Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based ...inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these "de-regulated" SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The importance of the bi-directional natural killer-dendritic cell crosstalk in coordinating anti-tumour and anti-microbial responses in vivo has been well established. However, physical parameters ...associated with natural killer-dendritic cell interactions have not been fully elucidated. We have previously used a simple "Y" shaped microfluidic device to study natural killer cell-migratory responses toward chemical gradients from a conditioned medium of dendritic cells. There are, however, limitations of the Y-shaped microfluidic devices that could not support higher throughput analyses and studies of cell-cell interactions. Here, we report two novel microfluidic devices (D
-Chip, T
-Chip) we applied in advanced studies of natural killer-cell migrations and their interactions with dendritic cells in vitro. The D
-Chip is an improved version of the previously published Y-shaped device that supports high-throughput analyses and docking of the cells of interest in the migration assay before they are exposed to a chemical gradient. The T
-Chip is created to support analyses of natural killer-dendritic cell cell-cell interactions without the requirement of promoting a natural killer cell to migrate long distances to find a loaded dendritic cell in the device. Using these two microfluidic platforms, we observe quantitative differences in the abilities of the immature and lipopolysaccharide-activated mature dendritic cells to interact with activated natural killer cells. The contact time between the activated natural killer cells and immature dendritic cells is significantly longer than that of the mature dendritic cells. There is a significantly higher frequency of an immature dendritic cell coming into contact with multiple natural killer cells and/or making multiple simultaneous contacts with multiple natural killer cells. To contrast, an activated natural killer cell has a significantly higher frequency of coming into contact with the mature dendritic cells than immature dendritic cells. Collectively, these differences in natural killer-dendritic cell interactions may underlie the differential maturation of immature dendritic cells by activated natural killer cells. Further applications of these microfluidic devices in studying natural killer-dendritic cell crosstalk under defined microenvironments shall enrich our understanding of the functional regulations of natural killer cells and dendritic cells in the natural killer-dendritic cell crosstalk.
Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that ...alternative splicing can be dynamically regulated in response to membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.
Background: Excitable cells show activity-dependent alternative splicing of ion channels.
Results: CaMKIV phosphorylates hnRNP L at Ser-513, which is essential for depolarization-repression of a Slo1 potassium channel exon and splicing factor U2AF65.
Conclusion: Depolarization controls alternative splicing of Slo1 channels through Ser-513 phosphorylation and inhibition of U2AF65.
Significance: This provides the first direct link between depolarization/CaMKIV and the constitutive spliceosome.
The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better ...prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs
compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis
, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.
The regulation of gene expression through alternative pre-mRNA splicing is common in metazoans and is often controlled by intracellular signaling pathways that are important in cell physiology. We ...have shown that the alternative splicing of a number of genes is controlled by membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) through CaMKIV-responsive RNA elements (CaRRE1 and CaRRE2); however, the trans-acting factors remain unknown. Here we show that the heterogeneous nuclear ribonucleoprotein (hnRNP) L is a CaRRE1 binding factor in nuclear extracts. An hnRNP L high affinity CA (cytidine-adenosine) repeat element is sufficient to mediate CaMKIV and hnRNP L repression of splicing in a location (3′-splice site proximity)-dependent way. Depletion of hnRNP L by RNA interference followed by rescue with coexpressed exogenous hnRNP L demonstrates that hnRNP L mediates the CaMKIV-regulated splicing through CA repeats in heterologous contexts. Depletion of hnRNP L also led to increased inclusion of the stress axis-regulated exon and a CA repeat-harboring exon under depolarization or with activated CaMKIV. Moreover, hnRNP L binding to CaRRE1 was increased by CaMKIV and, conversely, was reduced by pretreatments with protein phosphatases. Therefore, hnRNP L is an essential component of CaMKIV-regulated alternative splicing through CA repeats, with its phosphorylation likely playing a critical role.
CD3ζ has emerged as a clinically important immunological marker in head and neck squamous cell carcinoma (HNSCC) with reduced level of expression reported in both tumor infiltrating lymphocytes and ...peripheral blood lymphocytes. In this prospective study (power = 0.99, α = 0.05), CD3ζ expression was compared in 47 HNSCC patients and 53 controls using standardized flow cytometric method. There was no statistical difference in the percentages of the CD3 ε+ T‐cell subset present in the peripheral blood mononuclear cells of the HNSCC patients and the healthy controls; however, T cells from the HNSCC patients produced a significantly weaker IFN‐γ response in comparison to the healthy controls, when they were stimulated by the recall viral CEF peptide antigen. All patients were followed up for at least 3 years with a median follow‐up of 45 months. Levels of CD3ζ‐chain expression were measured at 117 follow‐up visits at six‐month intervals. Receiver operating characteristic curve identified the optimal cut off as a 12% increase in post treatment CD3ζ‐chain expression from the baseline levels to confirm absence of HNSCC with the area under curve of 0.81 (95% CI = 0.68–0.94) for predicting absence of HNSCC. The specificity, sensitivity and positive predictive value were 81.25% 79.21% and 97.56%, respectively. Three‐year disease specific survival (DSS) was significantly lower (p = 0.007) at 63.2% for patients who showed <12% increase in CD3ζ‐chain level as compared to 96.2% for patients who had ≥12% increase. Our results indicate that the change in CD3ζ‐chain expression from the baseline is an independent predictor of residual and recurrent HNSCC.
What's new?
There is an urgent need for biomarkers that allow early detection of relapse in head and neck squamous cell carcinoma (HNSCC). CD3ζ may be one such marker, as lymphocytes from HNSCC patients express reduced levels of this protein. In this study, the authors found that patients whose CD3ζ levels increased by less than 12% after treatment had significantly lower survival. These results indicate that measurement of CD3ζ over time may allow oncologists to detect and treat recurrence early enough to improve outcomes.
Effector functions and cellular properties of natural killer (NK) cells are regulated by cellular and extracellular factors shaped by the microenvironments. NK cells express specific chemokine and ...non-chemokine receptors to aid preferential migrations or localizations in tissues. Good understanding of how NK-cell migratory properties are regulated in physiological and pathological microenvironments will provide further insights into the development of NK cell-based therapeutic approaches. In contrast to the commonly used conventional in vitro migration assays such as Trans-well assays that measure movements of a population of the migratory cells, microfluidic-based devices support live-cell imaging of cell migrations under a well-defined chemical gradient(s) at microscale. Subsequent analyses at single-cell level provide quantitative measurements of cell-migration parameters such as speed and Chemotactic Index, and permit distinguishing chemotaxis, chemokinesis, and chemo-repulsion. Our recent work established the use of a Y-shaped microfluidic device to study NK cell migrations in vitro. In this chapter, we described the detailed method of acquiring and analyzing NK cell migration in the microfluidic devices.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common neoplasm in the world. The follow-up protocols currently available do not appear to diagnose treatment failures and recurrences ...early enough to provide the best treatment to improve the survival rates of patients. The identification of biomarkers may aid in diagnosing, monitoring the progression, or predicting treatment outcomes in HNSCC. The present study aimed to evaluate whether cluster of differentiation (CD) 3ζ chain expression may serve as a biomarker for the early detection of recurrent or persistent HNSCC. However, in a longitudinal study, a standardized method that allows consistent data comparisons in an inter-assay manner is critical. The present study reveals a method to monitor expression levels of CD3ζ over multiple time-points using flow cytometry. The present study validated the use of an internal control and normalization procedure for tracking alterations in CD3ζ expression in samples from patients with HNSCC, which were collected and assayed for a longitudinal study.
CD3zeta has emerged as a clinically important immunological marker in head and neck squamous cell carcinoma (HNSCC) with reduced level of expression reported in both tumor infiltrating lymphocytes ...and peripheral blood lymphocytes. In this prospective study (power=0.99, alpha=0.05), CD3zeta expression was compared in 47 HNSCC patients and 53 controls using standardized flow cytometric method. There was no statistical difference in the percentages of the CD3 epsi+ T-cell subset present in the peripheral blood mononuclear cells of the HNSCC patients and the healthy controls; however, T cells from the HNSCC patients produced a significantly weaker IFN-gamma response in comparison to the healthy controls, when they were stimulated by the recall viral CEF peptide antigen. All patients were followed up for at least 3 years with a median follow-up of 45 months. Levels of CD3zeta-chain expression were measured at 117 follow-up visits at six-month intervals. Receiver operating characteristic curve identified the optimal cut off as a 12% increase in post treatment CD3zeta-chain expression from the baseline levels to confirm absence of HNSCC with the area under curve of 0.81 (95% CI=0.68-0.94) for predicting absence of HNSCC. The specificity, sensitivity and positive predictive value were 81.25% 79.21% and 97.56%, respectively. Three-year disease specific survival (DSS) was significantly lower (p=0.007) at 63.2% for patients who showed <12% increase in CD3zeta-chain level as compared to 96.2% for patients who had ≥12% increase. Our results indicate that the change in CD3zeta-chain expression from the baseline is an independent predictor of residual and recurrent HNSCC. What's new? There is an urgent need for biomarkers that allow early detection of relapse in head and neck squamous cell carcinoma (HNSCC). CD3zeta may be one such marker, as lymphocytes from HNSCC patients express reduced levels of this protein. In this study, the authors found that patients whose CD3zeta levels increased by less than 12% after treatment had significantly lower survival. These results indicate that measurement of CD3zeta over time may allow oncologists to detect and treat recurrence early enough to improve outcomes.
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