Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation.

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

A sequence conserved between CD5 and CD6 binds an FERM domain and exerts a restraint on T-cell activation.

CD5 and CD6 are related surface receptors that limit and promote T-cell responses. Co-stimulatory effects of CD6 depend on binding a cell surface ligand, CD166, and recruitment of the intracellular adaptor proteins GADS and SLP-76 by C-terminal phosphotyrosines. We have continued to identify interactions of CD5 and CD6 to understand their roles in T-cell activation. In a screen to identify binding partners for peptides containing a cytoplasmic sequence, SDSDY conserved between CD5 and CD6, we identified ezrin radixin moesin (ERM) proteins, which link plasma membrane proteins to actin. Purified radixin FERM domain bound directly to CD5 and CD6 SDSDY peptides in a phosphorylation-dependent manner (KD = 0·5-2 µm) at 37°. In human T-cell blasts, mutation of the CD6 SDSDY sequence enhanced CD69 expression in response to CD3 monoclonal antibody. In this proximal readout, interactions of the SDSDY sequence were dominant compared with the C-terminal tyrosines of CD6. In contrast, in a more downstream readout, interleukin-2 expression, in response to immobilized CD3 and CD6 monoclonal antibodies, the C-terminal tyrosines were dominant. The data suggest that varying functional effects of CD6 and potentially CD5 depend on interactions of different cytoplasmic regions with the cytoskeleton and alter depending on the stimuli.

Addition of the C-terminus of CD6 to a chimeric antigen receptor enhances cytotoxicity and does not compromise expression.

T cells expressing chimeric antigen receptors (CARs) are a promising new cancer immunotherapy that has now reached the clinic. CARs are synthetic receptors that redirect T cells towards a tumour-associated antigen and activate them through various fused signalling regions, for example derived from CD3ζ, 4-1BB or CD28. Analysis of the optimal combination of CAR components including signalling domains is not yet comprehensive and may vary with the particular application. The C-terminus of the T-cell surface receptor CD6 is critical for its co-stimulatory effects and signals through two phospho-tyrosine motifs that bind to the intracellular adaptor proteins GADS and SLP-76. Addition of the C terminus of CD6 did not compromise CAR expression, showing it was a stable moiety that can be used independently of the native receptor. A third-generation CAR containing 4-1BB, CD3ζ and the C terminus of CD6 (4-1BBz-CD6) enhanced interferon-γ release and cytotoxicity when compared with the second-generation 4-1BB CD3ζ (4-1BBz) CAR. The CD6 C terminus is a valuable addition to potential components for modular design of CARs to improve effector function, particularly cytotoxicity.

Domain-specific CD6 monoclonal antibodies identify CD6 isoforms generated by alternative-splicing.

The characterization of the architecture, structure and extracellular interactions of the CD6 glycoprotein, a transmembrane receptor expressed in medullary thymocytes and all mature T-cell populations, has been enhanced by the existence of monoclonal antibodies (mAbs) that specifically recognize the various scavenger receptor cysteine-rich (SRCR) domains of the ectodomain. Using engineered isoforms of CD6 including or excluding each of the three SRCR domains, either expressed at the membranes of cells or in soluble forms, we provide conclusive and definitive evidence that domain 2 of CD6, previously not identifiable, can be recognized by the CD6 mAbs OX125 and OX126, and that OX124 targets domain 3 and can block the interaction at the cell surface of CD6 with its major ligand CD166. Alternative splicing-dependent CD6 isoforms can now be confidently identified. We confirm that following T-cell activation there is a partial replacement of full-length CD6 by the CD6Δd3 isoform, which lacks the CD166-binding domain, and we find no evidence for the expression of other CD6 isoforms at the mRNA or protein levels.

CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action.

CD6 is a type I T-cell surface receptor that modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab™), which has reached the clinic for treatment of autoimmune disease. We characterized molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action. Epitope mapping using the crystal structure of CD6 to design mutants identified two distinct binding sites on different faces of domain 1, one containing residue R77, crucial for MT605 and T12.1 binding and the other, E63, which is crucial for itolizumab and MEM98. Analysis of binding kinetics revealed that itolizumab has a lower affinity compared with other CD6 domain 1 mAbs. We compared potential agonistic (triggering) and antagonistic (blocking) properties of CD6 mAbs in assays where the mechanism of action was well defined. CD6 domain 1 and 3 mAbs were equally effective in triggering interleukin-2 production by a cell line expressing a chimeric antigen receptor containing the extracellular region of CD6. CD6 domain 1 mAbs hindered binding of multivalent immobilized CD166 but were inferior compared with blocking by soluble CD166 or a CD6 domain 3 mAb. Characterization of CD6 mAbs provides an insight into how their functional effects in vivo may be interpreted and their therapeutic use optimized.

The SIRP family of receptors and immune regulation.

The immune system must be highly regulated to obtain optimal immune responses for the elimination of pathogens without causing undue side effects. This tight regulation involves complex interactions between membrane proteins on leukocytes. Members of the signal-regulatory protein (SIRP) family, which are expressed mainly by myeloid cells, provide one example of these regulatory membrane proteins. There are three SIRP-family genes that encode proteins that have similar extracellular regions but different signalling potentials, and are therefore known as 'paired receptors'. In this Review, we describe recent studies defining the ligands of the SIRP-family members, with particular emphasis on relating the molecular interactions of these proteins to their role in immune-cell regulation.

A polymorphism in a phosphotyrosine signalling motif of CD229 (Ly9, SLAMF3) alters SH2 domain binding and T-cell activation.

Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome-wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val602 variant of the non-synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two-fold lower affinity compared with the SLE-associated Met602 variant for the small adaptor protein SAP. Comparison of the two variants in T-cell lines revealed the Val602 variant to be significantly more highly expressed than CD229 Met602 . Activation was diminished in cells expressing CD229 Val602 compared with CD229 Met602 as measured by up-regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine-tuning in the immune system with significant functional consequences.

Signal-regulatory protein α from the NOD mouse binds human CD47 with an exceptionally high affinity-- implications for engraftment of human cells.

One common way to study human leucocytes and cancer cells in an experimental in vivo situation is to use mice that have been genetically engineered to lack an immune system and prevent human cell rejection. These mice lack CD132 and either RAG2 or the catalytic subunit of the DNA-dependent protein kinase, to make the mice deficient in lymphocytes and natural killer cells. The NOD mouse strain provides a better background for engraftment than other strains due to stronger engagement of the signal-regulatory protein-α (SIRPα) inhibitory receptor with human CD47 (hCD47) resulting in a 'don't-eat-me' signal. To determine the molecular parameters that determine this major functional effect in the NOD mouse we measured the affinity of hCD47 for SIRPα from various mouse strains. Human CD47 bound SIRPα from the NOD mouse with an affinity 65 times greater than SIRPα from other mouse strains. This is due mainly to the NOD SIRPα lacking two amino acids in domain 1 compared with other mouse strains. Remarkably the SIRPα(NOD) binds hCD47 with 10 times the affinity of the syngeneic hCD47/hSIRPα interaction. This affinity is outside the normal range for affinities for leucocyte surface protein interactions and raises questions as to what is the optimal affinity of this interaction for engraftment and what other xenogeneic interactions involved in homeostasis may also not be optimal.

Cytomegalovirus e127 protein interacts with the inhibitory CD200 receptor.

CD200 is a cell surface glycoprotein that binds an inhibitory receptor (CD200R) on myeloid cells. CD200 orthologues are present in many species of virus, and we show that the rat cytomegalovirus CD200 orthologue (e127) is expressed at the cell surface on infected cells. It binds the host CD200R with the same affinity as that of the host protein, and thus this protein acts as a close mimic of the host protein and has the potential to downregulate immune responses to the virus.

A ligand for CD5 is CD5.

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro functional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions.

Downstream of tyrosine kinase 1 and 2 play opposing roles in CD200 receptor signaling.

The CD200 receptor (CD200R) negatively regulates myeloid cells by interacting with its widely expressed ligand CD200. CD200R signals through a unique inhibitory pathway involving a direct interaction with the adaptor protein downstream of tyrosine kinase 2 (Dok2) and the subsequent recruitment and activation of Ras GTPase-activating protein (RasGAP). Ligand engagement of CD200R also results in tyrosine phosphorylation of Dok1, but this protein is not essential for inhibitory CD200R signaling in human myeloid cells. In this paper, we show that CD200R-induced phosphorylation of Dok2 precedes phosphorylation of Dok1, and that Dok2 and Dok1 recruit different downstream proteins. Compared with Dok2, Dok1 recruits substantially less RasGAP. In addition to binding RasGAP, Dok2 recruits the adaptor molecule Nck in response to ligand engagement of CD200R. CD200R-induced phosphorylation of Dok1 results in the recruitment of CT10 sarcoma oncogene cellular homologue-like (CrkL), whereas the closely related CT10 sarcoma oncogene cellular homologue interacts constitutively with Dok1. Knockdown of Dok1 or CrkL expression in U937 cells resulted in increased Dok2 phosphorylation and RasGAP recruitment to Dok2. These data are consistent with a model in which Dok1 negatively regulates Dok2-mediated CD200R signaling through the recruitment of CrkL.

Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells.

The CD200 receptor (CD200R) acts as a negative regulator of myeloid cells by interacting with its widely expressed ligand CD200. Using mutants expressed in U937 cells, we show that inhibition is mediated by the PTB domain binding motif (NPLY) in the receptor's cytoplasmic region. The adaptor protein downstream of tyrosine kinase 2 (Dok2) bound directly to the phosphorylated NPLY motif with a 10-fold higher affinity (K(D) of approximately 1 microM at 37 degrees C) than the closely related Dok1. Both of these proteins have been suggested to play a role in CD200R signaling in murine cells. Dok2 was phosphorylated in response to CD200R engagement and recruited RAS p21 protein activator 1 (RasGAP). Knockdown of Dok2 and RasGAP by RNA interference revealed that these proteins are required for CD200R signaling, while knockdown of Dok1 and the inositol 5-phosphatase SHIP did not affect CD200R-mediated inhibition. We conclude that CD200R inhibits the activation of human myeloid cells through direct recruitment of Dok2 and subsequent activation of RasGAP, which distinguishes this receptor from the majority of inhibitory receptors that utilize ITIMs and recruit phosphatases.

T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand.

The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR-pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial. It has been proposed that TCR triggering is promoted by segregation at the T cell-APC interface of cell-surface molecules with small ectodomains (such as TCR-pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation. A similar dependence on receptor-ligand complex dimensions was observed with artificial TCR-ligand systems that span the same dimensions as the TCR-pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR-pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering.

Inhibition and activation by CD244 depends on CD2 and phospholipase C-gamma1.

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-gamma1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.

CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76.

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 muM at 37 degrees C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5.

Signal regulatory protein alpha ligation induces macrophage nitric oxide production through JAK/STAT- and phosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/H2O2-dependent pathways.

Signal regulatory protein alpha (SIRPalpha) is a glycoprotein receptor that recruits and signals via the tyrosine phosphatases SHP-1 and SHP-2. In macrophages SIRPalpha can negatively regulate the phagocytosis of host cells and the production of tumor necrosis factor alpha. Here we provide evidence that SIRPalpha can also stimulate macrophage activities, in particular the production of nitric oxide (NO) and reactive oxygen species. Ligation of SIRPalpha by antibodies or soluble CD47 triggers inducible nitric oxide synthase expression and production of NO. This was not caused by blocking negative-regulatory SIRPalpha-CD47 interactions. SIRPalpha-induced NO production was prevented by inhibition of the tyrosine kinase JAK2. JAK2 was found to associate with SIRPalpha in macrophages, particularly after SIRPalpha ligation, and SIRPalpha stimulation resulted in JAK2 and STAT1 tyrosine phosphorylation. Furthermore, SIRPalpha-induced NO production required the generation of hydrogen peroxide (H(2)O(2)) by a NADPH oxidase (NOX) and the phosphatidylinositol 3-kinase (PI3-K)-dependent activation of Rac1, an intrinsic NOX component. Finally, SIRPalpha ligation promoted SHP-1 and SHP-2 recruitment, which was both JAK2 and PI3-K dependent. These findings demonstrate that SIRPalpha ligation induces macrophage NO production through the cooperative action of JAK/STAT and PI3-K/Rac1/NOX/H(2)O(2) signaling pathways. Therefore, we propose that SIRPalpha is able to function as an activating receptor.

T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex.

The cell surface receptor CD6 regulates T cell activation in both activating and inhibitory manners. The adaptor protein SLP-76 is recruited to the phosphorylated CD6 cytoplasmic Y662 residue during T cell activation, providing an activating signal to T cells. In this study, a biochemical approach identified the SH2 domain-containing adaptor protein GADS as the dominant interaction partner for the CD6 cytoplasmic Y629 residue. Functional experiments in human Jurkat and primary T cells showed that both mutations Y629F and Y662F abolished costimulation by CD6. In addition, a restraint on T cell activation by CD6 was revealed in primary T cells expressing CD6 mutated at Y629 and Y662. These data are consistent with a model in which bivalent recruitment of a GADS/SLP-76 complex is required for costimulation by CD6.

CD6 as a Cell Surface Receptor and As a Target for Regulating Immune Responses.

CD6 has been exploited as a drug target as its expression is restricted, primarily to T cells, it has a well characterised cell surface ligand, CD166 and regulates T cell activation through a long cytoplasmic tail. CD6 can affect both the initiation and maintenance of T cell function in a negative and positive manner respectively so that it is important to understand these dual effects of a potential drug target. The effective mode of action of clinical monoclonal antibodies (mAbs) that recognise cell surface receptors including CD6 is commonly cytotoxic depletion of cells. It is not clear how current therapeutic strategies to target CD6 perturb function. With the benefit of new structural data, this review provides a critical analysis and interpretation of experiments in which various reagents have been tested and offers some suggestions as how more effective drugs may be developed.