The authors would like to acknowledge Michael Chou, Bingbing Dai, and Liang Xiao for contributing data for this manuscript. We also acknowledge Dr. John Gray for the generous gifts of reagents used in this study. P.B. is supported by a postdoctoral fellowship from the National Cancer Center. This research was supported by grants from the National Institute of Health (R01AI68978, P01CA132681 and RCA170820A), a grant from the Bill and Melinda Gates Foundation, a translational acceleration grant from the Joint Center for Translational Medicine and a grant from the California HIV/AIDS Research Program.

DC-LV stable producer cells are constructed based on the GPR packaging cell line 11 that contains the necessary lentiviral components gagpol, rev and the tet-off system. First, retroviral transduction is used to generate a GPRS packaging cell line that encodes a tet-dependent SVGmu glycoprotein. Then, concatemer array transfection is used to transfect the GPRS cell line with a lentiviral vector transgene such as GFP. This stable producer cell line, designated as LV-MGFP, can be tested in vitro and in vivo for its ability to produce a DC-LV vaccine against GFP.
1. Generating a Tet-dependent SVGmu Cell Line
Plasmid pRX-SVGmu is a construct in which DC-specific glycoprotein SVGmu is cloned downstream of the tTA-advanced promoter of retroviral plasmid pRetroX-Tet-off 1 (Figure 1C). Culture 293T cells in D10 (Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mM L-glutamine). Culture GPR packaging cell line in D10 with doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml). All cells are cultured in a humidified 37 &deg;C incubator with 5% CO2.
<ol>
	<li>16-18 hr before transient transfection, plate 2 x 106 293T cells in 4 ml of D10 in a 6-cm tissue culture dish such that the cells approach 90% confluency at the time of transfection.</li>
	<li>Transfection of retroviral plasmids: mix 100 &mu;l 1.25 M CaCl2 solution, sterile Milli-Q water and the following plasmids: 5 &mu;g pRX-SVGmu, 2.5 &mu;g pGag-Pol, 2.5 &mu;g pVSV-G. The final volume is 500 &mu;l. To a 5 ml polystyrene round-bottom tube, add 500 &mu;l 2X HBS (50 mM HEPES, 10 mM KCl, 12 mM Dextrose, 280 mM NaCl, 1.5 mM Na2HPO4*7H2O, pH 7.05). Add the plasmid mixture dropwise into the 2X HBS buffer while bubbling the buffer vigorously with a glass Pasteur pipette. After adding the mixture, continue bubbling for another 30 sec. Then, add the whole mixture onto the 293T cells in the 6-cm culture dish, and incubate at 37 &deg;C.</li>
	<li>4 hr after transfection, carefully replace the medium in the culture dish with 4 ml pre-heated D10.</li>
	<li>Spin infection: 48 hr after transfection, harvest SVGmu-encoding retroviral particles in the supernatant by passing the supernatant through a 0.45-&mu;m filter. Plate the GPR packaging cells in a 24-well dish at 2 x 104 cells/well. Add filtered supernatant to GPR packaging cells in the dish (2 ml/well) and centrifuge cells for 90 min at 1,050 x g and 25 &deg;C. Change medium into fresh D10 with doxycycline (1 ng/ml) after spin-infection (2 ml/well).</li>
	<li>72 hr post-transfection, expand the culture of transduced packaging cells in D10 with doxycycline (1 ng/ml) and puromycin (2 ng/ml).</li>
	<li>To confirm expression of SVGmu, culture the cells without doxycycline for 48 hr. Measure surface expression of SVGmu with flow cytometry using anti-Sindbis serum. These cells are designated as GPRS packaging cell line.</li>
</ol>
2. Construct DC-LV Producer Cells by Concatemer Array Transfection
Lentiviral transfer plasmid TL20-GFP is a self-inactivating lentiviral transfer vector plasmid based on pCL20c-MSCV-GFP with a Dox-regulatable viral RNA genome expression system and replacement of the cytomegalovirus (CMV) enhancer with 7 tet operators(Figure 1C) 11,12,15. Plasmid PGK-ble is a bleomycin resistant (ble) cassette driven by a weak PGK promoter 2.
<ol>
	<li>Digest 20 &mu;g plasmid TL20-GFP with the restriction enzyme SfiI at 50 &deg;C. Digest 20 &mu;g plasmid PGK-ble with PflMI at 37 &deg;C.</li>
	<li>Purify the DNA fragments (the PGK-ble cassette is 1011 bp and the vector TL20-GFP is 6861 bp) by agarose gel electrophoresis.</li>
	<li>Ligate the TL20-GFP vector and PGK-ble cassette at a molar ratio of 25:1 (NEB T4 DNA Ligase). Incubate overnight at room temperature.</li>
	<li>After overnight ligation, purify DNA from the ligation mixture (Qiagen DNeasy Blood and Tissue Kit). Ensure the amount of purified DNA is around 5 &mu;g.</li>
	<li>16-18 hr before transfection, plate GPRS packaging cells in a 6-cm tissue culture dish such that the confluency will be about 90% at the time of transfection.</li>
	<li>Use the calcium phosphate transfection method described in step 1.2 to transfect 5 &mu;g of the purified concatemeric DNA into the GPRS packaging cells in the 6-cm tissue culture dish. 4 hr after transfection, carefully remove the medium and replace with 4 ml pre-heated D10 with doxycycline (1 ng/ml).</li>
	<li>48 hr after transfection, change the medium into D10 with doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml). Select for transfected clones by adding zeocin (50 &mu;g/ml) in the culture medium. Culture the cells for about 2 weeks until cell colonies can be seen at the bottom of the dishes.</li>
	<li>Label the cell colonies at the bottom of the dishes. Take 24-well tissue culture dishes, number the wells and add to each well 2 ml D10 containing zeocin (50 &mu;g/ml), doxycycline (1 ng/ml), and puromycin (2 &mu;g/ml). Aspirate the medium of the transduced cells, then add one drop of trypsin onto each of the colonies for less than one minute. Then, add one or more drops of D10 on the same colonies. Pick up colonies one by one with a pipette and transfer them into separate wells of the 24-well tissue culture plate.</li>
	<li>Culture and expand all cell clones in D10 containing zeocin (50 &mu;g/ml), doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml) for the evaluation of viral producing ability.</li>
</ol>
3. Evaluate Viral Production of Each Cell Clone
<ol>
	<li>Trypsinize the producer cells and plate around 4x106 cells in 6-cm tissue culture dish in D10 without doxycycline such that the confluency exceeds 90%. Replace the medium with fresh pre-heated D10 daily and collect the medium for the titer assay 72 hr after removing dox.</li>
	<li>Harvest the viral supernatant by filtering the medium with a 0.45-&mu;m filter.</li>
	<li>Plate target cells expressing the DC-SIGN receptor (e.g. 293T.hDC-SIGN or mouse bone marrow derived dendritic cells 16) and 293T as a negative control in 96-well culture dishes, 1 x 104 cells / well. Add 2-fold serial dilutions of the viral supernatant to the wells (100 &mu;l/well). Usually, 6-8 dilutions are sufficient to reach the range in which transduced GFP-positive cells are in a linear relationship to the amount of virus added.</li>
	<li>Centrifuge cells and replace the medium as described in step 1.4. BMDCs should be cultured in RPMI medium with 10% FBS and GM-CSF (1:20 J558L conditioned medium).</li>
	<li>Measure the GFP expression in transduced cells by flow cytometry 4-6 days post-transduction. The lentiviral vector titer is calculated in the vector dilution range when the percentage of GFP-positive cells and vector amount are in a linear relationship. Choose the cell clone with the highest viral production for subsequent applications.</li>
</ol>
4. Produce and Concentrate Lentiviral Vectors
<ol>
	<li>Culture the producer cell line (LV-MGFP) in 15-cm tissue culture dishes.</li>
	<li>Trypsinize the producer cells and plate cells in 15-cm tissue culture dishes at greater than 90% confluency in fresh D10 without doxycycline. Change medium with fresh, pre-heated D10 daily.</li>
	<li>At the time of peak viral production (as determined by the user), harvest the viral supernatant and filter it using a 0.45-&mu;m filter. Load the filtered supernatant into thick wall 32.5 ml ultracentrifuge tubes, seal with parafilm and centrifuge at 50,000 x g, 4 &deg;C for 90 min. Thoroughly resuspend the pellet in 50 &mu;l or an appropriate volume of PBS or HBSS depending on the application.</li>
</ol>
5. Immunize Mice In vivo and Analyze Antigen-specific Immune Response
<ol>
	<li>Produce high-titer virus as described in section 4.</li>
	<li>Inject BALB/c mice (6-8 week old, female) via a footpad route with the vector suspension (25 &mu;l per footpad).</li>
	<li>2 weeks after immunization, isolate spleen and harvest splenocytes. Culture splenocytes with GFP epitope peptides and analyze the presence of GFP-specific CD8+ T cells using intracellular cytokine staining, as previously described 17 (Figure 3B).</li>
</ol>

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The authors declare that they have no competing financial interests.

Here, we have outlined a method for producing large quantities of lentiviral vectors using 293T cells stably transduced with all lentiviral components under the tet off regulatory system. To date, most protocols to produce lentiviral vectors rely on standard calcium phosphate transient transfection (see 18, for example). This approach has been successful on a clinical scale, but it may suffer from some limitations not likely to be present in scaling up production from stable cell lines. For example, co-transfe...

Many vector systems have been developed for gene delivery. Vectors based on lentiviruses have been among the most commonly studied viral system. These vectors are advantageous because they can efficiently transduce both dividing and non-dividing cells 1, achieve long-term expression due to integration into the host genome, exhibit low natural anti-vector immunity in most human populations 2, and have a low potential for genotoxicity from insertional mutagenesis 3,4.
Production of lentiviruses has always been colored by safety concerns. Lentiviral vectors are generally derived from HIV-1, the etiological agent of AIDS. Transient transfection of individual components of the lentivector (transfer, envelope, and packaging plasmids) is a common and flexible means of delivering genetic material in laboratory settings. However, scaling-up transient transfections for clinical applications is cumbersome and may lead to the development of replication-competent lentivirus 5,6. To overcome these hurdles, several stable packaging and producer cell lines have been developed 6-11. One of these lines, the GPR packaging line 11, has the attractive advantage of being regulated by tetracycline. In this paper, we demonstrate how to adapt this system to produce self-inactivating lentiviral vectors that are specifically targeted toward dendritic cells (DC-LVs) 12.
Dendritic cells (DCs) are the most robust antigen presenting cells of the immune system. They have been the subject of great interest in cancer vaccine development because they directly initiate, program, and regulate tumor-specific immune responses 13. Incorporating a vaccination protocol to include DCs has the potential to elicit a stronger antitumor immune response than peptide or DNA vaccines. Recently, we have developed a lentiviral vector that specifically targets dendritic cells through a modified sindbis virus glycoprotein, SVGmu 14. These vectors are unique in that they show high specificity to dendritic cells and generate stronger immune responses than nonspecific, VSVg-pseudotyped vectors.
Here, we report a method for producing large quantities of these DC-targeted lentiviral vectors. We demonstrate that these DC-LVs can infect DCs and generate strong CD8+ T cell immune responses. All procedures involving animals were performed humanely, under the approval of the USC Intitutional Animal Care and Use Committee. In order to perform in vivo and clinical experiments, it is critical to create a cell line that can produce virus at a high titer. Performing the transduction and transfection steps exactly as described will maximize the chances of generating such a clone.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

The stable cell line described in this method can produce large quantities of lentiviral vectors that are specifically targeted to dendritic cells. As shown in Figure 1B, isolation of individual clones yielded stable cell lines of varying quality 12. Among 26 clones tested, 8 produced lentiviral particles at a titer of greater than 106 transduction units per ml (TU/ml), which is a typical benchmark for SVG-pseudotyped lentiviral vectors produced by transient transfection. At the sam...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

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Immunology and Infection

14.5K Aufrufe.  University of Southern California, Los Angeles. Wir verwenden hier retrovirale Transduktion und Transfektion konkatemeren eine Zelllinie, die die Komponenten eines lentiviralen Vektors (LV) in der Abwesenheit von Tetracyclin Ausdruck zu erstellen. Das LV kodiert GFP und mit einem Glykoprotein, SVGmu, die spezifisch für einen Rezeptor auf dendritischen Zellen pseudotypisiert.

Serie: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...