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.

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.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.

Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.

Effects of CX3CR1 and CXCR2 antagonists on running-dependent intramuscular neutrophil recruitments and myokine upregulation.

Muscle contractile activity stimulates intramuscular recruitment of immune cells including neutrophils emerging to serve as a prerequisite for exerting proper muscular performance, although the underlying mechanisms and their contributions to myokine upregulation remain ill-defined. We previously reported that pharmacological inhibition of CX3CR1, a fractalkine receptor, dampens gnawing-dependent neutrophil recruitment into masseter muscles along with compromising their masticatory activity. By using a running exercise model, we herein demonstrated that hindlimb muscles require collaborative actions of both CX3CR1- and CXCR2-mediated signals for achieving neutrophil recruitment, upregulation of myokines including interleukin (IL)-6, enhanced GLUT4 translocation, and adequate endurance capability. Mechanistically, we revealed that a combination of CX3CR1 and CXCR2 antagonists, i.e., AZD8797 and SB2205002, inhibits exercise-inducible ICAM-1 and fractalkine upregulations in the area of the endothelium and muscle-derived CXCL1 upregulation, both of which apparently contribute to the intramuscular neutrophil accumulation in working muscles. Intriguingly, we also observed that 2 h of running results in intramuscular augmentation of innate lymphoid type 2 cells (ILC2s) markers, i.e., Bcl11b mRNA levels and anti-GATA-3-antibody-positive signals, and that these effects are completely abolished by administration of the combination of CX3CR1 and CXCR2 antagonists. Taken together, our findings strongly suggest that the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells including neutrophils and possibly ILC2s, mediated through these local factors, plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.NEW & NOTEWORTHY This study provides compelling evidence that running-dependent intramuscular neutrophil recruitment requires both CX3CR1- and CXCR2-mediated signals that prime not only myofiber-derived myokine upregulations but also endothelium ICAM-1 and fractalkine expressions. The results revealed the importance of the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells, including neutrophils and possibly ILC2s, which plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.

A proximity-tagging system to identify membrane protein-protein interactions.

The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

Rapid Fabrication of Patterned Gels via Microchannel-Conformal Frontal Polymerization.

From the perspective of both fundamental and applied science, it is extremely advisable to develop a facile and feasible strategy for fabricating gels with defined structures. Herein, the authors report the rapid synthesis of patterned gels by conducting frontal polymerization (FP) at millimeter-scale (2 mm), where a series of microchannels, including linear-, parallel-, divergent-, snakelike-, circular- and concentric circular channels, were used. They have investigated the effect of various factors (monomer mass ratio, channel size, initiator concentration, and solvent content) on FP at millimeter-scale, along with the propagating rule of the front during FP in these microchannels. In addition, we developed a new microfluidic-assisted FP (MFP) strategy by combining the FP and microfluidic technique. Interestingly, the MFP can realize the production of hollow-structured gel in a rapid and continuous fashion, which have never been reported. Our work not only offers an effective pathway towards patterned gels by the microchannel-conformal FP, but also gives new insight into the continuous production of hollow-structured materials. Such a method will be beneficial for fabricating vessel and scaffold materials in a flexible, easy-to-perform, time and energy saving way.

Electrophilic Properties of 2'-Deoxyadenosine···Thymine Dimer: Photoelectron Spectroscopy and DFT Studies.

The anion radical of the 2'-deoxyadenosine···thymine (dAT•-) pair has been investigated experimentally and theoretically in the gas phase. By employing negative-ion photoelectron spectroscopy (PES), we have registered a spectrum typical for the valence-bound anion, featuring a broad peak at the electron-binding energy (EBE) between ∼1.5 and 2.2 eV with the maximum at ∼1.9 eV. The measured value of the adiabatic electron affinity (AEA) for dAT was estimated to be ∼1.1 eV. Calculations performed at the M06-2X/6-31++G(d,p) level revealed that the structure, where thymine is coordinated to the sugar of dA by two hydrogen bonds, is responsible for the observed PES signal. The AEAG and the vertical detachment energy of 0.91 and 1.68 eV, respectively, calculated for this structure reproduce the experimental values well. The role of the possible proton transfer in the stabilization of anionic radical complexes is discussed.

Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.

Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.

Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

Simplified Optimal Estimation of Time-Varying Electromyogram Standard Deviation (EMGσ): Evaluation on Two Datasets.

To facilitate the broader use of EMG signal whitening, we studied four whitening procedures of various complexities, as well as the roles of sampling rate and noise correction. We separately analyzed force-varying and constant-force contractions from 64 subjects who completed constant-posture tasks about the elbow over a range of forces from 0% to 50% maximum voluntary contraction (MVC). From the constant-force tasks, we found that noise correction via the root difference of squares (RDS) method consistently reduced EMG recording noise, often by a factor of 5-10. All other primary results were from the force-varying contractions. Sampling at 4096 Hz provided small and statistically significant improvements over sampling at 2048 Hz (~3%), which, in turn, provided small improvements over sampling at 1024 Hz (~4%). In comparing equivalent processing variants at a sampling rate of 4096 Hz, whitening filters calibrated to the EMG spectrum of each subject generally performed best (4.74% MVC EMG-force error), followed by one universal whitening filter for all subjects (4.83% MVC error), followed by a high-pass filter whitening method (4.89% MVC error) and then a first difference whitening filter (4.91% MVC error)-but none of these statistically differed. Each did significantly improve from EMG-force error without whitening (5.55% MVC). The first difference is an excellent whitening option over this range of contraction forces since no calibration or algorithm decisions are required.

Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling.

The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.

Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity.

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

Boron-Made N2 : Realization of a B≡B Triple Bond in the B2 Al3 - Cluster.

Until now, all B≡B triple bonds have been achieved by adopting two ligands in the L→B≡B←L manner. Herein, we report an alternative route of designing the B≡B bonds based on the assumption that by acquiring two extra electrons, an element with the atomic number Z can have properties similar to those of the element with the atomic number Z+2. Specifically, we show that due to the electron donation from Al to B, the negatively charged B≡B kernel in the B2 Al3 - cluster mimics a triple N≡N bond. Comprehensive computational searches reveal that the global minimum structure of B2 Al3 - exhibits a direct B-B distance of 1.553 Å, and its calculated electron vertical detachment energies are in excellent agreement with the corresponding values of the experimental photoelectron spectrum. Chemical bonding analysis revealed one σ and two π bonds between the two B atoms, thus confirming a classical textbook B≡B triple bond, similar to that of N2 .

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.

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.

BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.

Antigen Receptor Nanoclusters: Small Units with Big Functions.

Adaptive lymphocytes express highly variable antigen receptors, allowing them to recognize a large variety of proteins, for example, derived from pathogens and tumor cells. Despite decades of investigations, the signaling mechanisms of these receptors are still incompletely understood. Super-resolution imaging studies revealed that antigen receptors, their coreceptors, and even some downstream signaling molecules tend to form dynamic nanometers-sized self-clusters in quiescent cells. Antigen stimulation induces the coalescence of these nanoclusters to form membrane proximal signalosomes that can mediate efficient signal transduction. In this review, we discuss the dynamic structures of T cell receptor and B cell receptor nanoclusters, the driving forces behind this spatial reorganization, as well as their potential relevance in the modulation of lymphocyte activation and function.

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor.

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.

Realization of an Al≡Al Triple Bond in the Gas-Phase Na3 Al2 - Cluster via Double Electronic Transmutation.

The discovery of homodinuclear multiple bonds composed of Group 13 elements represents one of the most challenging frontiers in modern chemistry. A classical triple bond such as N≡N and HC≡CH contains one σ bond and two π bonds constructed from the p orbitals perpendicular to the σ bond. However, the traditional textbook triple bond between two Al atoms has remained elusive. Here we report an Al≡Al triple bond in the designer Na3 Al2 - cluster predicted in silico, which was subsequently generated by pulsed arc discharge followed by mass spectrometry and photoelectron spectroscopy characterizations. Being effectively Al2- due to the electron donation from Na, the Al atoms in Na3 Al2 - undergo a double electronic transmutation into Group 15 elements, thus the Al2- ≡Al2- kernel mimics the P≡P and N≡N molecules. We anticipate this work will stimulate more endeavors in discovering materials using Al2- ≡Al2- as a building block in the gas phase and in the solid state.