This work was supported by: The Juvenile Diabetes Research Foundation [JDRF 5-CDA-2014-210-A-N] (S. M.). The National Health and Medical Research Council (NHMRC GNT123586) (S. M.), Diabetes Australia Research Trust Millennium Award (Y17M1-MANS) (S. M.), Operational Infrastructure Support Program of the Victorian Government (S. M., A. D., E. T., M. S.), and NHMRC Postgraduate Scholarship APP1094337 and JDRF PhD Top-up Scholarship (M. S.).

All subjects gave informed consent prior to the collection of peripheral blood. Ethical approval for experiments using PBMC was given by St. Vincent&#8217;s Hosptial (HREC-A 135/08, and HREC-A 161/15).
1. Reagent Preparation
<ol>
	<li>Human T cell media
	<ol>
		<li>Prepare RP-5 media for culturing PBMC, which consists of RPMI 1640, 1x non-essential amino acids, L-alanyl-L-glutamine dipeptide (2 mM), penicillin (100 U/mL)/streptomycin (0.1 mg/mL), and 5% pooled human serum (PHS).</li>
	</ol>
	</li>
	<li>CFSE stock solutions
	<ol>
		<li>Prepare a master stock by dissolving 25 mg of CFSE in ~9 mL of DMSO to achieve a final stock solution with a concentration of 5 mM.</li>
		<li>Prepare a working stock by diluting the master stock in PBS to achieve a working concentration of 10 &#181;M.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human PBMCs from Whole Blood
<ol>
	<li>There are generally between 0.5&#8211;1.5 x 106 PBMCs/mL of human peripheral blood. Therefore, the amount of blood required depends upon the desired number of PBMCs. Dilute human peripheral blood with PBS at least 1:2. Separate the PBMCs by adding 15 mL of density gradient medium to a 50 mL tube, then overlay 35 mL of diluted whole blood.</li>
	<li>Centrifuge at 850 x g for 15 min without deceleration at room temperature (RT). This will result in three clear layers: the bottom layer containing the red blood cell pellet, middle layer of density gradient medium with white blood cells lining its top interface, and top plasma layer14.</li>
	<li>Remove approximately 20 mL of the top plasma layer. Collect the white blood cell layer, being careful to avoid the red blood cell pellet. Wash 3x with PBS and count viable cells using trypan blue exclusion on a hemocytometer. Dilute to 1 x 106 PBMCs/mL in PBS.</li>
	<li>Non-CFSE-stained cells
	<ol>
		<li>These cells are used as compensation controls for flow cytometry, comprising of unstained and CD4+ single-stained cells. Add 300 &#181;L of each control sample PBMC suspension to a 10 mL tube, top with PBS, and centrifuge at 550 x g for 5 min at RT.</li>
		<li>Resuspend 1 x 106 cells/mL in RP-5 media. Incubate these unlabeled cells for 7 days in a 37 &#176;C/5% CO2 incubator with the CFSE-labeled cells (step 2.6.1).</li>
	</ol>
	</li>
	<li>CFSE-stained cells
	<ol>
		<li>Transfer the cells from step 2.3 into a 50 mL tube. Add 1.0 &#181;L of CFSE working stock solution (10 &#181;M) per 1 mL of cell suspension to the side of the tube. Mix quickly by inverting the tube several times. The final concentration of CFSE is 10 nM.</li>
		<li>Incubate for 5 min in a 37 &#176;C/5% CO2 incubator. To stop the staining, add 5 mL of RP-5 media, pellet the cells by centrifuging for 5 min at 550 x g. Resuspend the PBMCs at 1 x 106/mL in RP-5 media.</li>
		<li>Add 1.0 mL of cell suspension to a 10 mL tube. Use one tube for each antigen to be tested.</li>
	</ol>
	</li>
	<li>Antigenic stimulation of human PBMCs and cell culture
	<ol>
		<li>Culture human CFSE-labelled PBMCs with antigens in RP-5 media for 7 days in a 37 &#176;C/5% CO2 incubator. Culture 1 x 105 cells/well (100 &#181;L) in a 96 well plate with 100 &#181;L/well of RP-5 media containing diluted antigen. 
		NOTE: Antigens used, including working concentrations, are summarized in Table 1.</li>
	</ol>
	</li>
</ol>
3. Anti-CD4 Staining for FACS Analysis
<ol>
	<li>Pipette 200 &#181;L of the cultured cells into FACS tubes, wash cells 1x in 1.0 mL of PBS containing 0.1% FCS, and centrifuge for 5 min at 550 x g.</li>
	<li>Stain with anti-human CD4 Alexa Fluor 647 (0.25 &#956;g/mL) in 100 &#181;L of PBS/0.1% FCS. Keep aside a sample of CFSE-labelled cells, unstained with any other fluorophores, to use for setting the FACS CFSE compensation. Incubate the cells at 4 &#176;C protected from light for 20 min.</li>
	<li>Wash the cells by adding 1 mL of PBS/0.1% FCS, centrifuge at 550 x g for 5 min at RT, and resuspend in 100 &#181;L of PBS/0.1% FCS. Immediately before FACS analysis, add 1 &#181;L of propidium iodide (PI, 0.1 mg/mL) to all tubes to allow the dead cells to be excluded by flow cytometry.</li>
</ol>
4. Flow Cytometric Configuration and Gating Strategy
NOTE: Figure 1 shows the FACS configuration including compensation controls and gating strategy.
<ol>
	<li>Gate the forward scatter (FSC) vs. side scatter (SSC) (Figure 1A) population to include all lymphocytes.</li>
	<li>Gate the FSC vs. PI (Figure 1B) population on PI negative cells to exclude apoptotic cells.</li>
	<li>Use unstained cells to set a voltage baseline for non-fluorescent cells. Set the voltages for CD4-A647 and CFSE so that the fluorescence signal is below 1,000 for each (Figure 1C). Use the single color controls CFSE (Figure 1D) and CD4-A647 (Figure 1E) to confirm positive fluorescent signals (~10,000) for each color, using the voltages set with unstained cells.</li>
	<li>CFSE and PI have some spectral overlap; adjust the compensation to subtract PI fluoresence from CFSE fluoresence until the CFSE-only sample does not yield a signal in the PI channel. 
	NOTE: These gates were applied to all samples analyzed herein.</li>
</ol>
5. Calculation of Cell Division Index to Enumerate Antigen-specific CD4+ T Cell Proliferation
NOTE: Cell division index (CDI) refers to the number of divided cells (CD4+/CFSEdim) per 5,000 undivided cells (CD4+/ CFSEbright). When the number of undivided CD4+ cells is not exactly 5,000, the number of divided cells is corrected to express the number of divided cells per 5,000 undivided cells. For example, using the tetanus toxoid-specific proliferation (Figure 2D), there were 4,930 undivided cells (CFSEbright) and 3,268 divided cells (CFSEdim); therefore, the corrected number of divided cells is (5,000/4,930) x 3,268 = 3,304.3.
<ol>
	<li>Calculate the CDI (Table 1) by dividing the number of divided cells/5,000 undivided cells from the antigen-stimulated group by the mean number of divided cells (per 5,000 undivided cells) from the cells cultured without antigen (Table 2).</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> Journal of Immunological Methods. 171, 131-137 (1994).</li><li>Mannering, S. I., 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=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> Journal of Immunological Methods. 283, 173-183 (2003).</li><li>Quah, B. J. C., Parish, C. 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The authors declare no conflicts of interest.

CFSE-based proliferation is a simple and robust method for detecting and enumerating antigen-specific human CD4+ T cells. It has been previously demonstrated that using the optimal concentration of CFSE is essential for optimal results4. Too much CFSE abrogates proliferation, whereas too little does not allow for divided and undivided cells to be distinguished. In contrast, relatively high concentrations (5.0 &#181;M) of CFSE are used to label purified murine T cells3<...

The ability to detect and study antigen-specific T cells is important in studies of cell-mediated immunity. However, doing so is particularly challenging for autoantigen-specific CD4+ T-cell responses, which are very weak and difficult to detect. A common method used for the detection of antigen-specific lymphocyte proliferation is [3H]-thymidine, which is a radiolabeled nucleotide incorporated into the DNA of proliferating cells. Although the [3H]-thymidine assay can detect DNA synthesis, this method is an indirect measure of cell division, because DNA synthesis can initiate independently of mitosis (i.e., during gene duplication and apoptosis1). This issue is compounded by the fact that antigen-specific proliferation of cells can result in considerable apoptosis2, leading to potential overestimation of antigen-specific proliferation. Furthermore, the [3H]-thymidine method does not provide phenotypic information for proliferating lymphocytes, such as CD4+ or CD8+ lineage proliferation in PBMCs stimulated with antigenic peptides.
In 2003, we published the first dye dilution assay using CFSE, called the CFSE-based proliferation assay3,4. CFSE is a fluorescent dye that binds stably to intracellular proteins by forming a covalent bond to intracellular lysine residues. Since CFSE-labeled proteins are divided equally among daughter cells3, cells that have divided can be distinguished from undivided cells by flow cytometry, which also allows for the quantitative phenotyping of lymphocyte populations. Indeed, the number of divisions a cell has undergone from the time of CFSE-staining can be measured to some degree5. More recently, many similar dyes such as CellTrace Violet proliferation dye (VPD) and CytoTrack dye have been developed, which work in a similar way6. This protocol focuses on CFSE, but the principles apply equally to other related dyes.
Peptide-MHC tetramer staining is a widely used method for detecting and cloning antigen-specific CD8+ T cells. This is a well-established method7,8,9,10; however, tetramer-based cloning requires existing knowledge of the epitope/MHC restriction and each epitope requires its own tetramer11, which limits the scope of discovery and cloning of novel epitope-specific T cells. The CFSE-based proliferation can be used with peptides, proteins, or cell lysates. The protocol described herein is both simple and robust, and the responding CD4+ T cells can be sorted for use in downstream functional and biochemical characterization assays12,13.

<table>
	<tbody>
		<tr>
			<td>Anti-human CD4-AlexaFluor647</td>
			<td>Biolegend</td>
			<td>317422</td>
			<td>RRID:AB_2716180</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>ThermoFisher</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (1x)</td>
			<td>Gibco</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A H1N1 (PR8) Matrix protein 1</td>
			<td>Sino Biological</td>
			<td>40010-V07E</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential amino acids (1x)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin (1x)</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Pooled human serum</td>
			<td>Australian Red Cross</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proinsulin C-peptide PI33-63</td>
			<td>Purar Chemicals</td>
			<td>N/A</td>
			<td>Custom made synthetic peptide</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetanus Toxoid protein</td>
			<td>Statens Serum Intitut</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In vitro stimulation of human PBMCs with tetanus toxoid protein: PBMCs were stained with CFSE and stimulated for 7 days in the presence of tetanus toxoid. Almost all donors showed a strong T cell response to tetanus toxoid because they had been vaccinated, which makes tetanus toxoid a useful positive control antigen. Figure 2 demonstrates in triplicate, that the CFSE proliferation of CD4+ T cells from unstimulated PBMCs was minimal (~12 CFSEdi...

Watch this Scientific Journal Video about Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester at JoVE.com

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.

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 ...

quantification-proliferating-human-antigen-specific-cd4-t-cells-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.4K Views.  St. Vincent's Institute of Medical Research. 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.

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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&#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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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.

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

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

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Chapters in this video

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Assessing the Innate Sensing of HIV-1 Infected CD4⁺ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

In Vitro Differentiation of Human CD4⁺FOXP3⁺ Induced Regulatory T Cells (iTregs) from Naïve CD4⁺ T Cells Using a TGF-β-containing Protocol

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model