Lymphocyte-specific Kinase Research Articles

Abstract C156: Dasatinib targets an upregulated SRC family kinase gene in extramedullary leukemia, improving prognosis in these patients

Abstract Purpose: To review outcomes of pts with extramedullary leukemic tumors (EMLT) treated with the second-generation tryrosine kinase inhibitor (TKI) dasatinib. Procedures: Reported cases of extramedullary leukemic tumors in patients treated with TKIs were reviewed and survivals elicited from authors. RNA seq and RT-PCR for lymphocyte-specific kinase (LCK) mRNA were performed on cases of EMLT in breast. Summary: Leukemic cells growing as tumors in any organ lead to treatment failure and death, even if marrow is in remission. Median survival of patients with EMLT is 5 months, unchanged for the last 50 years. Like solid cancers, EMLT are invasive and metastatic, showing desmoplastic fibrosis and invasion of adipose tissue. Traditional anti-leukemia chemo has not reliably cured EMLT in any site, but excision of primary tumors followed by adjuvant chemo has achieved remissions of > 20 yrs. This suggests the benefit of removing tumor microenvironment (TME) containing cytokines and immune cells influencing tumor growth and resistance. TME is a logical target to ablate EMLT; TME is not affected by agents that kill replicating leukemia cells. RNAseq of very long-stored samples of EMLT in breast found upregulation of LCK compared to normal breast, confirmed by LCK RT-PCR analysis in 3 of 5 ALL tumors (2 Ph+ALL, 1 B-ALL) and 1 of 8 AML tumors. LCK is a target of DAS, a second-generation TKI that, like first-generation imatinab (IM), effectively controls BCR-ABL+ (Ph+) chronic myeloid leukemia (CML) marrow. However, IM cannot prevent or treat EM relapse, documented in 90 reported cases, mostly in CNS sites, with or without blastic marrow. CNS leukemia can lead to blindness, deafness, and paralysis. Responses to routine CNS therapy with RT and intrathecal or high-dose chemo +/- transplant, occur but lengthy DFS is rare. Since 2004, DAS +/- routine therapy has cleared CNS sites (meninges, brain, optic nerve) for up to 7+ yrs in Ph+ and T lymphoid leukemias. LCK is expressed diffusely in the CNS within neurons and ependymal cells. Tumors in non-CNS sites have also been cleared by DAS +/- routine therapy in pts with CML, Ph+ALL, T-ALL, and Ph+ B lymphoblastic lymphoma. Sites include skin, pancreas, stomach, testis, and epidural space, with DFS up to 9+ yrs. Among 137 of 153 DAS-treated pts with EMLT with followup after response, 23 have had progression in EM sites, while 47 were disease-free for 1 to 10 yrs (29 pts > 2 yrs). Conclusions: DAS appears to be an effective agent for treatment of EMLT. LCK is a potential DAS target in both Ph+ and non-Ph+ hematologic malignancies (HM), as EM tumors occur in leukemias, lymphomas, and myeloma. DAS combined with other therapies may benefit some HM patients and improve survival. Prospective clinical studies are needed to define which patients might benefit from DAS and the role of LCK. Routine scanning by PET/CT should be conducted in HM to find clinically unrecognized EMLT and to document response. Citation Format: Isabel Cunningham, Jianqi Yang, Herbert B Newton, Rory A Fisher. Dasatinib targets an upregulated SRC family kinase gene in extramedullary leukemia, improving prognosis in these patients [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C156.

Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase (Lck)

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure–function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck’s activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations using data available from different crystalographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck’s conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

Human Pegivirus Type 1: A Common Human Virus That Is Beneficial in Immune-Mediated Disease?

Two groups identified a novel human flavivirus in the mid-1990s. One group named the virus hepatitis G virus (HGV) and the other named it GB Virus type C (GBV-C). Sequence analyses found these two isolates to be the same virus, and subsequent studies found that the virus does not cause hepatitis despite sharing genome organization with hepatitis C virus. Although HGV/GBV-C infection is common and may cause persistent infection in humans, the virus does not appear to directly cause any other known disease state. Thus, the virus was renamed “human pegivirus 1” (HPgV-1) for “persistent G” virus. HPgV-1 is found primarily in lymphocytes and not hepatocytes, and several studies found HPgV-1 infection associated with prolonged survival in people living with HIV. Co-infection of human lymphocytes with HPgV-1 and HIV inhibits HIV replication. Although three viral proteins directly inhibit HIV replication in vitro, the major effects of HPgV-1 leading to reduced HIV-related mortality appear to result from a global reduction in immune activation. HPgV-1 specifically interferes with T cell receptor signaling (TCR) by reducing proximal activation of the lymphocyte specific Src kinase LCK. Although TCR signaling is reduced, T cell activation is not abolished and with sufficient stimulus, T cell functions are enabled. Consequently, HPgV-1 is not associated with immune suppression. The HPgV-1 immunomodulatory effects are associated with beneficial outcomes in other diseases including Ebola virus infection and possibly graft-versus-host-disease following stem cell transplantation. Better understanding of HPgV-1 immune escape and mechanisms of inflammation may identify novel therapies for immune-based diseases.

Integrated Molecular and Immune Phenotype of HER2-Positive Breast Cancer and Response to Neoadjuvant Therapy: A NeoALTTO Exploratory Analysis.

Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high). Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method. A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1-6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40-5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12-5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45-1.22 (P interaction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74-0.86) to 0.83 (95% CI, 0.78-0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49-0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30-0.86). Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Novel peptide-based inhibitor for targeted inhibition of T cell function.

Novel immunosuppressants are sought to overcome the side effects of currently used drugs. T cells play a central role in the functioning of the immune system; hence, drugs that specifically inhibit T cell function are expected to be better immunosuppressants with fewer side effects than the ones currently used. Peptides that interfere with crucial protein-protein interactions (PPIs) have been shown to influence cell physiology and have therapeutic potential. In this study, we designed a peptide, GVITAA, which specifically inhibits the function of lymphocyte-specific protein kinase (LCK), a signaling molecule that is mainly expressed in T cells and is responsible for positively regulating T cell function. Aspartate Histidine -Histidine Cysteine (DHHC21) -LCK is an important PPI present in T cells; DHHC21 interacts with LCK and targets the kinase to membrane rafts by adding a palmitoyl group. GVITAA is a ten amino acid peptide that interferes with the DHHC21-LCK interaction, prevents the membrane localization of LCK, and inhibits LCK-mediated initiation of complex signal transduction pathways required for T cell activation. In this study, we present evidence that the GVITAA peptide when conjugated with a cell-penetrating peptide-human immunodeficiency virus transactivator of transcription (TAT) and incubated with mouse T cells specifically inhibits LCK-mediated T cell receptor signaling, cytokine secretion, and T cell proliferation. This peptide does not affect other non-T cell functions and is non-toxic. A similar strategy was also tested and demonstrated in human peripheral T cells.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN‐γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin‐and‐lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory‐ and almost lost on effector memory T cells. MAL– T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL– T cells memory subtypes. Further, resting MAL– T cells harbor a larger pool of Ser59‐ and Tyr394‐ double phosphorylated lymphocyte‐specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation‐induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.

Identification of novel prognosis-related genes in the endometrial cancer immune microenvironment.

The incidence of endometrial cancer is increasing each year, and treatment effects are poor for patients with advanced and specific subtypes. Exploring immune infiltration-related factors in endometrial cancer can aid in the prognosis of patients and provide new immunotherapy targets. We downloaded immune metagene and functional data of patients with different subtypes of endometrial cancer from The Cancer Genome Atlas database and selected the lymphocyte-specific kinase (LCK) metagene as a representative genetic marker of the immune microenvironment in endometrial cancer. The results showed that LCK metagene expression is related to the prognosis of patients with endometrioid endometrial adenocarcinoma subtypes and highly correlated with the PTEN and PIK3CA mutational status. A search for LCK-related modules returned seven independent genetic predictors of survival in patients with endometrial cancer. The TIMER algorithm showed that the expression of these seven genes was positively correlated with the infiltration levels of six types of immune cells. The diagnostic value of these markers was validated using real-time quantitative PCR and immunohistochemical methods. Our results identified CD74, HLA-DRB5, CD52, HLA-DPB1 and HLA-DRB1 as possible valuable genetic markers for the diagnosis and prognosis of endometrial cancer and provided a theoretical basis for immunotherapy targets for its clinical treatment.

A common signaling pathway leading to degranulation in mast cells and its regulation by CCR1-ligand.

Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

Bioinformatic profiling of prognosis-related genes in the breast cancer immune microenvironment.

In the microenvironment of breast cancer, immune cell infiltration is associated with an improved prognosis. To identify immune-related prognostic markers and therapeutic targets, we determined the lymphocyte-specific kinase (LCK) metagene scores of samples from breast cancer patients in The Cancer Genome Atlas. The LCK metagene score correlated highly with other immune-related scores, as well as with the clinical stage, prognosis and tumor suppressor gene mutation status (BRCA2, TP53, PTEN) of patients in the four breast cancer subtypes. A weighted gene co-expression network analysis was performed to detect representative genes from LCK metagene-related gene modules. In two of these modules, the levels of the co-expressed genes correlated highly with LCK metagene levels, so we conducted an enrichment analysis to discover their functions. We also identified differentially expressed genes in samples with high and low LCK metagene scores. By examining the overlapping results from these analyses, we obtained 115 genes, and found that 22 of them were independent predictors of overall survival in breast cancer patients. These genes were validated for their prognostic and diagnostic value with external data sets and paired tumor and non-tumor tissues. The genes identified herein could serve as diagnostic/prognostic markers and immune-related therapeutic targets in breast cancer.

Lymphocyte-specific kinase expression is a prognostic indicator in ovarian cancer and correlates with a prominent B cell transcriptional signature.

To investigate the prognostic and biologic significance of immune-related gene expression in high grade serous ovarian cancer (HGSOC). Gene expression dependent survival analyses for a panel of immune related genes were evaluated in HGSOC utilizing The Cancer Genome Atlas (TCGA). Prognostic value of LCK was validated using IHC in an independent set of 72 HGSOC. Prognostic performance of LCK was compared to cytolytic score (CYT) using RNAseq across multiple tumor types. Differentially expressed genes in LCK high samples and gene ontology enrichment were analyzed. High pre-treatment LCK mRNA expression was found to be a strong predictor of survival in a set of 535 ovarian cancers. Patients with high LCK mRNA expression had a longer median progression free survival (PFS) of 29.4months compared to 16.9months in those without LCK high expression (p = 0.003), and longer median overall survival (OS) of 95.1months versus 44.5months (p = 0.001), which was confirmed in an independent cohort by IHC (p = 0.04). LCK expression was compared to CYT across tumor types available in the TCGA and was a significant predictor of prognosis in HGSOC where CYT was not predictive. Unexpectedly, LCK high samples also were enriched in numerous immunoglobulin-related and other B cell transcripts. LCK is a better prognostic factor than CYT in ovarian cancer. In HGSOC, LCK high samples were characterized by higher expression of immunoglobulin and B-cell related genes suggesting that a cooperative interaction between tumor infiltrating T and B cells may correlate with better survival in this disease.

Lymphocyte-specific protein tyrosine kinase expression predicts survival in ovarian high-grade serous carcinoma

Objective: Cellular immune response, specifically tumor-infiltrating lymphocytes (TILs), correlates with survival in ovarian carcinoma; however, specific gene expression patterns for this response remain poorly understood. Our objective was to evaluate the impact of a subset of immune-related gene expression on prognosis ovarian high-grade serous carcinoma (OHGSC), with a focus on lymphocyte specific kinase (LCK). Prognostic value of LCK status was compared to the previously validated metric of cytolytic activity score (CYT).

Bioinformatic Profiling of Prognosis-Related Genes in Breast Cancer Immune Microenvironment

Background: The infiltration of immune and stromal cells in breast cancer microenvironment is strongly related to prognosis. Mining potential immune-related prognostic markers or therapeutic targets is significant. Methods: We had applied multiple approaches to search for the lymphocyte-specific kinase (LCK) metagene scores and LCK metagenes were used as the objects of study to screen the representative genes using weighted gene co-expression network analysis (WGCNA); besides, the differentially ex-pressed genes (DEGs) in samples with high or low LCK metagenes scores were analyzed, and the co-expressed genes markedly correlated with the genes in LCK metagenes were mined jointly. Finally, survival analyses were applied to search for the genes, and gene functions were mined through en-richment analysis. Additionally, these genes were further verified using the external dataset and paired tumor and non-tumor tissues. Findings: LCK metagene scores have high correlations with other categories of immune-related scores, clinical stage, prognosis and the mutation levels of multiple tumor suppressor genes (BRCA1, BRCA2, TP53 and PTEN), from the four kinds of breast cancer subtypes. We screened 22 genes re-lated to immunity and prognosis in breast cancer according to the expression of immune metagenes. Interpretation: Potential immune-related therapeutic targets or markers for the diagnosis and prog-nosis of breast cancer were mined. Funding: Science and Technology Commission of Shanghai Municipality (No. 17411961100). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: The undertaken of all the samples from patients of this study was approved by the Ethics Committees of Obstetrics and Gynecology Hospital of Fudan University and consent with all the patients.

Novel Role of Lck in Leptin-Induced Inflammation and Implications for Renal Aging.

Aging is associated with increased fat mass and elevated serum leptin levels (hyperleptinemia), causing proinflammation in the kidneys where it plays a primary role in the removal of endogenous leptin from the circulation. Lymphocyte-specific kinase (Lck) is a positive regulator of inflammatory signaling and a potential treatment target for age-related diseases, but its role in leptin signaling is unknown. Here, we investigated how Lck influences hyperleptinemia-induced inflammation in kidney tissues from 6- and 21-month-old rats. Results indicate that Lck expression and activation increased significantly in aged rat kidneys, especially at renal tubules. Furthermore, we identified interactions between Lck and short leptin-receptor isoforms, suggesting that Lck is a protein tyrosine kinase regulating leptin signaling. We further investigated whether increased Lck expression in renal tubular epithelial cells and macrophage infiltration are associated with leptin-induced inflammation. We then demonstrated that leptin activates Lck and proinflammatory transcription factors (STAT3 and NF-κB), while Lck knockdown modulates the expression of both transcription factors. Collectively, these data implicate that Lck leads to development of leptin-induced renal inflammation during aging. Inhibition of this protein tyrosine kinase may therefore be an appropriate therapeutic option for protection against age-related hyperleptinemia.

CEACAM1 promotes CD8+ T cell responses and improves control of a chronic viral infection

Dysfunction of CD8+ T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV) infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8+ T cells, and the absence of CEACAM1 on virus-specific CD8+ T cells limits the antiviral CD8+ T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8+ T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8+ T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important regulator of virus-specific CD8+ T cell functions in mice and humans and represents a promising therapeutic target for modulating CD8+ T cells.

Revealing the Mechanical Basis of T Cell Signaling

Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts another cell, there are long-standing questions of whether T cells transmit defined forces to their receptors and whether chemo-mechanical coupling influences immune function. I will discuss the development of DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual T cell receptor forces during T-cell activation. We show that naive T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their receptors within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of receptor forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when forces are chemically abolished or physically “filtered” to a level of 12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune receptor mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Towards a better understanding of on and off target effects of the lymphocyte-specific kinase LCK for the development of novel and safer pharmaceuticals

In this work we have developed a multi-tiered computational platform to study protein-drug interactions. At the beginning of the workflow more efficient and less accurate methods are used to enable large libraries of proteins in many conformations and massive chemical libraries to be screened. At each subsequent step in the workflow a subset of input data is investigated with increased accuracy and more computationally expensive methods. We demonstrate the developed workflow with the investigation of the lymphocyte-specific kinase LCK, which is implicated as a drug target in many cancers and also known to have toxic effects when unintentionally targeted. Several LCK states and conformations are investigated using molecular docking and generalized Born and surface area continuum solvation (MM/GBSA). Different variations in the drug screening process provide unique results that may elucidate the biological mechanisms underlying the drug interactions.

Spectroscopic investigations and molecular docking study of (2E)-1-(4-Chlorophenyl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one using quantum chemical calculations

In this work, the vibrational spectral analysis was carried out using FT-IR and FT-Raman spectroscopy of (2E)-1-(4-Chlorophenyl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one. The computations were performed at DFT level of theory to get the optimizedgeometry and vibrational wave numbers of the normal modes of the title compound using Gaussian09 software. The complete vibrational assignments of wave numbers were made on the basis of potential energy distribution. The calculated HOMO and LUMO energies show chemical activity of the molecule. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The hyperpolarizability values are reported and the first hyperpolarizability of the title compound is 83.85 times that of standard NLO material urea. From the MEP plot, the negative electrostatic potential regions are mainly localized over the carbonyl group, the phenyl rings and are possible sites for electrophilic attack. The positive regions are localized over all the hydrogen atoms and are possible sites for nucleophilic attack. The molecular docking results suggest that the compound might exhibit inhibitory activity against lymphocyte-specific kinase and may results in design of novel T-cell immunosuppressants.

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Diffusion Analysis of Lymphocyte Specific Kinase Reveals Immobilisation Hot Spots in Live T-Cell Plasma Membranes

T-Lymphocytes initiate an adaptive immune response after specific binding of the T-cell receptor (TCR) to an antigenic peptide bound to the major histocompatibility complex (peptide-MHC) on an antigen presenting cell. Key events upon TCR-pMHC binding involve the phosphorylation of intracellular tyrosine residues of the TCR and recruitment of adapter molecules, which results in downstream signalling. Initial TCR phosphorylation is primarily carried out by the membrane associated protein lymphocyte specific kinase (Lck) making it a central molecule for T-cell signalling. Recent work on Lck, using superresolution microscopy techniques, has shown the organisation of Lck in nanoclusters in fixed as well as in live cells [1]. However, neither the structural mechanism nor the functional role of this non-random Lck distribution in the plasma membrane are well understood. Possible explanations for Lck clustering may be local lipid heterogeneities as well as interactions with other plasma membrane proteins or the cortical actin cytoskeleton. In order to acquire a better understanding of Lck dynamics in live cells, we carried out superresolution experiments on Lck-mEOS3.2, stably expressed in JCaM 1.6 cells. We used single molecule tracking to elucidate the diffusional behaviour of Lck. Based on the reported existence of nanoclusters, we implemented a simple and practical method to separately analyse Lck diffusion inside and outside these structures. Surprisingly, only Lck-molecules inside clusters were immobilised, whereas molecules outside of clusters exhibited pure Brownian motion. We further found that disruption of the actin cytoskeleton did not influence Lck immobilisations. [1] Rossy, J., et al., Conformational states of the kinase Lck regulate clustering in early T cell signalling. Nature immunology 14, 82-89 (2013). This work was supported by the Austrian Science Fund/FWF project P26337-B21 and P27941-B28.