Christopher E. Rudd

Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling

Many studies have shown the central importance of the co-receptors CD28, inducible costimulatory molecule (ICOS) and cytotoxic T lymphocyte antigen 4 (CTLA4) in the regulation of many aspects of T-cell function. CD28 and ICOS have both overlapping and distinct functions in the positive regulation of T-cell responses, whereas CTLA4 negatively regulates the response. The signalling pathways that underlie the function of each of the co-receptors indicate their shared and unique properties and provide compelling hints of functions that are as yet uncovered. Here, we outline the shared and distinct signalling events that are associated with each of the co-receptors and provide unifying concepts that are related to signalling functions of these co-receptors.

ADAP–SLP-76 Binding Differentially Regulates Supramolecular Activation Cluster (SMAC) Formation Relative to T Cell–APC Conjugation

T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production.

T-cell signalling and immune system disorders.

T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell proliferation, cytokine production and differentiation into effector cells. These cascades comprise an array of protein-tyrosine kinases, phosphatases, GTP-binding proteins and adaptor proteins that regulate generic and specialised functions. The integration of these signals is essential for the normal development, homeostasis and function of T cells. Defects in a single mediator can produce T cells that are unable to participate fully in an immune response and/or that mount an inappropriate response, which leads to immunodeficiency, autoimmunity or leukaemia/lymphomas. This review highlights some of the key players in T-cell signalling and their involvement in the development of various clinical disease states. Some of these immune-specific signalling proteins are attractive potential targets in the development of therapies to augment T-cell responses to antigen or tumours, and to treat immune cell disorders.

Membrane Rafts Play a Crucial Role in Receptor Activator of Nuclear Factor κB Signaling and Osteoclast Function

Membrane lipid rafts play a key role in immune cell activation by recruiting and excluding specific signaling components of immune cell surface receptors upon the receptor engagement. Despite this, the role of these microdomains in the regulation of osteoclasts as controlled by receptor activator of nuclear factor κB (RANK) has yet to be established. In this study, we demonstrate that the raft microdomain expression plays an essential role in osteoclast function and differentiation. Expression of raft component flotillin greatly increased during osteoclast differentiation, whereas engagement of RANK induced the translocation of tumor necrosis factor receptor-associated factor 6 to rafts where Src was constitutively resident. Disruption of rafts blocked TRAF6 translocation and Akt activation by RANK ligand in osteoclasts and further reduced the survival of osteoclasts. Actin ring formation and bone resorption by osteoclasts were also found to require the integrity of rafts. Our observations demonstrate for the first time that RANK-mediated signaling and osteoclast function are critically dependent on the expression and integrity of raft membrane microdomains.

Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Isoforms Alternatively Inhibit and Costimulate Human T Cell Function

Carcinoembryonic Ag-related cellular adhesion molecule 1 (CEACAM1) represents a group of transmembrane protein isoforms that consist of variable numbers of extracellular Ig-like domains together with either a long cytoplasmic (cyt) tail containing two immunoreceptor tyrosine-based inhibitory motifs or a unique short cyt tail. Although CEACAM1 has been reported to be expressed on the surface of T lymphocytes upon activation, its roles in T cell regulation are controversial due to the lack of functional characterization of each individual CEACAM1 isoform. We thus cotransfected Jurkat T cells with CEACAM1 isoform-encoding constructs and an IL-2 promoter-bearing plasmid or a small interference RNA targeting src homology domain 2 containing phosphatase 1. In a luciferase reporter assay and through measurements of cytokine secretion (IL-2, IL-4, and IFN-γ), CEACAM1 containing either a long or a short cyt tail inhibited or costimulated, respectively, TCR/CD3 complex plus CD28 mediated activation with the inhibitory functions of the long cyt tail dominating. The inhibitory function of CEACAM1, was dependent upon src homology domain 2 containing phosphatase 1 activity, required both tyrosine residues within the immunoreceptor tyrosine-based inhibitory motif domains of the cyt tail and was mediated through the mitogen-activated protein kinase pathway. CEACAM1-mediated inhibition could be functionally reconstituted by incubation of PBMC with either a CEACAM1-specific mAb or CEACAM1-Fc fusion protein in the presence of an allogeneic or mitogenic stimulus, respectively. These studies indicate that the long and short cyt tails of CEACAM1 serve as inhibitory and costimulatory receptors, respectively, in T cell regulation.

Lnk Adaptor: Novel Negative Regulator of B Cell Lymphopoiesis

Originally thought to have the functions now ascribed to the linker for activation of T cells protein (LAT), Lnk is coming into its own as an adaptor protein that mediates signaling through several receptor pathways. An essential role for Lnk in B cell development and maturation was recently uncovered by Perlmutter and colleagues. Rudd discusses the role of Lnk in B cells and hypothesizes a mechanism whereby Lnk, and its closely related protein family members, the adaptor molecules containing pleckstrin homology (PH) and Src-homology 2 (SH2) domains (APS), and Src-homology 2-B protein (SH2-B), may mediate signal promotion or attenuation.

Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)‐mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25– T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti‐CD3 cross‐linking resulted in a lower calcium influx and extracellular signal‐regulated kinase 1/2 phosphorylation. Examination of the CD3ζ chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of ζ‐associated protein‐70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain‐containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells.

MAPK p38: alternative and nonstressful in T cells

The MAP kinase p38 is normally regulated by the MAPKKK-MAPKK pathway in mammalian cells. However, analysis of T cell signaling shows an alternative pathway for p38 activation exists.

Hematopoietic Adaptors in T-Cell Signaling: Potential Applications to Transplantation

Recent advances have been made in understanding the basis of T‐cell signaling with the identification of hematopoeitic‐specific adaptor proteins, or molecular scaffolds that facilitate protein complex formation and the integration of signals from the surface of T cells. Their potential relevance as targets in the modulation of transplantation relates to their immune‐cell‐specific expression and their ability to integrate signals needed for T‐cell/APC conjugate formation, cytokine production and the clonal expansion of T cells. While LAT, GADS and SLP‐76 are needed for TcR‐induced cytokine production, the adaptors ADAP, VAV and SKAP‐55 play specialized roles in the regulation of integrin adhesion and conjugation. Given the importance of these functions to the reactivity of T cells to allodeterminants of tissue grafts (GvH), and in the recognition and destruction of leukemic cells (GvL), these adaptors represent a new generation of potential targets in the modulation of transplantation.