The alternative NF-kB pathway in mature B cell development

The nuclear factor-kB (NF-kB) signaling cascade is comprised of two branches, the canonical and alternative NF-kB pathways. Signaling through the alternative NF-kB pathway culminates in the activation of the downstream transcription factor subunits, RELB and NF-kB2. The biological roles of RELB and NF-kB2 within the B cell lineage have been obscured in constitutional knockout mice by the diverse functions of these subunits in non-B cell types. To overcome these limitations, conditional alleles were generated to investigate the roles of RELB and NF-kB2 in B cell development. These alleles allowed the identification of complex functional requirements for RELB and/or NF-kB2 in naïve B cells, germinal center (GC) B cells and plasma cells (PCs). These functional requirements may have implications for B cell malignancies that display mutations that constitutively activate the alternative NF-kB pathway. A large body of work has demonstrated that B cell activating factor (BAFF) signaling is critical for the maintenance of mature B cells. However, the contribution of the alternative NF-kB subunits that are activated downstream of BAFF remained unclear, especially in regards to their specific target genes. We have identified critical, B cell-intrinsic roles for RELB and NF-kB2 in the maintenance of mature B cells. In response to BAFF, these subunits were found to control the expression of anti-apoptotic genes, genes that ensure correct positioning within the B cell niche, and genes involved in promoting B–T cell interactions that allow effective antigen-mediated activation. During the GC B cell reaction, light zone (LZ) B cells undergo affinity-based selection mediated by T follicular helper (Tfh) cells. A subset of LZ B cells show activation of the NF-kB signaling cascade, suggesting a critical role for NF-kB in the selection of high-affinity GC B cells. We here report that GC B cell development occurred normally in mice with conditional deletion of either relb (RELB) or nfkb2 (NF-kB2) in GC B cells. In contrast, the simultaneous ablation of both subunits caused rapid involution of established GCs, similar to what has been observed for ablation of the canonical NF-kB transcription factor subunit c-REL. Intriguingly, RNA-sequencing analysis of relb/nfkb2-deleted GC B cells revealed no overlap between the genes controlled by RELB/p52 and c-REL within GC B cells. This suggests that signaling through the separate NF-kB pathways in GC B cells results in the expression of different biological programs that are independently required for the maintenance of the GC reaction. In addition, we observed that human PCs and PC precursors within the LZ showed high protein levels of NF-kB2 compared to surrounding lymphocytes, suggesting a biological role for this subunit in PCs. Indeed, ablation of nfkb2 alone in GC B cells led to a dramatic decrease in antigen-specific serum IgG1 and antigen-specific IgG1-secreting cells. Interestingly however, the mice developed normal frequencies of PCs, suggesting a role for NF-kB2 in PC physiology rather than differentiation

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.xºpost-print5505 K

Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development

The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway

Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B cells

Signaling through the canonical nuclear factor‐κB (NF‐κB) pathway is critical for the generation and maintenance of mature B cells and for antigen‐dependent B‐cell activation. c‐REL (rel) and RELA (rela) are the downstream transcriptional activators of the canonical NF‐κB pathway. Studies of B cells derived from constitutional rel knockout mice and chimeric mice repopulated with rela–/– fetal liver cells provided evidence that the subunits can have distinct roles during B‐cell development. However, the B cell‐intrinsic functions of c‐REL and RELA during B‐cell generation and antigen‐dependent B‐cell activation have not been determined in vivo. To clarify this issue, we crossed mice with conditional rel and rela alleles individually or in combination to mice that express Cre‐recombinase in B cells. We here report that, whereas single deletion of rel or rela did not impair mature B‐cell generation and maintenance, their simultaneous deletion led to a dramatic reduction of follicular and marginal zone B cells. Upon T cell‐dependent immunization, B cell‐specific deletion of the c‐REL subunit alone abrogated the formation of germinal centers (GCs), whereas rela deletion did not affect GC formation. T‐independent responses were strongly impaired in mice with B cell‐specific deletion of rel, and only modestly in mice with RELA‐deficient B cells. Our findings identify differential requirements for the canonical NF‐κB subunits c‐REL and RELA at distinct stages of mature B‐cell development. The subunits are jointly required for the generation of mature B cells. During antigen‐dependent B‐cell activation, c‐REL is the critical subunit required for the initiation of the GC reaction and for optimal T‐independent antibody responses, with RELA being largely dispensable at this stage

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMsInstituto de Salud Carlos IIIInstitut CurieInstitut national de la santé et de la recherche médicale (INSERM)Centre national de la recherche scientifique (CNRS)Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Germinal center B cell maintenance and differentiation are controlled by distinct NF-kappa B transcription factor subunits

Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-kappa B signaling pathway has been implicated; however, the distinct roles of the individual NF-kappa B transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-kappa B subunits c-REL or RELA, which are both activated by the canonical NF-kappa B pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-kappa B pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-kappa B in GC lymphomagenesis

Single-cell analysis reveals divergent responses of human dendritic cells to the MVA vaccine

International audienceModified vaccinia Ankara (MVA) is a live, attenuated human smallpox vaccine and a vector for the development of new vaccines against infectious diseases and cancer. Efficient activation of the immune system by MVA partially relies on its encounter with dendritic cells (DCs). MVA infection of DCs leads to multiple outcomes, including cytokine production, activation of costimulatory molecules for T cell stimulation, and cell death. Here, we examined how these diverse responses are orchestrated in human DCs. Single-cell analyses revealed that the response to MVA infection in DCs was limited to early viral gene expression. In response to the early events in the viral cycle, we found that DCs grouped into three distinct clusters. A cluster of infected cells sensed the MVA genome by the intracellular innate immunity pathway mediated by cGAS, STING, TBK1, and IRF3 and subsequently produced inflammatory cytokines. In response to these cytokines, a cluster of noninfected bystander cells increased costimulatory molecule expression. A separate cluster of infected cells underwent caspase-dependent apoptosis. Induction of apoptosis persisted after inhibition of innate immunity pathway mediators independently of previously described IRF-dependent or replication-dependent pathways and was a response to early MVA gene expression. Together, our study identified multiple mechanisms that underlie the interactions of MVA with human DCs

The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function

CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity