We thank the patients and volunteers who participated in our research.

Human cells are potentially infectious. Therefore, the experiments are performed strictly in accordance with the required precautions and procedures discussed as occupational safety and health administration (OSHA) and personal protection equipment (PPE).
1. Isolation of PBMCs from whole blood
<ol>
	<li>Collect all the materials needed from Table 1 and bring them to room temperature (RT). Warm RP10F to 37 &#176;C. Adjust centrifuge to RT. Except for centrifugations and cell counting, perform all the steps using viable cells in a biological safety cabinet.</li>
	<li>Obtain blood in five 10 mL tubes (desired amount) containing anticoagulant. Store the whole blood at ambient temperature (18&#8210;24 &#176;C). Label 50 mL separation tubes with the human research subject number for the blood sample to be processed.</li>
	<li>Transfer 10&#8210;15 mL of blood into each separation tube(s) with the matching subject number. Dilute the blood at least 2 fold with Hank&#8217;s balanced salt solution (HBSS). Do not exceed 35 mL of diluted blood per tube.</li>
	<li>Carefully and slowly underlay the blood with ~13 mL of density medium. Watch through the transparent density medium at the bottom of the tube, and stop pipetting when the pipette is almost empty (to prevent bubble release). Carefully remove the pipette to avoid mixing the blood and density medium layers.</li>
	<li>Carefully transfer the filled separation tubes to the centrifuge without disturbing the layers.</li>
	<li>Centrifuge at 500 x g for 30 min with the centrifuge brake off (deceleration set to zero). 
	NOTE: If the centrifuge only displays rpm, consult rotor specifications to estimate the rpm equivalent for 500 x g.</li>
	<li>Carefully remove separation tubes from the centrifuge without disturbing the layers. Observe the buffy coat, which has been formed in between the density medium and plasma layers.</li>
	<li>Pipette from the top to remove and discard most of the upper plasma fraction, so that 10 mL remains above the buffy coat. Carefully and slowly collect the buffy coat. Transfer the buffy coats from two separation tubes into a new pre-labeled and sterile 50 mL tube, as shown in Figure 1.</li>
	<li>Dilute the PBMCs at least 2 fold&#160;with HBSS, bringing the volume in each new tube up to 50 mL. Remember to switch the centrifuge brake to full. Pellet PBMCs by centrifugation at 400 x g for 10 min. Remove the supernatant as much as possible and tap the bottom of the tube to loosen the pellet.</li>
	<li>To lyse residual red blood cells (RBC), resuspend each cell pellet in 1&#8210;2 mL of ammonium-chloride-potassium (ACK) lysis buffer per 10 mL original blood volume. Incubate for exactly 5 min. Promptly stop lysis with an equal or greater volume of HBSS and adjust volume to 50 mL. Centrifuge at 400 x g for 10 min.</li>
	<li>Remove the supernatant and tap the bottom of tube to loosen the cell pellet. Pool cells from the same donor. Bring volume up to 50 mL with HBSS. Centrifuge at 400 x g for 10 min.</li>
	<li>Remove the supernatant and tap the bottom of tube to loosen the cell pellet. Resuspend cells in 10 mL of warm RP10F medium, and take an aliquot for viable cell counting using trypan blue.</li>
	<li>Calculate the total cell number in each sample using a hemocytometer.</li>
</ol>
2. Purification of CD4+CD45RO+ T cells from PBMCs&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
NOTE: Purification of CD4+CD45RO+ T cells from PBMCs is done by using commercially available magnetic separation (see Table of materials) with minor modifications. It is preferred to follow the kit manual for incubation time as each commercial kit has their own instructions.
<ol>
	<li>Wash the desired quantity of PBMCs in 10 mL of selection buffer. Centrifuge at 400 x g for 10 min. Remove the supernatant and tap the bottom of the tube to loosen the pellet.</li>
	<li>Dilute PBMCs to 5 x 107 cells/mL in selection buffer and transfer them to a 5 mL of polystyrene round-bottom tube (12 x 75 mm).</li>
	<li>Add 50 &#181;L of antibody cocktail per 1 mL of sample, and mix gently. Incubate at room temperature for 5 min.</li>
	<li>Immediately before use, vortex magnetic particles for 30 sec on high speed. Add 50 &#181;L of magnetic particles per 1 mL of sample to the tube containing PBMCs, and mix gently.</li>
	<li>Bring the volume up to 2.5 mL with selection buffer and mix gently. Place the tube (without lid) into the magnet, and incubate at RT for 2.5 min.</li>
	<li>Pick up the magnet, and in a continuous motion, invert the magnet and tube to pour the enriched cell suspension into a new sterile tube.</li>
	<li>To increase recovery, add 2.5 mL of selection buffer to the tube remaining in the magnet, without disturbing the immobilized beads. Keep in the magnet for another 2.5 min, and repeat step 2.6 to recover additional cells.</li>
	<li>Take an aliquot for viable cell counting using trypan blue. Calculate the total cell number using a hemocytometer or cell counter.</li>
	<li>Confirm the purity by flow cytometry (Figure 3).</li>
</ol>
3. Chemical activation
<ol>
	<li>Adjust CD4+CD45RO+ T cells to 5 x 106 cells/mL with warm RP10F medium, and distribute cells into culture dishes of the desired size. Rest cells in a humidified 37 &#176;C 5% CO2 incubator overnight.</li>
	<li>Pellet rested cells by centrifugation at 400 x g for 10 min. Remove the supernatant and tap the bottom of tube to loosen the cell pellet.</li>
	<li>Adjust cell concentration to 5 x 106 cells/mL with warm RP10F medium, and distribute 0.5&#8210;1 x 107 cells into each of three sterile, screw-cap tubes. 
	NOTE: If sufficient cells are available, duplicate stimulations or additional time points may be included in the experimental design.</li>
	<li>Stimulate cells in tubes 2 and 3 with PMA and A23187. Add PMA to 25 ng/mL and A23187 to 500 ng/mL and mix gently. Add an equal volume of dimethyl sulfoxide (DMSO) to the cells in tube 1 that will serve as a vehicle (control). For instance, if using 1 &#181;L of PMA and 1 &#181;L of A23187, then add 2 &#181;L of DMSO to the vehicle tube. Keep the final concentration of DMSO below 0.5% in all tubes.</li>
	<li>Loosen the caps on the tubes, and return cells to the 37 &#176;C 5% CO2 incubator for 2 h (tube 2) and 6 h (tubes 1 and 3). At the indicated time, centrifuge cells at 500 x g for 10 min.</li>
	<li>Prior to lysis, discard as much of the supernatant as possible, without disturbing the cell pellet.</li>
	<li>Promptly lyse the cells,&#160;as directed by instructions from the commercially available RNA isolation kit (see Table of Materials). Proceed with RNA isolation, or freeze the lysate at -80 &#176;C to process later with additional samples. 
	NOTE: RNA purity and integrity may be verified using&#160;microcapillary electrophoresis.</li>
	<li>Optional: To completely remove all traces of DNA from the purified RNA sample, use RNA clean up kit (see Table of Materials), according to manufacturer&#8217;s instructions.&#160;</li>
</ol>

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The authors have nothing to disclose.
Ethical Disclosure: 
This research protocol was approved by the Institutional Review Board (IRB) of the University of Arkansas for Medical Sciences (UAMS, Little Rock, AR) The microarray data presented in this study was done on the samples recruited under a research protocol approved by the IRB of Henry Ford Hospital (Detroit, MI).

Several ways of isolating PBMCs have been developed, and each has their own advantages and limitations28. We routinely collect up to 50 mL of blood in five 10 mL tubes containing anticoagulant. The volume of the blood for PBMC isolation depends on several factors such as health and age of the research subject and also on phlebotomist expertise. A critical procedural step in the protocol is the formation of the step gradient. Poor layering may result in partial or complete failure of PBMCs to sediment at the interface. We prefer the under-layering method described here, as it is easy to start the bottom layer. To completely dispense all of the density medium below the blood, it is critical to use a pipet aid with no air leaks. Contamination of the PBMC fraction by undesired cell types can be minimized by careful and consistent collection of the buffy coat, which should be performed in the same way for each isolation. If PBMCs will not be further fractionated, collecting different amounts of the density gradient and plasma layers between isolations should be avoided. RBC lysis is performed to minimize the potential impact of contaminating RBC- and reticulocyte-derived RNA on downstream gene expression analyses. Hypotonic lysis will be inhibited by excess isotonic&#160;buffer.
Further isolation of T cell subset is important for molecular studies. Here we described&#160;subsequent CD4+CD45RO+ T cells selection by negative selection to remove undesired cell types. Negative selection relies on antibodies recognizing specific cell surface markers for all undesired cells. Antibody coated cells are then removed by magnetic beads. This selection protocol removes unwanted cells while allowing untouched and unstimulated target cells remain free floating, which is essential in studying gene activation. However, care must be taken to avoid cell clumps, which&#160;reduce the final purity of selected CD4+ CD45RO+ T cells. Ethylenediaminetetraacetic acid (EDTA) present in the selection buffer minimizes cell clumping. Yield of T cells depends on factors such as initial volume of the blood, patient&#160;variables such as the treatment being administered to the patient and disease stage at the time of sample collection. Treatment given to the patients may also affect the cell viability. In addition, sample collection before any procedure such as photopheresis also have positive impact on CD4+CD45RO+ T cells purity. We have observed that sample collection after photopheresis treatment procedure has negative impact on CD4+CD45RO+ T cells yield.
Neoplastic T cell clones from SS patients most frequently express surface markers consistent with a mature, memory CD4 T cell phenotype29,30. However, phenotypic plasticity has been occasionally observed with respect to surface markers including CD4, CD45RO, CD45RA, CD7 and/or CD2631. Previous studies have also shown the heterogeneity in CD45RO and CD45RA expression among SS patients29, whereas the majority of SS cases are still CD45RO+. Roelens et al.31 also showed that SS may exhibit interindividual and intraindividual heterogeneity with mixed population of na&#239;ve (TN), central memory (TCM), transitional memory (TTM), effector memory (TEM), and terminal effector memory (TEMRA) subsets. However, their results clearly show that majority of the SS cells has TCM phenotype. We focused our study on the CD45RO+ surface immunophenotype&#160;most common in SS patients, and confirmed phenotype by flow cytometry. In planning studies of T cell subsets in patients, it is important to consider phenotypic heterogeneity of the disease being studied, and purification strategy may therefore be adjusted as needed to obtain the desired T cell population for analysis.
There are several ways to stimulate T cells and PBMCs to examine functional gene expression. We prefer chemical activation (PMA + A23187 ionophore), since we are interested in gene regulation in the nucleus. Chemical activation is a best option for this purpose because it acts as a broad activator and is more uniform&#160;compared to antigen specific stimulation. PMA is a small organic compound that diffuses through the cell membrane into the cytoplasm, and directly activates protein kinase C. A23187 allows calcium to pass through membranes. These compounds bypass surface receptors, and together mimic the effects of T cell receptor ligation with co-stimulation mediated by CD28. The chemicals activate several intracellular signaling pathways, resulting in nuclear transcription factor activation and upregulation of cytokine&#160;genes that are accessible to transcription activation. Although&#160;chemical activation and CD3CD28 ligation produce strikingly similar global gene expression profiles in normal cells32, chemical activation with PMA + A23187 is a good choice since SS T cells can lose expression of surface receptors including TCR components33. Chong et al.22 compared the activation of cytokine genes between PMA/A23187 to anti-CD3 and anti-CD28 antibodies in PBMCs from normal, early MF/CTCL, and late MF/CTCL patients. They reported that PMA/A23187&#160;caused more rapid and intense activation of the&#160;IL-2&#160;gene as compared to anti-CD3/CD28 stimulation. Additionally, they showed that the slower activation kinetics with anti-CD3/CD28 antibodies is potentially from cross-linking and membrane signaling necessary for stimulation. Furthermore, trends in expression of cytokines among the different cell populations studied were preserved with PMA/A23187. Since, we are interested in gene expression activation, chemical stimulation is an ideal approach because it acts as a broad activator and is more consistent compared to antigen specific stimulation. CD3/CD28 ligation is ideal to investigate pathways important in membrane based signal transduction. In addition, chemical activation is less expensive and does not require special equipment. In the current study, PMA + A23187 significantly activated cytokine genes in ND but not SS T cells, suggesting that SS T cells have functional&#160;deficiencies downstream of the TCR.
In summary, this protocol provides phenotypically pure T cells from precious patient-derived blood, and a method for assessing genome-wide changes in functional gene expression. We demonstrate that transcriptomic profiling of SS T cells compared to normal CD45RO+ T cells reveal profound differences in gene activation in fresh human T cells from patients with CTCL. These studies will aid the development of diagnostic biomarkers and therapeutic strategies targeting novel markers in CTCL. In addition, this strategy and protocol in studying primary human T cells may be valuable in adapting to studies of other T cell mediated diseases.

Cutaneous T-cell lymphoma (CTCL), including the most common subtypes mycosis fungoides (MF) and S&#233;zary syndrome (SS), is a heterogeneous group of diseases derived from transformation and uncontrolled proliferation of mature skin-homing T cells1,2. The neoplastic T cells have a mature CD4+CD45RO+, memory phenotype3, and express skin homing adhesion markers, increasing epidermotropism4, which manifests as a rash particularly in early disease. The clinical course of MF is often indolent when under routine managed care, but a subset of patients can progress to more advanced disease. In these MF cases, skin lesions grow and thicken into large tumors, and neoplastic T cells may disseminate to lymph nodes and visceral organs. In contrast, SS is a more aggressive, leukemic variant of CTCL5, characterized by a triad of symptoms: generalized erythroderma (defined as affecting &#62;80% of total body surface area), lymphadenopathy, and presence of more than 1000/mm3 circulating clonal atypical T cells with cerebriform nuclei, so called S&#233;zary cells6,7. The prognosis for SS patients is significantly worse than MF. SS is rare with an incidence rate of 0.1/100,000, and represents approximately 3% of total CTCL cases8,9. CTCL typically presents in older adults with a median age of about 60 years10. The incidence rate for CTCL had been increasing and while the cause is unclear, the rate has stabilized since 199811,12.
The molecular pathogenesis of SS remains unclear. Genetic, epigenetic, and gene expression studies have produced a wealth of novel data, however there remain inconsistent&#160;findings, primarily due to the small patient&#160;cohorts studied2, as well as differences in experimental design and control populations13,14. Improved genomic and transcriptomic characterization may shed light on both disease mechanisms and previously unexplored therapeutic targets. Therefore, more studies from a larger patient population are needed to better understand this heterogenous malignancy. Biomarker panels that are highly sensitive and specific in one SS cohort have performed less uniformly in other cohorts15, which represents a serious obstacle in the development of reliable diagnostic and prognostic biomarkers for SS16. Ideal diagnostic biomarkers will be consistently and highly over-expressed in malignant T cells, while absent or nearly absent in normal T cells17. The discovery of disease-specific biomarkers is important for the advancement of diagnostic and therapeutic protocols for SS.
High quality transcriptomic data for both malignant and normal T cells requires an efficient and reliable approach to sample preparation. Here, we will discuss a detailed yet simple strategy to obtain RNA samples from T cell populations relevant to SS. We will discuss the isolation of peripheral blood mononuclear cells (PBMC) from whole blood, negative magnetic selection of disease-relevant CD4+CD45RO+ T cell populations, chemical activation to reveal&#160;differences in functional responses, and preparation of RNA for transcriptomic profiling. In the current protocol, chemical activation has been performed using phorbol myristate acetate (PMA) and calcium ionophore (A23187)18,19, because previous studies have shown defective T-cell receptor signaling in CTCL, and stimulation with PMA/A23187&#160;bypasses the T-cell receptor20,21. Also, PMA/A23187&#160;permits a more direct proximal activation of nuclear signals needed for cytokine gene activation. Finally, stimulation of T cells provides an additional level of insight into the regulation of gene expression that could not be gained from resting T cells where dynamic change is absent.&#160;

<table><tbody><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisher Scientific</td><td>02-681-320</td><td></td></tr><tr><td>10 ml Disposable Plastic Pipette</td><td>Thermo Scientific</td><td>170356</td><td></td></tr><tr><td>1000 ul Pipet tips</td><td>VWR</td><td>10017-038</td><td></td></tr><tr><td>15 ml Conical Tubes</td><td>Corning</td><td>352196</td><td></td></tr><tr><td>25 ml Disposable Plastic Pipette</td><td>Thermo Scientific</td><td>170357</td><td></td></tr><tr><td>5 ml Disposable Plastic Pipette</td><td>Thermo Scientific</td><td>170355</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>Thermo Scientific</td><td>339652</td><td></td></tr><tr><td>A23187 ionophore</td><td>Fisher Scientific</td><td>BP595</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>75004381</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-5x5ml</td><td></td></tr><tr><td>EasySep Human Memory CD4+ T cell Enrichment Kit</td><td>StemCell</td><td>19157</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>16140-071</td><td></td></tr><tr><td>HBSS</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>HEPES</td><td>Fisher Bioreagents</td><td>BP310-500</td><td></td></tr><tr><td>KHCO3</td><td>Fisher Bioreagents</td><td>P184-500</td><td></td></tr><tr><td>Lymphocyte Separation Medium</td><td>Corning</td><td>25-072-CV</td><td></td></tr><tr><td>Na2EDTA</td><td>ACROS</td><td>10378-23-1</td><td></td></tr><tr><td>NaHCO3</td><td>Fisher Bioreagents</td><td>S233-500</td><td></td></tr><tr><td>NH4Cl</td><td>Fisher Bioreagents</td><td>A661-500</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>GIBCO</td><td>15140122</td><td></td></tr><tr><td>phorbol12-myristate13-acetate (PMA)</td><td>Sigma</td><td>P-8139</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-1000XLS+</td><td>RAININ</td><td>17014382</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-10XLS+</td><td>RAININ</td><td>17014388</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-200XLS+</td><td>RAININ</td><td>17014391</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-20XLS+</td><td>RAININ</td><td>17014392</td><td></td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>R1013</td><td></td></tr><tr><td>Rneasy Plus Mini Kit</td><td>Qiagen</td><td>74136</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>31800-022</td><td></td></tr><tr><td>T-25 Flask</td><td>Thermo Scientific</td><td>2024-10</td><td></td></tr></tbody></table>

isolating human peripheral blood mononuclear cells and cd4+ t cells from s&#233;zary syndrome patients for transcriptomic profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

This protocol includes procedures for the isolation of PBMCs from SS blood, purification of CD4+CD45RO+ T cells by negative selection,&#160;stimulation of purified T cells, and isolation of total RNA for transcriptomic profiling. Figure 1 describes the process of PBMC isolation from whole blood. Please note that the total yield of SS PBMCs will vary with starting blood volume and circulating tumor burden of each patient. In our laboratory, the average yield of SS PBMCs was 4.6&#8201;&#215;&#8201;106 cells/mL of whole blood (1.85&#8201;&#215;&#8201;106 &#8211; 3.25 x 107 cells/mL for 7 SS). The mean viability of isolated PBMCs was 95&#8210;99%. Figure 2 shows high purity and viability of selected CD4+CD45RO+ memory T cells. The average yield of CD4+CD45RO+ T cells from SS PBMCs was 75% (75.6% &#8211; 84%), compared to 15.9% (3% &#8211; 30%) from normal donors (ND) PBMCs obtained from leukoreduction system (LRS) chambers. The viability and purity of CD4+CD45RO+ T cells obtained by this negative selection protocol has been consistently high (Figure 3).
We previously combined the activation protocol above with microarrays to study the functional changes in the transcriptomes of both SS and ND T cells, and have demonstrated that SS memory T cells and SS PBMCs poorly express cytokine and other immune response genes compared to&#160;ND T cells and PBMCs19,22,23. Figure 4 shows the robust activation of several cytokine genes including IL4, IL 10, IL13 and IL22 in ND T cells, but not in SS T cells. This defect in functional&#160;gene expression in SS T cells have since been confirmed by other groups24. In addition, many genes not normally expressed in ND T cells are highly expressed in SS T cells, both at rest and following stimulation (Figure 4). These include the previously described SS biomarker genes DNM3, PLS3, TOX and TWIST125,26,27, as well as ANK1 and SGCE, which were first reported by our group. These positive biomarkers are highly&#160;expressed in SS, but not ND, and avoid technical pitfalls associated with negative biomarkers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61470/61470fig1v2.jpg" /> 
Figure 1: PBMC isolation from whole blood. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61470/61470fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61470/61470fig2.jpg" /> 
Figure 2: Negative selection of CD4+ memory T cells from isolated PBMCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61470/61470fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61470/61470fig3.jpg" /> 
Figure 3: Purity of CD4+CD45RO+ T cells was confirmed by flow cytometry. Lymphocytes were gated by light scatter (A), live lymphocytes excluded eFluor780 viability dye (B), and (C) represents non-selected normal donors (ND). Negative selection resulted in nearly pure populations of CD45RO+ T cells in ND (D) and SS patients (E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61470/61470fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61470/61470fig4.jpg" /> 
Figure 4: Differential gene expression in resting and activated CD4+CD45RO+ memory T cells from SS and ND. Gene expression z-score is represented by a color scale from red (high expression) to green (low expression). Colored bars at the top of the heat map represents cell treatments: mock/vehicle treated (red), 2 h stimulated (blue), and 6 h stimulated (yellow). Several SS biomarker genes are highly expressed and cytokine genes are poorly expressed in SS T cells compared to ND T cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61470/61470fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
		</tr>
		<tr>
			<td>Density Medium</td>
			<td>Lymphocyte Separation Medium, Ficoll-Hypaque, or equivalent density medium with density = 1.077-1.080g/ml at 20oC.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>1x Hank&#8217;s balanced salt solution, 4.2 mM NaHCO3, 10 mM HEPES, pH 7.2</td>
		</tr>
		<tr>
			<td>RP10F</td>
			<td>RPMI 1640 medium, 10 % heat inactivated fetal bovine serum (FBS), 1x penicillin-streptomycin solution, pH 7.2</td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, No need to adjust pH. It should be ~ 7.3.</td>
		</tr>
		<tr>
			<td>Selection buffer</td>
			<td>1x HBSS, 2% FBS, 2 mM EDTA</td>
		</tr>
		<tr>
			<td>phorbol12-myristate13-acetate (PMA)</td>
			<td>50 &#181;g/ml in DMSO</td>
		</tr>
		<tr>
			<td>A23187 ionophore</td>
			<td>500 &#181;g/ml in DMSO</td>
		</tr>
	</tbody>
</table>
Table 1: Reagents.

Watch this Scientific Journal Video about Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from SÃ©zary Syndrome Patients for Transcriptomic Profiling at JoVE.com

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

isolating-human-peripheral-blood-mononuclear-cells-cd4-t-cells-from

Nanyang Technological University

Research

JoVE Journal

Cancer Research

5.3K Views.  University of Arkansas for Medical Sciences. We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with Sézary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Video: Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from Sézary Syndrome Patients for Transcriptomic Profiling

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Genetics

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Immunology and Infection

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&#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 ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Biology

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

Source: Mily, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from Sézary Syndrome Patients for Transcriptomic Profiling

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