Name: isolation and transplantation of different aged murine thymic grafts.
Uploaded: 2015-05-13T14:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

This work was supported by funding received from the National Institutes of Health (1R01AI083269).

The murine studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina Chapel Hill and all animal care was in accordance with the IACUC guidelines.
1. Preparation of Newborn and Adult Thymi
<ol>
	<li>Prepare all reagents and equipment prior to euthanizing donor mice.</li>
	<li>Sterilize surgical instruments by autoclaving or other appropriate methods. All surgical procedures must be performed under a laminar flow hood to maintain sterile conditions and avoid contamination. Assemble the tools needed for extraction of thymi from donor mice.</li>
	<li>Fill a 60 mm dish with sterile 1x PBS (pH 7.4) and place on ice inside the hood. This will be used to briefly store thymi excised from donor mice prior to transplantation.</li>
	<li>As per ethical guidelines, euthanize newborn donor mice by decapitation and adult donor mice by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>For removal of the thymus: Lay the mouse in a dorsal recumbent position on a sterile absorbent paper towel and spray with 70% ethanol prior to making an incision.</li>
	<li>Expose the abdominal and thoracic cavity by making a midline incision through the skin. Fold the skin over the chest and forelimbs to reveal the thoracic cavity.</li>
	<li>Make two lateral incisions through the diaphragm and ribcage to expose the superior mediastinum and anterior thoracic cavity. The thymus should be visible as two white lobes immediately above and adjacent to the heart.</li>
	<li>Tease apart the connective tissue surrounding the thymus with fine forceps, making certain not to disrupt the capsule. While holding back the ribcage with forceps, use a second pair of forceps to extract the two lobes of the thymus by positioning curved forceps underneath the organ and pulling vertically. This can be done using a dissecting scope for extracting thymi from newborn mice.</li>
	<li>Place the thymus in the 60 mm dish containing sterile 1x PBS (pH 7.4) on ice and separate thymic lobes by cutting through the connective isthmus. Remove any debris from the thymic lobes making certain not to damage the capsule, and cut the thymus into the appropriate number of sections for transplantation.</li>
	<li>Do not manipulate the thymi obtained from newborn mice. 
	NOTE: Used for a maximum of two recipient mice (1 lobe per recipient).</li>
	<li>Transplant the thymi obtained from adult mice into a maximum of 4-6 recipient mice. Using a pair of forceps, carefully grasp one adult thymic lobe, as shown in Figure 1, cut the thymus into three equal sections using surgical scissors.</li>
	<li>Repeat steps 1.3-1.4 for each donor mouse. In order to limit the time thymi are exposed, prepare 1 donor thymus at a time.</li>
</ol>
2. Thymus Implantation Under the Kidney Capsule
<ol>
	<li>Assemble the pre-sterilized equipment listed in Table 1. Proper aseptic technique should be utilized during the course of the procedure to prevent exposing transplant-recipients to contaminated tools or reagents.</li>
	<li>Prior to transplantation, weigh and tag each recipient mouse.</li>
	<li>Set up the dissecting microscope and anesthesia circuit in the laminar flow hood.</li>
	<li>Using an electric razor, shave the left side of the recipient mouse and ensure no hair remains around the area used to make the incision.</li>
	<li>Turn on isofluorane vaporizer and anesthetize the mouse using a dose of between 1-2%. Ascertain proper anesthetization prior to starting the surgical procedure by checking for an absence of reflex following toe pinch.</li>
	<li>After the mouse is properly anesthetized, apply veterinary ointment to the eyes of the mouse to prevent dryness and prepare the mouse for transplantation as follows:</li>
	<li>Position the mouse under the dissecting microscope in a right lateral recumbent position so that the shaved side is facing up. Starting in the center of the surgical area, dispense in a circular motion using a disposable transfer pipette 70% ethanol, followed by povidone-iodine (betadine). Repeat the ethanol/betadine treatment 3 times prior to making an incision.</li>
	<li>Using dissecting scissors make a 1-2 cm flank incision directly above the kidney.</li>
	<li>Using medium forceps grasp connective tissue adjacent to the kidney and gently lift the kidney so that it lies atop the musculature. To keep the kidney exposed and in place over the musculature, insert one arm of medium forceps underneath the kidney, paying special attention not to disrupt the renal ilium.</li>
	<li>In order to prevent tissue desiccation, irrigate the kidney with sterile 1x PBS (pH 7.4) during each step until manipulation of the kidney and kidney capsule is complete.</li>
	<li>Using fine forceps pinch and lift the capsule near the edge of the kidney most distal to the adrenal glands to separate it from the kidney.</li>
	<li>Use an 18 gauge needle to make an incision large enough to insert the prepared piece of donor thymus (Figure 2A). Make certain to keep the capsular incision as small as possible to prevent dislocation of the graft over time.</li>
	<li>While keeping the capsule pulled away from the kidney, use a second pair of fine forceps to insert the graft underneath the capsule and push the thymus as far forward from the capsular incision as possible (Figure 2B).</li>
	<li>Remove the forceps from underneath the kidney, and gently return the kidney back into place through the incision.</li>
	<li>Close the musculature by suturing the peritoneal wall and applying betadine once closed. After suturing is complete, apply wound clips to close the dermis and apply betadine to the surrounding area.</li>
	<li>Return the mouse to a cage that has been warmed using a heat lamp.</li>
	<li>Monitor each post-operative mouse until it regains full consciousness and mobility and do not place mice that have not recovered following anesthesia into a cage with other animals.</li>
	<li>Treat mice with post-operative pain management drugs as needed. Provide mice with post-operative analgesics such as acetaminophen in the drinking water at a concentration of 1.6 mg/ml.</li>
	<li>Repeat steps 2.3-2.9 for each recipient mouse.</li>
	<li>Ensure that mice return to normal activity within 1 hr post-surgery. Monitor post-operative weight, motor skills, as well as the drinking and feeding activity of each mouse to determine complications arising as a result of the transplant procedure. 
	NOTE: Post-operative weight loss, when compared to pre-operative weight, as well as altered mobility or failure to eat or drink may indicate complications.</li>
	<li>Monitor T cell reconstitution in peripheral blood by bleeding mice via tail nick followed by separation of lymphocytes using Lympholyte cell separation media according to the manufacturer&#8217;s specifications. Accomplish T cell characterization by staining lymphocytes with&#160;fluorescently conjugated antibodies specific for&#160;T cell markers CD3, CD4, and CD8, as well as a live dead discriminator such as a fluorescently activated succinimidyl ester8. Analysis as shown in Figure 3 can be accomplished via gating on singlets, live cells, and CD3 positive cells.</li>
</ol>

<ol>
	<li>Kappler, J. W., Roehm, N., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+tolerance+by+clonal+elimination+in+the+thymus.">T cell tolerance by clonal elimination in the thymus.</a> Cell. 49 (2), 273-280 (1987).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat. Rev. Immunol. 6, 127-135 (2006).</li><li>Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+selection+of+the+T+cell+repertoire:+what+thymocytes+see+(and+don&#8217;t+see).">Positive and negative selection of the T cell repertoire: what thymocytes see (and don&#8217;t see).</a> Nat. Rev. Immunol. 14 (6), 377-391 (2014).</li><li>Schwartz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+immunologic+self-tolerance.">Acquisition of immunologic self-tolerance.</a> Cell. 57 (7), 1073-1081 (1989).</li><li>Kishimoto, H., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+central+tolerance+in+NOD+mice.">A defect in central tolerance in NOD mice.</a> Nat. Immunol. 2 (11), 1025-1031 (2001).</li><li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+central+tolerance+with+reaggregate+thymus+organ+cultures.">Investigating central tolerance with reaggregate thymus organ cultures.</a> Methods. Mol. Biol. 380, 185-196 (2007).</li><li>He, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+development+of+autoreactive+T+cells+in+NOD+mice+is+regulated+in+an+age-dependent+manner.">Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.</a> J. Immunol. 191 (12), 5858-5866 (2013).</li><li>Stewart, C. C., Stewart, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.+Immunophenotyping.">Chapter 6. Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Rice, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+transplantation+for+complete+DiGeorge+syndrome:+medical+and+surgical+considerations.">Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations.</a> J. Pediatr. Surg. 39 (11), 1607-1615 (2004).</li><li>Markert, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+thymus+tissue+in+complete+DiGeorge+Syndrome.">Transplantation of thymus tissue in complete DiGeorge Syndrome.</a> N. Engl. J. Med. 341, 1180-1189 (1999).</li><li>Market, M. L., Devlin, B. H., McCarthy, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation.">Thymus transplantation.</a> Clin. Immunol. 135 (2), 236-246 (2010).</li><li>Wells, A. D., Li, X., Strom, T. B., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+peripheral+T-cell+depletion+in+transplantation+tolerance.">The role of peripheral T-cell depletion in transplantation tolerance.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (1409), 617-623 (2001).</li><li>Albuquerque, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+FOXN1-Deficiency+is+associated+with+ab+double-negative+and+FoxP3++T-cell+expansions+that+are+distinctly+modulated+upon+thymic+transplantation.">Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.</a> PLoS One. 7 (5), e37042 (2012).</li><li>Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation+restores+the+repertoires+of+forkhead+box+protein+3+(FoxP3)++and+FoxP3-+T+cells+in+complete+DiGeorge+anomaly.">Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.</a> Clin Exp Immunol. 173 (1), 140-149 (2013).</li><li>Savino, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thymus+is+a+common+target+organ+in+infectious+diseases.">The thymus is a common target organ in infectious diseases.</a> PLoS Pathog. 2 (6), e62 (2006).</li></ol>

The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge syndrome, characterized by thymic hypoplasia or thymic aplasia9-10. Thymus transplantation, like most organ transplantation, is not free of complications, which are mostly due to transplant tolerance-related complications12 and development of autoimmune pathologies11. These issues, coupled with the need to better understand and characterize mechanisms involved in thymocyte tolerance induction led to the development of this method. The protocol described herein experimentally models thymic transplantation and demonstrates that efficient T cell reconstitution is obtained in recipients.
There are several key points to note in the protocol. It is necessary to be adept at surgery and aseptic technique prior to performing the transplantation surgery. Adherence to this will help minimize trauma, the time the mouse is anesthetized, and recovery time so that 100% post-surgery survival rate can be readily obtained. When preparing the donor thymus, it is imperative that the organ is kept on wet ice to minimize cell death. In addition the thymic lobes need to be cut to appropriate sized fragments to permit implantation without tearing the kidney capsule. Similarly, care is required to prevent any injury to the kidney upon making the incision of the renal capsule. Placement of the thymic graft underneath the kidney capsule is another important step in the process. Injury to the kidney or tearing of the renal capsule can be avoided with gentle handling, using appropriate fine-tipped surgical tools, and sufficient practice. One possibility to note is that successful T cell engraftment may also be achieved by implanting thymic lobes at sites other than under the kidney capsule, for instance at a subcutaneous site. The kidney however provides an ideal environment to receive thymic grafts due to a high level of vascularization, which promotes both graft survival as well as efficient T cell trafficking following thymic selection.
A distinct advantage of the above thymus transplant protocol is the technical ease in determining T cell engraftment. Using flow cytometry, success of the procedure is qualitatively and quantitatively assessed by measuring the frequency of T cells in peripheral blood. In addition to observing mature T cells in the blood, T cell engraftment is detected in various lymphoid organs. The latter permits further study of the expansion, activation, and differentiation of peripheral T cells in various host genetic environments under physiological conditions. One caveat that investigators may consider when interpreting results is that following thymic engraftment into an immunodeficient recipient, newly selected T cells will undergo lymphopenic expansion in the periphery. Although this rapid expansion will not alter thymic selection events, it may lead to preferential expansion of distinct T cell subsets in peripheral tissues. 
This protocol is widely applicable for studying mechanisms regulating T cell development and homeostasis in a variety of pathologies. For example, this approach can be used to study FoxP3 regulatory T cell generation and development7,13-14, as well as T cell renewal and export from the thymus in in vivo models of persistent viral, fungal, or bacterial infection15. In summary, this article describes a method of thymus transplantation that entails minimal manipulation of donor tissue, and a high success rate once the technique has been mastered.

The thymus is an organ in which critical events in T cell development occur1. Resident thymocytes, upon rearrangement of the T cell receptor (TCR) &#945; and &#946; genes, undergo a series of interactions with lymphostromal and antigen presenting cells (APC) in the cortical and medullary regions of the thymus2. Thymic positive selection is mediated by cortical thymic epithelial cells (TEC) to produce thymocytes that recognize antigenic peptides in the context of host major histocompatibility complex (MHC) class I and II molecules2-3. Subsequent thymic negative selection entails purging of autoreactive thymocytes, driven by an interaction with medullary TEC or dendritic cells (DC) that present peptides derived from self-proteins bound by MHC class I and II molecules3. The end result of these processes is the establishment of a pool of mature CD4+ and CD8+ T cells able to respond to a broad spectrum of foreign antigens while exhibiting minimal reactivity to self-proteins4.
The efficiency of thymic selection events is influenced by a host of factors, including thymic maturation, frequency of medullary and cortical TEC, subset composition of thymic DC, and the source of thymic precursors3. Notably, aberrant thymic selection can result in autoimmune5 or immunodeficient pathologies, which arise from impaired negative or positive selection, respectively. The molecular events regulating thymic selection, however, are poorly understood. In vitro approaches such as reaggregate thymic organ cultures (RTOC)6, have proven to be useful for analyzing basic events associated with thymic selection, but fail to fully recapitulate the dynamics of ongoing in vivo events. As a result, this thymic transplantation-based approach was established to better study thymocyte selection events in vivo7.
This protocol describes transplanting thymi from newborn and adult donor mice into immunodeficient scid recipient mice. This technique permits the study of mechanisms that regulate positive and negative thymic selection, as well as thymic output of various T cell subsets during ontogeny. Most recently, this approach has been used to demonstrate that the efficiency of thymic selection is limited early after birth in mice leading to increased development of autoreactive T cells, and a reduced T cell repertoire specific for foreign antigens7.

<table><tbody><tr><td>Ethanol&amp;nbsp;</td><td>Decon Laboratories</td><td>2705HC</td><td></td></tr><tr><td>PBS</td><td>GIBCO</td><td>14190-144</td><td>pH 7.4</td></tr><tr><td>Betadine Solution</td><td>Purdue Products</td><td>67618-150-17</td><td></td></tr><tr><td>18 gauge needle</td><td>BD</td><td>305195</td><td></td></tr><tr><td>Sutures (1.0 metric)</td><td>Ethicon</td><td>J493G</td><td>18&quot; (45cm)</td></tr><tr><td>AUTOCLIP Wound Clips (9mm)</td><td>Clay Adams&amp;reg; Brand</td><td>427631</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher Scientific</td><td>13-711-20</td><td>Sterile, disposable</td></tr><tr><td>Mouse anti-CD3 Ab</td><td>eBioscience</td><td>11-0031-85</td><td>Clone: 1452C11</td></tr><tr><td>Mouse anti-CD4 Ab</td><td>eBioscience</td><td>48-0042-82</td><td>Clone: RM4-5</td></tr><tr><td>Mouse anti-CD8 Ab</td><td>eBioscience</td><td>25-0081-82</td><td>Clone: 53-6.7</td></tr><tr><td>Lancet</td><td>Medipoint</td><td>Goldenrod 4mm</td><td></td></tr><tr><td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td><td>Invitrogen</td><td>P30253</td><td></td></tr><tr><td>96-well round botton polypropylene plates</td><td>Corning</td><td>3365</td><td></td></tr><tr><td>1.2 ml polypropylene cluster tubes</td><td>Corning</td><td>4401</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD</td><td>352002</td><td></td></tr><tr><td>40uM Nylon Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>16% Paraformaldehyde Solution, EM Grade</td><td>Electron Microscopy Services</td><td>15710</td><td>Hazardous</td></tr><tr><td>Puralube Optical Ointment</td><td>Fisher Scientific</td><td>NC0138063</td><td></td></tr><tr><td>Lympholyte</td><td>Cedarlane</td><td>CL5030</td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Corning</td><td>430196</td><td>60 mm x 15 mm, Sterile</td></tr></tbody></table>

isolation and transplantation of different aged murine thymic grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

The success of this procedure is dependent on minimal surgical trauma as well as the accurate positioning of the graft underneath the kidney capsule. The thymic graft should be cut to ensure appropriately sized thymic sections for subsequent transplantation as shown in Figure 1. Following the schematic in Figure 1, thymi from newborn or adult mice can be used for successful subcapsular transplantation with consistent and reproducible T cell engraftment results. As mentioned, appropriate positioning of the graft under the kidney capsule is important in maintaining long-term survival, function, and vascularization of the graft. As seen in Figure 2, the thymic graft should be placed atop the kidney, closest to the adrenal glands (anterior of kidney), on the opposite end from the capsular incision (posterior of kidney). A successful thymic transplant, when coupled with an appropriate hematopoietic environment, can remain productive in excess of 30 weeks7.
Following transplantation, engraftment is assessed by flow cytometric analyses of T cells obtained from peripheral blood. Figure 3 and Table 2 demonstrate the typical level of T cell engraftment observed in organs and peripheral blood in a transplant recipient 6 weeks post-transplantation. Using this technique, our group has previously shown the kinetics of CD4+ and CD8+ T cell reconstitution in peripheral blood over time in transplant recipients of newborn and adult thymi. Briefly, circulating T cells can be observed in the periphery within one week post-surgery and CD4+ and CD8+ T cell numbers continue to increase until the peripheral T cell compartment is fully reconstituted at 5-6 weeks post transplantation7.
<img alt="Figure 1" src="/files/ftp_upload/52709/52709fig1.jpg" /> 
Figure 1: Preparation of thymic lobes and sections from donor newborn and adult mice. Donor thymi are excised from 1 day-old (N.B.) and 4 week-old (Adult). Dashed lines are presented as a guide for preparing the maximum number of sections from one donor organ for subsequent transplantation.
<img alt="Figure 2" src="/files/ftp_upload/52709/52709fig2.jpg" /> 
Figure 2: Position of capsular incision and placement of thymic graft. An appropriately sized section prepared from the donor thymus is inserted into the subcapsular space created by a small incision in the renal capsule at the posterior end of the kidney (A). Anterior to the kidney, closest to the adrenal glands (not pictured) and at the opposite end from the capsular incision, is the optimal position for the graft that is inserted underneath the capsule as shown in (B).
<img alt="Figure 3" src="/files/ftp_upload/52709/52709fig3.jpg" /> 
Figure 3. Determining successful engraftment via T cell reconstitution in peripheral blood and lymphoid organs. Flow cytometric data are representative of T cell reconstitution observed 6 weeks post-transplantation in a scid recipient of a N.B. thymic graft. Similar levels of T cell engraftment are observed in peripheral blood, spleen, pancreatic lymph node (PLN), and the mesenteric lymph node (MLN) following successful transplantation of a thymic graft.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fine Dissecting forceps</td>
		</tr>
		<tr>
			<td>Medium Dissecting forceps (2)</td>
		</tr>
		<tr>
			<td>Fine dissecting forceps (curved tip)</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
		</tr>
		<tr>
			<td>70% Ethanol&#160;</td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
		</tr>
		<tr>
			<td>Dissecting microscope w/light source</td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes (2)</td>
		</tr>
		<tr>
			<td>Drugs for pain management: Acetaminophen</td>
		</tr>
		<tr>
			<td>Sterile wipes&#160;</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
		</tr>
		<tr>
			<td>Electric razor</td>
		</tr>
		<tr>
			<td>Isoflurane and isoflurane vaporizer</td>
		</tr>
		<tr>
			<td>Sutures (fast absorbing, plain gut)</td>
		</tr>
		<tr>
			<td>Dissecting Scissors&#160;</td>
		</tr>
		<tr>
			<td>9 mm Stainless Steel Wound clips</td>
		</tr>
		<tr>
			<td>Wound clip removal tool</td>
		</tr>
		<tr>
			<td>Sterile 1x PBS</td>
		</tr>
		<tr>
			<td>60 mm Dish</td>
		</tr>
	</tbody>
</table>
Table 1: Materials needed during surgical thymic transplantation. List of necessary materials used to implant the thymus underneath the kidney capsule of the recipient mouse. The quantity of specified materials is listed in parenthesis.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Total Number of CD4 T cells</td>
			<td>Total Number of CD8 T cells</td>
		</tr>
		<tr>
			<td>Blood</td>
			<td>1,652 cells/&#965;l</td>
			<td>351 cells/&#965;l</td>
		</tr>
		<tr>
			<td>Spleen</td>
			<td>11,546,617</td>
			<td>4,203,299</td>
		</tr>
		<tr>
			<td>PLN</td>
			<td>1,241,789</td>
			<td>364,918</td>
		</tr>
		<tr>
			<td>MLN</td>
			<td>2,182,266</td>
			<td>532,059</td>
		</tr>
	</tbody>
</table>
Table 2. Total number of T cells present in peripheral blood and lymphoid organs of a thymic transplant recipient. Total numbers of CD4+ and CD8+ T cells recovered from the organs as well as peripheral blood of a newborn thymic transplant recipient 6 weeks post-transplantation. These numbers are representative of CD4+ and CD8+ T cell cellularity that is typically observed in successful newborn and adult thymic transplant recipients.

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.7K Views.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

Video: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Developmental Biology

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Medicine

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video