determine the prognostic impact of extra MYC copies. The prevalence of cases with MYC copy number gain was 20.9%. PFS was shorter for patients with ≥5 MYC copies compared to controls (p = 0.0005, HR = 2.25). .MYC gain trended towards worse OS; patients with ≥7MYC copies had worse OS (p = 0.013), similar to patients with MYC translocations. We propose that MYC gain represents a dose-dependent prognostic factor for LBCLs.

Alternative genetic alterations of MYC, BCL2, and/or BCL6 in high-grade B-cell lymphoma (HGBL) and diffuse large B-cell lymphoma (DLBCL): Can we identify different prognostic subgroups?

High-grade B-cell lymphoma (HGBL)/diffuse large B-cell lymphoma (DLBCL) with rearrangements (R) in MYC and BCL2 and/or BCL6 are correlated with poor prognosis. Little is known about the impact of other genetic alterations (gain (G) or amplification (A)) of these genes. The aim of the study was to investigate whether we can identify new prognostic subgroups. Fluorescence in situ hybridization (FISH) results from 169 HGBL/DLBCL were retrospectively categorized into: (1) concurrent MYC-R and BCL2-R and/or BCL6-R-samples with MYC-R and BCL2-R (+/- BCL6-R); n = 21, and HGBL/DLBCL with MYC-R and BCL6-R; n = 11; (2) concurrent R and G/A in MYC and BCL2 and/or BCL6 called "alternative HGBL/DLBCL"-samples with (n = 16) or without (n = 6) BCL2 involvement; (3) BCL2 and/or BCL6 alterations without MYC involvement (n = 35); (4) concurrent G/A in MYC and BCL2 and/or BCL6 without R (n = 25); and (5) "No alterations" (n = 55). Patients with HGBL/DLBCL-MYC/BCL2 and "alternative" HGBL/DLBCL (with BCL2 involvement) had significantly worse survival rates compared to the "no alterations" group. G/A of these genes in the absence of rearrangements did not show any prognostic significance. HGBL/DLBCL with MYC-R and BCL6-R without BCL2 involvement showed a better survival rate compared to HGBL/DLBCL-MYC/BCL2. According to immunohistochemistry, "double/triple" expression (DEL/TEL) did not show a significantly worse outcome compared to absent DEL/TEL. This study highlights the continued value of FISH assessment of MYC, BCL2, and BCL6 in the initial evaluation of HGBL/DLBCL with different survival rates between several genetic subgroups.

Primary cardiac large B cell lymphoma.

A female patient in her mid-60s presented with progressive shortness of breath, pleuritic chest pain and bilateral leg swelling for 1 week. Initial diagnostic workup revealed pericardial effusion, and a localised pericardial tubular mass on CT chest. Pericardial fluid analysis showed elevated white cells, with predominance of medium-large sized atypical lymphoid cells. Atypical lymphocytes stained positive for CD79a, CD10, PAX-5, BCL-2 and BCL6. Fluorescence in situ hybridisation testing demonstrated MYC and BCL6 rearrangements without BCL2 gene rearrangement. The overall morphological, immunohistochemical and cytogenetic findings supported a diagnosis of high-grade B cell lymphoma with MYC and BCL6 rearrangements. After extensive staging workup, localised disease involving the pericardium with a diagnosis of primary cardiac large B cell lymphoma was established. She was treated with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab chemotherapy. Rituximab was discontinued owing to largely absent CD20 expression. Interim positron emission tomography-CT after three cycles revealed a complete response, and the patient completed six cycles of therapy.

Bcl6 is a subset-defining transcription factor of lymphoid tissue inducer-like ILC3.

Innate lymphoid cells (ILCs) are tissue-resident effector cells with roles in tissue homeostasis, protective immunity, and inflammatory disease. Group 3 ILCs (ILC3s) are classically defined by the master transcription factor RORγt. However, ILC3 can be further subdivided into subsets that share type 3 effector modules that exhibit significant ontological, transcriptional, phenotypic, and functional heterogeneity. Notably lymphoid tissue inducer (LTi)-like ILC3s mediate effector functions not typically associated with other RORγt-expressing lymphocytes, suggesting that additional transcription factors contribute to dictate ILC3 subset phenotypes. Here, we identify Bcl6 as a subset-defining transcription factor of LTi-like ILC3s in mice and humans. Deletion of Bcl6 results in dysregulation of the LTi-like ILC3 transcriptional program and markedly enhances expression of interleukin-17A (IL-17A) and IL-17F in LTi-like ILC3s in a manner in part dependent upon the commensal microbiota-and associated with worsened inflammation in a model of colitis. Together, these findings redefine our understanding of ILC3 subset biology.