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Protein-protein interactions (PPIs) play pivotal roles in directing T cell fate. One key player is the non-receptor tyrosine protein kinase Lck that helps to transduce T cell activation signals. Lck is mediated by other proteins via interactions that are inadequately understood. Here, we use the deep learning method AF2Complex to predict PPIs involving Lck, by screening it against ∼1,000 proteins implicated in immune responses, followed by extensive structural modeling for selected interactions. Remarkably, we describe how Lck may be specifically targeted by a palmitoyltransferase using a phosphotyrosine motif. We uncover "hotspot" interactions between Lck and the tyrosine phosphatase CD45, leading to a significant conformational shift of Lck for activation. Lastly, we present intriguing interactions between the phosphotyrosine-binding domain of Lck and the cytoplasmic tail of the immune checkpoint LAG3 and propose a molecular mechanism for its inhibitory role. Together, this multifaceted study provides valuable insights into T cell regulation and signaling., Competing Interests: Declaration of interests M.G. is a stockholder at AgnistaBio, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The interpretation of ligand-target interactions at atomistic resolution is central to most efforts in computational drug discovery and optimization. However, the highly dynamic nature of protein targets, as well as possible induced fit effects, makes difficult to sample many interactions effectively with docking studies or even with large-scale molecular dynamics (MD) simulations. We propose a novel application of Self-Organizing Maps (SOMs) to address the sampling and dynamic mapping tasks, particularly in cases involving ligand flexibility and induced fit. The SOM approach offers a data-driven strategy to create a map of the interaction process and pathways based on unbiased MD. Furthermore, we show how the preliminary SOM mapping is complementary to kinetic analysis, with the employment of both network-based approaches and Markov state models. We demonstrate the method by comprehensively mapping a large dataset of 640 μs of unbiased trajectories sampling the recognition process between the phosphorylated YEEI peptide and its high-specificity target lck-SH2. The integration of SOM into unbiased simulation protocols significantly advances our understanding of the ligand binding mechanism. This approach serves as a potent tool for mapping intricate ligand-target interactions with unprecedented detail, thereby enhancing the characterization of kinetic properties crucial to drug design., (© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/).)

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity., (©2024 American Association for Cancer Research.)

Sorting nexin 27 is a unique member of the sorting nexin family of proteins that mediates the endosome-to-plasma membrane trafficking of cargos bearing a PSD95/Dlg1/ZO-1 (PDZ)-binding motif. In brain, sorting nexin 27 regulates synaptic plasticity, and its dysregulation contributes to cognitive impairment and neuronal degeneration. In T lymphocytes, sorting nexin 27 partners with diacylglycerol kinase ζ to facilitate polarized traffic and signaling at the immune synapse. By silencing sorting nexin 27 expression in a human T-cell line, we demonstrate that sorting nexin 27 is a key regulator of the early T-cell tyrosine-based signaling cascade. Sorting nexin 27 transcriptionally controls CD4 abundance in resting conditions and that of its associated molecule, Lck. This guarantees the adequate recruitment of Lck at the immune synapse, which is indispensable for subsequent activation of tyrosine phosphorylation-regulated events. In contrast, reduced sorting nexin 27 expression enhances NF-κB-dependent induction of CXCR4 and triggers production of lytic enzymes and proinflammatory cytokines. These results provide mechanistic explanation to previously described sorting nexin 27 function in the control of immune synapse organization and indicate that impaired sorting nexin 27 expression contributes to CD4 T-cell dysfunction., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Preeclampsia (PE) is a heterogeneous disease that seriously affects the health of mothers and fetuses. Lack of detection assays, its diagnosis and intervention are often delayed when the clinical symptoms are atypical. Using personalized pathway-based analysis and machine learning algorithms, we built a PE diagnosis model consisting of nine core pathways using multiple cohorts from the Gene Expression Omnibus database. The model showed an area under the receiver operating characteristic (AUROC) curve of 0.959 with the data from the placental tissue samples in the development cohort. In the two validation cohorts, the AUROCs were 0.898 and 0.876, respectively. The model also performed well with the maternal plasma data in another validation cohort (AUROC: 0.815). Moreover, we identified tyrosine-protein kinase Lck (LCK) as the hub gene in this model and found that LCK and pLCK proteins were downregulated in placentas from PE patients. The pathway-level model for PE can provide a novel direction to develop molecular diagnostic assay and investigate potential mechanisms of PE in future studies., (© 2024. The Author(s), under exclusive licence to The Japanese Society of Hypertension.)

The serine/threonine phosphatase calcineurin is a component of the T cell receptor (TCR) signalosome, where it promotes T cell activation by dephosphorylating Lck S59 . Using small interfering RNA (siRNA)-mediated knockdown and CRISPR-Cas9-targeted genetic disruption of the calcineurin A chain α and β isoforms, we find that calcineurin also functions as an adaptor in TCR-signaled human T cells. Unlike inhibition of its phosphatase activity, in the absence of calcineurin A, TCR signaling results in attenuated actin rearrangement, markedly reduced TCR-Lck microcluster formation and recruitment of the adaptor RhoH, and diminished phosphorylation of critical targets downstream of Lck such as TCRζ and ZAP-70. Reconstitution of deficient T cells with either calcineurin Aα or Aβ restores TCR microcluster formation and signaling, as does reconstitution with a phosphatase-inactive Aα chain. These results assign a non-enzymatic adaptor function to calcineurin in the formation and stabilization of a functional TCR signaling complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Despite significant advances over recent years, the treatment of T cell acute lymphoblastic leukemia (T-ALL) remains challenging. We have recently shown that a subset of T-ALL cases exhibited constitutive activation of the lymphocyte-specific protein tyrosine kinase (LCK) and were consequently responsive to treatments with LCK inhibitors and degraders such as dasatinib and dasatinib-based PROTACs. Here we report the design, synthesis and in vitro/vivo evaluation of SJ45566, a potent and orally bioavailable LCK PROTAC.

The tyrosine kinase Lck is mandatory for initiating signaling responses downstream the antigenic T cell receptor (TCR). Numerous studies have shown that a prerequisite for efficient and well-balanced Lck regulation and function is its finely orchestrated spatial distribution pattern, especially at the plane of the plasma membrane. There is a wealth of knowledge on Lck localization sites, preference for specialized lipid microenvironments and colocalization partners. However, several questions concerning the spatial organization of its differentially phosphorylated conformers and the dynamics of their juxtaposition in relation to ligated and non-ligated TCRs remain elusive. In this brief report we introduce a non-invasive nanobody-based approach for mapping Lck subcellular allocation with high precision. Our initial data using this methodology, provide insight into the topology of Lck in resting T cells and its confined localization in a strictly delimited environment within the plane of the plasma membrane., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tyritidis, Tsioupros, Christou, Koutras, Morfos and Nika.)

The lymphocyte-specific protein tyrosine kinase (LCK) plays a crucial role in both T-cell development and activation. Dysregulation of LCK signaling has been demonstrated to drive the oncogenesis of T-cell acute lymphoblastic leukemia (T-ALL), thus providing a therapeutic target for leukemia treatment. In this study, we introduced a sophisticated virtual screening strategy combined with biological evaluations to discover potent LCK inhibitors. Our initial approach involved utilizing the PLANET algorithm to assess and contrast various scoring methodologies suitable for LCK inhibitor screening. After effectively evaluating PLANET, we progressed to devise a virtual screening workflow that synergistically combines the strengths of PLANET with the capabilities of Schrödinger's suite. This integrative strategy led to the efficient identification of four potential LCK inhibitors. Among them, compound 1232030-35-1 stood out as the most promising candidate with an IC 50 of 0.43 nM. Further in vitro bioassays revealed that 1232030-35-1 exhibited robust antiproliferative effects on T-ALL cells, which was attributed to its ability to suppress the phosphorylations of key molecules in the LCK signaling pathway. More importantly, 1232030-35-1 treatment demonstrated profound in vivo antileukemia efficacy in a human T-ALL xenograft model. In addition, complementary molecular dynamics simulations provided deeper insight into the binding kinetics between 1232030-35-1 and LCK, highlighting the formation of a hydrogen bond with Met319. Collectively, our study established a robust and effective screening strategy that integrates AI-driven and conventional methodologies for the identification of LCK inhibitors, positioning 1232030-35-1 as a highly promising and novel drug-like candidate for potential applications in treating T-ALL.

Objective: To identify the biomarkers for early rheumatoid arthritis (RA) diagnosis and explore the possible immune regulatory mechanisms., Methods: The differentially expressed genesin RA were screened and functionally annotated using the limma, RRA, batch correction, and clusterProfiler. The protein-protein interaction network was retrieved from the STRING database, and Cytoscape 3.8.0 and GeneMANIA were used to select the key genes and predicting their interaction mechanisms. ROC curves was used to validate the accuracy of diagnostic models based on the key genes. The disease-specific immune cells were selected via machine learning, and their correlation with the key genes were analyzed using Corrplot package. Biological functions of the key genes were explored using GSEA method. The expression of STAT1 was investigated in the synovial tissue of rats with collagen-induced arthritis (CIA)., Results: We identified 9 core key genes in RA (CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL), which regulate synovial inflammation primarily through cytokines-related pathways. ROC curve analysis showed a high predictive accuracy of the 9 core genes, among which STAT1 had the highest AUC (0.909). Correlation analysis revealed strong correlations of CD3G, ITGAL, LCK, CD8A, and STAT1 with disease-specific immune cells, and STAT1 showed the strongest correlation with M1-type macrophages ( R =0.68, P =2.9e-08). The synovial tissues of the ankle joints of CIA rats showed high expressions of STAT1 and p-STAT1 with significant differential expression of STAT1 between the nucleus and the cytoplasm of the synovial fibroblasts. The protein expressions of p-STAT1 and STAT1 in the cell nuclei were significantly reduced after treatment., Conclusion: CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL may serve as biomarkers for early diagnosis of RA. Gene-immune cell pathways such as CD3G/CD8A/LCK-γδ T cells, ITGAL-Tfh cells, and STAT1-M1-type macrophages may be closely related with the development of RA.

Regulatory T (Treg) cells are indispensable in maintaining the immune homeostasis and preventing autoimmune diseases. Regulatory T (Treg) cells include thymus derived Treg cells (tTregs) and peripherally induced Treg cells (iTreg), which are differentiated from antigen stimulated CD4 + naïve T cells in presence of TGFβ. tTregs are quite stable, and more immune suppressive, while iTreg cells are less stable, and are prone to differentiate into inflammatory T cells. Therefore, identification of small molecules that could promote the differentiation of iTreg cells is an attractive strategy for autoimmune diseases. Inhibition of AKT/mTOR pathway promotes their differentiation. Whether inhibition of Lck/Fyn kinase activity (upstream of AKT/mTOR pathway) can be used to promote the differentiation of iTreg cells has not been determined. Here, we showed that Srci1, a small molecular inhibitor of Lck/Fyn, promoted the differentiation of FOXP3 + iTreg cells. Srci1 treatment resulted in inhibition of phosphorylation of key components of AKT/mTOR pathway, including mTOR, p70 S6K, 4EBP1, and promoted the expression of Foxp3 and its target genes, thereby promoted differentiation of in vitro iTreg cells. Srci1 treated iTreg cells showed more similar gene expression profile to that of tTreg cells. Our results thus suggest that inhibition of Lck/Fyn kinase activity can promote the differentiation of iTreg cells, and may have implication in autoimmune diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome., Methods: We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM., Results: Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK., Conclusions: The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Woessner, Brandl, Hartmann, Schamel, Hartl and Minguet.)

CD4 + T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4 + T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors., Competing Interests: ML, PT, CK, PY, KL, HP, KV No competing interests declared, MK Has disclosed an outside interest in Module Therapeutics to the University of Arizona. Conflicts of interest resulting from this interest are being managed by the University of Arizona in accordance with their policies, (© 2023, Lee et al.)

Lymphocyte-specific protein tyrosine kinase (Lck) plays vital roles in the T-cell receptor- mediated development, function, and differentiation of T-cells. Given its substantial involvement in T cell signaling, irregularities in the expression and functionality of Lck may lead to various diseases, including cancer. In this study, we found that compound 12a exerted significant inhibitory potency against Lck with an IC 50 value of 10.6 nM. In addition, 12a demonstrated high efficacy in various colon cancer cell lines as indicated by GI 50 values ranging from 0.24 to 1.26 μM. Notably, 12a inhibited the phosphorylation of Lck in Colo201 cells. Overall, the anti-proliferative effects of 12a on diverse cancer cell lines highlights its potential application for the treatment of various cancer types., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The CD4 or CD8 co-receptors' interaction with the protein-tyrosine kinase Lck initiates the tyrosine phosphorylation cascade leading to T cell activation. A critical question is: to what extent are co-receptors and Lck coupled? Our contribution concerns Zn 2+ , indispensable for CD4 - and CD8-Lck formation. We combined biochemical and cellular approaches to show that dynamic fluctuations of free Zn 2+ in physiological ranges influence Zn(CD4) 2 and Zn(CD4)(Lck) species formation and their ratio, although the same Zn(Cys) 2 (Cys) 2 cores. Moreover, we demonstrated that the affinity of Zn 2+ to CD4 and CD4-Lck species differs significantly. Increased intracellular free Zn 2+ concentration in T cells causes higher CD4 partitioning in the plasma membrane. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn 2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly., Competing Interests: Declaration of interests There is no competing interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lymphocyte-specific protein tyrosine kinase (LCK), a member of the Src family of tyrosine kinases, is implicated in the pathogenesis of almost all types of leukemia via T cells activation and signal transduction. LCK is highly expressed in acute lymphoblastic leukemia (ALL), and knockdown of the LCK gene can significantly inhibit the proliferation of leukemia cell lines. Here, we designed and synthesized a series of benzothiazole derivatives as novel LCK inhibitors using both docking-based virtual screening and activity assays for structural optimization. Among these compounds, 7 m showed a strong inhibitory activity in the proliferation of leukemia cell lines and LCK kinase activity. Moreover, we found that compound 7 m could induce apoptosis while simultaneously blocking cell cycle via decreasing its phosphorylation at Tyr394 of the LCK. Collectively, these findings shed new light on compound 7 m that would be utilized as a promising drug candidate with apoptosis-triggered and cell cycle arrest activities for the future ALL therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation., (© 2024. The Author(s).)

Signal-transducing adaptor protein-2 (STAP-2) is a unique scaffold protein that regulates several immunological signaling pathways, including LIF/LIF receptor and LPS/TLR4 signals. STAP-2 is required for Fas/FasL-dependent T cell apoptosis and SDF-1α-induced T cell migration. Conversely, STAP-2 modulates integrin-mediated T cell adhesion, suggesting that STAP-2 is essential for several negative and positive T cell functions. However, whether STAP-2 is involved in T cell-antigen receptor (TCR)-mediated T cell activation is unknown. STAP-2 deficiency was recently reported to suppress TCR-mediated T cell activation by inhibiting LCK-mediated CD3ζ and ZAP-70 activation. Using STAP-2 deficient mice, it was demonstrated that STAP-2 is required for the pathogenesis of Propionibacterium acnes-induced granuloma formation and experimental autoimmune encephalomyelitis. Here, detailed functions of STAP-2 in TCR-mediated T cell activation, and how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases, are reviewed.

Mutations affecting T-cell receptor (TCR) signaling typically cause combined immunodeficiency (CID) due to varying degrees of disturbed T-cell homeostasis and differentiation. Here, we describe two cousins with CID due to a novel nonsense mutation in LCK and investigate the effect of this novel nonsense mutation on TCR signaling, T-cell function, and differentiation. Patients underwent clinical, genetic, and immunological investigations. The effect was addressed in primary cells and LCK-deficient T-cell lines after expression of mutated LCK. RESULTS: Both patients primarily presented with infections in early infancy. The LCK mutation led to reduced expression of a truncated LCK protein lacking a substantial part of the kinase domain and two critical regulatory tyrosine residues. T cells were oligoclonal, and especially naïve CD4 and CD8 T-cell counts were reduced, but regulatory and memory including circulating follicular helper T cells were less severely affected. A diagnostic hallmark of this immunodeficiency is the reduced surface expression of CD4. Despite severely impaired TCR signaling mTOR activation was partially preserved in patients' T cells. LCK-deficient T-cell lines reconstituted with mutant LCK corroborated partially preserved signaling. Despite detectable differentiation of memory and effector T cells, their function was severely disturbed. NK cell cytotoxicity was unaffected. Residual TCR signaling in LCK deficiency allows for reduced, but detectable T-cell differentiation, while T-cell function is severely disturbed. Our findings expand the previous report on one single patient on the central role of LCK in human T-cell development and function., (© 2023. The Author(s).)

Eosinophilia is a common finding in drug hypersensitivity reactions (DHR). Its cause is unclear, as neither antigen/allergen-driven inflammation nor clonal expansion is involved. Most delayed-DHRs are due to p-i (pharmacologic interaction of drugs with immune receptors). These are off-target activities of drugs with immune receptors that result in various types of T-cell stimulation, some of which involve excessive IL-5 production. Functional and phenotypic studies of T-cell clones and their TCR-transfected hybridoma cell lines revealed that some p-i-induced drug stimulations occur without CD4/ CD8 co-receptor engagement. The CD4/CD8 co-receptors link Lck (lymphocyte-specific protein tyrosine kinase) and LAT (linker for activation of T cells) to the TCR. Alteration of Lck or LAT can result in a TCR signalosome with enhanced IL-5 production. Thus, if a more affine TCR-[drug/peptide/HLA] interaction allows bypassing the CD4 co-receptor, a modified Lck/LAT activation may lead to a TCR signalosome with elevated IL-5 production. This "IL-5-TCR-signalosome" hypothesis could also explain eosinophilia in superantigen or allo-stimulation (graft-versus-host disease), in which evasion of CD4/CD8 co-receptors has also been described. It may open new therapeutic possibilities in certain eosinophilic diseases by directly targeting the IL-5-TCR signalosome., (© 2023 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The protein tyrosine phosphatase PTPN22 inhibits T cell activation by dephosphorylating some essential proteins in the T cell receptor (TCR)-mediated signaling pathway, such as the lymphocyte-specific protein tyrosine kinase (Lck), Src family tyrosine kinases Fyn, and the phosphorylation levels of Zeta-chain-associated protein kinase-70 (ZAP70). For the first time, we have successfully produced PTPN22 CS transgenic mice in which the tyrosine phosphatase activity of PTPN22 is suppressed. Notably, the number of thymocytes in the PTPN22 CS mice was significantly reduced, and the expression of cytokines in the spleen and lymph nodes was changed significantly. Furthermore, PTPN22 CS facilitated the positive and negative selection of developing thymocytes, increased the expression of the TCRαβ-CD3 complex on the thymus cell surface, and regulated their internalization and recycling. ZAP70, Lck, Phospholipase C gamma1(PLCγ1), and other proteins were observed to be reduced in PTPN22 CS mouse thymocytes. In summary, PTPN22 regulates TCR internalization and recycling via the modulation of the TCR signaling pathway and affects TCR expression on the T cell surface to regulate negative and positive selection. PTPN22 affected the development of the thymus, spleen, lymph nodes, and other peripheral immune organs in mice. Our study demonstrated that PTPN22 plays a crucial role in T cell development and provides a theoretical basis for immune system construction.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function., Competing Interests: Declaration of interests National University of Singapore has filed patents based on these findings. P.A.M. is a shareholder and advisory board member of Gen Y Biologics Pte., Ltd. (company registration number: 202005553Z). Patent title: Engineered Immune Cells (PCT/SG2020/050090). Published September 10, 2020: WO 2020/180243 (L.W., N.R.J.G., and J.B.)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8 + T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation., (© 2022. The Author(s).)

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (Lck A ) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that Lck A is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady Lck A shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing Lck A . We then challenged steady Lck A formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in Lck A generation, except for the CD45 transmembrane domain that drastically reduced Lck A due to its excessive lateral proximity to CD45. Comprehensively, Lck A formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymphocyte-specific protein tyrosine kinase (LCK) is common in a variety of hematologic malignancies but comparatively less common in solid tumors. This study aimed to explore the potential diagnostic and prognostic value of LCK across tumors through integrative and comprehensive pan-cancer analysis, as well as experimental validation. Multiple databases were used to explore the expression, alteration, prognostic value, association with immune infiltration, and potential functional pathways of LCK in pan-cancers. The results were further validated by western blotting and qPCR of patient samples as well as tumor cell lines. High LCK expression typically represents a better prognosis. Notably, drug sensitivity prediction of LCK identified P-529 as a candidate for drug development. Gene Annotations (GO) and KEGG analyses showed significant enrichment of PD-L1 and the T-cell receptor pathway. The results from patient samples and tumor cell lines confirmed these conclusions in LIHC. In conclusion, LCK is differentially expressed in multiple tumors and normal tissues. Further analysis highlighted its association with prognostic implications, pan-cancer genetic alterations, and immune signatures. Our data provide evidence for a diagnostic marker of LCK and the possible use of LCK as a target for the treatment of tumors.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4 + CD8 + double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Lymphocyte-specific protein tyrosine kinase (Lck) is a member of the Src family of tyrosine kinases. The significance of Lck inhibition in lung fibrosis has not yet been fully elucidated, even though lung fibrosis is commonly preceded by inflammation caused by infiltration of T-cells expressing Lck. In this study, we examined the effect of Lck inhibition in an experimental mouse model of lung fibrosis. We also evaluated the effect of Lck inhibition on the expression of TGF-β1, an inhibitory cytokine regulating the immune function, in regulatory T-cells (Tregs)., Methods: Lung fibrosis was induced in mice by intratracheal administration of bleomycin. A-770041, a Lck-specific inhibitor, was administrated daily by gavage. Tregs were isolated from the lung using a CD4+CD25+ Regulatory T-cell Isolation Kit. The expression of Tgfb on Tregs was examined by flow cytometry and quantitative polymerase chain reaction. The concentration of TGF-β in bronchoalveolar lavage fluid (BALF) and cell culture supernatant from Tregs was quantified by an enzyme-linked immunosorbent assay., Results: A-770041 inhibited the phosphorylation of Lck in murine lymphocytes to the same degree as nintedanib. A-770041 attenuated lung fibrosis in bleomycin-treated mice and reduced the concentration of TGF-β in BALF. A flow-cytometry analysis showed that A-770041 reduced the number of Tregs producing TGF-β1 in the lung. In isolated Tregs, Lck inhibition by A-770041 decreased the Tgfb mRNA level as well as the concentration of TGF-β in the supernatant., Conclusions: These results suggest that Lck inhibition attenuated lung fibrosis by suppressing TGF-β production in Tregs and support the role of Tregs in the pathogenesis of lung fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs., (© 2022 by The American Society of Hematology.)

Ethnopharmacological Relevance: Hypericum japonicum Thunb. ex Murray (Hypericaceae), named 'Tianjihuang' is a traditional Chinese medicine with hepatoprotective, antibacterial, and antitumour effects. Betulinic acid (BA) is its active constituent and has been found to have a number of biological effects, including antiviral, anti-inflammatory, and anti-malarial therapeutic properties. Non-alcoholic fatty liver disease and acute alcoholic liver injury have both been proven to benefit from BA. BA's effects and mechanism on liver fibrosis are still unknown., Aim of the Study: The purpose of this study was to explore the influence of BA on lymphocyte-specific protein tyrosine kinase (Lck), a non-receptor Src family kinase, that reduces liver fibrosis by inhibiting the phosphorylation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways through the interaction of Lck and SOCS1., Materials and Methods: A liver fibrosis model was established in vivo with CCl 4 using haematoxylin and eosin (HE) staining, Masson staining, immunohistochemical staining, and immunofluorescence staining. Hepatic stellate cells were induced with transforming growth factor (TGF)-β1 in vitro, using Western blotting, immunofluorescence staining, and a cell scratch assay., Results: In a CCl 4 -induced mouse hepatic fibrosis model and in TGF-β1-activated HSC-T6 cells, BA markedly reduced fibrosis, as demonstrated by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in vivo and in vitro. BA significantly suppressed the activity and expression of Lck in vitro. Overexpression of Lck may diminish the effect of BA on liver fibrosis. In vitro, BA also greatly increased the expression of suppressor of cytokine signalling 1 (SOCS1) while it considerably inhibited the expression of p-JAK and p-STAT1., Conclusions: These findings suggest that BA promotes the expression of SOCS1 by the inhibiting the interaction between Lck and SOCS1, followed by the inhibition of JAK/STAT phosphorylation to prevent the progression of liver fibrosis. Therefore, BA could be used as a promising natural supplement for the treatment of liver fibrosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Alterations in both the expression and function of the non-receptor tyrosine kinase Zap70 are associated with numerous human diseases including immunodeficiency, autoimmunity, and leukemia. Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLCγ1 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70 C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70 C564R , is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70 C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70 C564A displayed the hyperphosphorylation of TCR-ζ and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and there is an unmet need for targeted therapies, especially for patients with relapsed disease. We have recently identified pre-T cell receptor and lymphocyte-specific protein tyrosine kinase (LCK) signaling as a common therapeutic vulnerability in T-ALL. LCK inhibitor dasatinib showed efficacy against T-ALL in preclinical studies and in patients with T-ALL; however, this is transient in most cases. Leveraging the proteolysis targeting chimera (PROTAC) approach, we developed a series of LCK degraders using dasatinib as an LCK ligand and phenyl-glutarimide as a cereblon-directing moiety. Our lead compound SJ11646 exhibited marked efficiency in cereblon-mediated LCK degradation in T-ALL cells. Relative to dasatinib, SJ11646 showed up to three orders of magnitude higher cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples in vitro, with drastically prolonged suppression of LCK signaling. In vivo pharmacokinetic and pharmacodynamic profiling indicated a 630% increase in the duration of LCK suppression by SJ11646 over dasatinib in patient-derived xenograft models of T-ALL, which translated into its extended leukemia-free survival over dasatinib in vivo. Last, SJ11646 retained a high binding affinity to 51 human kinases, particularly ABL1, KIT, and DDR1, all of which are known drug targets in other cancers. Together, our dasatinib-based phenyl-glutarimide PROTACs are promising therapeutic agents in T-ALL and valuable tools for developing degradation-based therapeutics for other cancers.

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., (© 2022 The Authors.)

CD4 + T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ~435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that enhance pMHCII responses, and covary with residues in an intracellular motif that inhibits responses. Importantly, while CD4 interactions with the Src kinase, Lck, are viewed as key to pMHCII responses, our data indicate that CD4-Lck interactions derive their importance from the counterbalancing activity of the inhibitory motif, as well as motifs that direct CD4-Lck pairs to specific membrane compartments. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering., Competing Interests: ML, PT, CK, KL, HP, KV No competing interests declared, MK has disclosed an outside interest in Module Therapeutics to the University of Arizona. Conflicts of interest resulting from this interest are being managed by the University of Arizona in accordance with their policies, (© 2022, Lee et al.)

Lck and Zap70, two non-receptor tyrosine kinases, play a crucial role in the regulation of membrane proximal TCR signaling critical for thymic selection, CD4/CD8 lineage choice and mature T cell function. Signal initiation upon TCR/CD3 and peptide/MHC interaction induces Lck-mediated phosphorylation of CD3 ITAMs. This is necessary for Zap70 recruitment and its phosphorylation by Lck leading to full Zap70 activation. In its native state Zap70 maintains a closed conformation creating an auto-inhibitory loop, which is relieved by Lck-mediated phosphorylation of Y315/Y319. Zap70 is differentially expressed in thymic subsets and mature T cells with CD8 T cells expressing the highest amount compared to CD4 T cells. However, the mechanistic basis of differential Zap70 expression in thymic subsets and mature T cells is not well understood. Here, we show that Zap70 is degraded relatively faster in DP and mature CD4 T cells compared to CD8 T cells, and inversely correlated with relative level of activated Zap70. Importantly, we found that Zap70 expression is negatively regulated by Lck activity: augmented Lck activity resulting in severe diminution in total Zap70. Moreover, Lck-mediated phosphorylation of Y315/Y319 was essential for Zap70 degradation. Together, these data shed light on the underlying mechanism of Lck-mediated differential modulation of Zap70 expression in thymic subsets and mature T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Damen, Tebid, Viens, Roy and Dave.)

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca 2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in Lck Y192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4 + and CD8 + T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5 + ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5 + ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5 - B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5 + ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5 + ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5 + ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5 + ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5 + ILBs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhou, Blevins, Crawford and Kaminski.)

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis and the destruction of small joints. Emerging evidence shows that immunoglobulin D (IgD) stimulation induces T-cell activation, which may contribute to diseases pathogenesis in RA. In this study, we investigated the downstream signaling pathways by which IgD activated T cells as well as the possible role of IgD in the T-B interaction. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. We demonstrated that IgD activated T cells through IgD receptor (IgDR)-lymphocyte-specific protein tyrosine kinase (Lck)-zeta-associated protein 70 (ZAP70)/phosphatidylinositol 3-kinase (PI3K)/nuclear factor kappa-B (NF-κB) signaling pathways; IgD-induced CD4 + T cells promoted the proliferation of CD19 + B cells in RA patients. A novel fusion protein IgD-Fc-Ig (composed of human IgD-Fc domain and IgG 1 Fc domain, which specifically blocked the IgD-IgDR binding) inhibited the coexpression of IgDR and phosphorylated Lck (p-Lck) and the expression levels of p-Lck, p-ZAP70, p-PI3K on CD4 + T cells, and decreased NF-κB nuclear translocation in Jurkat cells. Meanwhile, IgD-Fc-Ig downregulated the expression levels of CD40L on CD4 + T cells as well as CD40, CD86 on CD19 + B cells in RA patients and healthy controls. It also decreased the expression levels of CD40L on CD4 + T cells and CD40 on CD19 + B cells from spleens of collagen-induced arthritis (CIA) mice and reduced IL-17A level in mouse serum. Moreover, administration of IgD-Fc-Ig (1.625-13 mg/kg, iv, twice a week for 4 weeks) in CIA mice dose-dependently decreased the protein expression levels of CD40, CD40L, and IgD in spleens. IgD-Fc-Ig restrains T-cell activation through inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting B-cell activation. Our data provide experimental evidences for application of IgD-Fc-Ig as a highly selective T cell-targeting treatment for RA., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Lymphocyte-specific protein tyrosine kinase (Lck), a non-receptor Src family kinase, has a vital role in various cellular processes such as cell cycle control, cell adhesion, motility, proliferation, and differentiation. Lck is reported as a key factor regulating the functions of T-cell including the initiation of TCR signalling, T-cell development, in addition to T-cell homeostasis. Alteration in expression and activity of Lck results in numerous disorders such as cancer, asthma, diabetes, rheumatoid arthritis, atherosclerosis, and neuronal diseases. Accordingly, Lck has emerged as a novel target against different diseases. Herein, we amass the research efforts in literature and pharmaceutical patents during the last decade to develop new Lck inhibitors. Additionally, structure-activity relationship studies (SAR) and docking models of these new inhibitors within the active site of Lck were demonstrated offering deep insights into their different binding modes in a step towards the identification of more potent, selective, and safe Lck inhibitors.

Under physiological conditions, CD8 + T cells need to recognize low numbers of antigenic pMHC class I complexes in the presence of a surplus of non-stimulatory, self pMHC class I on the surface of the APC. Non-stimulatory pMHC have been shown to enhance CD8 + T cell responses to low amounts of antigenic pMHC, in a phenomenon called co-agonism, but the physiological significance and molecular mechanism of this phenomenon are still poorly understood. Our data show that co-agonist pMHC class I complexes recruit CD8-bound Lck to the immune synapse to modulate CD8 + T cell signaling pathways, resulting in enhanced CD8 + T cell effector functions and proliferation, both in vitro and in vivo . Moreover, co-agonism can boost T cell proliferation through an extrinsic mechanism, with co-agonism primed CD8 + T cells enhancing Akt pathway activation and proliferation in neighboring CD8 + T cells primed with low amounts of antigen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Wu, Ng, Leow, Wei, Gascoigne and Brzostek.)

TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr 394 and the inhibitory pTyr 505 , control Lck activity. Recently, pTyr 192 in the Lck SH2 domain emerged as a third regulator. How pTyr 192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr 192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr 394 and pTyr 505 are controlled by Lck Tyr 192 Lck with a nonphosphorylated SH2 domain (Lck Phe 192 ) displayed hyperactivity, possibly by promoting Lck Tyr 394 transphosphorylation. Lck Glu 192 mimicking stable Lck pTyr 192 was inhibited by Tyr 505 hyperphosphorylation. To overcome this effect, we further mutated Tyr 505 The resulting Lck Glu 192 /Phe 505 displayed strongly increased amounts of pTyr 394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr 192 may be to protect Lck pTyr 394 and/or pTyr 505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In this Research Highlight, we summarized the results from a study by Wu et al. published in Nature Cell Biology which uncovered that asparagine (Asn), a non-essential amino acid in mammalians, is able to enhance the TCR-mediated activation and efficacy of CD8 + T cells towards tumour through lymphocyte-specific protein tyrosine kinase (LCK) signalling. This study provides insights into the physiological function of Asn in T cell activation and effector functions, showing the encouraging possibility of key metabolites for empowering cancer immunotherapy., (© 2021 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRβ-variable (TRBV) 17 + TCRs from the naïve mouse CD8 + T cell repertoire that recognizes the H-2D b -NP 366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Amphioxus (e.g., Branchiostoma belcheri , Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhou, Xiao, Zhan, Qu, Mou, Deng, Zhang, Lan, Huang and Li.)

Objective: Perivascular adipose tissue (PVAT) contributes to vascular homeostasis and is increasingly linked to vascular pathology. PVAT density and volume were associated with abdominal aortic aneurysm (AAA) presence and dimensions on imaging. However, mechanisms underlying the role of PVAT in AAA have not been clarified. This study aimed to explore differences in PVAT from AAA using gene expression and functional tests., Methods: Human aortic PVAT and control subcutaneous adipose tissue were collected during open AAA surgery. Gene analyses and functional tests were performed. The control group consisted of healthy aorta from non-living renal transplant donors. Gene expression tests were performed to study genes potentially involved in various inflammatory processes and AAA related genes. Live PVAT and subcutaneous adipose tissue (SAT) from AAA were used for ex vivo co-culture with smooth muscle cells (SMCs) retrieved from non-pathological aortas., Results: Adipose tissue was harvested from 27 AAA patients (n [gene expression] = 22, n [functional tests] = 5) and five control patients. An increased inflammatory gene expression of PTPRC (p = .008), CXCL8 (p = .033), LCK (p = .003), CCL5 (p = .004) and an increase in extracellular matrix breakdown marker MMP9 (p = .016) were found in AAA compared with controls. Also, there was a decreased anti-inflammatory gene expression of PPARG in AAA compared with controls (p = .040). SMC co-cultures from non-pathological aortas with PVAT from AAA showed increased MMP9 (p = .033) and SMTN (p = .008) expression and SAT increased SMTN expression in these SMC., Conclusion: The data revealed that PVAT from AAA shows an increased pro-inflammatory and matrix metallopeptidase gene expression and decreased anti-inflammatory gene expression. Furthermore, increased expression of genes involved in aneurysm formation was found in healthy SMC co-culture with PVAT of AAA patients. Therefore, PVAT from AAA might contribute to inflammation of the adjacent aortic wall and thereby plays a possible role in AAA pathophysiology. These proposed pathways of inflammatory induction could reveal new therapeutic targets in AAA treatment., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)