Immune checkpoints are inhibitory receptors on T cells that tumors exploit to suppress immune responses thereby achieving immune escape. Checkpoint inhibitors, which block these receptors and activate the anti-tumor immune response, have revolutionized cancer treatment—a breakthrough recognized with the 2018 Nobel Prize in Physiology or Medicine. Following PD-1 and CTLA-4, LAG3-targeted drugs receive FDA approval in 2023, the third approved immune checkpoint inhibitors, marking another milestone in cancer immunotherapy. However, like other immune checkpoint therapies, LAG3-targeted treatments benefit only a subset of patients. Identifying those most likely to respond remains a pressing clinical challenge. This requires a deeper understanding of LAG3’s functional mechanisms—a question that has remained unresolved since LAG3’s discovery in 1990. Until now, how ligand binding triggers LAG3 activation has been a major unsolved mystery in the field.

A recent study published in Cell unveils a groundbreaking discovery about the immune checkpoint receptor LAG3. Led by an international team of researchers—including Prof. Xu Chenqi from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences; Prof. Wang Haopeng from ShanghaiTech University; Prof. Dario Vignali from the University of Pittsburgh School of Medicine; Prof. Kong Yan from Peking University Cancer Hospital and Research Institute; and Dr. Shen Zhirong from BeiGene—this study reveals for the first time the molecular switch mechanism underlying LAG3 activation. The team develops a predictive system based on functional biomarkers, providing new strategies for precision immunotherapy.

To investigate how LAG3 is activated, the research team conducts a mass spectrometry-based proteomic analysis of its post-translational modifications (PTMs). Their findings reveal that ligand binding triggers rapid polyubiquitination at the K498 site of the LAG3 receptor. Unlike conventional ubiquitination, which often marks proteins for degradation, this modification plays a pivotal functional role in LAG3 activation. Further studies demonstrate that polyubiquitination acts as a molecular switch to unleash LAG3’s immunosuppressive function. LAG3’s intracellular region contains a Basic residue Rich Sequence (BRS motif), followed by a key signaling domain with the FSALE motif and the polyubiquitination site. In its resting state, LAG3’s critical intracellular signaling motifs are sequestered within the membrane, preventing signal transduction. Upon ligand binding, polyubiquitination facilitates the release of these motifs from the membrane, thereby enabling LAG3’s immune checkpoint function (Figure 1). This “sequestering-unleashing” regulatory mechanism, mediated by polyubiquitination, represents a previously unknown mode of receptor activation.

Building on their discovery of LAG3 activation, the researchers develop a novel biomarker for predicting treatment efficacy. The results show that this functional biomarker (Functionality Biomarker) for characterizing the activated state of LAG3 has significant advantages over traditional biomarkers that merely assess LAG3 expression levels (Expression Biomarker). In clinical studies, the functional biomarker demonstrates superior predictive power: its expression level is 51.7 times higher in responders compared to non-responders (p = 0.0379). In contrast, traditional expression-based biomarkers show only a 6.5-fold difference and lack statistical significance. These findings highlight the functional biomarker’s potential to enhance patient selection for LAG3-based therapies, paving the way for more precise and effective immunotherapy strategies.

Beyond unveiling LAG3’s activation mechanism, this study underscores the broader biological significance of BRS motifs. This work is another significant example showing the biological functions of BRS motifs. Prof. Xu Chenqi’s team has been studying at the forefront of BRS signaling for decades, revealing their crucial roles in diverse immune receptors, including TCR, BCR, CD28, LAG3, PD-L1, and IL7R. Approximately 70% of single-transmembrane proteins have been found to contain BRS motifs in their intracellular juxtamembrane regions. BRS motifs interact with acidic phospholipids and specific protein residues, forming a dynamic electrostatic network that is influenced by factors such as ions and membrane properties. Recent studies from Prof. Xu’s group show that BRS motifs regulate key biological processes, including phosphorylation, ubiquitination, liquid-liquid phase separation, and mechanotransduction. For further details, visit Xu Lab website at https://xulab.sibcb.ac.cn/. Representative publications include Cell (2008), Nature (2013), Cell Research (2017), Nature Structural & Molecular Biology (2017), Cell (2020), and Immunity (2024).

Reference：https://www.cell.com/cell/fulltext/S0092-8674(25)00199-0

The signal transduction mechanism of immunoreceptors and its clinical application has always been a frontier hotspot in biology and medicine, which can help us understand the fundamental immune principles and develop innovative immunotherapies.

Current immunotherapies are essentially based on the signal regulation of immunoreceptors, such as immune checkpoint blockade therapy, CAR-T and TCR-T cell therapies, etc. Immunoreceptors are all transmembrane proteins, and the existence of juxta-membrane positively charged residues in transmembrane proteins is a known conclusion in classic biochemistry textbooks. However, the signal mechanism, physiological and pathological functions, and application prospects of these sequences have not been systematically studied.

A perspective article published in Nature Reviews Immunology, researchers led by Prof. XU Chenqi and HE Xing from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, along with SHI Xiaoshan from the Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. The article focuses on a general signaling motif—the basic-residue-rich sequence (BRS), defines the BRS motif, summarizes its juxta-membrane signal transduction mechanisms, discusses the relation between BRS mutations in immunoreceptors and human diseases, and explores the potential of BRS in innovative immunotherapies.

The article first systematically analyzed the sequence characteristics of human single-transmembrane proteins and found that 70% of human single-transmembrane proteins carry BRS in the intracellular juxta-membrane region. BRS is usually 10 amino acids long, carries two or more net positive charges, and is often located in the intracellular juxta-membrane region, but can also be distributed in the distal-membrane position. To date, BRS in various immunoreceptors such as antigen receptors (T cell receptors, B cell receptors), co-stimulatory receptors, co-inhibitory receptors, NK cell receptors, Fc receptors, and cytokine receptors has been experimentally reported.

Based on existing research reports, this article summarized the juxta-membrane electrostatic regulation network theory mediated by BRS. BRS can interact electrostatically with negatively charged or π-electron-carrying lipid and protein molecules in the cell membrane and juxta-membrane region, and environmental factors can further regulate these interactions, forming a spatiotemporally dynamic juxta-membrane electrostatic network.

Current known network members include: BRS, acidic phospholipids (such as PS, PI(4,5)P2), steroid molecules (such as cholesterol, hydroxycholesterol), cations (such as calcium ions), and membrane proteins or peripheral membrane proteins carrying negative charges or π-electrons rich regions (such as LCK, p85, LAG3, PLCγ1), etc. Through the juxta-membrane electrostatic network, BRS regulates the phosphorylation, ubiquitylation, liquid-liquid phase separation, and mechanical signal transduction of immunoreceptors, covering the life cycle of immunoreceptors from rest, triggering, signal amplification, signal decay, and degradation.

Next, the article systematically organized the relevance between BRS mutations and diseases, as well as the prospects for the translational application of BRS. Through systematic analysis of the UniProt database, more than 100 disease-related BRS mutations have been recorded, but the specific pathogenic mechanisms of most of them still need to be studied. The prospects for the translational application of BRS include the signal regulation of BRS in natural immunoreceptors and the design of synthetic immunoreceptors using BRS.

The steroid metabolite 7α-hydroxycholesterol can loosen the packing of cell membrane lipid molecules, helping the BRS of the TCR signaling subunit CD3ε to bind stronger with the membrane. Thus, in the preparation of TCR-T cells, it inhibits the basal phosphorylation signal of TCR, increases the proportion of memory cells, and improves the longevity of immunotherapy.

On the other hand, the E-CAR molecule formed by adding the CD3ε signaling region to the second-generation CAR molecule has better signal transduction ability, where BRS can mediate the formation of liquid-liquid phase separation through cation-π bonds, helping cells form more mature and efficient immune synapses, thereby enhancing the antigen sensitivity and longevity of E-CAR-T cells. In addition to CAR, BRS is also crucial for the efficient signal transduction of another synthetic receptor, SNIPR.

Finally, the article points out that the human proteome has a rich library of BRS, which potentially has a broad application. However, at the current stage, the understanding and application of BRS signal transduction mechanisms are still very limited, and there are a series of important issues that need to be resolved. For example, can BRS be divided into subclasses? Do different subclasses have different signaling patterns? How do various BRS mutations lead to human diseases? How to rationally manipulate BRS signals or rationally design synthetic receptors containing BRS? The answers to these questions will greatly enhance our understanding of the immune system and help the development of immunotherapies.

Reference:https://www.nature.com/articles/s41577-024-01105-6

In a study published in Cell Chemical Biology, teams led by Dr. XU Chenqi from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, collaborated with Dr. WANG Haopeng from ShanghaiTech University and Dr. LOU Jizhong from the Institute of Biophysics, Chinese Academy of Sciences, discovered that the natural sterol metabolite 7a-hydroxycholesterol (7a-HC) can directly inhibit TCR (T Cell Receptor) signal transduction. Utilizing this function, the researchers demonstrated that short-term treatment with 7a-HC can reduce basal TCR-T cell signaling, increase the proportion of memory cells, and thereby enhance the long-term efficacy of immunotherapy.

Cholesterol molecules can be oxidized at multiple sites, producing various hydroxycholesterols (oxidized cholesterols). Cholesterol can enhance the immune function of CD8 T cells; however, hydroxycholesterols exhibit significant immunosuppressive functions, inhibiting normal T cell immune responses in the tumor microenvironment . Nevertheless, the immune system operates as a balanced system where unfavorable factors can be converted into beneficial ones in appropriate contexts, creating new immunotherapy strategies.

The CD3ε subunit in the TCR/CD3 complex contains a juxtamembrane basic residue-rich sequence (JM-BRS) that can electrostatically interact with negatively charged phospholipids in the plasma membrane. This interaction inserts the JM-BRS and the adjacent tyrosine-based signaling motifs (ITAMs) into the membrane, restricting JM-BRS's recruitment of Lck kinase and Lck's phosphorylation of ITAMs. Due to the presence of hydroxyl groups at positions 3 and 7, 7a-HC can reduce the packing density of lipid molecules in the cell membrane, making the membrane structure looser. This helps the intracellular region of CD3ε insert better into the membrane, thereby inhibiting TCR signal transduction.

TCR signaling is a double-edged sword: it activates and proliferates T cells, granting them effector functions, but also accelerates terminal differentiation, weakening T cell memory. Therefore, TCR signaling needs to be controlled at an appropriate level to achieve sufficient T cell effector functions while maintaining a degree of stemness, ensuring a robust and long-lasting T cell immune response. Leveraging 7a-HC's TCR signaling inhibitory function, the research team conducted several short-term treatments (each lasting 20 minutes) during the in vitro expansion of TCR-T cells. This significantly suppressed basal T cell signaling and markedly increased the proportion of memory T cells. 7a-HC-treated TCR-T cells demonstrated significant advantages in long-term tumor killing, as evidenced by better tumor control in animal models. This study reveals a new regulatory mechanism of TCR signaling and proposes a new strategy for TCR-T cell therapy.

Contact: cqxu@sibcb.ac.cn  

Reference: https://www.sciencedirect.com/science/article/pii/S2451945624001673

In a study published in EMBO Molecular Medicine, Prof. XU Chenqi's team from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, Prof. ZHU Zheng-Jiang's team from the Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and Prof. WANG Zhigang's team from the Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital reported that the asynchronous cholesterol biosynthesis in MSS CRC shapes tumor immune landscape through a Th17-modulation mechanism.

Cancer immunotherapies have experienced significant success in recent years. However, many types of cancers exhibit poor responses to current immunotherapies. Colorectal cancer with microsatellite stability (MSS CRC), constituting the majority subtype of CRC (approximately 85% of patients), demonstrates insensitivity to immune checkpoint blockade therapies. Additionally, MSS CRC is reported to exhibit an enrichment of a pro-inflammatory CD4+ T cell subset known as Th17 in the tumor microenvironment. This subset has been implicated in mediating PD-1 resistance in various cancer types. Therefore, there is a strong interest in investigating the mechanisms behind the Th17 enrichment observed in MSS CRC.

In this study, a metabolic cue responsible for the Th17 enrichment in MSS CRC is reported through the examination of large human cohorts and animal models. The researchers discovered that MSS CRC cells can induce T cell polarization toward the Th17 lineage by secreting distal cholesterol precursors (DCPs). The cholesterol biosynthesis pathway is asynchronously upregulated in MSS CRC cells, resulting in the abnormal accumulation of DCPs. By inhibiting Cyp51, the cholesterol biosynthesis upstream enzyme, and thereby reducing intratumoral DCPs, tumor progression of MSS CRC was suppressed through a Th17-modulation mechanism.

This study reveals a novel mechanism of cancer-immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

Contact: cqxu@sibcb.ac.cn

Reference: https://www.embopress.org/doi/full/10.1038/s44321-023-00015-9