MYC is essential for the formation and maintenance of germinal centers

Germinal centers (GC) are sites of intense B cell proliferation, central for T cell dependent antibody responses. However, the role of MYC, a key cell cycle regulator, in this process has been questioned. Here, we identified MYC positive B cell subpopulations in immature and mature GCs, and show through genetic ablation of Myc that they play indispensable roles in GC formation and maintenance. The identification of these functionally critical cellular subsets has important implications for human B cell lymphomagenesis, which mostly originates from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may be at a particularly high risk for malignant transformation

Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination

Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (C(H)) exons with downstream C(H) exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation

Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.Peer reviewe

RAC1(P29S) Induces a Mesenchymal Phenotypic Switch via Serum Response Factor to Promote Melanoma Development and Therapy Resistance

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61. Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch. Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma. When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis. RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors. These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target

Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma

Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells

BAFF-R-dependent activation of the alternative NF-κB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-κB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-κB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked

A preclinical model of peripheral T‐cell lymphoma GATA3 reveals DNA damage response pathway vulnerability

Abstract Peripheral T‐cell lymphoma (PTCL) represents a rare group of heterogeneous diseases in urgent need of effective treatments. A scarcity of disease‐relevant preclinical models hinders research advances. Here, we isolated a novel mouse (m)PTCL by serially transplanting a lymphoma from a germinal center B‐cell hyperplasia model (Cγ1‐Cre Blimp1fl/fl) through immune‐competent mice. Lymphoma cells were identified as clonal TCRβ+ T‐helper cells expressing T‐follicular helper markers. We also observed coincident B‐cell activation and development of a de novo B‐cell lymphoma in the model, reminiscent of B‐cell activation/lymphomagenesis found in human PTCL. Molecular profiling linked the mPTCL to the high‐risk “GATA3” subtype of PTCL, showing GATA3 and Th2 gene expression, PI3K/mTOR pathway enrichment, hyperactivated MYC, and genome instability. Exome sequencing identified a human‐relevant oncogenic β‐catenin mutation possibly involved in T‐cell lymphomagenesis. Prolonged treatment responses were achieved in vivo by targeting ATR in the DNA damage response (DDR), a result corroborated in PTCL cell lines. This work provides mechanistic insight into the molecular and immunological drivers of T‐cell lymphomagenesis and proposes DDR inhibition as an effective and readily translatable therapy in PTCL

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and beta-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/beta-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system

The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers

Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell-dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc(+) B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc(+) GC subpopulations may be at a particularly high risk for malignant transformation