Paul M. Allen

Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.

The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.

A Novel Adaptor Protein Orchestrates Receptor Patterning and Cytoskeletal Polarity in T-Cell Contacts

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.

Partial T cell signaling: Altered phospho-ζ and lack of zap70 recruitment in APL-induced T cell anergy

Studies of T cell responses to altered peptide ligands (APLs) have provided functional evidence that a T cell receptor (TCR) can interpret subtle changes in its ligand, resulting in different phenotypic outcomes. One dramatic effect of APL stimulation with live antigen-presenting cells (APCs) is the induction of energy as opposed to proliferation. We investigated the intracellular signaling events involved in generating this unresponsiveness by comparing protein-tyrosine phosphorylation patterns after stimulation with anergy-inducing APL or the immunogenic peptide. In resting T cell clones, presentation with APL/live APC stimulated a unique pattern of TCR phospho-zeta species and a subsequent lack of association with zap70. This demonstrates that the TCR-CD3 complex can engage selective intracellular biochemical signaling pathways as a direct consequence of the nature of the ligand recognized and the initial phosphotyrosine pattern of the TCR-CD3 proteins, leading to different phenotypes.

Essential Role of Neutrophils in the Initiation and Progression of a Murine Model of Rheumatoid Arthritis

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/B×N mouse RA model, transfer of autoreactive Ig from the K/B×N mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/B×N serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/B×N transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/B×N serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91phox genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.

Tickling the TCR: selective T-cell functions stimulated by altered peptide ligands

Recent observations of T-cell responses following T-cell receptor (TCR) interaction with altered peptide ligands have highlighted the complexity of this signalling system. The indications are that the TCR responds to minor changes in ligand with gradations of T-cell activation and effector functions. Brian Evavold, Joanne Sloan-Lancaster and Paul Allen review these studies and present a model in which partial T-cell activation and TCR antagonism are related events in a continuum of signalling through the TCR.

HIGH- AND LOW-POTENCY LIGANDS WITH SIMILAR AFFINITIES FOR THE TCR : THE IMPORTANCE OF KINETICS IN TCR SIGNALING

We have examined binding characteristics for a single TCR interacting with five of its different peptide/MHC ligands using surface plasmon resonance. We find that very small structural changes produce ligands with similar equilibrium binding affinities (K(D)) for the TCR, but vastly different potencies for T cell activation. Ligands with similar K(D)s induce similar amounts of total phospho-zeta but distinct patterns of zeta phosphorylation. Lower potency ligands induce only incomplete phosphorylation of TCR zeta and generally have faster off-rates. Therefore, the potency of TCR ligands is primarily determined by the half-life of the TCR-ligand complex and the consequent ability to induce complete phosphorylation of zeta.

Eradication of Established Tumors by CD8+ T Cell Adoptive Immunotherapy

We generated the DUC18 T cell receptor transgenic mouse expressing an H-2Kd -restricted transgenic T cell receptor specific for the syngeneic CMS5 fibrosarcoma rejection antigen mutated ERK2(136-144). DUC18 mice were capable of specifically eliminating lethal CMS5 tumor challenges, and transfer of DUC18 splenocytes to naive nontransgenic recipients conferred protection from subsequent and established CMS5 tumor burdens. Eradication of established tumor burdens by adoptive transfer of DUC18 splenocytes was dose and time dependent. Transferred tumor-specific T cells remained functional in vivo and capable of rejecting small tumors even in the presence of large, established tumor burdens. These findings highlight the kinetic battle between tumor growth and the production of a tumor-specific response and have critical implications for effective adoptive immunotherapy.

Regulation of Lck activity by CD4 and CD28 in the immunological synapse

Although the Src family tyrosine kinase Lck is essential for T cell receptor (TCR) signaling, whether or how Lck is activated is unknown. Using a phosphospecific antiserum to Lck, we show here that Lck becomes autophosphorylated when T cells are stimulated by antigen-presenting cells (APCs). We found that TCR cross-linking alone could not stimulate Lck autophosphorylation and CD45 was not required for this process. Instead, the T cell accessory molecules CD4 and CD28 cooperated to induce autophosphorylation of Lck. CD4 recruited Lck to the T cell–APC interface, whereas CD28 sustained Lck activation. These data show how the multiple interactions afforded by the immunological synapse drive efficient and highly specific signaling.

Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease.

The intestinal microbiota is important for induction of inflammatory bowel disease (IBD). IBD is associated with complex shifts in microbiota composition, but it is unclear whether specific bacterial subsets induce IBD and, if so, whether their proportions in the microbiota are altered during disease. Here, we fulfilled Kochs postulates in host-genotype-specific fashion using a mouse model of IBD with human-relevant disease-susceptibility mutations. From screening experiments we isolated common commensal Bacteroides species, introduced them into antibiotic-pretreated mice, and quantitatively reisolated them in culture. The bacteria colonized IBD-susceptible and -nonsusceptible mice equivalently, but induced disease exclusively in susceptible animals. Conversely, commensal Enterobacteriaceae were >100-fold enriched during spontaneous disease, but an Enterobacteriaceae isolate failed to induce disease in antibiotic-pretreated mice despite robust colonization. We thus demonstrate that IBD-associated microbiota alterations do not necessarily reflect underlying disease etiology. These findings establish important experimental criteria and a conceptual framework for understanding microbial contributions to IBD.

Specific T cell recognition of minimally homologous peptides: Evidence for multiple endogenous Ligands

The T cell receptor (TCR) can interact with a spectrum of peptides as part of its ligand, including the immunogenic peptide, variants of this peptide,and apparently unrelated peptides. The basis of this broad specificity for ligand was investigated by substitution analysis of a peptide antigen and functional testing using a B cell apoptosis assay. A peptide containing as few as 1 aa in common with this peptide could stimulate a specific T cell response. Two endogenous ligands, an agonist and a partial agonist, were readily identified from a search of the SwissProt database, indicating that multiple endogenous ligands likely exist for a given T cell. These findings strongly support the concept that one TCR has the ability to interact productively with multiple different ligands, and provide evidence that such ligands exist in the endogenous peptide repertoire.