Nicolai S. C. van Oers

ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR ζ in murine thymocytes and lymph node T cells

Studies with T cell lines and clones have shown that engagement of the TCR results in the tyrosine phosphorylation of the TCR subunits. This leads to the recruitment of the ZAP-70 protein tyrosine kinase, an interaction involving the two SH2-domains of ZAP-70 with tyrosine-phosphorylated zeta and CD3. However, as previously described, murine thymocytes and lymph node T cells express a constitutively tyrosine-phosphorylated zeta subunit in the basal state. Here, we show that a fraction of ZAP-70 molecules are constitutively associated with tyrosine-phosphorylated zeta. TCR ligation promotes a large increase in the tyrosine phosphorylation of ZAP-70 as well as other TCR subunits. Genetic studies reveal that the constitutive ZAP-70 association with tyrosine-phosphorylated zeta does not absolutely require either TCR or coreceptor interactions with MHC molecules.

Scalable signaling mediated by T cell antigen receptor–CD3 ITAMs ensures effective negative selection and prevents autoimmunity

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

The Antibiotic-Resistance Arrow of Time: Efflux Pump Induction is a General First Step in the Evolution of Mycobacterial Drug-Resistance

ABSTRACT We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an “antibiotic resistance arrow of time.”

The 21- and 23-kD forms of TCRζ are generated by specific ITAM phosphorylations

The T cell receptor (TCR) ζ subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCRζ is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCRζ.

Spatiotemporal patterning during T cell activation is highly diverse.

The timing and the distribution of signaling intermediates reflect the efficiency and nature of T cell receptor activation. Right Times and Right Places Signals downstream of receptor activation can be effectively regulated by controlling both the distribution of signaling intermediates within the cell and the time at which they get to their destinations. Such spatiotemporal patterning of receptors, kinases, and adaptor molecules can have a substantial effect on their local concentrations and on the probabilities that they will interact with each other. Previous studies have characterized the patterning of individual or small groups of molecules; however, systems-level analyses of such processes are lacking. Singleton et al. have studied the spatiotemporal patterning of some 30 signaling intermediates in various mouse T cells activated by antigen-presenting cells under different conditions. As well as establishing that the patterning of these molecules regulates the efficiency of T cell receptor signaling, this study also shows that different T cell activation conditions generate different spatiotemporal patterns. Temporal and spatial variations in the concentrations of signaling intermediates in a living cell are important for signaling in complex networks because they modulate the probabilities that signaling intermediates will interact with each other. We have studied 30 signaling sensors, ranging from receptors to transcription factors, in the physiological activation of murine ex vivo T cells by antigen-presenting cells. Spatiotemporal patterning of these molecules was highly diverse and varied with specific T cell receptors and T cell activation conditions. The diversity and variability observed suggest that spatiotemporal patterning controls signaling interactions during T cell activation in a physiologically important and discriminating manner. In support of this, the effective clustering of a group of ligand-engaged receptors and signaling intermediates in a joint pattern consistently correlated with efficient T cell activation at the level of the whole cell.

PTPH1 Is a Predominant Protein-tyrosine Phosphatase Capable of Interacting with and Dephosphorylating the T Cell Receptor ζ Subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor ζ subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor ζ subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR ζ substrate (specific activity ranging from 0.23 to 40 pmol/min/μg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-ζ. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ζ ITAMs, with PTPH1 directly dephosphorylating ζ. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR ζ.

The CD3 ζ Subunit Contains a Phosphoinositide-Binding Motif That Is Required for the Stable Accumulation of TCR–CD3 Complex at the Immunological Synapse

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell–APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS–phosphoinositide interactions in supporting T cell activation.

The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions

The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology.

The formation and functions of the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits.

The interaction between the T cell receptor (TCR) and its cognate antigen/major histocompatibility complex (MHC) complex activates a cascade of intracellular protein phosphorylations within the T cell. The signals are initiated by the specific phosphorylation of two tyrosine residues located in a conserved sequence motif termed an ITAM (immune receptor‐based tyrosine activation motif). There are 10 ITAMs in the TCR complex, and 6 of these ITAMs are present in the TCR ζ homodimer. Following TCR stimulation, the TCR ζ subunit forms two tyrosine‐phosphorylated intermediates of 21‐ and 23‐kDa, respectively. The dramatic and diverse biological responses of T cells are proposed to be partly regulated by the relative ratios of the 21‐ vs. 23‐kDa phosphorylated forms of TCR ζ that are induced following TCR ligation. In this review, we describe a stepwise model of ζ phosphorylation required for the formation of these two phosphorylated derivatives. We describe the kinases and phosphatases controlling these phosphorylation processes. In addition, we present some preliminary findings from ongoing studies that discuss the contributions of each phosphorylated form of ζ on T cell development, TCR signaling, T cell anergy induction, and T cell survival.

SYK MUTATION IN JURKAT E6-DERIVED CLONES RESULTS IN LACK OF P72SYK EXPRESSION

The human leukemic Jurkat cell line is commonly used as a model cellular system to study T lymphocyte signal transduction. Various clonal derivatives of Jurkat T cells exist which display different characteristics with regard to responses to external stimuli. Among these, the E6-1 clone of Jurkat T cells has been used as a parental line from which numerous important somatic mutant clones have been generated. During the course of experiments examining signals initiated by the T cell antigen receptor in an E6-1-derived Jurkat cell clone J.CaM1, we observed that the 72-kilodalton Syk protein tyrosine kinase previously found in other Jurkat cells was not detected. Upon further analysis it was determined that Syk transcripts from the J.CaM1 cells as well as the parental E6-1 cells contain a single guanine nucleotide insertion at position 92. This nucleotide insertion results in a shift in the Syk open reading frame leading to alternate codon usage as well as the generation of a termination codon at position 109. Thus, Syk transcripts in E6-1 cells and E6-1-derived clones are predicted to be capable of encoding only the first 33 amino acids of the 630-amino acid wild type Syk. These findings are incompatible with a recently proposed model of T cell antigen receptor signal transduction based, in part, on experiments conducted using E6-1-derived cells, suggesting that Syk might play a role upstream of Lck and Zap70.