An analysis of the putative role of ROQUIN as a T cell RING-directed ubiquitin ligase

An analysis of the putative role of ROQUIN as a T cell RING-directed ubiquitin ligase Abstract: Acquiring long-term protective immunity involves the generation and recall production of high affinity antibodies by B cells in response to foreign antigen. This occurs only with the help of T cells. T follicular helper (Tfh) cells in particular are specialised in homing to B cell-enriched follicles and germinal centres (GCs) of secondary lymphoid organs where they provide selective paracrine signals to support high affinity surface immunoglobulin expression on mutating B cells that differentiate into long-lived plasma cells and memory B cells. Deregulation of Tfh cells leads to a breakdown in the negative selection of pathogenic GC B cell mutants that seed lymphomas or produce disease-causing autoantibodies. It is thus in this context, for the potential to realise biomedical innovations, important to characterise signalling networks that control Tfh cell development and effector function. Here, we sought to map the signalling network of an incompletely understood Tfh cell immunomodulator, ROQUIN, encoded by the Rc3h1 locus. ROQUIN, along with its only mammalian paralogue, ROQUIN2 harbours a unique ROQ domain indispensible in RNA regulation and in preventing unrestricted Tfh cell accumulation that causes systemic autoimmunity. Upstream of the ROQ domain is the ROQUIN amino terminus containing the most evolutionarily conserved sequence and a predicted but uninspected RING domain associated with E3 ubiquitin ligase activity. To determine the putative role of this ROQUIN RING domain in T cell-dependent B cell immunity, we generated a novel mouse model (Tringless), harbouring a T cell-specific genetic deletion of Rc3h1 exon 2, encoding for the RING domain. In vivo analysis of Tringless mice revealed minimal perturbations in ROQUIN ROQ-RNA signalling but an unexpected requirement for T cell ROQUIN RING activity in selectively promoting Tfh cell and GC responses to T-dependent antigens. The development of other effector T cell subsets (Th1, Th2, Th17 and Treg) was not affected. In order to identify potential substrates of the ROQUIN RING domain, we systematically screened for protein partners able to directly bind ROQUIN. Adenosine Monophosphate- activated Protein Kinase (AMPK) was discovered to be targeted by ubiquitin-dependent ROQUIN RING signalling, which was required to limit aberrantly high AMPK activity. Well established as an antagonist to mTOR metabolic signalling and as a central regulator of cellular bioenergetics and proliferation, AMPK activity has not been previously described in Tfh cells. Using an mTOR-deficient mutant mouse strain (chino), we confirmed mTOR as an essential pathway in Tfh cell formation. Consequently, we qualify ROQUIN as a bone fide RING-driven E3 ubiquitin ligase and report a novel ROQUIN-AMPK-mTOR metabolic nexus that our data suggests is important in promoting Tfh cell responses

IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

During T cell–dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell–intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell–autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1+ GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21

B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells

Bcl-6 expression in CD4+ T cells is required to generate extrafollicular antibody responses

T-cell subsets in the germinal center

T cells are known to migrate to B-cell-enriched follicles and germinal centers within secondary lymphoid organs to provide help to B cells. Cognate T:B interactions that take place at the T:B border and subsequently within germinal centers are essential for B-cell priming, differentiation into germinal center B cells, and selection of mutated cells into memory B cells or memory plasma cells. In recent years, different stages of maturation within B-cell helper T cells, collectively known as B-follicular helper T (Tfh) cells, as well as heterogeneity amid germinal center T cells are becoming clear. Indeed, germinal centers support not only bona fide Tfh cells but also CD4+ and CD8+ follicular regulatory T (Tfr) cells that act to suppress germinal center responses and B-cell helper natural killer T cells. There is a growing need for more precise phenotypic and functional distinction of these specialized T-cell subsets. In this review, we summarize current knowledge on the ontogeny, molecular identity, and functional relevance of the various subsets of germinal center T cells

ROQUIN signalling pathways in innate and adaptive immunity

ROQUIN is an RNA-binding protein that plays important roles in both the innate and adaptive immune systems. ROQUIN binds to several key immune-relevant messenger RNA (mRNA) targets through its ROQ domain modulating their stability and influencing macrophage function and the peripheral homeostasis of T cells and B cells. More recently, the E3 ubiquitin ligase activity of the ROQUIN RING domain has been shown to be crucial for T-cell-dependent B-cell responses against infection. Defective ROQUIN activity can culminate in a range of diseases, such as systemic autoimmunity, immunodeficiency, and inflammatory bowel disorder. Here, we provide a current overview of the immunomodulatory role of ROQUIN defined by its ribonucleoprotein-like structure, its repertoire of mRNA targets shared by related RNA-binding enzymes, and its involvement in a range of intracellular signalling pathways central to shaping immune responses.C.G.V. is a recipient of an NHMRC ElizabethBlackburn Fellowship, and funding from NHMRC program andproject grants

ROQUIN-2 shares overlapping functions with its paralog ROQUIN-1 in the repression of mRNAs controlling Tfh cell accumulation and systemic inflammation

Accumulation of T follicular helper (Tfh) cells and proinflammatory cytokines drive autoantibody-mediated diseases. The RNA-binding protein Roquin-1 (Rc3h1) represses the inducible costimulator ICOS and interferon- (IFN-) in T cells to prevent Tfh cell accumulation. Unlike Rc3h1san mice with a mutation in the ROQ domain of Roquin-1, mice lacking the protein, paradoxically do not display increased Tfh cells. Here we have analyzed mice with mutations that eliminate the RING domain from Roquin-1 or its paralog, Roquin-2 (Rc3h2). RING or ROQ mutations both disrupted Icos mRNA regulation by Roquin-1, but, unlike the ROQ mutant that still occupied mRNA-regulating stress granules, RING-deficient Roquin-1 failed to localize to stress granules and allowed Roquin-2 to compensate in the repression of ICOS and Tfh cells. These paralogs also targeted tumor necrosis factor (TNF) in nonlymphoid cells, ameliorating autoantibody-induced arthritis. The Roquin family emerges as a posttranscriptional brake in the adaptive and innate phases of antibody responses.This work was funded by an Elizabeth Blackburn NH&MRC Senior Research Fellowship to C.G.V. and NH and MRC program and project grants to C.G.V., M.C.C. and C.C.G