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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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. Due to variability between batches, it is recommended that each batch be titrated to determine the optimal concentration. We use our current batch at a much lower concentration (10 nM) than in the original publication (200 nM)4.
Another important aspect of this method is optimization of the antigen dose. It has been well established that T cells cultured with less antigen can be more sensitive and exhibit enhanced function. This was originally demonstrated in the context of HIV peptides15. The amount of tetanus toxoid used in this protocol has also been optimized previously4 to elicit highly sensitive and proliferative CD4+ T cells. Interestingly, it has been shown previously that excess antigen reduces antigen-specific proliferation15. Therefore, to detect and enumerate rare T cell populations in peripheral blood, optimizing both antigen and CFSE concentration is of utmost importance.
Since we published the first human dye dilution-based assay4, many similar dyes have been developed. We found that CD4+ T cell proliferation was detected less effectively when PKH-26, a lipophilic dye, was used compared to CFSE (unpublished). We have not systematically tested other dyes on the market, so there is no comment on their relative strengths. However, the protocol for quantifying T cell proliferation is equally applicable to all dye dilution proliferation assays.
Expressing proliferation as a cell division index (CDI) incorporates the background proliferation into calculation of the magnitude of responses. In our hands, the background is usually low (e.g., &#60;15 events/5,000 CD4+ CFSEbright). Background proliferation does vary between individuals and experiments. Using a ratio (in this case, the CDI) to express the strength of proliferation is somewhat effective in reducing its impact on the results. Background responses to proteins in the media can be problematic. It is recommended to use tissue culture media that contain human serum, not bovine serum, as bovine proteins can stimulate a weak background response that diminishes sensitivity of the assay. A high background proliferation has been found in people who have a mild infection, presumably due to proliferation that is primed in vivo in response to infection. In such cases, the assay needs to be repeated once the donor has cleared the infection.
The CFSE-based proliferation assay can be modified to efficiently clone human antigen-specific CD4+ T cells directly from PBMCs12. For example, this approach has been used to clone CD4+ T cells specific for insulin12. More recently, cloning and functional analyses of proinsulin C-peptide-specific CD4+ T cells has provided remarkable insight into the pathogenesis of T1D, particularly in patients with recent onset T1D13.
In conclusion, the CFSE-based proliferation assay is both sensitive and robust. It is quick and technically simple and can be performed using fresh or cryopreserved PBMC. The ability to incorporate CFSE into multicolor FACS analysis provides a quantitative measure of the immunophenotype of proliferating T cells from PBMC containing many cell types, and this degree of detail is not possible with assays utilizing DNA incorporation such as [3H]-thymidine.

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 CFSEdim cells; Figure 2A,B,C), while there was a marked proliferation of CD4+ T cells in response to tetanus toxoid (&#62;3,000 CFSEdim cells; Figure 2D,E,F).
In vitro stimulation of human PBMCs with antigenic peptides: PBMCs were stained with CFSE and stimulated for 7 days in the presence of human proinsulin C-peptide. Figure 3 demonstrates the detection of proinsulin C-peptide-specific CD4+ T cells in the peripheral blood of an individual with type 1 diabetes. Proliferation was not observed in the healthy donor&#8217;s PBMC (data not shown). In Figure 4, PBMCs stimulated with influenza A virus (H1N1 PR8) matrix protein demonstrated proliferation; however, the tetanus-specific response for this donor was relatively weak, and responses are generally variable between donors.
Taken together, these results demonstrate that the assay can be performed using full-length proteins and short synthetic peptides.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59545/59545fig1.jpg" /> 
Figure 1: Compensation controls and gating strategy for CFSE-based proliferation of CD4+ T cells. Unstimulated PBMCs following 7 days of in vitro cell culture. Lymphocytes (A) were stained with propidium iodide, and propidium iodide-negative cells were gated to exclude dead cells (B). FACS compensation controls included unstained cells (C), cells stained with CFSE alone (D), and cells stained with CD4 alone (E). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59545/59545fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59545/59545fig2.jpg" /> 
Figure 2: Proliferation of CFSE-stained tetanus-specific CD4+ T cells. PBMCs were CFSE-stained and cultured for 7 days in the presence of 145 ng/mL (166 LfU/mL) tetanus toxoid. Cells were stained with CD4. CFSE dilution is shown without antigen (A-C) and in response to tetanus toxoid protein (D-F). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59545/59545fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59545/59545fig3.jpg" /> 
Figure 3: Proliferation of CFSE-stained human CD4+ T cells in response to proinsulin C-peptide. CFSE-stained PBMCs were cultured for 7 days in the presence of 10 &#181;M proinsulin C-peptide. Cells were stained with CD4. CFSE dilution is shown using PBMC from a donor with type 1 diabetes. CFSE dilution in unstimulated PBMCs (A), tetanus toxoid protein stimulated (B), and human proinsulin C-peptide stimulated PBMCs (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59545/59545fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59545/59545fig4.jpg" /> 
Figure 4: Proliferation of CFSE-stained human CD4+ T cells in response to influenza A virus (H1N1 PR8) matrix protein 1. PBMCs were CFSE-stained and cultured for 7 days in the presence of 0.08 &#181;g/mL influenza A virus matrix protein 1. Cells were stained with CD4. CFSE dilution is shown for unstimulated cells (A), tetanus toxoid stimulated cells (B), and influenza A virus matrix protein-stimulated cells (C). &#160;&#160;&#160;&#160; <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59545/59545fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Working Concentration</td>
		</tr>
		<tr>
			<td>Tetanus Toxoid protein</td>
			<td>145 ng/mL</td>
		</tr>
		<tr>
			<td>Influenza A H1N1 (PR8) Matrix protein 1</td>
			<td>0.08&#8211;10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Proinsulin C-peptide PI33-63</td>
			<td>10 nM</td>
		</tr>
	</tbody>
</table>
Table 1: Enumeration of tetanus-specific CD4+ proliferation. CDI calculations using 5,000 recorded flow cytometry events from unstimulated and tetanus-stimulated human PBMC.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Undivided cells CD4+CFSEbright</td>
			<td>Divided cells CD4+CFSEdim</td>
			<td>Number of divided cells CFSEdim/5,000 CFSEbright</td>
			<td>Mean divided cells CFSEdim/5,000 CFSEbright</td>
			<td></td>
		</tr>
		<tr>
			<td>Nil 1</td>
			<td>5,004</td>
			<td>11</td>
			<td>11</td>
			<td>12.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Nil 2</td>
			<td>4,995</td>
			<td>14</td>
			<td>14</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nil 3</td>
			<td>5,006</td>
			<td>12</td>
			<td>12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Undivided cells CD4+CFSEbright</td>
			<td>Divided cells CD4+CFSEdim</td>
			<td>Number of divided cells CFSEdim/ 5000 CFSEbright</td>
			<td>CDI Number of divided cells (Tetanus) Mean divided cells (Nil) </td>
			<td>Mean CDI</td>
		</tr>
		<tr>
			<td>Tetanus 1</td>
			<td>4,930</td>
			<td>3,258</td>
			<td>3,304.3</td>
			<td>268</td>
			<td>263.7</td>
		</tr>
		<tr>
			<td>Tetanus 2</td>
			<td>4,928</td>
			<td>3,205</td>
			<td>3,251.8</td>
			<td>263.7</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetanus 3</td>
			<td>4,910</td>
			<td>3,142</td>
			<td>3,199.6</td>
			<td>259.5</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Antigens used to simulate CD4+ proliferation.

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 ...

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Universidad San Sebastian

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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

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 ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Environment

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Biology

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Cancer Research

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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Advanced Biology

Immunology

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Source: Ghassemi, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59949">Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy.</a> J. Vis. Exp. (2019)
This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

Technika ex vivo do wytwarzania chimerycznych limfocytów T receptora antygenowego

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, ...

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Immunotherapy

Cellular Immunotherapy

Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

Source: Hickey, J. W., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58640">Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles.</a> J. Vis. Exp. (2018).
This video describes a protocol for isolating rare-antigen-specific CD8+ T cells using magnetic nanoparticle-based artificial antigen-presenting cells (aAPCs) functionalized with antigenic peptide-loaded MHC-Igs and anti-CD28 antibodies. Interaction of the aAPCs with CD8+ T cells specifically expressing the antigenic peptide-targeting T cell receptors, followed by their expansion in vitro, results in the enrichment and expansion of this CD8+ T cell population.

Wzbogacanie i ekspansja limfocytów T CD8+ specyficznych dla antygenu

Source: Hickey, J. W., et al. Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles. J. Vis. Exp. (2018).
This video describes a ...

Immune Systems and Components

Assays and Functional Profiling

An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells

Source: Simonetti, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61867">A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice.</a> J. Vis. Exp. (2021)
This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and a nucleic acid stain.

Test do analizy cyklu komórkowego limfocytów T CD8+ specyficznych dla antygenu

1. Preparation of medium and staining solution

	Prepare Complete Medium: Roswell Park Memorial Institute (RPMI) medium with 2 mM glutamine, 100 U/mL ...

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

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

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Technika ex vivo do wytwarzania chimerycznych limfocytów T receptora antygenowego

Wzbogacanie i ekspansja limfocytów T CD8+ specyficznych dla antygenu

Test do analizy cyklu komórkowego limfocytów T ^CD8+ specyficznych dla antygenu