measuring alloreactivity in a mixed population of t cells

Measuring Alloreactivity in a Mixed Population of T Cells

Begin by seeding 0.5 times 10 to the sixth dendritic cells into each well of a 96-well cell culture plate, followed by 1 times 10 to the sixth T cells in a final volume of up to 50 microliters total of culture medium per well. Incubate the cold cultures for four hours at 37 degrees Celsius and 5% carbon dioxide.
Then, add three times the culture volume of 1.5% formaldehyde in PBS to each well for 30 minutes at room temperature. At the end of the fixation, transfer the cells from each well into a corresponding 5-milliliter polystyrene tube, and stain in the cells with the appropriate fluorochrome-conjugated antibodies in 100 microliters of wash buffer for 30 minutes at room temperature, protected from light.
Next, wash the cells in 1 milliliter of wash buffer and resuspend the pellets in perm wash buffer containing phalloidin-FITC. After another 30 minutes at room temperature protected from light, wash the cells in 1 milliliter of fresh perm wash buffer. Resuspend the pellets in the nuclear dye of interest in perm wash buffer for a final 30-minute incubation at room temperature protected from light, and wash the cells in 1 milliliter of perm wash buffer, followed by one wash in wash buffer alone. Then, resuspend the cells in 50 to 100 microliters of fresh wash buffer, and transfer the cells into small-capped microcentrifuge tubes.
To analyze the cell-to-cell interactions, first, reserve one channel for brightfield image acquisition, and load the first control tube. With the brightfield channel turned off, in the Workspace window, create a new scatterplot for each channel used with the aspect ratio over the area, and gate the singlets where the aspect ratio is close to one.
For each fluorochrome, check the positive population, and adjust the laser voltage in the illumination box as necessary. In the Channels box, select the appropriate channels for the fluorochromes used in the staining. Then, click Record. When the number of events reaches the specified threshold, the acquisition will stop automatically.
After all of the single stain controls have been acquired, load the first experimental sample tube, and acquire several tens of thousands of events under the appropriate analysis channels, including the brightfield channel.

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1. Prepare Reagents and Materials Required
<ol>
	<li>Prepare phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) (&#34;wash buffer&#34;). Prepare PBS with 2% fetal calf serum (FCS) containing 0.1% nonionic detergent (&#34;perm-wash buffer&#34;; see the Table of Materials). Prepare PBS with 1.5% formaldehyde. 
	NOTE: Formaldehyde is corrosive and potentially carcinogenic and must be handled while wearing appropriate personal protective equipment.</li>
	<li>Prepare PBS containing 2% FBS and 0.5 mM ethylenediaminetetraacetic acid (EDTA) for magnetic cell separation (&#34;MCS buffer&#34;).</li>
	<li>Prepare 50 &#181;g/mL phalloidin-fluorescein isothiocyanate (Phalloidin-FITC) in dimethyl sulfoxide (DMSO). Prepare a 1 mg/mL nuclear stain (e.g.&#160;1 mg/mL 7-amino actinomycin D (7-AAD) in DMSO or bis-benzimide dye; see the Table of Materials) in DMSO. Prepare fluorochrome-labeled antibodies appropriate for the cells of interest and the imaging flow cytometer.</li>
	<li>Obtain animal tissues (e.g.,&#160;lymph nodes and spleen) as a source of T cells and allogeneic animal tissues (e.g.,&#160;spleen and bone marrow) as a source of antigen-presenting cells or progenitors.</li>
	<li>Prepare cell culture medium (e.g.,&#160;Roswell Park Memorial Institute medium (RPMI) 1640 or Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FBS), 50 &#181;M 2-mercaptoethanol, penicillin, and streptomycin and obtain 24- and/or 96-well cell culture plates.</li>
</ol>
2. Prepare Antigen-presenting Cells (APCs)
NOTE:&#160;In theory, any APC population could be examined with this method. Immature mouse bone marrow-derived dendritic cells (DCs) as APCs were sued in this case. Many protocols exist for generating these cells. Briefly, the following protocol was used.
<ol>
	<li>Flush marrow from femurs and tibias into RPMI 1640 or DMEM.</li>
	<li>Pass the suspended cells through a 70-&#181;m cell strainer to remove small pieces of bone and debris.</li>
	<li>Pellet the cells by centrifugation and then lyse red cells using an ammonium chloride buffer for 5 min at room temperature.</li>
	<li>Pellet the cells by centrifugation (400 x g, 5 min) and resuspend the cell pellet.</li>
	<li>Wash cells in 5 - 10 mL of wash buffer, pellet by centrifugation (400 x g, 5 min), and re-suspend the cell pellet.</li>
	<li>Enrich hematopoietic precursors over a cell separation column by labeling the cells with biotinylated anti-CD3 (5 &#181;g/mL), anti-B220 (5 &#181;g/mL), anti-MHC class II (1 &#181;g/mL), and anti-CD11b (5 &#181;g/mL) antibodies.</li>
	<li>Pellet the cells by centrifugation (400 x g, 5 min) and re-suspend the cell pellet.</li>
	<li>Incubate the cells with anti-biotin magnetic microbeads (see the Table of Materials) at 4 &#176;C for 10 min.</li>
	<li>Wash and pellet the cells (400 x g, 5 min) and re-suspend them in 1 mL of MCS buffer before removing the labeled cells using a large positive selection (LS) magnetic cell separation column primed with 3 mL of MCS buffer and placed in its magnet. Wash the column 3 times with 3 mL of MCS buffer; the flow-through will contain the desired cells.</li>
	<li>Culture the cells that pass through the column for 6 days in RPMI 1640 or DMEM supplemented with 2 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and 2 ng/mL of recombinant human transforming growth factor &#946;1 (TGF&#946;1). Replace half the medium every 2 days with a fresh complete medium containing 2 ng/mL GM-CSF and TGF&#946;1.&#160; 
	NOTE:&#160;Human TGF&#946;1 has activity in mouse cells. Data have been generated using these immature DCs. Other cells (e.g.,&#160;B cells and mature DCs) may be suitable as APCs but have not been tested in this assay.</li>
	<li>Cryopreserve DCs in 90% serum/10% DMSO and store in liquid nitrogen; recover on the day of use. Prior to use, count the number of viable DCs in a hemocytometer using trypan blue exclusion. Re-suspend the cell pellet in culture medium at the appropriate density (see step 4.1) prior to use in section 4.</li>
</ol>
3. Prepare T Cells
<ol>
	<li>Use negative selection methods to avoid inadvertently transmitting activating or inhibitory signals to the cells. 
	NOTE:&#160;In this example, CD4+&#160;T cells are prepared for analysis.
	<ol>
		<li>To prepare CD4+&#160;T cells from the mouse spleen, mash the spleen through a 70-&#181;m cell strainer using the plunger of a syringe. Wash the cell strainer with wash buffer.</li>
		<li>Pellet the suspended cells by centrifugation (400 x g, 5 min) and then lyse the red cells by re-suspending the pellet in an ammonium chloride buffer for 5 min at room temperature.</li>
		<li>Pellet the cells by centrifugation (400 x g, 5 min) and re-suspend the pellet.</li>
		<li>Wash the cells in 5 - 10 mL of wash buffer and pellet by centrifugation (400 x g, 5 min). Re-suspend the pellet.</li>
		<li>Stain the cells with biotinylated antibodies to CD8, major histocompatibility complex class II (MHC II, 1 &#181;g/mL), and CD19 (5 &#181;g/mL). Incubate for 10 min at 4 &#176;C.</li>
		<li>Wash the cells in 10 mL of wash buffer and pellet by centrifugation (400 x g, 5 min). Re-suspend the pellet.</li>
		<li>Incubate the cells with anti-biotin magnetic microbeads (see the Table of Materials) according to the manufacturer&#39;s directions.</li>
		<li>Wash the cells in 10 mL of wash buffer and pellet by centrifugation (400 x g, 5 min); re-suspend the pellet.</li>
		<li>Resuspend the cells in 1 mL of MCS buffer and enrich the CD4+&#160;T cells over a magnetic cell separation column primed with 3 mL of MCS buffer on a magnet. Wash the column 3 times with 3 mL of MCS buffer. Column flow-through will contain the enriched T cells.</li>
	</ol>
	</li>
	<li>By standard flow cytometry, assess T cell purity using an aliquot of the negatively selected cells. Stain the cells using a fluorochrome-streptavidin conjugate (to identify any biotin-labeled cells that should have been removed on the column) and an antibody or antibodies to identify the T-cell population of interest (CD4 in this case); a purity of &#8805;85% is acceptable.
	<ol>
		<li>Count the T cells in a hemocytometer by trypan blue exclusion (&#8805;90% viability is acceptable). 
		NOTE:&#160;MCS buffer contains EDTA, which must be removed prior to the assay. To accomplish this, pellet the cells by centrifugation (400 x g, 5 min) and wash them in 1 mL of wash buffer. Pellet the cells again (400 x g, 5 min) and re-suspend in culture medium at the appropriate density (see step 4.1).</li>
	</ol>
	</li>
</ol>
4. Co-incubate T Cells and DCs
<ol>
	<li>Seed T cells and DCs at a 2:1 T: DC ratio in a 24-well or 96-well cell culture plate. Ensure that the final culture volume is &#8804;500 &#181;L for 24-well plates or &#8804;50 &#181;L for 96-well plates.&#160;&#160; 
	NOTE:&#160;Smaller volumes encourage cell-cell interactions and allow space for subsequent fixation buffers.
	<ol>
		<li>Adjust precise cell numbers empirically, but as a general guide, use 1 x 106&#160;T cells and 0.5 x 106&#160;DCs per well (96-well plate). These should be considered the minimum numbers of cells because using fewer cells makes the enumeration of immune synapses difficult.
		<ol>
			<li>To increase cell numbers, set up replicate wells and pool&#160;after&#160;step 5 (fixation). 
			NOTE:&#160;When setting up replicate wells, it is advisable to seed DCs in all of the wells first and then seed T cells in all of the wells; this minimizes discrepancies in incubation time between wells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubate the plate for 4 h at 37 &#176;C in a 5% CO2&#160;atmosphere.</li>
</ol>
5. Fix Cells in Plate
<ol>
	<li>Add 3 times the culture volume of 1.5% formaldehyde in PBS to each well and incubate at room temperature for 30 min; it is important to fix cells prior to removing them from the plate to minimize the disruption of cell-cell interactions.</li>
	<li>Transfer cells in the plate into tubes for subsequent washing and staining. At this stage, set aside additional cells for single-stain controls. Apart from these controls, the entire culture should be stained with the antibody cocktail (see step 4.1.1).</li>
</ol>
6. Stain Cells
<ol>
	<li>Stain cells in 100 &#181;L of wash buffer containing a cocktail of the desired fluorochrome-conjugated antibodies for 30 min at room temperature, protected from light. 
	NOTE:&#160;The cocktail includes T cell-specific and APC-specific antibodies. Fluorochromes should be chosen such that they can be distinguished using the configuration of the imaging flow cytometer. In the experiments shown here, blue fluorophore-conjugated CD11b (5 &#181;g/mL, see the Table of Materials) and APC-conjugated CD90.2 (5 &#181;g/mL) were used.</li>
	<li>Wash the cells in 1 mL of wash buffer and centrifuge for 5 min at 400 x g.&#160;Decant the supernatant. Resuspend the cells in perm-wash buffer containing phalloidin FITC at 0.05-0.5 &#181;g/mL and incubate for 30 min at room temperature protected from light.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;Phalloidin FITC concentrations of about 0.1 &#181;g/mL work well for mouse cells, but the appropriate concentration is expected to vary by supplier, cell type, and imaging flow cytometer.</li>
	<li>Wash the cells in 1 mL of perm/wash buffer and centrifuge for 5 min at 400 x g. Decant the supernatant. Resuspend the cells in perm/wash buffer containing nuclear dye at the appropriate concentration (e.g.,&#160;approximately 25 &#181;g/mL 7-AAD) and incubate for 30 min at room temperature protected from light.</li>
	<li>Wash the cells in 1 mL of perm/wash buffer and centrifuge for 5 min at 400 x g. Decant the supernatant. Wash the cells once in wash buffer, pellet, and re-suspend in 50 - 100 &#181;L of wash buffer, transferring the cells to small, capped microcentrifuge tubes.</li>
	<li>Proceed to data acquisition immediately or store the cells at 4 &#176;C protected from light for up to several days prior to acquisition on the imaging flow cytometer. 
	NOTE:&#160;Cells have been successfully stored in this way for up to 7 days. Longer storage may be possible but has not been tested.</li>
</ol>
7. Acquire Data
<ol>
	<li>Initialize and configure the imaging flow cytometer according to the manufacturer&#39;s instructions. Ensure stability of the flow core prior to collecting any data.</li>
	<li>Reserve one channel for brightfield image acquisition. Acquire single-stain control data with the brightfield channel turned off.
	<ol>
		<li>Click the &#34;Load&#34; button and insert a tube containing a fully stained sample (a sample that has been stained with all required fluorochromes) into the holder.</li>
		<li>In the &#34;workspace&#34; window, select and create a new scatterplot with the aspect ratio over the area and gate singlets where the aspect ratio is close to 1. Create a new scatterplot for each channel used (intensity of the channel in the horizontal axis).</li>
		<li>For each fluorochrome, check the positive population and, if required, adjust the laser voltage in the &#34;Illumination&#34; box.</li>
		<li>Unload the tube and load the first single-stained tube.</li>
		<li>In the &#34;Acquisition Settings&#34; box, type the sample name and set the number of events that should be collected; if it is a single stain (for compensation controls), 1,000 - 2,000 events are sufficient.</li>
		<li>In the &#34;Channels&#34; box, select the channels that each sample has been stained with. For single-stain controls, all channels should be selected with brightfield and side scatter off. Click the &#34;Record&#34; button under the &#34;Acquisition&#34; box; when the number of events reaches the specified threshold, the acquisition will stop automatically.</li>
		<li>Click the &#34;Return&#34; button to unload the tube. Repeat steps 7.2.4 - 7.2.6 for each single-stain control. Depending on the cytometer and software, it may not be possible to set all of the gates shown in&#160;Figure 1&#160;during acquisition (more precise gating is performed during analysis; see section 8).</li>
	</ol>
	</li>
	<li>Acquire samples as for single-stained samples (in step 7.2), but in the &#34;Channels&#34; box, select all channels that are required, including the brightfield channel.
	<ol>
		<li>Before recording data, check to confirm that the channel intensity is appropriate for identifying the desired cell populations. If not, adjust the laser settings and re-record single-stain controls using the new settings, as described in step 7.2.</li>
		<li>For each sample, acquire several tens of thousands of events. 
		NOTE:&#160;Under most conditions, cell-cell contact events are a small minority of the total cell number (most are single cells). In general, it is desirable to have at least 100 events in the final membrane contact gate.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Various</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamenetetraacetic acid, 0.5M solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 nonionic detergent</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td>Solution is 37% formaldehyde and so must be diluted 25 times for 1.5% solution</td>
		</tr>
		<tr>
			<td>Cell strainers, 70 &#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>P5282</td>
			<td></td>
		</tr>
		<tr>
			<td>7-aminoactinomycin D</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1310</td>
			<td>Reconstitute in DMSO</td>
		</tr>
		<tr>
			<td>Allophycocyanin-conjugated anti-mouse CD90.2</td>
			<td>eBioscience</td>
			<td>17-0902</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific blue-conjugated anti-mouse CD11b</td>
			<td>eBioscience</td>
			<td>48-0112</td>
			<td>Pacific blue has been replaced by eFluor 450</td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse CD3</td>
			<td>eBioscience</td>
			<td>13-0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse MHC class II</td>
			<td>eBioscience</td>
			<td>13-5321</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse B220</td>
			<td>eBioscience</td>
			<td>13-0452</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse CD8</td>
			<td>eBioscience</td>
			<td>13-0081</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse CD19</td>
			<td>eBioscience</td>
			<td>13-0193</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-biotin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS magnetic cell separator</td>
			<td>MIltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse GM-CSF</td>
			<td>Peprotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF&#946;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td>Human TGF&#946;1 has activity on mouse cells</td>
		</tr>
		<tr>
			<td>Amnis ImageStream X Mark II</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>Imaging flow cytometer; details available at http://www.emdmillipore.com/</td>
		</tr>
		<tr>
			<td>IDEAS Software</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>Free download (registration required): https://www.amnis.com/index.php/page/Display/login%20%20</td>
		</tr>
		<tr>
			<td>Cell culture medium&#160;</td>
			<td>Various</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Various</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates</td>
			<td>Various</td>
			<td>Varies</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Juvet, S. C. et al., <a href="https://app.jove.com/v/55283">Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates a method for assessing alloreactivity in mixed T cell populations by observing interactions with alloantigen-carrying dendritic cells, leading to the formation of immunological synapses. Differential staining and quantification of synapse-containing doublets via flow cytometry reveal T cell alloreactivity.

<img alt="Figure 1" src="/files/ftp_upload/21913/21913_Figure1.jpg" /> 
Figure 1. Gating Strategy Used to Identify Alloreactive Immune Synapses. A.&#160;In-focus events are gated from all events by reviewing cell images based on the root mean square of the rate of change of the image intensity profile (Gradient RMS) using the brightfield channel (Channel 4, Ch04), as described in the text.&#160;B.&#160;Among in-focus events, doublets are distinguishe...

1. Prepare Reagents and Materials Required

	Prepare phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) (&#34;wash buffer&#34;). ...

Begin with a multi-well plate containing mouse dendritic cells, or DCs carrying alloantigens or genetic variants of self-antigens.
Introduce mouse T cells, including alloreactive T cells that are non-reactive to self-antigens, while recognizing foreign antigens.
The alloreactive T cells recognize these alloantigens and interact with DCs, triggering cytoskeleton rearrangement, including F-actin reorganization.
This promotes receptors on T cells to engage with co-stimulatory molecules on DCs, forming a cellular connection &#8212; an immunological synapse.
Fix the cells, transfer them to a tube, and introduce a cocktail of fluorochrome-labeled antibodies specific to T cell and DC receptors, imparting distinct fluorescence.
Remove unbound antibodies and resuspend the cells in a buffer containing a permeabilizing agent that permeabilizes the cells and green fluorophore-tagged protein markers that bind to F-actin.
Centrifuge and resuspend the cells in a buffer containing nuclear dye, staining the nuclei red.
Using imaging flow cytometry, identify the doublets &#8212; corresponding to T cell and DC conjugates with green crosshairs of F-actin &#8212; confirming synapse formation.
Quantify the doublets representing the degree of alloreactivity.

18 次观看. 测量混合 T 细胞群中的同种异体反应性

视频: 测量混合 T 细胞群中的同种异体反应性 - 实验

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

科研

JoVE Journal

癌症研究

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

免疫与感染

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Measuring Alloreactivity in a Mixed Population of T Cells

成績單