Abstract

SLAT: A Novel GEF Coordinating Actin Cytoskeleton Reorganization and Ca²⁺ Signaling in T Cells

Stephane Becart, Ann. J. Canonigo Balancio and Amnon Altman

SWAP-70-like adaptor of T cells (SLAT) is a novel protein abundantly expressed in T cells, which we have originally isolated based on its selectively high expression in polarized Th2 cells. Beginning at its N-terminus, SLAT displays a potential EF hand domain, an ITAM-like motif, a PH domain and a DH-like domain, which was later found to possess GEF activity toward Cdc42 and Rac1. Thus, SLAT and its homologue, SWAP-70, which is abundantly expressed in B cells, constitute a novel subfamily of Rho family GEFs. Antigen stimulation causes SLAT to translocate to the immunological synapse (IS). Our recent characterization of Slat^-/- mice revealed a defect in the expansion of DN1 cells, which carries over to all subsequent steps in T cell development, resulting in an overall decrease of ~50% in the number of thymocytes and peripheral T cells, without a significant alterations of CD4/CD8 T cell subsets. In vivo, Slat^-/- mice are resistant to lung inflammation and EAE, experimental diseases mediated by Th2, Th1 and Th17 cells. Peripheral T cells of Slat^-/- mice display a ~50% reduction in proliferation, IL-2 production, and in vitro Th1 and Th2 differentiation. The genetic defect of Slat^-/- peripheral T cells was mapped to Ca²⁺ release from intracellular ER stores, thereby acting upstream of the site of action of Stim1 and Orai1, and preventing TCR-stimulated Ca²⁺ influx. Our more recent structure-function analysis of SLAT revealed a novel mechanism for its activation and IS recruitment. Consistent with its obligatory role in Ca²⁺ signaling, ectopic SLAT expression led to activation of an NFAT, but not NF-κB or AP-1, reporter gene. The TCR-induced translocation of SLAT to the IS required Lck-mediated phosphorylation of tyrosine residues 133 and 144 located in an ITAM-like sequence, but was independent of the SLAT PH domain. This sub-cellular relocalization was coupled to, and necessary for, activation of the NFAT pathway. Furthermore, membrane targeting of the SLAT DH (catalytic) domain was sufficient to trigger TCR-mediated NFAT activation and Th1 and Th2 differentiation in a Cdc42-dependent manner, and a constitutively active Cdc42 mutant restored these events in primary Slat^-/- peripheral T cells. Therefore, tyrosine phosphorylation-mediated relocalization of SLAT to the site of antigen recognition is required to exert its pivotal role in NFAT-dependent CD4⁺ T cell differentiation. Hence, SLAT is novel T cell signaling protein, which couples the activation of small GTPases (primarily Cdc42) to Ca²⁺ signaling. Current studies are aimed at elucidating the mechanism through which SLAT controls Ca²⁺ release from the ER, and how it couples actin cytoskeleton reorganization to Ca²⁺ signaling in T cells.

Protein Ubiquitination in Immune Regulation: Regulation of Treg Generation and Lung Inflammation by the E3 ubiquitin Ligase Itch via Modulating TGF-b Signaling

Yun-Cai Liu

The process of ubiquitin conjugation to a protein substrate is catalyzed by a cascade of enzymatic reactions including E1, E2, and E3s. The E3s, which can be classified into RING (really interesting new gene)-type, and HECT (homologous to the E6-associated protein carboxyl-terminus)-type families, determine the specificity of substrate recognition via well-defined protein-protein interactions. In addition to the proteosome-dependent degradation, protein ubiquitination also serves as a means of functional modification such as protein-protein interaction, receptor downmodulation, or transcriptional regulation. In the immune system, it is involved in lymphocyte development, activation, and tolerance, antigen presentation, or viral infection. In this talk, I will focus on our latest results on how the E3 ligase Itch regulates T cell differentiation and anergy induction, and how these regulatory processes are critically involved in the development of allergic inflammation. Mice deficient in the E3 ubiquitin ligase Itch develop severe airway inflammation in response to antigen challenge. However, the detailed molecular mechanisms remain unclear. Here we found that Itch plays a critical role in the development of Tregs in the periphery. Itch^-/-CD4⁺CD25^- naïve T cells are resistant to TGF-β-mediated suppression and fail to express Foxp3. Retroviral transduction of the negative modulator Smad7 into Itch^+/+CD4⁺CD25^- cells mimics Itch deficiency. In addition, ectopic FoxP3 expression induces the inhibitory function of Itch^-/-CD4⁺CD25^-cells. Adoptive transfer of FoxP3-transduced OVA-specific Itch^-/-Th2 cells prevents airway inflammation. The results suggest an important role for Itch in regulating TGF-β-induced Treg generation and allergic responses.

Development of Immune Systems by ITAM Receptors and Their Role in Host Defense

Sho Yamasaki

ITAM is immunoreceptor tyrosine-based activation motif that is identified in the cytoplasmic region of many immune receptors such as T cell receptor (TCR), B cell receptor (BCR), Fc receptor (FcR) or NK receptors (NKR). Upon stimulation, tyrosine residues in ITAM are phosphorylated by Src family protein tyrosine kinases (PTKs) and deliver downstream signals through recruiting Syk family PTKs. ITAM-coupled receptors is often called "multi-task" receptor because they have ability to discriminate the quality of the ligand. TCR and BCR can recognize the difference of antigens to determine multiple cellular responses. Recently, some C-type lectin is reported to utilize ITAM as well. Among them is Macrophage-inducible C-type lectin (Mincle). We found that Mincle selectively associated with the Fc receptor γ-chain (FcRγ) and activated macrophages to produce inflammatory cytokines/chemokines. Mincle-expressing cells were activated in the presence of dead cells, and a component of small nuclear ribonucleoprotein (snRNP) was identified as a Mincle ligand that is released from dead cells. We induced thymocyte death by irradiating mice and found that neutrophil infiltration into the thymus was blocked by injection of anti-Mincle mAb. Thus, Mincle is a receptor that senses non-homeostatic cell death and thereby induces the production of inflammatory cytokines to drive the infiltration of neutrophils into damaged tissue. In addition, we have recently found that Mincle also recognizes non-self pathogenic fungus. Thus, Mincle may function as a dual receptor for the danger; "damaged self" and "invading non-self". The physiological consequences of the recognition of these ligands by Mincle will be discussed.

Genetics and Biochemistry of Endogenous Glycosphingolipid Antigens for NKT Cells

Yunsen Li, Prakash Thapa, David Hawke, Yuji Kondo, Keiko Furukawa, Koichi Furukawa, Fong-Fu Hsu, Dietlind Adlercreutz, Joel Weadge, Monica M Palcic, Peng G. Wang, Steven B Levery and Dapeng Zhou

Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, which exert critical regulatory functions in the innate arm of immunity. Pathological activation of NKT cells is a causative factor in mouse models of cancer, asthma, and autoimmune diseases. The thymic development and peripheral activation of NKT cells are dependent on their recognition of natural ligands presented by CD1d, a non-polymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that β-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. Furthermore, we and others have found a β-linked glycosphingolipid, isoglobotriaosylceramide (iGb3) is a stimulatory NKT ligand. The iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. In this study, we have performed a glycosphingolipidomics study of mouse thymus, and studied mice mutants which are deficient in β-hexosaminidase b or α-galactosidase A, two glycosidases that are up- and down-stream agents of iGb3 turnover, respectively. Our mass spectrometry methods generated a first database for glycosphingolipids expressed by mouse thymus, which are specifically regulated by rate-limiting glycosidases. Of identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large scale fractionation, enrichment and characterization of minor species of glycosphingolipids, be necessary for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids.

New Insights into Aggrecan and Collagen Degradation

Amanda J Fosang, Stephanie J Gauci and Leonie M Tutolo

Accelerated catabolism of aggrecan and type II collagen is a feature of cartilage destruction in arthritis. ADAMTS-5 is the major aggrecanase in mouse cartilage and MMP-13 is the major cartilage collagenase in several species including humans. One approach to studying the activity of these enzymes is to mutate the substrates, rendering them enzyme-resistant. We have generated the Bailey mouse with type II collagen resistant to collagenase cleavage and the Jaffa mouse with aggrecan resistant to ADAMTS cleavage in the interglobular domain.Degradation of fibrillar collagens is initiated by collagenase cleavage at a highly conserved site in the triple helix. We mutated the mouse col2a1 gene to change amino acids PQG⁷⁷⁵↓⁷⁷⁶LAG to PPG⁷⁷⁵↓⁷⁷⁶MPG in collagen II. Bailey collagen II is resistant to all collagenases. The Jaffa mouse whose aggrecan is resistant to ADAMTS cleavage was made by mutating the agc1 gene to change amino acids EGE³⁷³↓³⁷⁴ALG to EGE³⁷³↓³⁷⁴ NVY. Aggrecanases do not recognise this sequence as a cleavage site. The enzyme-resistant Jaffa and Bailey mice offer distinct advantages over the ADAMTS-5 and MMP-13 null mice for studying aggrecanolysis and collagenolysis, because the consequences of targeted mutations in the aggrecan or collagen substrates are not confounded by the effects of null mutations in enzymes, on other substrates, or compensation by other enzymes. We have compared the extent of aggrecan loss and cartilage erosion in inflammatory arthritis, between Jaffa and Bailey mice. Insights gleaned from comparing aggrecanolysis with collagenolysis will help prioritise strategies for blocking cartilage erosion in arthritic disease.