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 border="0" cellpadding="0" cellspacing="0" style="width:706px;" width="706">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:269px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">02-681-320</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">10 ml Disposable Plastic Pipette</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">170356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1000 ul Pipet tips</td>
			<td style="width:151px;">VWR</td>
			<td style="width:119px;">10017-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">15 ml Conical Tubes</td>
			<td style="width:151px;">Corning</td>
			<td style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25 ml Disposable Plastic Pipette</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">170357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">5 ml Disposable Plastic Pipette</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">170355</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">50 ml Conical Tubes</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">A23187 ionophore</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">BP595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Centrifuge</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">75004381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMSO</td>
			<td style="width:151px;">Sigma</td>
			<td style="width:119px;">D2650-5x5ml</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">EasySep Human Memory CD4+ T cell Enrichment Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:119px;">19157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:151px;">GIBCO</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">HBSS</td>
			<td style="width:151px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">HEPES</td>
			<td style="width:151px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">KHCO3</td>
			<td style="width:151px;">Fisher Bioreagents</td>
			<td style="width:119px;">P184-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Lymphocyte Separation Medium</td>
			<td style="width:151px;">Corning</td>
			<td style="width:119px;">25-072-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Na2EDTA</td>
			<td style="width:151px;">ACROS</td>
			<td style="width:119px;">10378-23-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">NaHCO3</td>
			<td style="width:151px;">Fisher Bioreagents</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">NH4Cl</td>
			<td style="width:151px;">Fisher Bioreagents</td>
			<td style="width:119px;">A661-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin solution</td>
			<td style="width:151px;">GIBCO</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">phorbol12-myristate13-acetate (PMA)</td>
			<td style="width:151px;">Sigma</td>
			<td style="width:119px;">P-8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td style="width:151px;">RAININ</td>
			<td style="width:119px;">17014382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pipet-Lite LTS Pipette L-10XLS+</td>
			<td style="width:151px;">RAININ</td>
			<td style="width:119px;">17014388</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td style="width:151px;">RAININ</td>
			<td style="width:119px;">17014391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pipet-Lite LTS Pipette L-20XLS+</td>
			<td style="width:151px;">RAININ</td>
			<td style="width:119px;">17014392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RNA Clean &#38; Concentrator-5</td>
			<td style="width:151px;">Zymo Research</td>
			<td style="width:119px;">R1013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Rneasy Plus Mini Kit</td>
			<td style="width:151px;">Qiagen</td>
			<td style="width:119px;">74136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RPMI 1640</td>
			<td style="width:151px;">GIBCO</td>
			<td style="width:119px;">31800-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">T-25 Flask</td>
			<td style="width:151px;">Thermo Scientific</td>
			<td style="width:119px;">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/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/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/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/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 SÃƒÂ©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

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Cancer Research

5.1K 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

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as valency, spatial arrangement, interdomain distance, and Fc mutations affect the anti-tumor efficacy of these therapies, commonly by influencing the homing of T cells to tumors, which remains a major challenge. Here, we describe a method to transduce activated human T cells with luciferase, allowing in vivo tracking of T cells during T-BsAb therapy studies. The ability of T-BsAbs to redirect T cells to tumors can be quantitatively evaluated at multiple time points during treatment, allowing researchers to correlate the anti-tumor efficacy of T-BsAbs and other interventions with the persistence of T cells in tumors. This method alleviates the need to sacrifice animals during treatment to histologically assess T cell infiltration and can be repeated at multiple time points to determine the kinetics of T cell trafficking during and after treatment.

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as ...