Ionic CD3-Lck interaction regulates the initiation of T-cell receptor signaling.

Antigen-triggered T-cell receptor (TCR) phosphorylation is the first signaling event in T cells to elicit adaptive immunity against invading pathogens and tumor cells. Despite its physiological importance, the underlying mechanism of TCR phosphorylation remains elusive. Here, we report a key mechanism regulating the initiation of TCR phosphorylation. The major TCR kinase Lck shows high selectivity on the four CD3 signaling proteins of TCR. CD3ε is the only CD3 chain that can efficiently interact with Lck, mainly through the ionic interactions between CD3ε basic residue-rich sequence (BRS) and acidic residues in the Unique domain of Lck. We applied a TCR reconstitution system to explicitly study the initiation of TCR phosphorylation. The ionic CD3ε-Lck interaction controls the phosphorylation level of the whole TCR upon antigen stimulation. CD3ε BRS is sequestered in the membrane, and antigen stimulation can unlock this motif. Dynamic opening of CD3ε BRS and its subsequent recruitment of Lck thus can serve as an important switch of the initiation of TCR phosphorylation.

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor.

T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3εCD). At the single-molecule level, we found that CD3εCD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS. These conformations were generated because different regions of CD3εCD had heterogeneous lipid-binding properties and therefore had heterogeneous dynamics. Live-cell imaging experiments demonstrated that different antigen stimulations could stabilize CD3εCD at different conformations. Lipid-dependent conformational dynamics thus provide structural basis for the versatile signaling property of TCR.

Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism.

CD8(+) T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme, led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Lipid in T-cell receptor transmembrane signaling.

T-cell receptor (TCR) is a key receptor in the immune system that can recognize antigen and initiate adaptive immune response. TCR activity needs to be regulated in a precise manner to trigger sufficient response to foreign pathogens but avoid unnecessary harm to the host. Despite of its importance, the molecular mechanism of TCR transmembrane signaling still remains elusive. Emerging studies show that lipid can play sophisticated roles in regulating the structure and function of TCR. This review mainly discusses how acidic phospholipids regulate TCR signaling through ionic protein-lipid interaction.

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.

The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms.

Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids.

Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.