06-807 Sigma-AldrichAnti-LAT Antibody

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

Caspase/Granzyme B mediated protein degradation is involved in elimination of activated T cell receptor (TCR) signaling molecules during processes of thymocyte selection and maintenance of peripheral homeostasis of T cells. Key components of TCR signaling cassette including LAT undergo biological inactivation in response to pro-apoptotic or anergy inducing environmental stimuli. Although available Western immunoblotting-based techniques are appropriate for detection of protein degradation in bulk populations of target cells, quantitative assessment of this process at a single cell level requires a different approach. Here we report on a novel, flow cytometry-based method for assessment of LAT integrity. This method exploits a loss of an anti-LAT antibody epitope recognition following proteolytic degradation of C-terminal domain of the LAT. We show that the LAT degradation precedes phosphatidylserine translocation to the outer leaflet of the plasma membrane and thus may constitute an early marker of T cell apoptosis. When used in conjunction with multi-parameter flow cytometry, our method revealed that FoxP3(+)CD4(+)CD8(low) thymocytes i.e. precursors of thymus derived CD4(+) regulatory T cells, in contrast to Foxp3(-)CD4(+)CD8(low) thymocytes are resistant to LAT degradation in response to CD3ε crosslinking. This finding can be used as an additional marker for T regulatory cell lineage.

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγ1 and PLCγ2. It is known that PLCγ1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγ1/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγ1 single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγ1/PLCγ2 double-deficient T cells compared with PLCγ1 single-deficient T cells. TCR-mediated signal transduction, including Ca²⁺ mobilization and Erk activation, was further impaired in PLCγ1/PLCγ2 double-deficient relative to PLCγ1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγ1 heterozygous- and PLCγ2 homozygous-deficient (PLCγ1⁺/⁻PLCγ2⁻/⁻) relative to wild-type, PLCγ2 single-deficient (PLCγ2⁻/⁻), or PLCγ1 heterozygous-deficient (PLCγ1⁺/⁻) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection.

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

T lymphocyte activation requires signal 1 from the TCR and signal 2 from costimulatory receptors. For long-lasting immunity, growth and survival signals imparted through the Akt/protein kinase B (PKB) pathway in activated or effector T cells are important, and these can be strongly influenced by signaling from OX40 (CD134), a member of the TNFR superfamily. In the absence of OX40, T cells do not expand efficiently to Ag, and memory formation is impaired. How most costimulatory receptors integrate their signals with those from Ag through the TCR is not clear, including whether OX40 directly recruits PKB or molecules that regulate PKB. We show that OX40 after ligation by OX40L assembled a signaling complex that contained the adapter TNFR-associated factor 2 as well as PKB and its upstream activator phosphoinositide 3-kinase (PI3K). Recruitment of PKB and PI3K were dependent on TNFR-associated factor 2 and on translocation of OX40 into detergent-insoluble membrane lipid microdomains but independent of TCR engagement. However, OX40 only resulted in strong phosphorylation and functional activation of the PI3K-PKB pathway when Ag was recognized. Therefore, OX40 primarily functions to augment PKB signaling in T cells by enhancing the amount of PI3K and PKB available to the TCR. This highlights a quantitative role of this TNFR family second signal to supplement signal 1.

Cytotoxic lymphocytes kill target cells through polarized release of the content of lytic granules at the immunological synapse. In human NK cells, signals for granule polarization and for degranulation can be uncoupled: Binding of β(2) integrin LFA-1 to ICAM is sufficient to induce polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. In this study, we investigated the basis for this difference. IL-2-expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR ζ-chain, tyrosine kinase Syk, and phospholipase C-γ. Whereas CD16 activated Ca(2+) mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, phospholipase C-γ, and protein kinase C, as well as by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca(2+)-dependent degranulation and paxillin-dependent granule polarization.

Despite the defined function of the β-catenin pathway in thymocytes, its functional role in peripheral T cells is poorly understood. We report that in a mouse model, β-catenin protein is constitutively degraded in peripheral T cells. Introduction of stabilized β-catenin into primary T cells inhibited proliferation and cytokine secretion after TCR stimulation and blunted effector cell differentiation. Functional and biochemical studies revealed that β-catenin selectively inhibited linker for activation of T cells phosphorylation on tyrosine 136, which was associated with defective phospholipase C-γ1 phosphorylation and calcium signaling but normal ERK activation. Our findings indicate that β-catenin negatively regulates T cell activation by a previously undescribed mechanism and suggest that conditions under which β-catenin might be inducibly stabilized in vivo would be inhibitory for T cell-based immunity.

Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation.

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3 zeta and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.