All the buffy coat bags used for this protocol were irradiated thanks to availability of Dr. Peter Rosenthal, Dr. Dirk B&#246;hmer and the whole team of the Radiology Department of Charit&#233; Benjamin Franklin. Scholarship Holder/Mary Roxana Christopher, this work is supported by a scholarship from the German Cardiac Society (DGK).

The study was conducted according to the Statute of the Charit&#233; for Ensuring Good Scientific Practice and the legal guidelines and provisions on privacy and ethics were respected. The Ethics Committee approved all human experiments and the privacy and anonymity of the patients were maintained in accordance with the rules reported on the Ethic Form.
NOTE: For the protocol described below fresh human stenotic valve samples were used.
1. Reagent preparation
<ol>
	<li>Prepare the enriched RPMI complete medium by adding in RPMI 1640 Medium: Non-essential amino acids; Sodium Pyruvate, L- Glutamine, &#223;-Mercaptoethanol and Penicillin-Streptomycin (Pen/Strep). Filter it before use. Store at +4 &#176;C and use within two months.</li>
	<li>Prepare the cell culture medium (CCM) for the cloning phase using the enriched RPMI 1640 complete medium added heat-inactivated Fetal Bovine Serum (FBS), HB basal medium, 50 U of Interleukin 2 (IL-2) and Human Serum (HS). Filter and use it within the day. Do not store.</li>
	<li>Prepare the cell culture medium for the refeeding phase using the enriched RPMI 1640 complete medium with added FBS, HB basal medium, 30 U of IL-2 and HS. Filter and use it within the day. Do not store.</li>
	<li>Prepare the wash buffer containing 250 mL of RPMI and 5 mL Pen/Strep. Store at +4 &#176;C, use within two months.</li>
</ol>
2. Human T lymphocyte isolation and culture into T-25 flasks
<ol>
	<li>Place the stenotic valve sample into a sterile Petri dish filled with Wash Buffer, ensuring that the entire valve sample is covered with it. Let stand for 15 minutes.</li>
	<li>Cut the stenotic valve in small pieces using a scalpel and let it stand again for 10 minutes.</li>
	<li>Prepare the cell culture medium with 50 U of IL-2, as previously described, and filter it.</li>
	<li>Add the CCM inside the T25 flasks and using a sterile plastic plier, place the valve pieces inside the flasks.</li>
	<li>Store the flasks in a vertical position inside a CO2 incubator for one week, taking care to observe the flask&#39;s status under the microscope. T cell clones should be visible approximately three days after the culture step; for better observation it is advisable to place the flasks in horizontal position for 20 min before the microscopic analysis. 
	&#8203;NOTE: The quantity of cell culture medium depends on the amount of sample present and on how many T25 flasks will be used. Considering that each piece of valve requires 10 mL of CCM inside a flask, 25 mL of CCM in a T25 flask is appropriate for 3 pieces of valve.</li>
</ol>
3. Cloning phase
NOTE: For the cloning phase of T cells, it is necessary to start with the isolation of feeder cells from an irradiated buffy coat (BC), following the PBMCs protocol.
<ol>
	<li>Open the BC bag under the laminar flow hood using the bag spike with needle-free valve&#160;and transfer 50 mL of blood in a T150 flask. Add 70 mL of PBS to dilute blood inside the flask.</li>
	<li>Take four 50 mL conical tubes and place 20 mL of density gradient medium in each and carefully layer 30 mL of blood over it.</li>
	<li>Centrifuge for 25 min at&#160;800 x g and 20 &#176;C with the brake off.</li>
	<li>Carefully aspirate the supernatant and discard it as waste. Collect the PBMCs ring in a new 50ml conical tube adding inside the PBS solution up to a volume of 50ml.</li>
	<li>Centrifuge for 10 min at 400 x g and 20 &#176;C.</li>
	<li>Discard the supernatant and suspend the pellet by adding inside the PBS solution up to a volume of 50 mL. Repeat the centrifugation phase for 10 min at 400 x g and 20 &#176;C.</li>
	<li>Discard the supernatant and suspend the FCs in 10 mL of cell culture medium.</li>
	<li>Take a new collection tube and dilute the FCs with PBS 1:10 by adding inside 9.9 mL of PBS and 100 &#181;L from the tube with FCs and cell culture medium. Rinse well with a pipette and take 7 &#181;L from this dilution and count the cells under the microscope.</li>
	<li>Prepare the cell culture medium for the cloning phase: 70 mL with 50 U of IL-2, and then divide it in two 50 mL conical tubes, each tube will contain 25 mL of cell culture medium, as describe below:
	<ul style="list-style-type: disc;">
		<li>Tube 1: 25 mL of CCM + FCs + 0.6% of phytohemagglutinin (PHA)</li>
		<li>Tube 2: 25 mL of CCM + T lymphocytes 
		NOTE: The aim of the PMBCs method is to obtain 2 x 106 cells for each mL, so the 25 mL of CCM prepared (Tube 1) needs to be calculated accordingly. The general formula used is: Number of counted cells x 106: 1 mL = CCM x 106: X</li>
	</ul>
	</li>
</ol>
4. T Lymphocytes culture in multiwell plates
<ol>
	<li>Using the electronic pipette, collect all the cell culture medium inside the flasks and transfer it into a 50 mL conical tube. The empty flasks can be thrown away.</li>
	<li>Pellet the cells by centrifugation for 10 min at 400 x g and 20 &#176;C.</li>
	<li>Discard the supernatant, suspend the pellet in 50 mL of PBS and collect the cells by centrifugation for 10 min at 400 x g and 20 &#176;C. Repeat this step twice.</li>
	<li>Discard the supernatant and suspend the pellet in 1 mL of CCM. Take 7 &#181;L from this dilution and count the cells under the microscope. 
	NOTE: The number of counted cells needs to be multiplied for the volume of the chamber and for dilution applied.</li>
	<li>Proceed to dilute the cells counted from 105 to 103 in cell culture medium and then take 500 &#181;L from 103 and put it inside the Tube 2.</li>
	<li>Pour the 25 mL cell culture medium of Tube 1 inside a 100 mm x 15 mm plastic Petri dish.</li>
	<li>Take four 96-U bottom multiwell plates and using a multichannel pipette set to 100 &#181;L seed FCs in each well.</li>
	<li>Take the Tube 2 and pour the cell culture medium inside a Petri dish. Using a multichannel pipette set to 100 &#181;L, seed T cells in each well.</li>
	<li>Store the multiwell plates inside a CO2 incubator for one week.</li>
</ol>
5. First refeeding phase
<ol>
	<li>Using an irradiated buffy coat bag, follow the PBMCs method to obtain FCs as described in the previous method.</li>
	<li>Prepare the suitable quantity of CCM without PHA and filter it.</li>
	<li>Using a multichannel pipette, remove 100 &#181;L from each well, ensuring not to touch the bottom of the well.</li>
	<li>Add 100 &#181;L of FCs in all the wells as a feeding layer to provide cell culture nutrients and change the medium. 
	&#8203;NOTE: For each multiwell it is advised to consider 6 mL of CCM, so the recommended quantity for 4 multiwells is about 30 mL. PHA (0.4% of the total CCM quantity) needs to be added to the CCM once every two weeks.</li>
</ol>
6. Second refeeding phase
<ol>
	<li>Repeat the refeeding phase following the passage described above. 0.4% PHA needs to be added to the cell culture medium.</li>
</ol>
7. First splitting phase from 1 well/sample to 2 wells/sample
<ol>
	<li>Check all the four multiwells under the microscope and go ahead splitting those wells with T cell clones (TCCs).</li>
	<li>Follow the PBMCs protocol to isolate feeder cells from an irradiated buffy coat bag.</li>
	<li>Prepare the cell culture medium with 30 U of IL-2. Add the FCs and take a new 96-well multiwell plate with U bottom.</li>
	<li>Using a pipette set to 100 &#181;L, rinse well inside the wells of the selected samples and divide the 200 &#181;L of volume contained in each well into 2 new wells of the new multiwell plate, in order to have 100 &#181;L of volume for each of the new 2 wells.</li>
	<li>Add 100 &#181;L of FCs inside all the new wells. Each well must contain a total volume of 200 &#181;L.</li>
	<li>Set the pipette at 100 &#181;L and add 100 &#181;L into an additional two wells, containing only FCs to serve as a control. 
	&#8203;NOTE: Use light microscopy to observe all multiwell plates in order to determine which wells have successfully grown TCC. To make the selection of clones easier, a comparison can be made to the control (the two wells containing only FCs), with the clone appearing darker and larger compared to the control. The wells to be selected are those with a large, clear, round clone, with lymphocytes densely packed together. If no TCCs are present after two weeks of refeeding, it is recommended to wait one more week and perform an additional refeeding phase.</li>
</ol>
8. Second splitting phase from 2 wells/sample to 4 wells/sample
<ol>
	<li>Set the pipette to 100 &#181;L, rinse well inside the two wells of each sample and seed two new wells of 100 &#181;L for each one.</li>
	<li>Prepare the cell culture medium with 30 U of IL-2 and follow the PBMCs technique to extract the FCs from the buffy coat bag and place the FCs inside the CCM.</li>
	<li>Set the pipette to 100 &#181;L and seed the FCs in all the wells. Store the multiwells in a CO2 incubator for one week.</li>
</ol>
9. Third splitting phase from 4 wells/sample to 8 wells/sample:
<ol>
	<li>Using a multichannel pipette set to 100 &#181;L, rinse well inside all four wells of each sample and seed 100 &#181;L into each of the four new wells in a new 96-well plate.</li>
	<li>Prepare the cell culture medium and follow the PBMCs technique to extract the FCs from an irradiated buffy coat bag.</li>
	<li>Place the FCs inside the CCM. Set the pipette to 100 &#181;L and seed the FCs in all the wells. Store the multiwells in a CO2 incubator for one week</li>
</ol>
10. Cytofluorimetric analysis
<ol>
	<li>Take the multiwell plate with the oldest date from the CO2 incubator and identify the collection tubes with the sample numbers to analyze.</li>
	<li>Using a multichannel pipette set to 200 &#181;L with 2 tips, thoroughly rinse inside two wells of the same sample and put both inside the same collection tube. Repeat this step for all the samples. 
	&#8203;NOTE: The FCs samples will not be analyzed, as they only serve as controls.</li>
</ol>
11. Antibody Staining Panel Preparation for cytofluorimetric analysis
<ol>
	<li>Add 1 mL of PBS solution for each tube and centrifuge for 10 min at 400 x g and 20 &#176;C.</li>
	<li>After the centrifugation phase discard the supernatant. 
	NOTE: The antibody staining panel can be found in Table 1. Each single antibody concentration calculated is about 2 &#181;L/sample. It is recommended to briefly spin the antibody tubes before use. To protect the fluorophore-conjugated antibodies from light, all steps should be performed in the dark.</li>
	<li>Prepare the antibody mix inside a 1.5 mL tube and vortex it.</li>
	<li>Add the antibodies inside the samples and leave samples to incubate in the dark at room temperature for 15 min.</li>
	<li>Add 1 mL of PBS in each sample and centrifuge for 10 min at 400 x g and 20 &#176;C.</li>
	<li>Discard the supernatant and suspend the pellet with 500 &#181;L of PBS solution.</li>
	<li>Proceed with the cytofluorimetric analysis (FACS analysis, Table 1). 
	NOTE: The stained samples can be fixed using 0.5% paraformaldehyde (PFA) diluted in PBS solution and analyzed up to 15 days later. 
	&#8203;CAUTION: PFA is toxic and must be handled carefully.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Marker</td>
			<td>Fluorophore</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>PE/Cy7</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Alexa Fluor 488</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Brilliant violet 510</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>Brilliant violet 421</td>
		</tr>
		<tr>
			<td>CD25</td>
			<td>PE</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>Brilliant violet 711</td>
		</tr>
	</tbody>
</table>
Table 1. Antibody staining panel to detect the T lymphocytes population in calcification aortic valve disease.

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The authors have nothing to disclose.

Here we present a method to characterize T lymphocyte subpopulations isolated from stenotic aortic valve samples, using flow cytometry. This method requires the use of irradiated buffy coat to isolate the PBMCs. The radiation frequency to which the buffy coat bags must be subjected is 9000 Rad/90 Gray (Gy) and it represents a crucial step to halt the proliferation of the feeder cells. The role of the cells isolated from the buffy coat bags is to act only as feeder cells and provide nutrients for the T cells isolated from...

Calcific aortic valve disease (CAVD) is one of the most common heart valve disorders, with a heavy impact on healthcare. The frequency of aortic valve replacement in the last years has increased dramatically and is expected to increase further, due to the growing elderly population1.
The underlying pathophysiology of CAVD is only partially known and the current therapeutic strategies are limited to conservative measures or aortic valve replacement, either through surgical or percutaneous procedures. To date, no effective medical treatment can hinder or reverse CAVD progression and high mortality is associated with early symptom-onset, unless aortic valve replacement (AVR) is performed2. In patients with severe symptomatic aortic stenosis, the 3-year symptom-free survival was reported as low as 20%3. Transcatheter aortic valve replacement (TAVR) represents a new option, revolutionizing treatment for high-risk patients, especially among the elderly and has dramatically reduced the mortality, which was intrinsically high in this population4,5,6. Despite the promising results of TAVR, further research is necessary to understand CAVD pathophysiology to identify novel early therapeutic targets7,8,9.
Previously thought to be a passive, degenerative process, CAVD is now recognized as an active progressive disease, characterized by an osteoblastic phenotype switch of the aortic valve interstitial cells10. This disease involves progressive mineralization, fibrocalcific changes and reduced motility of the aortic valve leaflets (sclerosis), which ultimately obstruct blood flow leading to narrowing (stenosis) of the aortic valve opening11.
Inflammation is considered a key process in CAVD pathophysiology, similar to the process of vascular atherosclerosis. Endothelial injury enables the deposition and accumulation of lipid species, especially oxidized lipoproteins in the aortic valve12. These oxidized lipoproteins provoke an inflammatory response, as they are cytotoxic, with the inflammatory activity leading to mineralization. The role of innate and adaptive immunity in CAVD development and disease progression has been recently highlighted13. The activation and clonal expansion of specific subsets of memory T cells have been documented in patients with CAVD and mineralized aortic valve leaflets, so that inflammatory processes are assumed to be involved at least in the development of CAVD and presumably in disease progression as well14. In fact, although antigen-presenting cells and macrophages are present in both the healthy and diseased valve, the presence of T lymphocytes is indicative of an aged and diseased aortic valve. This lymphocytic infiltrate along with an increase in neovascularization and metaplasia are characteristic histological signs of CAVD15.
We hypothesize the existence of an interaction between the aortic valve interstitial cells and the activation of the immune system, which potentially triggers the initiation of a chronic inflammatory process in the aortic valve. The assaying of T cells from stenotic aortic valve tissue could be an efficient way to clarify their role in calcification development, as it can provide a close representation of the aortic valve cells in vivo. In the present work, using aortic valve tissue, we isolate T-lymphocytes, culture and clone them, and subsequently characterize them using fluorescence-activated cell sorting (FACS). Fresh aortic valve samples were excised from CAVD patients who received surgical valve replacement for severe aortic stenosis. After the surgical excision, the fresh valve sample was dissected in small pieces and the T cells were cultured, cloned then analyzed using flow cytometry. The staining procedure is simple and the stained tubes can be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The data generated from the staining panel can be used to track changes in T lymphocyte distribution over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subsets of interest.
The extraction of calcified tissue, the isolation of leukocytes from calcified tissue and particularly the use of flow cytometry on this type of tissue can be challenging, due to issues such as autofluorescence. Few publications exist with protocols for this specific purpose16,17,18. Herein we present a protocol designed specifically for the direct isolation and culture of T lymphocyte from human aortic valve samples. Clonal expansion of lymphocytes is a hallmark of adaptive immunity. Studying this process in vitro provides insightful information on the level of lymphocyte heterogeneity19. After a three-week incubation period, the T cell clones are ready to be explanted, as an adequate amount of T cells from each clone was obtained, so as to allow the phenotypic and functional study. Subsequently the phenotype of T clones is studied by cytofluorometry.
This immunological protocol is an adaptation of a method previously developed by Amedei et al. for T cell isolation and characterization from human tissue, especially designed for calcified human tissue, such as in CAVD20,21,22. The protocol here for the isolation of PBMCs (peripheral blood mononuclear cells) using irradiated buffy coat describes an effective way to obtain feeder cells (FC), specifically adjusted for the cloning phase of T lymphocytes isolated from valve interstitial cells. The feeder layer consists of in growth arrested cells, which are still viable and bioactive. The role of feeder cells is important to support in vitro survival and growth of T lymphocytes isolated from valve interstitial cells23. In order to avoid feeder cell proliferation in culture, these cells must undergo a growth arrest. This can be achieved in two ways: through physical methods such as irradiation, or through treatment with cytotoxic chemicals, such as mitomycin C (MMC), an antitumoral antibiotic that can be applied directly to the culture surface24. Here we show feeder cell growth arrest achieved through cell irradiation.
This method presents an efficient, cost-effective way to isolate and characterize T cells from aortic valve tissue, contributing to broadening the spectrum of immunological methods for exploring CAVD pathophysiology.

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			<td>Falcon Round-Bottom Polystyrene Tubes</td>
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			<td>Fast read 102 plastic counting chamber</td>
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			<td>Filters for culture medium 500 mL</td>
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			<td>HB Basal Medium</td>
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			<td>Human IL-2 IS</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-744</td>
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			<td>Gibco</td>
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			<td>Thermo Fisher Scientific</td>
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			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
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			<td>PHA (phytohemagglutinin)</td>
			<td>Stem Cell Technologies</td>
			<td>7811</td>
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investigating aortic valve calcification via isolation and culture of t lymphocytes using feeder cells from irradiated buffy coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

We used a simple and cost-effective method to characterize the leukocyte population of fresh aortic valve samples derived from human patients with severe aortic valve stenosis (refer to protocol). The method for isolating PBMCs is a vital step in obtaining feeder cells, which are used in every step of the experiment (cloning, refeeding and splitting phases) and enable the detection and characterization of infiltrating leukocytes in aortic valve samples. The key steps of this method are shown in Figur...

Watch this Scientific Journal Video about Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat at JoVE.com

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

investigating-aortic-valve-calcification-via-isolation-culture-t

Research

JoVE Journal

Immunology and Infection

2.4K Views.  Charité Universitätsmedizin Berlin and German Centre for Cardiovascular Research (DZHK). In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis. 

Video: Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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 ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.