Lck Gene Research Articles

Bilateral Congenital Cytomegalovirus Retinitis Secondary to LCK Gene Mutation

Bilateral Congenital Cytomegalovirus Retinitis Secondary to LCK Gene Mutation

Decoding Behcet's Uveitis: an In-depth review of pathogenesis and therapeutic advances.

Behcet's disease (BD) is a rare but globally distributed vasculitis that primarily affects populations in the Mediterranean and Asian regions. Behcet's uveitis (BU) is a common manifestation of BD, occurring in over two-thirds of the patients. BU is characterized by bilateral, chronic, recurrent, non-granulomatous uveitis in association with complications such as retinal ischemia and atrophy, optic atrophy, macular ischemia, macular edema, and further neovascular complications (vitreous hemorrhage, neovascular glaucoma). Although the etiology and pathogenesis of BU remain unclear, numerous studies reveal that genetic factors (such as HLA-B51), dysregulated immune responses of both the innate and adaptive immune systems, infections (such as streptococcus), and environmental factors (such as GDP) are all involved in its development. Innate immunity, including hyperactivity of neutrophils and γδT cells and elevated NK1/NK2 ratios, has been shown to play an essential role in this disease. Adaptive immune system disturbance, including homeostatic perturbations, Th1, Th17 overaction, and Treg cell dysfunction, is thought to be involved in BU pathogenesis. Treatment of BU requires a tailored approach based on the location, severity of inflammation, and systemic manifestations. The therapy aims to achieve rapid inflammation suppression, preservation of vision, and prevention of recurrence. Systemic corticosteroids combined with other immunosuppressive agents have been widely used to treat BU, and beneficial effects are observed in most patients. Recently, biologics have been shown to be effective in treating refractory BU cases. Novel therapeutic targets for treating BU include the LCK gene, Th17/Treg balance, JAK pathway inhibition, and cytokines such as IL-17 and RORγt. This article summarizes the recent studies on BU, especially in terms of pathogenesis, diagnostic criteria and classification, auxiliary examination, and treatment options. A better understanding of the significance of microbiome composition, genetic basis, and persistent immune mechanisms, as well as advancements in identifying new biomarkers and implementing objective quantitative detection of BU, may greatly contribute to improving the adequate management of BU patients.

The Alternative Splicing of Lck in Treg Induction in Immune Thrombocytopenia

Background： Immune intolerance, causing the production of autoantibodies, is thought to be one of the main pathogenesis of immune thrombocytopenia (ITP). As regulatory T cells (Tregs) are one of the most important cells to maintain immune tolerance and the phenotype, function, and importance of Tregs have been well defined over the past decade, extensive efforts have been devoted to unveiling the dysregulation of Tregs in ITP. It has been reported that the proportion of Tregs in peripheral blood of ITP patients is reduced and the immunosuppressive function of Tregs is impaired, but the mechanism remains unclear. As peripheral Treg production can be induced from CD4+ naive T cells (NTC) with TCR-stimulated activation and TGF-β treatment, how peripheral Treg induction is impaired in patients with ITP remains to be further investigated. Method： We isolated and performed high-throughput sequencing of the CD4+ NTC from the peripheral whole blood of ITP patients and healthy controls. After analyzing the differential expression and splicing pattern, we screened several genes of interest. In addition, a minigene plasmid of the target gene was constructed and RNA-chromatin immunoprecipitation (RNA-ChIP) was performed to explore the upstream splicing factor. PCR，Real-time quantitative PCR and Western Blot were used to validate the expression level and splicing pattern of various genes. Results： Our results revealed the proportion of Tregs induced from CD4+ NTC of ITP patients was decreased in vitro, suggesting the abnormal Treg induction in ITP patients. The differentially expressed genes of the CD4+ NTC between ITP patients and healthy controls were significantly enriched in the alternative splicing pathway based on high-throughput RNA-seq data. Further exploration of the differentially spliced genes revealed significant differences in the exon 8 skipping in the LCK gene between healthy controls and ITP patients, as LCK plays a crucial role in TCR signaling and TCR signaling is essential for Treg induction. Overexpressing the exon 8 spliced LCK, other than overexpressing the exon 8 included LCK in T cells limited the TCR activation. Furthermore, we found splicing factor X could recognize the exon 8 of LCK and promote the inclusion of exon 8 of LCK. Splicing factor X was lowly expressed in the CD4+ NTC of ITP patients. Accordingly, we proposed the hypothesis that the lower expressed splicing factor X in the CD4+ NTC of ITP patients may limit peripheral Treg induction by regulating LCK alternative splicing in ITP. By using RNA-CHIP, we found splicing factor X specifically bound to the exon 8 of LCK. Moreover, the administration of antisense oligos, which could mask the binding site, suppressed the recognition of the exon 8 of LCK and enhanced the exon skipping events of LCK transcripts. As expected, ASO transfection inhibited the Treg induction in CD4+ NTCs from healthy controls, which may mimic the Treg induction in ITP patients. Conclusion： Here we found splicing factor X was downregulated in the CD4+ NTC of ITP patients, which resulted in the exon 8 skipping in the LCK gene and a down-regulated TCR signaling, thereby limiting the induction of peripheral Tregs. This study will explore the pathogenesis of ITP from a novel perspective, and provide new ideas and targets for the clinical treatment of ITP.

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia.

Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. Typical T-ALL cells had increased dependence on BCL-xL, whereas early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling upregulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.

Patterns of immune infiltration and survival in endocrine therapy-treated ER-positive breast cancer: A computational study of 1900 patients

Tumor-infiltrating immune cells (TIICs) play a critical role in breast cancer (BC) prognosis, but little is known regarding the efficacy of endocrine therapy in patients with ER-positive BC with diverse immunological phenotypes. To investigate whether TIICs affect survival after endocrine therapy in patients with different BC molecular subtypes, data were gathered from six studies totaling 1900 samples. CIBERSORTx was used to analyze the invasion of 22 immune cell subpopulations using a bulk gene expression profile. The relationships of immune-related metagenes and immune cell subsets with survival (distant metastasis-free survival, relapse-free survival, and overall survival) were studied using Cox regression models with cell proportions modeled in quartiles. The immune score and IGHG3 and LCK gene activity were linked to a better prognosis. Among the immune cells, monocytes, resting CD4+ memory T cells and plasma cells were correlated with prolonged survival, while neutrophils, Tregs, M0 macrophages, and M2 macrophages were associated with an unfavorable prognosis. Similar effects were reported for the luminal A subtype. In the luminal B subtype, γδ T cells and eosinophils were favorable prognostic factors. Covariate-adjusted multivariate Cox regression analysis revealed that high proportions of resting CD4+ memory T cells and resting dendritic cells were correlated with a good prognosis. Meanwhile, neutrophils were associated with an unfavorable prognosis. Understanding how monocytes and macrophages interact in the tumor microenvironment may be a promising study focus. Comprehensive research on the cellular immune response in tumors could help facilitate the development of new treatments.

Distal Lck Promoter-Driven Cre Shows Cell Type-Specific Function in Innate-like T Cells.

Innate-like T cells, including invariant NKT cells, mucosal-associated invariant T (MAIT) cells, and γ δ T (γδT) cells, are groups of unconventional T lymphocytes. They play important roles in the immune system. Because of the lack of Cre recombinase lines that are specific for innate-like T cells, pan–T cell Cre lines are often used to study innate-like T cells. In this study, we found that distal Lck promoter–driven Cre (dLckCre) in which the distal Lck gene promoter drives Cre expression in the late stage of thymocyte development has limited function in the innate-like T cells using ROSA26floxed-Stop-tdTomato reporter. Innate-like T cells differentiate into mature functional subsets comparable to the CD4+ Th subsets under homeostatic conditions. We further showed that dLckCre-expressing γδT cells are strongly biased toward γδT1 phenotype. Interestingly, the γδT cells residing in the epidermis and comprising the vast majority of dendritic epidermal T cells nearly all express dLckCre, indicating dLckCre is a useful tool for studying dendritic epidermal T cells. Taken together, these data suggest that Lck distal promoter has different activity in the conventional and unconventional T cells. The use of dLCKcre transgenic mice in the innate-like T cells needs to be guided by a reporter for the dLckCre function.

Lck Is a Crucial Anergy Regulator in CLL

NFAT is a group of highly phosphorylated transcription factors which can be found in the cytoplasm of resting cells. Following activation via dephosphorylation, they translocate to the nucleus where they regulate developmental and activation programs in various cell types. The tyrosine kinase Lck plays an essential role in T cell activation. Lck expression in CLL cells has previously been described, but its function remains largely elusive. With respect to prognosis CLL constitutes a very heterogeneous disease. While some patients exhibit an indolent course without requiring specific treatment for lengthy periods, others progress rapidly and demonstrate limited survival. Patients featuring aggressive disease show an enhanced responsiveness to stimulation of the B cell receptor (BCR) complex, whereas those with a more indolent course are characterized by an anergic phenotype with B cell unresponsiveness to IgM ligation, PRDM1 (BLIMP-1) depression and an essential lack of phosphotyrosine induction as well as calcium flux. In up to 10% of CLL patients a transformation to aggressive lymphoma (Richter´s syndrome) can be observed which is associated with a significantly worse treatment outcome. We have preciously shown in a mouse model and in primary human CLL samples that NFAT2 is a crucial anergy regulator in this disease and defined a genetic anergy signature consisting of Lck and the E3 ligases Cbl-b and Grail (Märklin et al., Nature Commun., in press). Here, we show that Lck is a direct NFAT2 target and substantially contributes to the induction of anergy in CLL cells.Sequence analysis of the Lck gene locus revealed a potential NFAT binding site with the sequence TTTCATCAG in the Lck promotor. To biochemically define Lck as an unequivocal NFAT2 target, we performed chromatin immunoprecipitation (ChIP) assays in primary human CLL cells using a monoclonal antibody against NFAT2 and subsequent qRT-PCR. Our results demonstrate that NFAT2 binds to the CD40L gene locus which is a well-defined NFAT2 target gene in B cells and thus served as a positive control. We could further demonstrate that NFAT2 also binds to the Lck promotor and thus constitutes a direct NFAT2 target in human CLL cells. To assess the role of Lck in the induction of anergy in CLL, we made use of the Eµ-TCL1 transgenic mouse model. These mice exhibit TCL1 expression limited to the B cell lineage and develop a human-like CLL within 60 days. By mouse breeding with conventional Lck knockout mice (Lck-/-), we generated animal cohorts with B cell specific TCL1 expression and concomitant deletion of Lck (n=10). Eµ-TCL1 transgenic mice with intact Lck expression served as controls (n=10). After development of manifest CLL, animals were sacrificed and CD5+CD19+ CLL cells were isolated from bone marrow and spleens for further analysis. While CLL cells from Eµ-TCL1 transgenic exhibited an anergic phenotype characterized by compromised calcium flux as well as low IgM and PRDM1 (BLIMP-1) expression, Lck-deficient CLL cells showed evidence of enhanced calcium mobilization upon BCR stimulation and significantly increased expression of sIgM and PRDM1 (BLIMP-1) as assessed by flow cytometry or qRT-PCR. Utilizing immunofluorescence-based colocalization assays we were further able to show that Lck is recruited to the BCR in CLL cells from TCL1 mice upon BCR stimulation while in TCL1 mice with NFAT2 deletion no Lck recruitment to the BCR could be detected.Taken together, our data demonstrate that Lck is a direct NFAT2 target in anergic CLL cells and that its deletion contributes to the loss of the anergic phenotype which is generally associated with an indolent clinical course. Therefore our data identify Lck as a crucial regulator of the anergic phenotype in CLL. DisclosuresNo relevant conflicts of interest to declare.

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.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αβ and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αβ T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αβ T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Evidence for activated Lck protein tyrosine kinase as the driver of proliferation in acute myeloid leukemia cell, CTV-1

Acute myeloid leukemia (AML) is a heterogeneous group of fast growing cancers of myeloid progenitor cells, for which effective treatments are still lacking. Identification of signaling inhibitors that block their proliferation could reveal the proliferative mechanism of a given leukemia cell, and provide small molecule drugs for targeted therapy for AML. In this study, kinase inhibitors that block the majority of cancer signaling pathways are evaluated for their inhibition of two AML cell lines of the M5 subtypes, CTV-1 and THP-1. While THP-1 cells do not respond to any of these inhibitors, CTV-1 cells are potently inhibited by dasatinib, bosutinib, crizotinib, A-770041, and WH-4-23, all potent inhibitors for Lck, a Src family kinase. CTV-1 cells contain a kinase activity that phosphorylates an Lck-specific peptide substrate in an Lck inhibitor-sensitive manner. Furthermore, the Lck gene is over-expressed in CTV-1, and it contains four mutations, two of which are located in regions critical for Lck negative regulation, and are confirmed to activate Lck. Collectively, these results provide strong evidence that mutated and overexpressed Lck is driving CTV-1 proliferation. While Lck activation and overexpression is rare in AML, this study provides a potential therapeutic strategy for treating patients with a similar oncogenic mechanism.

The use of alternative promoters in T cell development

Abstract Many mammalian genes, including a number involved in immune development and function, use multiple promoters encoding expression of the same protein product. To understand the function of alternative promotors during T cell development, we carried out genome-wide RNA-Seq analysis of DN, DP, CD4 and CD8 thymocytes. 26% (2469/9595) of genes expressed by thymocytes used two or more alternative promotors at some point in T cell development. However, only 3.2% (80/2469) of the genes which utilized two or more promoters showed the difference of promotor activity among different T cell developmental stages which were validated by selected representative genes with qPCR assay. To analyze mechanisms of alternative promoter regulation, DNase hypersensitivity sites were mapped, and ChIP-Seq with anti-H3K4me3 antibodies was performed in the same thymic subpopulations. To determine the in vivo functional role of alternative promotors for T cell development, several genes were selected for study by deletion of individual promoters by CRISPR technology. We have initially generated mice with deletion of distal or proximal promotor of the lck gene and demonstrated distinct functional roles of the two promoters: the proximal lck promoter is critical for early stages of thymic T cell development while the distal promoter was necessary for normal T cell activation. Ongoing studies will characterize more broadly the functional role of alternative promoter expression in thymocyte development, and the mechanisms mediating selective promoter activity.

Identification of genetic pathways driving Ebola virus disease in humans and targets for therapeutic intervention

Introduction: LCKgene, also known as lymphocyte-specific proto-oncogene, is expressed in lymphocytes, and associated with coordinated expression of MHC class I and II in response to physiological stimuli, mediated through a combined interaction of promoters, suppressors, and enhancers. Differential usage ofLCKpromoters, transcribes dysfunctional transcript variants leading to leukemogenesis and non-induction of MHC class I gene variants. Viruses use C-type lectins, likeCD209,to penetrate the cell, and inhibit Pattern Recognition Receptors (PRR), hence evading immune destruction. Given that Ebolavirus (EBOV) disease burden could result from a dysfunctional LCK pathway, identification of the genetic pathway leading to proper immune induction is a major priority.Methods:Data for EBOV related virus samples were obtained from Gene Expression Omnibus database and RMEAN information per gene per sample were entered into a table of values. R software v.3.3.1 was used to process differential expression patterns across samples forLCK, CD209and immune-related genes. Principal component analysis (PCA) using ggbiplot v.0.55 was used to explain the variance across samples.Results:Data analyses identified three viral clusters based on transmission patterns as follows:LCK-CD209dependent, LCK-dependent specific to EBOV, and CD209 dependent. Compared to HLA class II gene variants, HLA class I (A, B and C) variants were <2 fold expressed, especially for EBOV samples. PCA analyses classifiedTYRO3,TBK1andLCKgenes independent of the data, leading to identification of a possible pathway involvingLCK, IL2, PI3k, TBK1, TYRO3andMYBgenes with downstream induction of immune T-cells.Discussion: This is the first study undertaken to understand the non-functional immune pathway, leading to EBOV disease pathogenesis and high fatality rates. Our lab currently exploits, through cutting edge genetic technology to understand the interplay of identified genes required for proper immune induction. This will guide antiviral therapy and possible markers for viral disease identification during outbreaks.

Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients.To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCγ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCγ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity.We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells.Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. DisclosuresIndraccolo:OncoMed Pharmaceuticals, Inc.: Research Funding.

Lck-Driven Cre Expression Alters T Cell Development in the Thymus and the Frequencies and Functions of Peripheral T Cell Subsets.

Conditional gene targeting using the bacteriophage-derived Cre recombinase is widely applied for functional gene studies in mice. Mice transgenic for Cre under the control of the lck gene promoter are used to study the role of loxP-targeted genes in T cell development and function. In this article, we show a striking 65% reduction in cellularity, preferential development of γδ versus αβ T cells, and increased expression of IL-7R in the thymus of mice expressing Cre under the proximal lck promoter (lck-cre(+) mice). The transition from CD4/CD8 double-negative to double-positive cells was blocked, and lck-cre(+) double-positive cells were more prone to apoptosis and showed higher levels of Cre expression. Importantly, numbers of naive T cells were reduced in spleens and lymph nodes of lck-cre(+) mice. In contrast, frequencies of γδ T cells, CD44(+)CD62L(-) effector T cells, and Foxp3(+) regulatory T cells were elevated, as was the frequency of IFN-γ-secreting CD4(+) and CD8(+) T cells. A literature survey of 332 articles that used lck-cre(+) mice for deletion of floxed genes indicated that results are statistically influenced by the control used (lck-cre(+) or lck-cre(-)), more frequently resembling the lck-cre(+) phenotype described in this article if lck-cre(-) controls were used. Altogether, care should be taken when interpreting published results and to properly control targeted gene deletions using the lck-cre(+) strain.

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke

The protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.

Long-range transcriptional effects and specific epigenetic marks in regulation of transcription from Lck proximal and distal promoters

Abstract Lck is a critical molecule for T cell development and activation. The T cell-specific expression of Lck is controlled by two distinct promoters separated by 15kb. The proximal promoter contributes the majority of Lck transcripts in immature thymic T cells, while the distal promoter expresses the majority of Lck transcripts in mature T cells. To study the regulation of these two promoters, promoter-mutated mouse models were generated by CRISPR technology, resulting in multiple mouse lines with distinct promoter deletions. The activity of the proximal promoter was reduced by 95% in the DP thymocytes of PROX[mut] mice with ~100bp deletion in the proximal promoter. Expression of distal-promoter-driven Lck mRNA was completely abrogated in immature and mature T cells in Lck distal promoter DIST[mut] mice with partial deletion of the distal promoter and exon 1. Distal-promoter-driven Lck mRNAs were not altered in PROX[mut] mice. However, proximal-promoter-driven Lck mRNAs were dramatically increased in the mature T cells of DIST[mut] mouse, suggesting that there is a long-range transcriptional interaction between the two promoters. To further understand the epigenetic influences in Lck gene expression, we analyzed histone modifications in the regions of Lck promoters. Our preliminary data showed the distal promoter was highly H3K9 acetylated in peripheral T cells but not immature T cells, and that this acetylation was decreased in DIST[mut] mice. Therefore, distal promoter H3K9 acetylation was correlated with promoter activity and may be involved in the developmental stage specificity of Lck promoters. The findings of this study on Lck promoters may provide important information on gene expression and its tissue-specificity.

Molecular characterization and expression of Lck in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus.

Lymphocyte-specific protein tyrosine kinase (Lck) plays a critical role in effective signal transductions that are fundamental to T cell differentiation, proliferation, and effector functions. In this paper, the Lck gene of Nile tilapia, Oreochromis niloticus (designated as On-Lck), was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed important structural characteristics required for T cell receptor (TCR) signal transduction were detected in the deduced amino acid sequence of On-Lck, and the deduced genomic structure of On-Lck was similar to the known Lck. In healthy Nile tilapia, the On-Lck transcripts were mainly detected in the thymus, spleen, head kidney, and gill. When immunized with inactivated S. agalactiae, the On-Lck mRNA expression was significantly upregulated in the thymus, spleen, and head kidney. Moreover, there was a clear time-dependent expression pattern of On-Lck after immunization, and the expression reached the highest level at 48 h in the spleen and thymus and at 72 h in the head kidney, respectively. This is the first report on the expression of Lck induced by intracellular bacteria vaccination in teleosts. These findings indicated that On-Lck may play an important role in the immune response to intracellular bacteria in Nile tilapia.

Discovery of Consensus Gene Signature and Intermodular Connectivity Defining Self-Renewal of Human Embryonic Stem Cells

Molecular markers defining self-renewing pluripotent embryonic stem cells (ESCs) have been identified by relative comparisons between undifferentiated and differentiated cells. Most of analysis has been done under a specific differentiation condition that may present significantly different molecular changes over others. Therefore, it is currently unclear if there are true consensus markers defining undifferentiated human ESCs (hESCs). To identify a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions, we have performed microarray analysis on undifferentiated hESCs (H1 and H9) and differentiated EBs and validated our results using publicly available expression array datasets. We constructed consensus modules by Weighted Gene Coexpression Network Analysis and discovered novel markers that are consistently present in undifferentiated hESCs under various differentiation conditions. We have validated top markers (downregulated: LCK, KLKB1, and SLC7A3; upregulated: RhoJ, Zeb2, and Adam12) upon differentiation. Functional validation analysis of LCK in self-renewal of hESCs using LCK inhibitor or gene silencing with siLCK resulted in a loss of undifferentiation characteristics-morphological change, reduced alkaline phosphatase activity, and pluripotency gene expression, demonstrating a potential functional role of LCK in self-renewal of hESCs. We have designated hESC markers to interactive networks in the genome, identifying possible interacting partners and showing how new markers relate to each other. Furthermore, comparison of these datasets with available datasets from induced pluripotent stem cells (iPSCs) revealed that the level of these newly identified markers was correlated to the establishment of iPSCs, which may imply a potential role of these markers in gaining of cellular potency.

Effects of Rho GTPase CDC42 on Th2 and Th17 differentiation (CCR6P.277)

Abstract We have reported that cell-division-cycle 42 (CDC42) of Rho GTPase family was important for T cell development and homeostasis with the use of conditional CDC42-deficient mice (CDC42flox/LckCre), in which CDC42 was deleted in CD4-CD8- double negative stage of thymocytes. Recently, we generated another line of conditional CDC42-deficient mice with selective expression of the Cre recombinase beginning at a late stage of T cell development (after positive selection of CD4+CD8+) using the distal promoter of the Lck (dLck) gene. We found that CDC42-deficiency (CDC42flox/dLckCre) did not affect T cell development, as expected. The development of natural regulatory T cells (nTreg) was also not affected by CDC42-deficiency. However, CDC42-deficiency impaired in vitro activation of both CD4+ and CD8+ T cells. When naïve CD4+ T cells were cultured under non-polarized conditions, CDC42-deficiency significantly reduced Th1 and Th2 cytokine secretion, but slightly increased Th17 cytokine secretion from CD4+ T cells. Under Th1/Th2/Th17-polarized conditions, CDC42-deficiency significantly downregulated Th2-differentiation, but was less effective to Th1 and Th17 differentiation. CDC42 has no significant role on the induction of Treg in vitro. The ongoing studies are trying to explore the underlying molecular mechanisms. Our findings suggest that CDC42 of Rho GTPase is not only crucial for T cell development and activation, but also involved in Th differentiation, especially Th2 cells.

Peripheral T cell-specific role of CDC42 of Rho GTPase family in T cell homeostasis and activation (P1286)

Abstract We have reported that deletion of CDC42 of Rho GTPase family in T cells at early developmental stage (CD4-CD8-CD44-CD25+) affects T cell homeostasis by the use of CDC42flox/flox/LckCre mice. Here, we generated CDC42-deficient mice with selective expression of the Cre recombinase beginning at a late stage of T cell development (after positive selection of CD4+CD8+) using the distal promoter of the Lck (dLck) gene. As expected, CDC42 deficiency (CDC42flox/flox/dLckCre) did not affect T cell development. However, splenic CD8+, but not CD4+, T cells were significantly reduced in CDC42- deficient mice. Further analysis revealed that there was a marked increase of both proliferation and apoptosis in vivo in naïve CD8+ T cells from CDC42-deficient mice. IL-7/IL-7R signaling is critical for T cell survival. The expression of IL-7R in naïve CDC42-deficient CD8+ T cells was down-regulated. These changes were not significant in naïve CDC42-deficient CD4+ T cells. CDC42 deficiency impaired in vitro activation of both CD4+ and CD8+ T cells. Furthermore, CDC42 deficiency significantly reduced Th1 and Th2 cytokine secretion, but increased Th17 cytokine secretion from CD4+ T cells under non-polarized culture conditions. These results are significantly different from that obtained by using CDC42flox/flox/LckCre mice and suggest a peripheral T cell-specific role of CDC42 in T cell homeostasis and activation.