EJV is supported by a fellowship from The Crohn s &amp; Colitis Foundation of America (CCFA). JRM is supported by grants from CCFA, Cancer Research Institute (CRI), Howard H. Goodman (MGH), Massachusetts Life Science Center (MLSC) and NIH Director s New Innovator Award.

1. Ex vivo generation of gut-homing and control T cells (see Figure 1)
<ol>
	<li>Isolate splenocytes by mashing one spleen from wild type mice. Resuspend the cells suspension in PBS and centrifuge for 5' at 400 x g. Remove the supernatant and lyse the red blood cells by resuspending the cell pellet in 4 ml of ACK lysis buffer for 2-3 minutes. After that, add 5 ml of PBS. Centrifuge for 5' at 400 x g and remove the supernatant.</li>
	<li>Resuspend total splenocytes at 1 x 106/mL in IMDM + 10% FBS + 50 mM 2-mercaptoethanol + penicillin/streptomycin (complete IMDM). Separate the cells in two groups, one of which is supplemented with RA (or synthetic RAR-agonists, such as Am80 or Am580) to a final concentration of 100-200 nM 3. T cells activated in the presence of RA or RAR-agonists will upregulate a4b7and CCR9 and will become gut-tropic T cells, whereas T cells activated without RA will become control T cells.</li>
	<li>Add 1.5-2.0 mL of the cell suspension to each well of a 24-well plate previously coated with anti-CD3 (10 mg/ml) and anti-CD28 (10 mg/ml) and incubate at 37&deg;C in 5% CO2 (for coating the plate add 500 ml/well of anti-CD3 plus anti-CD28 in PBS and incubate for 2 hours at 37&#730;C and then wash twice with 2 ml PBS).</li>
	<li>In order to avoid T cell overstimulation, after 2-3 days transfer the cell suspensions into a new uncoated 24-well plate. Incubate for additional 2-3 days. Depending on cell density and proliferation the media might become acid (yellow), in which case it might be necessary to replace half of the media with fresh complete IMDM. Harvest the cells after 4-5 days (counting from day 0).</li>
	<li>Typical final yields are 1-2 x 106 effector T cells/well. Therefore, a minimum of 9-12 wells of each gut-tropic T cells and control T cells should be plated in order to end up with 10-20 x 106 T cells per condition.</li>
</ol>
2. Analysis of gut-homing receptors on activated T cells
After 4-5 days of culture we should observe a high expression of gut-homing receptors a4b7and CCR9 on T cells activated in the presence of RA or RAR agonists, whereas control T cells should express very low levels of these receptors 4.
Flow cytometry (FACS) analysis.
<ol>
	<li>Collect between 0.2-0.5 x106 cells and centrifuge for 5 min at 300 g at 4&deg;C.</li>
	<li>Incubate with anti-CD4-FITC, anti-a4b7-PE (BD Biosciences), anti-CCR9-APC (eBiosciences) and anti-CD8-PercP for 15 min at 4&deg;C in dark.</li>
	<li>After the incubation, centrifuge for 5 min at 300 g 4&deg;C. Wash and resuspend the cells in staining buffer and analyze by FACS.</li>
</ol>
3. T cell labeling with CFSE and CMTMR cell trackers
<ol>
	<li>Keep all solutions at 37&deg;C before starting. Wash gut-tropic T cells (a4b7HighCCR9High) and control T cells (a4b7Low/Neg CCR9Low/Neg) twice with PBS to remove the serum. Adjust T cell concentration to 10-15 x 106/mL in PBS.</li>
	<li>Add CFSE (Carboxyfluorescein succinimidyl ester) or CMTMR (chloromethyl-benzoyl-amino-tetramethylrhodamine) fluorophores to either gut-tropic T and control T cells to a final concentration of 5 mM and 10 mM, respectively, gently vortex and incubate for 20 min at 37&deg;C. It is recommended to swap the labelings in the same or in a separate experiment in order to exclude potential intrinsic effects of fluorophores on T cell migration.</li>
	<li>After the incubation, quench with 1 volume of FBS and incubate for 1-5 min at room temperature. Dilute 10 times with warm PBS and centrifuge for 5 min at 300 g. Wash twice with PBS and resuspend in complete IMDM.</li>
	<li>Ideally, 10-20 x 106 cells of each T population should be mixed in a 1:1 ratio and then centrifuged at 300 x g for 5 min. Finally, resuspend the cells in 200-250 mL of warm PBS and inject via the tail vein.</li>
	<li>To calculate the exact input ratio (ideally close to 1) save 5-10 mL of the injected cell suspension and dilute with 300 mL of PBS and analyze by FACS.</li>
</ol>
4. Analysis of T cell homing
Although mice can be analyzed at various time points (between 1 h and several days post T cell injection), homing of gut-tropic T cells is best documented between 12-24 h post-injection. Longer time points increase the potential effects of other variables affecting the final cell numbers/ratios, such as T cell apoptosis and T cell exit from the tissue. After the mice are euthanized, cell suspensions from several tissues should be isolated without delay in order to decrease cell death, which might affect the final yields and results. Tissues of interest are the small intestine, colon, spleen, lymph nodes, Peyer's patches, liver, lungs and blood. If the injected T cells are bona fide gut-tropic T cells, they should home on average 5-10 times better (or higher) to the small intestine mucosa as compared to control T cells. However, other tissues, such as blood and the spleen, should not exhibit a preferential T cell migration. Isolation of lymphocytes from the intestinal lamina propria and the intraepithelial compartment has been previously described 5.
<ol>
	<li>FACS staining: Samples are resuspended depending on the numbers of cells collected, at a density no higher than 3.0 x 106 cells/ml. If congenic CD45.1+ or Thy1.1+ mice were used as recipients, the cells are stained with the corresponding congenic marker (e.g., CD45.2 or Thy1.2) combined with some lineage marker (e.g., TCRbchain, CD4, CD8, CD45.1+/CD45.2+).</li>
	<li>During FACS analysis, cells are gated on the congenic marker (e.g. CD45.1) and then on the specific lineage markers (e.g. CD4/CD8) and analyzed for the ratios between CMTMR and CFSE positive cells.</li>
	<li>The data is usually expressed as the Homing Index(HI), which is calculated as the ratio CFSE/CMTMR (or CMTMR/CFSE) in each tissue divided by the corresponding input ratio (see below). If the input ratio is very close to 1, then the tissue ratios will be equivalent to the HI.</li>
</ol>
HI = CFSEtissue / CMTMRtissue : CFSEinput / CMTMRinput
Blood should also be analyzed in order to determine whether both T cell populations are equally represented in the circulation or if one T cell population is decreased respect to the other (e.g., by preferential trapping in lungs/liver or by decreased viability). This might happen when comparing na&iuml;ve or resting memory T cells versus recently activated effector T cells, since the latter might be trapped to a greater extent in the lungs. If the HI in blood is significantly different than 1, one can normalize HI in tissues by the HI in blood. Blood can be obtained via cardiac puncture from mice anesthetized (with avertin or isofluorane prior euthanasia) and should be lysed twice with ACK buffer before using it for FACS staining.
Blood normalization = HItissue / HIBlood
5. Representative Results
Recipient mice (Thy1.1+) were euthanized18h post cell injection. Cell suspensions were generated from the spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and small intestine lamina propia (LP). After that the cells were stained for Thy1.2 and CD8 and then analyzed by FACS by gating on viable Thy1.2+CD8+ cells, which were finally analyzed for the ratio between CMTMR+ cells (gut-tropic T cells) and CFSE+ cells (control T cells) divided by the input ratio. The results can be displayed as shown in Figure 2, in which it can be appreciated that gut-tropic and control T cells homed equally to the spleen (HI close to1, indicated with a red line). By contrast, gut-tropic T cells migrated around 10 times more efficiently to the LP as compared to control T cells.
<img src="/files/ftp_upload/2619/2619fig1.jpg" alt="Figure 1" /> 
Figure1: Diagram showing the generation and labeling of gut-tropic and non-gut tropic T cells. T cells are isolated from wild type mice and activated with anti-CD3/anti-CD28 and in the presence or absence of 100-200 nM all-trans retinoic acid (RA). After 4-5 days RA-treated T cell acquire the expression of gut homing receptors CCR9 and a4b7. Then, gut-tropic and control T cells are differentially labeled with CFSE or CMTMR, washed, mixed in a 1:1 ratio and injected into a recipient mouse via the tail vein. Some labeled cells are used to determine the CFSE/CMTMR input ratio (should be close to 1).
<img src="/files/ftp_upload/2619/2619fig2.jpg" alt="Figure 2" /> 
Figure2: Example of homing analysis. Mice analyzed 12-18 h post T cell injection and single cell suspensions are obtained from the organs of interest. The cells are stained with anti-Thy1.2 (congenic marker) plus anti-CD8 and then Thy1.2+CD8+ T cells are analyzed for the ratio of CMTMR+ and CFSE+ cells in each tissue by FACS. The tissue CMTMR/CFSE ratios are then normalized by the Input CMTMR/CFSE ratio to obtain the homing indices (HI). In this example CMTMR+ and CFSE+ cells are gut-tropic and control effector T cells, respectively.

<ol>
	<li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+Three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.</a> Cell. 67, 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76, 301-314 (1994).</li><li>Iwata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21, 527-538 (2004).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+and+dynamic+control+of+CD8+T+cell+homing+by+dendritic+cells+from+skin-+and+gut-associated+lymphoid+tissues.">Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues.</a> J Exp Med. 201, 303-316 (2005).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imprinting+of+gut-homing+T+cells+by+Peyer's+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.</a> Nature. 424, 88-93 (2003).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li></ol>

No conflicts of interest declared.

Even though homing experiments provide very valuable information about the migration of total cell populations in any given tissue, it should be kept in mind that these assays do not directly analyze endothelial adhesion and therefore do not discriminate in which step(s) of the multistep adhesion cascade (tethering/rolling, activation or sticking) a given homing receptor is acting. The gold standard to define the specific role of homing receptors in the adhesion cascade is intravital microscopy (IVM) in which individual ...

Animals: C57BL/6mice are commonly used for T cell isolation. In addition, CD45.1 and Thy1.1 congenic strains are available through Jackson Laboratories (Bar Harbor, ME).
Culture media: IMDM (Iscove's Modified Dulbecco's Medium + L-Glutamine + Hepes) plus 10% heat-inactivated FBS (Fetal Bovine Serum, low endotoxin, Gibco&reg;, Invitrogen, Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (HyClone Antibiotics, Waltham, MA), 0.5 mg/ml fungizone/amphotericin B (Gibco), and 50 mM b-mercaptoethanol.
ACK Red Blood Cell Lysis buffer(RBC, 10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA, pH 8.0), adjust to pH 7.2-7.4 and store at room temperature).
PBS (Phosphate Buffered Saline, Hyclone, Waltham, MA).
Flow cytometry (FACS) media(PBS or IMDM + 2% FBS + 5 mM EDTA). When staining using Selectin-Fc chimeras, media with 2 mM Ca++ should be used in all steps (including FACS acquisition). IMDM is recommended in this case.
T cell labeling and adoptive transfer: CFSE(carboxyfluorescein diacetate, succinimidyl ester), CMTMR ((5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) from Molecular Probes&reg;, Invitrogen, Carlsbad, CA). 1000 x stocks should be made in DMSO (5 mM CFSE, 20 mM CMTMR) and stored at -20&deg;C.
Polyclonal T cell activation: 24-well or 96-well plates (tissue-culture treated, polystyrene, flat-bottom with lid, BD Falcon, Franklin Lakes, NJ) are incubated for 2 hours at 37&deg;C with 50 ml PBS, respectively, containing anti-CD3 plus anti-CD28 antibodies (10 mg/mL each). Then, culture plates are washed twice with PBS and used immediately for T cell culture. Alternatively, Dynabeads coated with anti-CD3/anti-CD28 (Dynal, Invitrogen, Carlsbad, CA) can be used for T cell activation instead of plate-bound antibodies.
Flow cytometry (FACS) staining: Polyclonal activation: CD3 (1452C11), CD28 (37.51). Lineage mAb: CD4 (L3T4), CD8a (Ly-2), Thy1.2 (CD90.2/53-1.2), CD45.2 (104). Gut-homing receptors: purified CCR9 (CD199/eBioCW-1.2, eBioscience, San Diego, CA), a4b7(LPAM-1/DATK3), isotype control (IgG2a, k). Skin-homing receptors: P-selectin-Fc (Purified Mouse P-Selectin - IgG Fusion Protein, BD Pharmingen, San Jose, CA), E-selectin-Fc (Recombinant Mouse E-Selectin/Fc Chimera, R&amp;D Systems, Minneapolis, MN) plus corresponding secondary reagent goat F(ab')2 anti-human IgG R-PE (Invitrogen, Carlsbad, CA).
All-trans retinoic acid(Sigma, St. Louis, MO)is resuspended in absolute ethanol or DMSO using a yellow bulb or an indirect source of light during the preparation. Store aliquots in glass vials at -80&deg;C and protected from light at all times. Synthetic RAR-agonists: Am80(Wako Chemicals, Richmond, VA).

competitive homing assays to study gut-tropic t cell migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Watch this Scientific Journal Video about Competitive Homing Assays to Study Gut-tropic T Cell Migration at JoVE.com

Competitive Homing Assays to Study Gut-tropic T Cell Migration

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

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Immunology and Infection

12.4K 조회수.  Massachusetts General Hospital, Harvard Medical School. 경쟁 유도 실험 직접 하나의 마우스로 두 개의 서로 다른 세포 인구의 철새 속성을 평가하실 수 있습니다. 여기 우리는 전 생체내 생성 인내심 트로픽 대 비 직감 트로픽 T 세포의 마이 그 레이션을 비교하여이 절차를 설명합니다.

비디오: Competitive Homing Assays to Study Gut-tropic T Cell Migration

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...