Emanuele Giurisato

Lipid rafts and T cell receptor signaling: a critical re-evaluation.

The current model suggesting that raft integrity is required for T cell activation is mostly (but not exclusively) based on the use of drugs, such as methyl‐β ‐cyclodextrin (Mβ CD), that disorganize rafts and inhibit T cell receptor (TCR)‐induced Ca2 + influx. Here we show that conditions that disrupt lipid raft integrity do not inhibit TCR triggering in Jurkat cells andnormal T lymphocytes. Indeed, we found that the reported inhibition of TCR‐induced Ca2 + influx by Mβ CD treatment is mainly due to (a) nonspecific depletion of intracellular Ca2 + stores and (b) plasma membrane depolarization of T cells. When these side‐effects are taken into account, raft disorganization does not alter TCR‐dependent Ca2 + signaling. In line with these results, also TCR‐induced tyrosine phosphorylation is not inhibited by dispersion of lipid rafts. By contrast, in the same conditions, Ca2 + signaling via the glycosylphosphatidylinositol (GPI)‐anchored protein CD59 is totally abolished. These results indicate that, while signaling through GPI‐anchored proteins requires lipid raft integrity, CD3‐dependent TCR activation occurs independently of cholesterol extraction.

T Cell Receptor Can Be Recruited to a Subset of Plasma Membrane Rafts, Independently of Cell Signaling and Attendantly to Raft Clustering

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3ε, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3ε amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca2+mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM1. We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.

Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse

ABSTRACT The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP3. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.

KSR1 Modulates the Sensitivity of Mitogen-Activated Protein Kinase Pathway Activation in T Cells without Altering Fundamental System Outputs

ABSTRACT Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that regulate cell fate decisions. They generate a wide range of signal outputs, including graded and digital responses. In T cells, MAPK activation is digital in response to T-cell-receptor stimulation; however, whether other receptors on T cells that lead to MAPK activation are graded or digital is unknown. Here we evaluate MAPK activation in T cells at the single-cell level. We show that T cells responded digitally to stimulation with superantigen-loaded antigen-presenting cells, whereas they responded in a graded manner to the chemokine SDF-1, demonstrating that the system output of the MAPK module is highly plastic and determined by components upstream of the MAPK module. These findings also confirm that different MAPK system outputs are used by T cells to control discrete biological functions. Scaffold proteins are essential for proper MAPK signaling and function as they physically assemble multiple components and regulators of MAPK cascades. We found that the scaffold protein KSR1 regulated the threshold required for MAPK activation in T cells without affecting the nature of the response. We conclude that KSR1 plays a central role in determining the sensitivity of T-cell responses and is thus well positioned as a key control point.

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE).

KSR2 is required for store-operated calcium entry (SOCE). KSR2 deficiency affects STIM1/ORAI1 puncta formation and cytoskeleton organization. In addition, KSR2-associated calcineurin is crucial for SOCE. These findings identify the KSR2-calcineurin complex to be crucial for store-dependent STIM1-ORAI1 dynamics.

Ligand-Dependent Activation of EGFR in Follicular Dendritic Cells Sarcoma is Sustained by Local Production of Cognate Ligands

Purpose: The aim of this study was to investigate the biological and clinical significance of epidermal growth factor receptor (EGFR) signaling pathway in follicular dendritic cell sarcoma (FDC-S). Experimental Design: Expression of EGFR and cognate ligands as well as activation of EGFR signaling components was assessed in clinical samples and in a primary FDC-S short-term culture (referred as FDC-AM09). Biological effects of the EGFR antagonists cetuximab and panitumumab and the MEK inhibitor UO126 on FDC-S cells were determined in vitro on FDC-AM09. Direct sequencing of KRAS, BRAF, and PI3KCA was conducted on tumor DNA. Results: We found a strong EGFR expression on dysplastic and neoplastic FDCs. On FDC-AM09, we could show that engagement of surface EGFR by cognate ligands drives the survival and proliferation of FDC-S cells, by signaling to the nucleus mainly via MAPK and STAT pathways. Among EGFR ligands, heparin-binding EGF-like growth factor, TGF-α and Betacellulin (BTC) are produced in the tumor microenvironment of FDC-S at RNA level. By extending this finding at protein level we found that BTC is abundantly produced by FDC-S cells and surrounding stromal cells. Finally, direct sequencing of tumor-derived genomic DNA showed that mutations in KRAS, NRAS, BRAF, and PI3KCA, which predicts resistance to anti-EGFR MoAb in other cancer models, are not observed in FDC-S. Conclusion: Activation of EGFR by cognate ligands produced in the tumor microenvironment sustain viability and proliferation of FDC-S indicating that the receptor blockade might be clinically relevant in this neoplasm. Clin Cancer Res; 19(18); 5027–38. ©2013 AACR.