Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

PD-1: A Driver or Passenger of T Cell Exhaustion?

Okazaki and colleagues report that PD-1 signaling mainly restrains effector function at the early stage of T cell activation. The authors find that cytokine genes require strong antigen stimulation and are more susceptible to PD-1 inhibition.

Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition.

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy.

Charging CAR by electrostatic power.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising approach for cancer treatment. CAR is a synthetic immune receptor that recognizes tumor antigen and activates T cells through multiple signaling pathways. However, the current CAR design is not as robust as T cell receptor (TCR), a natural antigen receptor with high sensitivity and efficiency. TCR signaling relies on specific molecular interactions, and thus electrostatic force, the major force of molecular interactions, play critical roles. Understanding how electrostatic charge regulates TCR/CAR signaling events will facilitate the development of next-generation T cell therapies. This review summarizes recent findings on the roles of electrostatic interactions in both natural and synthetic immune receptor signaling, specifically that in CAR clustering and effector molecule recruitments, and highlights potential strategies for engineering CAR-T cell therapy by leveraging charge-based interactions.

Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling.

Activation of the T-cell antigen receptor (TCR)-CD3 complex is critical to induce the anti-tumor response of CD8+ T cells. Here, we found that disulfiram (DSF), an FDA-approved drug previously used to treat alcohol dependency, directly activates TCR signaling. Mechanistically, DSF covalently binds to Cys20/Cys23 residues of lymphocyte-specific protein tyrosine kinase (LCK) and enhances its tyrosine 394 phosphorylation, thereby promoting LCK kinase activity and boosting effector T cell function, interleukin-2 production, metabolic reprogramming, and proliferation. Furthermore, our in vivo data revealed that DSF promotes anti-tumor immunity against both melanoma and colon cancer in mice by activating CD8+ T cells, and this effect was enhanced by anti-PD-1 co-treatment. We conclude that DSF directly activates LCK-mediated TCR signaling to induce strong anti-tumor immunity, providing novel molecular insights into the therapeutic effect of DSF on cancer.

Self-programmed dynamics of T cell receptor condensation.

A common event upon receptor-ligand engagement is the formation of receptor clusters on the cell surface, in which signaling molecules are specifically recruited or excluded to form signaling hubs to regulate cellular events. These clusters are often transient and can be disassembled to terminate signaling. Despite the general relevance of dynamic receptor clustering in cell signaling, the regulatory mechanism underlying the dynamics is still poorly understood. As a major antigen receptor in the immune system, T cell receptors (TCR) form spatiotemporally dynamic clusters to mediate robust yet temporal signaling to induce adaptive immune responses. Here we identify a phase separation mechanism controlling dynamic TCR clustering and signaling. The TCR signaling component CD3ε chain can condensate with Lck kinase through phase separation to form TCR signalosomes for active antigen signaling. Lck-mediated CD3ε phosphorylation, however, switched its binding preference to Csk, a functional suppressor of Lck, to cause the dissolvement of TCR signalosomes. Modulating TCR/Lck condensation by targeting CD3ε interactions with Lck or Csk directly affects T cell activation and function, highlighting the importance of the phase separation mechanism. The self-programmed condensation and dissolvement is thus a built-in mechanism of TCR signaling and might be relevant to other receptors.

INPP5K controls the dynamic structure and signaling of wild-type and mutated, leukemia-associated IL-7 receptors.

The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.

Paracoccus actinidiae sp.nov., a novel bacterium isolated from kiwi tree rhizosphere soil.

Strain M09T was isolated from the rhizoshere of kiwi fruit trees from an orchard located in Fangshan, Beijing, PR China (39° 49' 25.1″ N, 116° 4' 44.5″ E,). It is a short rod-shaped, Gram-stain-negative, facultatively anaerobic bacterium that tests positive for both oxidase and catalase. The strain exhibited growth within the temperature range of 15-45 °C (optimal growth at 30 °C) and the pH range of 4.0-10.0 (optimal growth at pH 7.0) and without NaCl. It also grew in a sodium chloride-free nutrient agar (NA) medium. The results of phylogenetic analysis of the 16S rRNA gene sequences indicated that M09T represents a member of the genus Paracoccus and shares high similarity with Paracoccus everestensis S8-55T (98.46%) and Paracoccus aerius 011410T (97.58%). The average nucleotide identity values between M09T and P. everestensis S8-55T, P. aerius 011410T, Paracoccus marinaquae X HP0099T and Paracoccus fontiphilus MVW-1T were 95.56, 84.51, 79.83 and 83.68%, respectively. The digital DNA-DNA hybridisation values between between M09T and P. everestensis S8-55T, P. aerius 011410T, P. marinaquae X HP0099T and P. fontiphilus MVW-1T were 56.40, 29.30, 21.60 and 28.60%, respectively. The major fatty acids identified were C10 : 0 3-OH (51.8%) and C18 : 1ω7c (35.5%). The major respiratory quinone was Q-10, with Q-8 present as a minor component. Polar lipids were mainly comprised of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Genome sequencing revealed that the strain has a DNA G+C content of 64.31 mol%. On the basis of this comprehensive taxonomic characterisation data, M09T represents a novel species within the genus Paracoccus and has been named Paracoccus actinidiae sp. nov. The type strain is designated as M09T (=GDMCC 1.4157T=KCTC 8143T).

FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells.

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer1-4. The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1+ T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

The relationship between thiamin, folic acid and cognitive function in a rat model of uremia.

End-stage renal disease is a worldwide health burden, but the pathogenesis of uremia-associated cognitive impairment (CI) is poorly recognized. We hypothesized that uremia brings about deficiency of thiamin and folic acid and causes CI by inducing oxidative stress. Therefore, 24 Sprague-Dawley rats were randomly divided into two groups: a 5/6 nephrectomy group (n = 12) and a sham-operated group (n = 12). The Morris water maze was used to assess the cognitive function eight weeks post-surgery, and serum levels of thiamin, folic acid and homocysteine were detected subsequently. Brain and kidney tissues were collected for pathological examination and 8-Hydroxy-2'-deoxyguanosine (8-OHdG) immunochemistry staining. Results showed that the escape latency on training days 1-2 was longer, and the time in quadrant IV on experimental day 6 was significantly shorter in 5/6 nephrectomy group. Meanwhile, the uremic rats showed decreased thiamin, folic acid and increased homocysteine. We also found the time in quadrant IV was positively correlated with thiamin and folic acid level, while negatively correlated with the blood urea nitrogen and 8-OHdG positive cell proportion. Furthermore, in 5/6 nephrectomy group, the hippocampal neuron count was significantly reduced, and a greater proportion of 8-OHdG positive cells were detected. Pretreating LPS-stimulated rat microglial cells with thiamin or folic acid in vitro alleviated the inflammatory impairment in terms of cell viability and oxidative stress. In summary, we applied a uremic rat model and proved that uremia causes serum thiamin and folic acid deficiency, homocysteine elevation, along with neuron reduction and severe oxidative stress in hippocampus, finally leading to CI.

Palladium Enhanced Iron Active Site - An Efficient Dual-Atom Catalyst for Oxygen Electroreduction.

Metal-nitrogen-carbon (M-C/N) electrocatalysts have been shown to have satisfactory catalytic activity and long-term durability for the oxygen reduction reaction (ORR). Here, a strategy to prepare a new electrocatalyst (Fe&Pd-C/N) using a unique metal-containing ionic liquid (IL) is exploited, in which Fe & Pd ions are positively charged species atomically dispersed by coordination to the N of the N-doped C substrate, C/N. X-ray absorption fine structure, XPS and aberration-corrected transmission electron microscopy results verified a well-defined dual-atom configuration comprising Fe+2.x -N4 coupled Pd2+ -N4 sites and well-defined spatial distribution. Electronic control of a coupled Fe-Pd structure produces an electrocatalyst that exhibits superior performance with enhanced activity and durability for the ORR compared to that of commercial Pt/C (20%, Johnson Matthey) in both alkaline and acid media. Density functional theory calculations indicate that Pd atom can enhance the catalytic activity of the Fe active sites adjacent to Pd sites by changing the electronic orbital structure and Bader charge of the Fe centers. The excellent catalytic performance of the Fe&Pd-C/N electrocatalyst is demonstrated in zinc-air batteries and hydrogen-air fuel cells.

Hydroxyl-/Carboxyl-Rich Graphitic Carbon Nitride/Graphene Oxide Composites for Efficient Photodegradation of Reactive Red 195 and Antibacterial Applications.

In this work, a protonated graphitic carbon nitride (P-g-C3N4)-coated graphene oxide (GO) composite (GO/P-g-C3N4) was prepared via wet-chemistry exfoliation, followed by a freeze-drying process. The GO/P-g-C3N4 composite was found to have an outstanding photodegradation performance effect on the reactive red 195 (RR195) dye and very strong antibacterial properties. Both the GO structure and the dispersed state of P-g-C3N4 were found to play a significant role in enhancing the photocatalytic activity of GO/P-g-C3N4. The GO/P-g-C3N4 obtained via freeze-drying retained a large number of oxygen-containing groups and showed higher catalytic activity and reusability than the reduced GO (rGO)/g-C3N4 obtained via thermal reduction. Characterization of the samples indicates that GO/P-g-C3N4 has a higher specific surface area and photocurrent density than rGO/g-C3N4; it is likely that these properties lead to the superior photocatalytic activity observed in GO/P-g-C3N4. Adsorption energy calculations indicate that O2 can be readily adsorbed onto the GO surface, which results in stronger oxidizing superoxide anion radicals (•O2-) and holes (h+); these active radicals can rapidly degrade RR195 dyes. Moreover, broad-spectrum antibacterial activity (demonstrated against Staphylococcus aureus and Escherichia coli) was observed in the case of the GO/P-g-C3N4 composite irradiated with visible light. This work offers new insights into the design of cost-effective g-C3N4-based photocatalysts for environmental remediation.

Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

Chromatin assembly factor 1B critically controls the early development but not function acquisition of invariant natural killer T cells in mice.

CD4+ CD8+ double-positive thymocytes give rise to both conventional TCRαß+ T cells and invariant natural killer T cells (iNKT cells), but these two kinds of cells display different characteristics. The molecular mechanism underlying iNKT cell lineage development and function acquisition remain to be elucidated. We show that the loss of chromatin assembly factor 1B (CHAF1b) maintains the normal development of conventional TCRαß+ T cells but severely impairs early development of iNKT cells. This dysregulation is accompanied by the impairment in chromatin activation and gene transcription at Vα14-Jα18 locus. Notably, ectopic expression of a Vα14-Jα18 TCR rescues Chaf1b-deficient iNKT cell developmental defects. Moreover, cytokine secretion and antitumor activity are substantially maintained in Vα14-Jα18 TCR transgene-rescued Chaf1b-deficient iNKT cells. Our study identifies CHAF1b as a critical factor that controls the early development but not function acquisition of iNKT cells via lineage- and stage-specific regulation.

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.

Intramembrane ionic protein-lipid interaction regulates integrin structure and function.

Protein transmembrane domains (TMDs) are generally hydrophobic, but our bioinformatics analysis shows that many TMDs contain basic residues at terminal regions. Physiological functions of these membrane-snorkeling basic residues are largely unclear. Here, we show that a membrane-snorkeling Lys residue in integrin αLß2 (also known as lymphocyte function-associated antigen 1 [LFA-1]) regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells. The amino group of the conserved Lys ionically interacts with the phosphate group of acidic phospholipids to stabilize αLß2 transmembrane association, thus keeping the integrin at low-affinity conformation. Intracellular Ca2+ uses its charge to directly disrupt this ionic interaction, leading to the transmembrane separation and the subsequent extracellular domain extension to increase adhesion activity. This Ca2+-mediated regulation is independent on the canonical Ca2+ signaling or integrin inside-out signaling. Our work therefore showcases the importance of intramembrane ionic protein-lipid interaction and provides a new mechanism of integrin activation.