Kong-Peng Lam

Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA

Immune responses are normally targeted against microbial pathogens and not self-antigens by mechanisms that are only partly understood. Here we define a newly discovered pathway that prevents autoimmunity by limiting the levels on T lymphocytes of a co-stimulatory receptor, the inducible T-cell co-stimulator (ICOS). In sanroque mice homozygous for an M199R mutation in the ROQ domain of Roquin (also known as Rc3h1), increased Icos expression on T cells causes the accumulation of lymphocytes that is associated with a lupus-like autoimmune syndrome. Roquin normally limits Icos expression by promoting the degradation of Icos messenger RNA. A conserved segment in the unusually long ICOS 3′ untranslated mRNA is essential for regulation by Roquin. This segment comprises a 47-base-pair minimal region complementary to T-cell-expressed microRNAs including miR-101, the repressive activity of which is disrupted by base-pair inversions predicted to abrogate miR-101 binding. These findings illuminate a critical post-transcriptional pathway within T cells that regulates lymphocyte accumulation and autoimmunity, and highlights the therapeutic potential of partially antagonising the ICOS pathway.

Cutting Edge: The Dependence of Plasma Cells and Independence of Memory B Cells on BAFF and APRIL

Memory B (BMEM) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting BMEM cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived BMEM cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, BMEM cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.

ICOS Controls the Pool Size of Effector-Memory and Regulatory T Cells

ICOS is an important regulator of T cell effector function. ICOS-deficient patients as well as knockout mice show severe defects in T cell-dependent B cell responses. Several in vitro and in vivo studies attributed this phenomenon to impaired up-regulation of cell surface communication molecules and cytokine synthesis by ICOS-deficient T cells. However, we now could show with Ag-specific T cells in a murine adoptive transfer system that signaling via ICOS does not significantly affect early T cell activation. Instead, ICOS substantially contributes to the survival and expansion of effector T cells upon local challenge with Ag and adjuvant. Importantly, the observed biological function of ICOS also extends to FoxP3+ regulatory T cells, as can be observed after systemic Ag delivery without adjuvant. In line with these findings, absence of ICOS under homeostatic conditions of nonimmunized mice leads to a reduced number of both effector-memory and FoxP3+ regulatory T cells. Based on these results, we propose a biological role for ICOS as a costimulatory, agonistic molecule for a variety of effector T cells with differing and partly opposing functional roles. This concept may reconcile a number of past in vivo studies with seemingly contradictory results on ICOS function.

Macrophage polarization to a unique phenotype driven by B cells

Regulation of adaptive immunity by innate immune cells is widely accepted. Conversely, adaptive immune cells can also regulate cells of the innate immune system. Here, we report for the first time the essential role of B cells in regulating macrophage (Mϕ) phenotype. In vitro B cell/Mϕ co‐culture experiments together with experiments in transgenic mice models for B‐cell deficiency or overexpression showed B1 cells to polarize Mϕ to a distinct phenotype. This was characterized by downregulated TNF‐α, IL‐1β and CCL3, but upregulated IL‐10 upon LPS stimulation; constitutive expression of M2 Mϕ markers (e.g. Ym1, Fizz1) and overexpression of TRIF‐dependent cytokines (IFN‐β, CCL5). Mechanistically, this phenotype was linked to a defective NF‐κB activation, but a functional TRIF/STAT1 pathway. B1‐cell‐derived IL‐10 was found to be instrumental in the polarization of these Mϕ. Finally, in vivo relevance of B1‐cell‐induced Mϕ polarization was confirmed using the B16 melanoma tumor model where adoptive transfer of B1 cells induced an M2 polarization of tumor‐associated Mϕ. Collectively, our results define a new mechanism of Mϕ polarization wherein B1 cells play a key role in driving Mϕ to a unique, but M2‐biased phenotype. Future studies along these lines may lead to targeting of B1 cells to regulate Mϕ response in inflammation and cancer.

Phospholipase Cγ2 Is Critical for Dectin-1-mediated Ca2+ Flux and Cytokine Production in Dendritic Cells

Dectin-1 is a C-type lectin that recognizes β-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-κB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) γ-inhibitor U73122, suggesting that Dectin-1 signals via a PLCγ pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCγ1 and PLCγ2. However, we show that PLCγ2 is the critical enzyme for Dectin-1 signaling in DCs. PLCγ2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor α. PLCγ2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCγ2-deficient DCs are also impaired in their activation of NF-κB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKα/β activation and IκBα degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCγ2 is a critical player in the Dectin-1 signal transduction pathway.

The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes.

B lymphocytes lacking the adaptor protein B cell linker (BLNK) do not proliferate in response to B cell antigen receptor (BCR) engagement. We demonstrate here that BCR-activated BLNK− /− B cells fail to enter the cell cycle, and this is due to their inability to induce the expression of the cell cycle regulatory proteins such as cyclin D2 and cyclin-dependent kinase 4. BCR-stimulated BLNK− /− B cells also do not up-regulate the cell survival protein Bcl-xL, which may be necessary for the cells to complete the cell cycle. In addition, BLNK− /− B cells exhibit a high rate of spontaneous apoptosis in culture. Examination of the various BCR-activated signaling pathways in mouse BLNK− /− B cells reveals the intact activation of Akt and mitogen-activated protein kinases but the impaired activation of nuclear factor (NF)-κB that is known to regulate genes involved in cell proliferation and survival. The inability to activate NF-κB in BCR-stimulated BLNK− /− B cells is due to a failure to induce the degradation of the inhibitory κB protein. In all these aspects, BLNK− /− B cells resemble xid B cells that have a mutation in Brutons tyrosine kinase (Btk). Recently, phospholipase C (PLC)-γ2 has also been demonstrated to be essential for NF-κB activation. Since BLNK has been shown separately to interact with both Btk and PLC-γ2, our finding of normal Btk but impaired PLC-γ2 activation in BCR-stimulated BLNK− /− B cells strongly suggests that BLNK orchestrates the formation of a Btk-PLC-γ2 signaling axis that regulates NF-κB activation. Taken together, the NF-κB activation defect may be sufficient to explain the similar defects in BCR-induced B cell proliferation and T cell-independent immune responses in BLNK− /−, Btk− /−, and PLC-γ2− /− mice.

Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon–β (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Brutons tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-β upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

The RNase III enzyme Dicer is essential for germinal center B-cell formation.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are important for pre-B and follicular B lymphopoiesis as demonstrated, respectively, by mb-1-Cre- and cd19-Cre-mediated deletion of Dicer, the RNase III enzyme critical for generating mature miRNAs. To explore the role of miRNAs in B-cell terminal differentiation, we use Aicda-Cre to specifically delete Dicer in activated B cells where activation-induced cytidine deaminase is highly expressed. We demonstrate that mutant mice fail to produce high-affinity class-switched antibodies and generate memory B and long-lived plasma cells on immunization with a T cell-dependent antigen. More importantly, germinal center (GC) B-cell formation is drastically compromised in the absence of Dicer, as a result of defects in cell proliferation and survival. Dicer-deficient GC B cells express higher levels of cell cycle inhibitor genes and proapoptotic protein Bim. Ablation of Bim could partially rescue the defect in GC B-cell formation in Dicer-deficient mice. Taken together, our data suggest that Dicer and probably miRNAs are critical for GC B-cell formation during B-cell terminal differentiation.

Cbp Deficiency Alters Csk Localization in Lipid Rafts but Does Not Affect T-Cell Development

ABSTRACT The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.

The ROQUIN family of proteins localizes to stress granules via the ROQ domain and binds target mRNAs

Roquin is an E3 ubiquitin ligase with a poorly understood but essential role in preventing T‐cell‐mediated autoimmune disease and in microRNA‐mediated repression of inducible costimulator (Icos) mRNA. Roquin and its mammalian paralogue membrane‐associated nucleic acid binding protein (MNAB) define a protein family distinguished by an ∼ 200 amino acid domain of unknown function, ROQ, that is highly conserved from mammals to invertebrates and is flanked by a RING‐1 zinc finger and a CCCH zinc finger. Here we show that human, Drosophila and Caenorhabditis elegans Roquin and human MNAB localize to the cytoplasm and upon stress are concentrated in stress granules, where stalled mRNA translation complexes are stored. The ROQ domain is necessary and sufficient for localization to arsenite‐induced stress granules and to induce these structures upon overexpression, and is required to trigger Icos mRNA decay. Gel‐shift, SPR and footprinting studies show that an N‐terminal fragment centred on the ROQ domain binds RNA from the Icos 3′‐untranslated region comprising the minimal sequence for Roquin‐mediated repression, adjacent to the miR‐101 sequence complementarity. These findings identify Roquin as an RNA‐binding protein and establish a specific function for the ROQ protein domain in mRNA homeostasis.