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.

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.

Polybasic RKKR motif in the linker region of lipid droplet (LD)-associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids.

Lipid droplets (LDs) are intracellular organelles and a central site for lipid synthesis, storage, and mobilization. The size of LDs reflects the dynamic regulation of lipid metabolism in cells. Previously, we found that cell death-inducing DFFA-like effector C (CIDEC) mediates LD fusion and growth by lipid transfer through LD-LD contact sites in adipocytes and hepatocytes. The CIDE-N domains of CIDEC molecules form homodimers, whereas the CIDE-C domain plays an important role in LD targeting and enrichment. Here, using targeted protein deletions and GFP expression coupled with fluorescence microscopy, we identified a polybasic RKKR motif in the linker region that connects the CIDE-N and CIDE-C domains of CIDEC and functions as a regulatory motif for LD fusion. We found that deletion of the linker region or mutation of the RKKR motif increases the formation of supersized LDs compared with LD formation in cells with WT CIDEC. This enhanced LD fusion activity required the interaction between CIDE-N domains. Mechanistically, we found that the RKKR motif interacts with acidic phospholipids via electrostatic attraction. Loss of this motif disrupted the protein-lipid interaction, resulting in enhanced lipid droplet fusion activity and thus formation of larger LDs. In summary, we have uncovered a CIDEC domain that regulates LD fusion activity, a finding that provides insights into the inhibitory regulation of LD fusion through CIDEC-lipid interactions.

Shaping immune landscape of colorectal cancer by cholesterol metabolites.

Cancer immunotherapies have achieved unprecedented success in clinic, but they remain largely ineffective in some major types of cancer, such as colorectal cancer with microsatellite stability (MSS CRC). It is therefore important to study tumor microenvironment of resistant cancers for developing new intervention strategies. In this study, we identify a metabolic cue that determines the unique immune landscape of MSS CRC. Through secretion of distal cholesterol precursors, which directly activate RORγt, MSS CRC cells can polarize T cells toward Th17 cells that have well-characterized pro-tumor functions in colorectal cancer. Analysis of large human cancer cohorts revealed an asynchronous pattern of the cholesterol biosynthesis in MSS CRC, which is responsible for the abnormal accumulation of distal cholesterol precursors. Inhibiting the cholesterol biosynthesis enzyme Cyp51, by pharmacological or genetic interventions, reduced the levels of intratumoral distal cholesterol precursors and suppressed tumor progression through a Th17-modulation mechanism in preclinical MSS CRC models. Our study therefore reveals a novel mechanism of cancer-immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

Exhaustion-associated cholesterol deficiency dampens the cytotoxic arm of antitumor immunity.

The concept of targeting cholesterol metabolism to treat cancer has been widely tested in clinics, but the benefits are modest, calling for a complete understanding of cholesterol metabolism in intratumoral cells. We analyze the cholesterol atlas in the tumor microenvironment and find that intratumoral T cells have cholesterol deficiency, while immunosuppressive myeloid cells and tumor cells display cholesterol abundance. Low cholesterol levels inhibit T cell proliferation and cause autophagy-mediated apoptosis, particularly for cytotoxic T cells. In the tumor microenvironment, oxysterols mediate reciprocal alterations in the LXR and SREBP2 pathways to cause cholesterol deficiency of T cells, subsequently leading to aberrant metabolic and signaling pathways that drive T cell exhaustion/dysfunction. LXRß depletion in chimeric antigen receptor T (CAR-T) cells leads to improved antitumor function against solid tumors. Since T cell cholesterol metabolism and oxysterols are generally linked to other diseases, the new mechanism and cholesterol-normalization strategy might have potential applications elsewhere.

Ionic protein-lipid interactions at the plasma membrane regulate the structure and function of immunoreceptors.

Adaptive lymphocytes express a panel of immunoreceptors on the cell surface. Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids forming microdomains in the plasma membrane can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. We have shown that negatively charged acidic phospholipids can interact with positively charged juxtamembrane polybasic regions of immunoreceptors, such as TCR-CD3, CD28 and IgG-BCR, to regulate protein structure and function. Furthermore, we pay our attention to protein transmembrane domains. We show that a membrane-snorkeling Lys residue in integrin αLß2 regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells, thus providing a new mechanism of integrin activation. Here, we review our recent progress showcasing the importance of both juxtamembrane and intramembrane ionic protein-lipid interactions.

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.

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.