The potential of ¹¹C-acetate PET for monitoring the Fatty acid synthesis pathway in Tumors.

Positron emission tomography (PET) is a molecular imaging modality that provides the opportunity to rapidly and non-invasively visualize tumors derived from multiple organs. In order to do so, PET utilizes radiotracers, such as ¹8F-FDG and ¹¹C-acetate, whose uptake coincides with altered metabolic pathways within tumors. Increased expression and activity of enzymes in the fatty acid synthesis pathway is a frequent hallmark of cancer cells. As a result, this pathway has become a prime target for therapeutic intervention. Although multiple drugs have been developed that both directly and indirectly interfere with fatty acid synthesis, an optimal means to assess their efficacy is lacking. Given that ¹¹Cacetate is directly linked to the fatty acid synthesis pathway, this probe provides a unique opportunity to monitor lipogenic tumors by PET. Herein, we review the relevance of the fatty acid synthesis pathway in cancer. Furthermore, we address the potential utility of ¹¹C-acetate PET in imaging tumors, especially those that are not FDG-avid. Last, we discuss several therapeutic interventions that could benefit from ¹¹C-acetate PET to monitor therapeutic response in patients with certain types of cancers.

The CD3 zeta 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)P(2), and PtdIns(3,4,5)P(3) 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 epsilon subunit contains a phospholipid-binding motif that regulates T cell functions.

The CD3 epsilon 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 epsilon, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 epsilon to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P(3), and PI(4,5)P(2). 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 epsilon localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 epsilon lipid-binding domain in T cell biology.

The membrane-proximal portion of CD3 epsilon associates with the serine/threonine kinase GRK2.

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.

The constitutive tyrosine phosphorylation of CD3zeta results from TCR-MHC interactions that are independent of thymic selection.

The TCR complex, when isolated from thymocytes and peripheral T cells, contains a constitutively tyrosine-phosphorylated CD3zeta molecule termed p21. Previous investigations have shown that the constitutive phosphorylation of CD3zeta results from TCR interactions with MHC molecules occurring in both the thymus and the periphery. To determine what contribution the selection environment had on this constitutive phosphorylation, we analyzed CD3zeta from several distinct class I- and II-restricted TCR-transgenic mice where thymocyte development occurred in either a selecting or a nonselecting MHC environment. Herein, we report that constitutively phosphorylated CD3zeta (p21) was present in thymocytes that developed under nonselecting peptide-MHC conditions. These findings strongly support the model that the TCR has an inherent avidity for MHC molecules before repertoire selection. Biochemical analyses of the TCR complex before and after TCR stimulation suggested that the constitutively phosphorylated CD3zeta subunit did not contribute to de novo TCR signals. These findings may have important implications for T cell functions during self-MHC recognition under normal and autoimmune circumstances.