This work was supported by American Gastroenterological Association (AGA) Research Scholars Award and Crohn&#8217;s and Colitis Foundation of America (CCFA) Career Development Award (to S.Z.S.), NIH NIDDK F30 DK089692 (to E.C.S.), and University of North Carolina Center for Gastrointestinal Biology and Disease Grant P30 DK34987 (Histology Core). The UNC Flow Cytometry Core Facility is supported in part by an NCI Center Core Support Grant (P30CA016086) to the UNC Lineberger Comprehensive Cancer Center. We thank Luke B. Borst from North Carolina State University College of Veterinary Medicine for his help with histopathological analysis and immunohistochemistry.

NOTE: Ensure that all animal protocols are approved by and in compliance with Institutional Animal Care and Use Committee (IACUC) regulations and the National Research Council&#8217;s Guide for the Care and Use of Laboratory Animals. Donor mice may be either male or female, but recipient mice should be male. If female recipients are to be used, the donor mice must be female5. Maintain colonies using regular, non-sterile bedding and non-acidified water, as these may impact the intestinal microbiota, and, thus, the colitis phenotype of the mice5,6.
1. Experimental Preparation
<ol>
	<li>Use ice-cold media and buffers. Keep cells on ice throughout the experiment.</li>
	<li>Perform the experiment in sterile biohazard hood using sterile technique.</li>
</ol>
2. Isolation of Splenic T cells
<ol>
	<li>Euthanize donor mouse/mice in CO2 chamber followed by cervical dislocation. Spray abdomen with 70% ethanol.</li>
	<li>Make a horizontal incision in the abdomen and peel away skin to expose peritoneum. Hold peritoneum away from the internal organs with the forceps and make an incision in the left abdominal peritoneum to expose and excise the spleen.</li>
	<li>Place the spleen in 10 ml of Complete Media in a Petri dish. Remove and discard excess tissue from spleen.</li>
	<li>Use 2 sterilized glass slides to crush and tease apart spleen into single-cell suspension. Filter cell suspension through a 70 &#956;m strainer into a 50 ml conical tube and rinse strainer with 5 ml of Complete Media. Place up to 5 spleens in one 50 ml conical tube.</li>
	<li>Centrifuge cells at 450 x g for 7 min. Discard supernatant by pouring off or by vacuum suction into waste container.</li>
	<li>Gently resuspend cells in 5 ml per spleen of Lysis Buffer to lyse red blood cells for 10 min at room temperature. Add an equal volume of Complete Media (5 ml per spleen) to the tube.</li>
	<li>Centrifuge cells at 450 x g for 7 min. Discard supernatant by pouring off or by vacuum suction into waste container.</li>
	<li>Gently resuspend cells in 10 ml of Labeling Buffer.</li>
	<li>Count cells by trypan blue exclusion.
	<ol>
		<li>Remove 20 &#956;l of cell suspension and add to 180 &#956;l trypan blue to a microfuge tube and mix thoroughly. After 5 min, add 10 &#956;l labeled cells to hemocytometer and count non-blue cells under the microscope. Determine total number of viable cells. Discard cell/trypan blue mix.</li>
	</ol>
	</li>
	<li>Centrifuge cells in 10 ml of Labeling Buffer at 450 x g for 7 min. Discard supernatant by pouring off or by vacuum suction into waste container.</li>
</ol>
3. Enrichment of CD4+ T cells
NOTE: Follow manufacturer&#8217;s instructions for specific products used in this section.
<ol>
	<li>Gently resuspend cells to a single-cell suspension of 20 x 106 cell/ml in cold Labeling Buffer.</li>
	<li>Add 5 &#956;l per 1 x 106 cells of biotinylated CD4 T cell enrichment antibodies. Incubate cells on ice for 15 min.</li>
	<li>Add 10x volume of Labeling Buffer. Centrifuge cells at 450 &#215; g for 7 min. Carefully aspirate all the supernatant using vacuum suction into waste container.</li>
	<li>Thoroughly vortex streptavidin-conjugated magnetic particles. Add 5 &#956;l of particles per 1 x 106 cells.</li>
	<li>Mix thoroughly but gently. Keep mix at 6-12 &#176;C for 30 min.</li>
	<li>Add Labeling Buffer to a concentration of 20-80 x 106 cells/ml. Transfer up to 1.0 ml labeled cells per 12 x 75 mm round-bottom test tube (referred to as the &#8220;positive-fraction tube&#8221;).</li>
	<li>Place each positive-fraction tube on magnet for 6-8 min.</li>
	<li>With the positive-fraction tubes still on the magnet, carefully transfer supernatant from positive-fraction tube with glass Pasteur pipet to a new sterile 50 ml conical tube (referred to as the &#8220;enriched fraction&#8221;). This enriched fraction contains CD4+ T cells. Be careful not to disrupt the labeled cells attracted&#160;to the magnet.</li>
	<li>Resuspend cells left in the positive-fraction tubes in the same volume of Labeling Buffer as in step 3.6 by pipetting up and down vigorously. Place positive-fraction tube back on magnet for 6-8 min.</li>
	<li>With positive-fraction tubes still on the magnet, carefully transfer supernatant (enriched fraction, CD4+) from positive-fraction tube with glass Pasteur pipet to sterile 50 ml conical tube from step 3.8 without disrupting cells attached to magnet.</li>
	<li>Repeat steps 3.9-3.10 to increase the yield of CD4+ T cells obtained. Continue protocol using enriched fraction (CD4+ cells).</li>
</ol>
4. Labeling and Sorting Cells7
<ol>
	<li>Centrifuge enriched cells at 450 x g for 7 min. Discard supernatant by pouring off or vacuum suction into waste container.</li>
	<li>Resuspend cells in 1 ml of Labeling Buffer. Remove aliquot of cells to count and to assess for cell viability by trypan blue exclusion as in step 2.9.</li>
	<li>Add a volume of Labeling Buffer to 10 x 106 cells/ml; if cells are already &#60;10 x 106, centrifuge at 450 x g for 7 min, discard supernatant by pouring off or vacuum suction into waste container, and add volume of Labeling Buffer to 10 x 106 cells/ml.
	<ol>
		<li>Set up separate aliquots of approximately 5-10 &#215; 105 cells each for unstained, isotype-stained, and single-stained control cells in microfuge tubes.</li>
	</ol>
	</li>
	<li>Add 5 &#956;g/ml CD4-FITC and 1 &#956;g/ml CD45RB-PE to cells. Add isotype control stains and single stains at the same concentration to appropriate aliquots in microfuge tubes. Mix well but gently and incubate on ice protected from light for 30 min.</li>
	<li>Add 10x volume of Labeling Buffer to cells and controls. Centrifuge 450 x g for 7 min. Discard supernatant by vacuum suction into waste container.</li>
	<li>Resuspend in Labeling Buffer to volume in step 4.5. Centrifuge 450 x g for 7 min. Discard supernatant by vacuum suction into waste container.</li>
	<li>Resuspend in Labeling Buffer to 10 x 106 cells/ml. Pass cells through 70 &#956;m strainer into FACS tube. Keep on ice protected from light until ready for FACS.</li>
	<li>Set up and determine appropriate compensation on the cell sorter with unstained cells and single-stained controls.</li>
	<li>Exclude nonviable cells with forward- and side-scatter gating (Figure 1A). Set up gating for CD4+ and CD45RB+ cells with isotype-stained controls. Gate cells on CD4+ population.</li>
	<li>Sort CD4+ cells into CD45RBhigh and CD45RBlow populations using a simple histogram for PE-stained cells into tubes with 2 ml of Complete Media. The CD45RBhigh population represents the highest 40% of CD4+CD45RB+ cells (CD45RBhigh), and the CD45RBlow population is the lowest 20% of CD4+CD45RB+ cells (CD45RBlow;&#160;Figure 1B).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> 
Figure 1: Representative flow cytometry plots of CD4+CD45RB T cell populations during FACS analysis. (A-C) FITC CD4- and PE CD45RB-stained splenocytes from donor C57BL/6 mice were sorted by FACS into CD4+CD45RBhigh and CD4+CD45RBlow T cell populations. (A) Doublet events were excluded on the forward scatter plot. (B) Lymphocytes were gated in the forward and side scatter plot. (C) CD4+ T cells were gated, and (D) CD4+CD45RB+ T cells were plotted on a PE versus Events histogram. CD4+CD45RBlow cells were considered to be the lowest 20% of CD45RB+ cells. CD4+CD45RBhigh cells were defined as the highest 40% of CD45RB+ cells.
<ol start="11">
	<li>Run an aliquot of each cell population on the FACS machine to assess purity of populations.</li>
	<li>Centrifuge sorted cells at 450 x g for 7 min. Resuspend in 1 ml PBS. Remove aliquot of cells to count and to assess for cell viability by trypan blue exclusion as in step 2.9.</li>
</ol>
5. Injection of Cells into Recipients
<ol>
	<li>Resuspend sorted cells to 4 x 106 cells/ml (CD45RBhigh) or 2 x 106 cells/ml (CD45RBlow) in PBS.</li>
	<li>Transfer 100 &#956;l of CD45RBhigh cells per recipient to new sterile tube. Add 100 &#956;l of PBS per recipient to this tube. Thus, total injection volume per animal is 200 &#956;l, and total amount of cells per recipient is 4 x 105 CD4+CD45RBhigh na&#239;ve T cells.</li>
	<li>If experimental group receiving T regulatory cells is desired, transfer 100 &#956;l of CD45RBhigh cells per recipient to new sterile tube. Add 100 &#956;l of CD45RBlow cells per recipient to the same tube. Total injection volume per animal is 200 &#956;l; ratio of CD45RBhigh:CD45RBlow cells is 2:1.</li>
	<li>Inject 100 &#956;l of CD45RBhigh or CD45RBhigh/CD45RBlow CD4+ cells intraperitoneally into the right and left side of the abdomen (total of 200 &#956;l) of each recipient.</li>
</ol>
6. Monitoring of Disease Progression in Recipient Animals
<ol>
	<li>To assess the clinical status of the recipient animals, assign aggregate clinical scores for the following parameters8 on the day of injection, weekly thereafter, and at time of sacrifice:
	<ol>
		<li>Determine wasting by measuring weight loss: 0 &#8211; no weight loss; 1 &#8211; 0.1-10% loss of initial body weight; 2 &#8211; more than 10% loss of initial body weight (Figure 2A).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> 
Figure 2: Clinical and gross pathological signs of inflammation occur after transfer of wild type CD4+CD45RBhigh T cells into Rag1-/- and NRDKO recipient mice11. (A) NRDKO recipients lost on average 10% of their initial body weights by 5 weeks post-transfer, whereas Rag1-/- recipients did not exhibit clinical signs of disease at this time. Each point represents the mean percentage of initial body weight for the cohort &#177; SEM. **, p &#60;0.005. (B) Some mice developed severe intestinal inflammation, as demonstrated by the presence of rectal prolapse. This is a representative picture of rectal prolapse in a NRDKO recipient mouse. (C) Grossly, colons from both Rag1-/- and NRDKO recipient mice are thickened and shortened compared to colons from Rag1-/- and NRDKO mice without T cell adoptive transfer. Colons from NRDKO recipient mice show severe inflammation and increased colon weights (data not shown). <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>
	<ol start="2">
		<li>Determine quality of stool by placing animal in clean container until it has passed stool: 0 &#8211; none; 1 &#8211; soft stool; 2 &#8211; watery and/or bloody stool.</li>
		<li>Determine general health of animal by presence of the following signs of disease: 0 &#8211; no hunched posture, bristled fur, or skin lesions; 1 &#8211; any one of the following present: hunched posture, bristled fur, or skin lesions.</li>
		<li>Determine presence of rectal prolapse: 0 &#8211; absent; 1 &#8211; present (Figure 2B).</li>
	</ol>
	</li>
	<li>Sacrifice animals by CO2 inhalation followed by cervical dislocation when they have lost 20% of their starting body weight or at desired time point, whichever comes first. Clinical disease is typically apparent starting at week 5 post-repletion.</li>
	<li>Assess for disease severity as previously described5,7.
	<ol>
		<li>Assign clinical scores as in step 6.18.</li>
		<li>Measure colon length and weight (Figure 2C).</li>
		<li>Perform histological analysis of inflammation5.</li>
		<li>Determine spontaneous cytokine expression in intestinal tissue explant cultures9, mesenteric lymph nodes10, and/or serum11.
		<ol>
			<li>Briefly, for explant cultures9, remove colons after sacrifice, open longitudinally, and clean with PBS. Incubate colons on an orbital shaker set at 250 rpm in Complete Media for 30 min at room temperature.</li>
			<li>Chop tissue into small pieces and incubated at 37 &#176;C in Complete Media for 24 hr. Collect the supernatant and use for quantitation of cytokines per 100 mg tissue by ELISA.</li>
		</ol>
		</li>
		<li>Perform ex vivo characterization of T cell phenotypes and/or function10.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Here we describe a step-by-step protocol inducing colonic inflammation in mice by adoptive transfer of CD4+CD45RB+ T cells into immunodeficient mice. We used C57BL/6 donor spleens and syngeneic Rag1-/- recipient mice, although other strains (e.g., BALB/c, 129S6/SvEv, non-obese diabetic (NOD)) and genetic models of immunodeficiency (e.g., SCID, Rag2-/-) may also be used4,14-16. It is well established that background strain affects e...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

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

Approximately 10 x 106 CD4+CD45RBhigh T cells from 10 spleens from adult C57BL/6 donor mice are reliably isolated. This number will vary depending on the age and strain of the donor mouse and the proficiency of the researcher. When 4 x 105 C57BL/6 CD4+CD45RBhigh T cells are transferred into C57BL/6 Rag1-/- recipient mice, clinical signs of disease emerge around week 5 post-repletion or sooner if mice are genetically susceptible to more ...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

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

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

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

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

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

16.2K Views.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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

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

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

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

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...