Name: preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy
Uploaded: 2016-11-08T13:00:00
Description: Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

We are grateful to Dr. Jos&#233; Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscop&#236;a Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnolog&#236;a (CONACYT), Sociedad Latinoamericana de Glicobiolog&#236;a, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Tem&#225;tica Glicociencia en Salud - CONACYT (253596).

Peripheral blood from healthy male donors was obtained with informed consent and approval of the Bioethics Committee of the Centro de Investigaci&#242;n en Din&#225;mica Celular- Universidad Aut&#242;noma del Estado de Morelos.
1. Isolation and Activation of Human Na&#239;ve CD4+ T Cells
<ol>
	<li>Collect 2 ml of peripheral blood derived from healthy human donors through informed consent and dilute with 2 ml of sterile PBS-EDTA (1x Phosphate Buffered Saline (PBS), 2 mM EDTA, pH 7....

<ol>
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C., Barral, A., Barral Netto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+human+peripheral+blood+monocyte+isolation.">A simple method for human peripheral blood monocyte isolation.</a> Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).</li><li>O'Neil-Andersen, N. J., Lawrence, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modulation+of+surface+and+intracellular+protein+expression+by+T+cells+after+stimulation+in+the+presence+of+monensin+or+brefeldin+A.">Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A.</a> Clin Diagn Lab Immunol. 9 (2), 243-250 (2002).</li><li>Mannering, S. I., Zhong, J., Cheers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+activation,+proliferation+and+apoptosis+in+primary+Listeria+monocytogenes+infection.">T-cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection.</a> Immunology. 106 (1), 87-95 (2002).</li><li>Marconi, S., Acler, M., 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=Anti-GD2-like+IgM+autoreactivity+in+multiple+sclerosis+patients.">Anti-GD2-like IgM autoreactivity in multiple sclerosis patients.</a> Mult Scler. 12 (3), 302-308 (2006).</li><li>Park, J. E., Wu, D. 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=Fine+specificity+of+natural+killer+T+cells+against+GD3+ganglioside+and+identification+of+GM3+as+an+inhibitory+natural+killer+T-cell+ligand.">Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.</a> Immunology. 123 (1), 145-155 (2008).</li><li>Simon, B. M., Malisan, F., Testi, R., Nicotera, P., Leist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disialoganglioside+GD3+is+released+by+microglia+and+induces+oligodendrocyte+apoptosis.">Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.</a> Cell Death Differ. 9 (7), 758-767 (2002).</li><li>Beske, O., Reichelt, M., Taylor, M. P., Kirkegaard, K., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poliovirus+infection+blocks+ERGIC-to-Golgi+trafficking+and+induces+microtubule-dependent+disruption+of+the+Golgi+complex.">Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex.</a> J Cell Sci. 120 (18), 3207-3218 (2007).</li><li>Zuber, C., Spiro, M. J., Guhl, B., Spiro, R. G., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+apparatus+immunolocalization+of+endomannosidase+suggests+post-endoplasmic+reticulum+glucose+trimming:+implications+for+quality+control.">Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.</a> Mol Biol Cell. 11 (12), 4227-4240 (2000).</li><li>Yamashiro, S., Okada, M., 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=Expression+of+(GD3+synthase)+gene+in+human+cancer+cell+lines:+high+level+expression+in+melanomas+and+up-regulation+in+activated+T+lymphocytes.">Expression of (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.</a> Glycoconj J. 12 (6), 894-900 (1995).</li><li>Wipfler, D., Srinivasan, G. V., 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=Differentially+regulated+expression+of+9-O-acetyl+GD3+(CD60b)+and+7-O-acetyl-GD3+(CD60c)+during+differentiation+and+maturation+of+human+T+and+B+lymphocytes.">Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes.</a> Glycobiology. 21 (9), 1161-1172 (2011).</li><li>Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., Old, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD3,+a+prominent+ganglioside+of+human+melanoma.+Detection+and+characterisation+by+mouse+monoclonal+antibody.">GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody.</a> J Exp Med. 155 (4), 1133-1147 (1982).</li></ol>

The described protocol can be used to localize gangliosides or proteins in cell suspensions of CD4+ T cells or other immune cells (e.g., PMBCs, Figure 5) starting from a small number of cells. Because of the small size of T cells and non-adherent properties, the acquisition of fluorescence microscopic images results in poor information or low quality if the cells are not correctly adhered.
This protocol combined with a good quality confocal microscopy analy...

The CD4+ T cells orchestrate the immune response through their effector functions after activation by antigen presenting cells1. The study of the cellular mechanisms that are modulated during activation allows insight into a basic process of immune function. However, the study of na&#239;ve CD4+ T cells can be complicated because they represent a very small population of cells in the blood periphery2.
Through fluorescence microscopy several reports have studied the localization of different molecules involved in CD4+ T cell activation, mainly proteins associated to the plasma membrane3. The gangliosides are sialic acid containing glycosphingolipids and although they have been extensively studied in nerve cells where they are abundant, other cells such as immune cells also express gangliosides with biologically relevant functions4,5. We previously reported that during activation of human na&#239;ve CD4+ T cells there is an upregulation of the &#945;2,8 sialyltransferase ST8Sia 1 (GD3 synthase) and the GM2/GD2 synthase, that induce the significant surface neoexpression of GD2 and the upregulation of GD3 ganglioside6. Further study of GD3, GD2 and other gangliosides in immune cells is necessary to complement a protein-based partial view of immune function.
Commonly, the study of ganglioside expression is based on techniques such as Thin Layer Chromatography (TLC)7, but this technique does not allow the spatial localization of gangliosides at the plasma membrane or in subcellular compartments, limiting biological analysis.
In this work, we describe a protocol for the antibody-mediated identification and localization of GD3 and GD2 gangliosides in human na&#239;ve CD4+ T cells and PBMCs after anti-CD3/anti-CD28 activation. With this protocol it is also possible to analyze the gene and molecular expression of gangliosides in a low number of cells in suspension, with acquisition of high quality images6, considering the small size of lymphocytes (9 &#181;m).

<table border="0" cellpadding="0" cellspacing="0" width="1052">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Advanced RPMI 1640&#160;</td>
			<td style="width:241px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">12633-012</td>
			<td style="width:193px;">Suplemented with 3% FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD3 antibody</td>
			<td>eBioscience</td>
			<td>16-0037-81</td>
			<td>OKT3 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD28 antibody</td>
			<td>ebioscience</td>
			<td>16-0288-81</td>
			<td>CD28.6 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD3&#160; antibody</td>
			<td>Abcam</td>
			<td>ab11779</td>
			<td>R24 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD2 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53831</td>
			<td>14G2a clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">FITC-conjugated anti-mouse IgG3 
			antibody</td>
			<td>Abcam</td>
			<td>ab97259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 333258</td>
			<td>Sigma-Aldrich</td>
			<td>861405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Ficoll Paque-Plus</td>
			<td>GE</td>
			<td>17-1440-02&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naive CD4+ T Cell Isolation Kit II, human</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Sorting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-tek chamber slide system</td>
			<td>Sigma-Aldrich</td>
			<td>C7182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV 1000 Laser Confocal Microscope (Olympus)&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Upright BX61WI and IX81&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GD3 synthase</td>
			<td>5&#180;-GAGCGTTCAGGAAACAAATGG- 3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GD3 synthase</td>
			<td>5&#180;-CCTGTGGGAAGAGAGAGTAAG-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-CAACACAGCAGACACAGTCC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-GTGGCAATCGTGACTAGAGC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
	</tbody>
</table>

preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

The protocol described in this manuscript renders a good quality of cultured and adhered resting and activated human na&#239;ve CD4+ T cells (Figure 1). The activated CD4+ T cells show the characteristic proliferative profile (Figure 1B) in comparison to the resting condition (Figure 1A). The CD25 late activation marker is useful to evaluate the efficient activation at 72 hr observed by confocal microscopy (F...

Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

The overall goal of this protocol is to demonstrate the preparation of a CD4+T cell suspension for staining to visualize GD3 and GD2 ganglioside localization on the cell surface using a low number of cells. This method visualization of membrane glycolipids in a low number of small suspension cells, as is the case of mononuclear cells. The main advantage of this technique is that you can evaluate ganglioside expression and localization in low amounts of CD4+T cells by optimizing sample use.The implications of this technique could extend toward research and diagnosis of some types of T cell leukemia. As we have found, some leukemia cell lines upregulate GD3 and GD2 ganglioside expression. Begin by diluting two milliliters of peripheral blood from healthy human donors with two milliliters of sterile PBS EDTA.Slowly dispense the diluted blood on top of three milliliters of 1.077 density sucrose solution. Then, centrifuge for 30 minutes at 250 times G at 21 degrees Celsius with no break to generate the gradient of peripheral blood mononuclear cells. After centrifugateion the PBMCs can be clearly observed as a white ring.Collect the PBMCs with a pipette and transfer to a sterile tube for washing. After washing the cells with PBS EDTA, purify naive CD4+T cells by negative selection with magnetic beads using a commercially available kit. Once a population of greater than 95%purity CD4+T cells has been obtained, prepare 24 well cell culture plates by adding 250 microliters of five micrograms per milliliter anti-CD3 monoclonal antibody in PBS to each well.Incubate the plate at 37 degrees Celsius for at least two hours. Following the incubation, remove the anti-CD3 antibody dilution from the plates and wash twice using sterile 1x PBS. Then, dilute the naive CD4+T cells with supplemented advanced RPMI 1640 medium to a density of one times 10 to the sixth cells per milliliter.Next, add 500 microliters of diluted cells to the anti-CD3 coated wells of the 24 well plate. Then complete the stimulation of naive CD4+T cells in anti-CD3 coated wells by adding one microgram per milliliter anti-CD28 antibody. Incubate the resting and activated CD4+T cells for zero to 72 hours at 37 degrees Celsius and 5%carbon dioxide.After the allotted incubation time has elapsed, collect the resting and activated naive CD4+T cells from the 24 well plates. Perform a cell count and if necessary, dilute the cells with fresh supplemented medium to a concentration of one times 10 to the sixth cells per milliliter. Seed 500 microliters of diluted cells into the wells of a 24 well plate containing poly-L-lysine coated cover slips or a slide chamber and incubate at 37 degrees Celsius and 5%carbon dioxide for a minimum of six hours.Following the incubation, the CD4+T cells will be adhered to the cover slips. Carefully remove the medium from the wells and then slowly add 500 microliters of sterile PBS. PBS addition during washing should be done very carefully, especially with slide chambers with less than 50, 000 cells per well are being prepared.Aspirate the PBS and add another 500 microliters of PBS to the wells to thoroughly remove the culture medium. After removing all of the PBS, add 500 microliters of filtered 4%paraformaldehyde and incubate for 30 minutes at room temperature to fix the cells. Following fixation, remove the fixative and wash at least twice with PBS.If desired, the plate can be stored with 500 microliters of PBS in each well for several days at four degrees Celsius. First, remove the PBS from the fixed cells and add 500 microliters of cold blocking buffer. Incubate for 20 minutes on ice.During incubation, it is critical not to vigorously shake the plate, as shaking will take off the cells. Carefully aspirate the blocking buffer and add blocking buffer containing 2.5 micrograms per milliliter of one of the primary antibodies or PE conjugated anti-CD25, APC conjugated anti-TCR, or an isotype control. Incubate for two hours on ice and protect from light or overnight at four degrees Celsius.Following the incubation, carefully remove the antibody solution and slowly add 500 microliters of PBS. Then add the appropriate secondary antibodies diluted in blocking buffer to 0.1 micrograms per milliliter. Protect from light and incubate for one hour on ice while taking care to avoid shaking.After washing three times in PBS as before, add Hoecht stain diluted in PBS to 0.1 micrograms per milliliter and incubate protected from light for 15 minutes at room temperature. Then place the washed slides on a paper towel and add 20 microliters of mounting solution or commercial mounting solution to prevent fading and quenching of fluorescence. Finally, pick up a cover slip with fine tweezers, turn it face down and place it on top of the mounting solution so that the side of the cover slip where cells are attached is in contact with the solution.Seal the cover slips with nail polish and analyze using fluorescence or confocal microscopy. This image shows bright field visualization of fixed CD4+T cells 72 hours after activation. Validation of efficient activation after staining is assessed using anti-CD25 antibody shown in red.The GD3 ganglioside is localized intracellularly and on the cell surface of activated CD4+T cells, as assessed by staining with anti-GD3 antibody shown in green. GD3, again shown in green, does not co-localize with the TCR, shown again in red, as assessed with anti-GD3 and anti-TCR antibody staining. However, the GD2 ganglioside does co-localize with the TCR and CD4+T cells, as assessed by staining with anti-GD2, shown in green, and anti-TCR antibody staining, shown in red.The GD3 ganglioside staining shows the intracellular and cell surface localization in a subset of activated PBMCs. While the GD2 ganglioside staining shows intracellular and cell surface localization in all activated PBMCs. Following this procedure of adherence and manipulation of the CD4+T cells, other methods like intracellular compartment staining can be performed in order to answer additional questions like the specific intracellular localization or processing of gangliosides.After watching this video, you should have a good understanding of how to handle and adhere a low number of CD4+T cells to obtain high quality fluorescent microscopy images to analyze ganglioside expression.

preparation-cd4-t-cells-for-analysis-gd3-gd2-ganglioside-membrane

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Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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

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

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

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

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...