Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes.

Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex.

Distinct signaling pathways regulate TLR2 co-stimulatory function in human T cells.

Toll-like receptor 2 (TLR2) serves as a co-stimulatory receptor for human T cells by enhancing T cell receptor (TCR)-induced cytokine production and proliferation. However, it is unknown where signals from the TCR and TLR2 converge to enhance T cell activation. To address this gap, we examined changes in TCR-induced signaling following concurrent TLR2 activation in human T cells. Both proximal TCR-mediated signaling and early NFκB activation were not enhanced by TCR and TLR2 co-activation, potentially due to the association of TLR2 with TLR10. Instead, TLR2 co-induction did augment Akt and Erk1/Erk2 activation in human T cells. These findings demonstrate that TLR2 activates distinct signaling pathways in human T cells and suggest that alterations in expression of TLR2 co-receptors may contribute to aberrant T cell responses.

Examiner reliability of fluorosis scoring: a comparison of photographic and clinical examination findings.

OBJECTIVE: To assess and compare examiner reliability of clinical and photographic fluorosis examinations using the Fluorosis Risk Index (FRI) among children in the Iowa Fluoride Study (IFS). METHODS: The IFS examined 538 children for fluorosis and dental caries at age 13 and obtained intraoral photographs from nearly all of them. To assess examiner reliability, duplicate clinical examinations were conducted for 40 of the subjects. In addition, 200 of the photographs were scored independently for fluorosis by two examiners in a standardized manner. Fluorosis data were compared between examiners for the clinical exams and separately for the photographic exams, and a comparison was made between clinical and photographic exams. For all three comparisons, examiner reliability was assessed using kappa statistics at the tooth level. RESULTS: Interexaminer reliability for the duplicate clinical exams on the sample of 40 subjects as measured by kappa was 0.59, while the repeat exams of the 200 photographs yielded a kappa of 0.64. For the comparison of photographic and clinical exams, interexaminer reliability, as measured by weighted kappa, was 0.46. FRI scores obtained using the photographs were higher on average than those obtained from the clinical exams. Fluorosis prevalence was higher for photographs (33 percent) than found for clinical exam (18 percent). CONCLUSION: Results suggest that interexaminer reliability is greater and fluorosis scores are higher when using photographic compared with clinical examinations.

Comparison of T cell receptor-induced proximal signaling and downstream functions in immortalized and primary T cells.

BACKGROUND: Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCgamma1, Vav1, and Erk1/Erk2 and substantially more Ca2+ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation. CONCLUSIONS/SIGNIFICANCE: Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line.

PI3 kinase function is vital for the function but not formation of LAT-mediated signaling complexes.

The induction of the T cell receptor (TCR) is necessary for the activation and function of human T cells. TCR activation results in the tyrosine phosphorylation of LAT, leading to the direct interaction with several proteins, including PLC-gamma 1, Grb2 and Gads. These direct ligands then mediate the indirect interaction of LAT with proteins, such as SLP-76, Vav1 and Itk. PLC-gamma 1, Vav1 and Itk contain pleckstrin homology (PH) domains that interact with the enzymatic product of phosphoinositide-3-kinase (PI3K), suggesting the function of PI3K may modulate LAT-mediated complexes. Therefore, we characterized the poorly understood role of PI3K activity in the formation and function of multiprotein signaling complexes that form at LAT. Inhibition of PI3K catalytic function had little effect on the phosphorylation of LAT, SLP-76, Vav1 or PLC-gamma 1 or on the ability of PLC-gamma 1 to interact with LAT or SLP-76. However, PI3K activity appeared to be required for the induction of downstream signaling events. These data indicate that the formation of LAT-mediated complexes do not appear to depend on PI3K activity, whereas the optimal downstream function of these complexes requires the catalytic function of PI3K.