This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

Research with human blood samples was approved by Concordia University Ethics Review Board, certificate number 10000618. The work on mouse muscle was approved by Concordia University Ethics Review Board, certificate number 2010BERG.
1. PBMC Isolation from Whole Blood
NOTE: Carry out this procedure in a biosafety cabinet using sterile technique and manufacturer-sterilized equipment.
<ol>
	<li>Carefully pour 10-15 ml of whole blood from the heparinized (anti-coagulant) blood collection tubes into a 50 ml sterile conical tube. For minor blood spills, wipe with ddH2O and 70% EtOH using cleaning tissue.</li>
	<li>Dilute the blood in the conical tube to a 1:1 ratio with Phosphate Buffer Saline (PBS 1x) pH 7.4. Ensure that the maximum total volume does not exceed 30 ml.</li>
	<li>Mix gently using a serological pipette gun avoiding bubbles. 
	NOTE: Do NOT mix by inverting the tube to avoid blood accumulation in the cap and subsequent seepage during centrifugation.</li>
	<li>Add 13 ml of Ficoll-paque&#160;(see Materials and Equipment table), to a new 50 ml capacity conical tube. Keep the tube upright in the rack.</li>
	<li>Using a transfer pipette, take diluted blood, touch the transfer pipette tip to the inside wall of the tube near the top. With slow and steady pressure, transfer the blood along the inside wall of the tube forming a blood layer on top of the sucrose layer. Do this step several times until all blood has been transferred.</li>
	<li>Cap the tube tightly and centrifuge the tube at room temperature for 30 min at 700 x g in a swing rotor with medium acceleration set to 5, and deceleration set to ZERO.</li>
	<li>Slowly take out the tube and without disturbing the layers, take it back to the biosafety cabinet.</li>
	<li>Carefully collect the buffy coat (thin cloudy white layer), where PBMCs are located, placed between the PBS/plasma and ficoll layers using a transfer pipette. Avoid collecting sucrose layer and do not disturb the red blood cell layer.</li>
	<li>Transfer the buffy coat into a new 50 ml sterile conical tube. It may take several times of repeating step 1.8 to collect all the PBMC.</li>
	<li>Add PBS pH 7.4 to the tube with the PBMC and fill up to the 45 ml mark. Shake tube thoroughly. Do not vortex.</li>
	<li>Wash 1: Centrifuge for 15 min at 480 x g with both maximum acceleration and deceleration set to 9. Observe formation of a pellet of PBMCs at the bottom of the conical tube.</li>
	<li>Discard the PBS (supernatant) into a plastic beaker with 10 ml of bleach.</li>
	<li>Loosen cell pellet by gently &#8220;racking&#8221; against an undulated surface (i.e., empty rack). Do not vortex.</li>
	<li>Add 25 ml of fresh PBS pH 7.4 to the tube. If more than one tube is being processed, pool the pellets together in this step.</li>
	<li>Wash 2: Centrifuge the tube for 12 min at 480 x g with both maximum acceleration and deceleration set to 9.</li>
	<li>Discard the PBS (supernatant) into the plastic beaker with a splash of bleach.</li>
	<li>Gently &#8220;rack&#8221; against an undulated surface (i.e., empty rack). Do not vortex.</li>
	<li>Add 25 ml of fresh PBS to the tube.
	<ol>
		<li>Take out 50 &#181;l of the cells and transfer into a microcentrifuge tube for viability count.</li>
		<li>Add an equal amount (50 &#181;l) of trypan blue (a viability stain) and pipette up and down to mix gently.&#160;</li>
	</ol>
	</li>
	<li>Take out 10 &#181;l and transfer it to a hemocytometer to check the viability of cells per ml. Record the number of cells/ml.</li>
	<li>Take out the desired amount of cells and centrifuge the tube for 12 min at 480 x g with both maximum acceleration and deceleration set to 9.</li>
	<li>Discard the PBS (supernatant) into the beaker with bleach.</li>
	<li>Gently &#8220;rack&#8221; against an undulated surface (i.e., empty rack). Add 80 &#181;l of PBS into the tube.</li>
</ol>
2. Making the PBMC Slide
<ol>
	<li>Place 80 &#181;l of the cells onto a microscope slide. Smear the drop with the help of another slide or place 2 drops of 40 &#181;l each on both ends of the slide.</li>
	<li>Leave slide in the biological safety cabinet to dry. Label the slide with a pencil on the frosted side.</li>
</ol>
3. Fixing the Samples on the Slides
<ol>
	<li>Prepare the fixative solution by mixing 0.5 ml of 37% formaldehyde to 4.5 ml of 99% ethanol.</li>
	<li>After the slides are dried, take out 2 ml of the freshly made fixative solution and pour it on the slide so that the entire surface of the slide is covered.</li>
	<li>Leave the solution on the slide for 1 min. Rinse the slide for 1 min with tap water and leave it to air dry.</li>
</ol>
4. Making the Amylase Solution for Negative Control
<ol>
	<li>Take 0.25 g of amylase powder and dissolve in 50 ml of distilled water. Pour the solution in a clean 100 ml beaker.</li>
	<li>Immerse the slide in the beaker so that half of the slide receives the treatment and the other half remains untreated and then incubate for 15 min at room temperature (Figure 1C).</li>
	<li>Make a note on which side of the slide is receiving the amylase treatment. Draw a line on the back of the slide indicating the border between the treatment and control.</li>
	<li>Wash the slides with ddH2O to remove the amylase solution and leave the slide to air dry.</li>
</ol>
5. Perform Periodic Acid Schiff (PAS) Staining and Imaging
NOTE: PAS reagents are toxic by inhalation and are corrosive, so the steps need to be done in a chemical fume hood, and the waste products must be properly disposed of according to institutional guidelines.
<ol>
	<li>Place the slide on a flat surface and pour 1.50-2.00 ml of Periodic Acid Solution on the sample. Incubate for 5 min at room temperature.</li>
	<li>Rinse the slides in several charges of distilled water.</li>
	<li>Pour 1.50-2.00 ml of Schiff&#8217;s reagent on the slide and incubate it at room temperature for 15 min.</li>
	<li>Wash the slide with distilled water for 5 min and leave it to air dry.</li>
	<li>Apply 10 &#181;l mounting media on the slide and cover with two small coverslips, or apply 50 &#181;l and use one large coverslip.</li>
	<li>Apply clear nail polish on the edges of the coverslip, let dry overnight.</li>
</ol>
6. Obtain Images with the Binocular Light Microscope Using the 100X Objective

<ol>
	<li>Rich, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+machinery+of+keilin's+respiratory+chain.">The molecular machinery of keilin's respiratory chain.</a> Biochem. Soc. Trans. 31 (Pt 6), 1095-1105 (2003).</li><li>Peter, J. B., Barnard, R. J., Edgerton, V. R., Gillespie, C. A., Stempel, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiles+of+three+fiber+types+of+skeletal+muscle+in+guinea+pigs+and+rabbits).">Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits).</a> Biochemistry. 11 (14), 2627-2633 (1972).</li><li>Jones, R. V., Goffi, G. P., Hutt, M. S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+glycogen+content+in+various+disease.">Lymphocyte glycogen content in various disease.</a> J. Clin. Pathol. 15 (1), 36-39 (1962).</li><li>Scott, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+in+human+peripheral+blood+leukocytes.+I.+characteristics+of+the+synthesis+and+turnover+of+glycogen+in+vitro.">Glycogen in human peripheral blood leukocytes. I. characteristics of the synthesis and turnover of glycogen in vitro.</a> J. Clin. Invest. 47 (2), 344-352 (1968).</li><li>Fedele, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=positive+index+of+lymphocytes+and+metabolic+control+in+insulin-treated+and+type+II+diabetes+mellitus.">positive index of lymphocytes and metabolic control in insulin-treated and type II diabetes mellitus.</a> Diabete Metab. 9 (3), 188-192 (1983).</li><li>Breli&#324;ska-Peczalska, R., Mackiewicz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+peripheral+blood+granulocytes+and+lymphocytes+in+patients+with+systemic+lupus+erythematosus.">Cytochemical studies of peripheral blood granulocytes and lymphocytes in patients with systemic lupus erythematosus.</a> Pol.Med.Sci.Hist.Bull. 15 (2), 231-234 (1976).</li><li>Yunis, A. A., Arimura, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+of+glycogen+metabolism+in+white+blood+cells.+I.+glycogen+phosphorylase+in+normal+and+leukemic+human+leukocytes.">Enzymes of glycogen metabolism in white blood cells. I. glycogen phosphorylase in normal and leukemic human leukocytes.</a> Cancer, Res. 24, 489-492 (1964).</li><li>Hagemans, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAS-positive+lymphocyte+vacuoles+can+be+used+as+diagnostic+screening+test+for+pompe+disease.">PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for pompe disease.</a> J. Inherit. Metab. Dis. 33 (2), 133-139 (2010).</li><li>Totsuka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+performance+and+soleus+muscle+fiber+composition+in+wild-derived+and+laboratory+inbred+mouse+strains.">Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.</a> 95 (2), 720-727 (2003).</li><li>Murat, J. C., Serfaty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+enzymatic+determination+of+polysaccharide+(glycogen)+content+of+animal+tissues.">Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues.</a> Clin. Chem. 20 (12), 1576-1577 (1974).</li><li>Arrizabalaga, O., Lacerda, H. M., Zubiaga, A. M., Zugaza, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+protein+regulates+glycogen+phosphorylase+activation+and+controls+interleukin+(IL)-2-dependent+T+cell+proliferation.">Rac1 protein regulates glycogen phosphorylase activation and controls interleukin (IL)-2-dependent T cell proliferation.</a> J. Biol. Chem. 287 (15), 11878-11890 (2012).</li><li>Pelletier, J., G, J., Mazure, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+titration+of+glycogen+in+vitro.">Biochemical titration of glycogen in vitro.</a> J.Vis.Exp. (81), (2013).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagliabracci+V.S.+Glycogen+and+its+metabolism:+Some+new+developments+and+old+themes.">Tagliabracci V.S. Glycogen and its metabolism: Some new developments and old themes.</a> Biochem.J. 441 (3), 763-787 (2012).</li><li>Salmoral, E. M., Tolmasky, D. S., Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+presence+of+glycogen+in+rat+thymus.">Evidence for the presence of glycogen in rat thymus.</a> Cell Mol.Biol. 36 (2), 163-174 (1990).</li><li>Darlington, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+Th17+(not+Th1)+responses+underlie+multiple+sclerosis+disease+abrogation+after+hematopoietic+stem+cell+transplantation.">Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation.</a> Ann.Neurol. 73 (3), 341-354 (2013).</li></ol>

The authors have nothing to disclose.

The critical steps of this video article were during washing and amylase treatment of the cells. While washing the slides, the key step was using a plastic squeezable washing bottle and letting the water gently run through the sample on the slide and not aiming directly onto the samples. Even the slightest direct water pressure would cause the cells to come off the slide. Another key step was to use the same slide for &#177; amylase conditions. After the PBMCs were adhered to the slide, the slide was carefully placed int...

Periodic acid Schiff (PAS) staining is an immunohistochemical technique that is widely used in muscle research and diagnostics. It is also utilized as a diagnostic tool on blood samples. The technique works by applying periodic acid solution to the sample, which oxidizes units within the polysaccharide creating aldehyde groups that react with the colourless Schiff&#39;s reagent thereby producing a deep magenta product. The steps of this procedure are shown in Figure 1. The stain turns anything with polysaccharides magenta, including glycogen, glycoproteins, glycolipids, mucins, or other molecules with polysaccharide moieties.
PAS staining is often used to measure glycogen levels in muscle fibers. Muscles tissue sections are ideal for the technique as they firmly attach to the slide and withstand multiple washing and staining steps. Glycogen is most present in fast twitch Type II muscle fibers, which have a high demand for rapid ATP production requiring glycogen for maximum performance1,2. Glycogen is a branched polymer of glucose that can be broken into free glucose through the action of glycogen phosphorylase enzymes. In times of rest and nutritional-sufficiency, glycogen is replenished through the process of glycogenesis, while in times of nutritional insufficiency or high-energy demand; glycogen is broken down into glucose by glycogenolysis. From as early as the 1950&#39;s clinician scientists have explored PAS staining on blood samples to analyze glycogen content in various diseases3-7. For example, in Pompe&#160;disease-a bonafide glycogen storage disease- white blood cells accumulate large amounts of glycogen that differs significantly from healthy controls8.
This video-article demonstrates an adapted version of PAS staining for use on peripheral blood mononuclear cells (PBMC) samples from venous blood of healthy human subjects. PBMCs contain mostly lymphocytes of the T lymphocyte and B lymphocyte families, as well as other immune cells such as natural killer cells and monocytes. The first purification step removes erythrocytes, neutrophils, and other granulocytes. This technique provides data on a concentrated proportion of lymphocytes allowing for more robust enumeration of PAS-positive cells as compared to using whole blood smears.
<img alt="Figure 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> 
Figure 1: Step by step methodology of PAS staining on PBMC. (A) First, isolation of PBMC is achieved through ficoll gradient, the left panel shows the preparation before centrifugation, the right panel shows it after centrifugation where the buffy coat containing the PBMC is observed in the center of the tube. (B) Isolated PBMCs are fixed onto the slide using formalin-ethanol fixative solution. The slide is gently rinsed with distilled water from a plastic wash bottle. (C) The slide is then placed in a 100 ml beaker half way filled with amylase solution, which will dissolve glycogen. The slide is gently rinsed. (D) The slide is treated with periodic acid solution, where oxidation of saccharides takes place. Slides are gently rinsed; this will remove the excess periodic acid and stop the oxidation step. (E) When the Schiff reagent is added to the slides, it will react with aldehydes created during the oxidation step. This colorless reagent will then result in a deep red magenta product. Slides are gently rinsed to remove the excess Schiff reagent.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

To validate the reagents and basic technique, PAS staining was performed according to manufacturer&#8217;s instructions on mouse soleus muscle sections. The staining was done same day as the sacrifice and in the last step of the staining, the sections were fixed by xylene. The soleus muscle is known to contain ~35% glycogen-positive cells9. The stained muscle cells displayed two distinct PAS-positive features- punctate granules within the cell, and a continuous line demarking the cell membrane (Figure ...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

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JoVE Journal

Immunology and Infection

21.1K 조회수.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

비디오: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

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 ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...