Name: transplantation of tail skin to study allogeneic cd4 t cell responses in mice
Uploaded: 2014-07-25T13:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

In this video publication and protocol, all animal procedures were conducted in accordance to the animal protocol approved by the Cantonal Authority Basel-Stadt, Switzerland. Perform all procedures in sterile conditions where possible.
1. Preparation of Surgery
<ol>
	<li>Autoclave all surgical instruments and gauze before use.</li>
	<li>Heat the warm pad and organize surgical instruments on the table (Table of Materials/Methods).</li>
	<li>Open the finger strip bandages. Apply petroleum jelly on the wound pad with a cotton swab (make sure the gauze is completely covered).</li>
	<li>Weigh the mice and administer analgesia Buprenorphine 0.05 mg/kg body weight. Note: this will provide analgesia immediately after transplantation.</li>
</ol>
2. Preparation of the Tail-skin for Transplantation
<ol>
	<li>Euthanize donor mice (e.g., I-Abm12) per CO2 asphyxiation and disinfect the tails with disinfection solution before excision.</li>
	<li>Position the tail with the black stripe down on a clean plastic board and cut it longitudinally throughout the middle line, making a superficial incision involving only the skin layer.</li>
	<li>Pull off the skin from the tail using gripping forceps.</li>
	<li>Put the tail-skin in a 10 cm cell culture dish filled with 10 ml HBSS.</li>
</ol>
3. Surgical Procedure
<ol>
	<li>Anesthetize recipient mice (e.g., C57BL/6) by inhalation of a 3% isofluorane solution in a transparent induction box. Note: It takes 3-5 min for the mouse to reach surgical plane anesthesia. Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Place mouse on the warm pad and apply 1.5% isofluorane via a mouth mask and asses each animal reflex by toe pinching.</li>
	<li>Shave the dorsal site of the recipient mice (e.g., C57BL/6) and remove contaminating hairs using a dry gauze swab.</li>
	<li>Disinfect shaved transplantation site with aseptic solution.</li>
	<li>Pinch the skin with forceps in the middle/right site of the dorsum and use curved scissors to make a round incision of about 1 x 0.6 cm.</li>
	<li>If bleeding occurs, then clean the wound with a sterile cotton swab.</li>
	<li>Evaluate the size of the incision and cut a transplant of the estimated size from the tail-skin using the scalpel.</li>
	<li>Trim corners to round the transplant.</li>
	<li>Load the transplant on the scalpel, dry excess of HBSS on a sterile cotton swab and place it in the graft bed of the recipient mouse.</li>
	<li>Avoid overlapping of donor and host skin and apply tissue adhesive on the contact zone exclusively.</li>
	<li>Apply a plaster without creases around the mouse waist.</li>
	<li>Apply adhesive non-elastic bandage to fix the plaster to the mouse (3 mm shifted).</li>
	<li>Incise with the scissors about 3 mm in the upper ventral site of the bandage. Note: This is to avoid trapping of the mouse&#8217;s teeth during the recovery phase.</li>
	<li>Remove the anesthetic-mouth-mask and keep the transplanted mice on the heating pad until awake.</li>
	<li>Return transplanted mice to their cages only when fully recovered and assess their mobility to ensure that the bandage is not too tight.</li>
	<li>Sterilize all surgical instruments with 70% ethanol before proceeding with next surgery.</li>
</ol>
4. Post-operative Care
<ol>
	<li>Add paracetamol-containing syrup to the drinking water (4 mg/ml) and administer it for 7 days.</li>
	<li>Return mouse to the animal room once it has recovered.</li>
	<li>Closely monitor mice for clinical signs including depression or other behavioral changes (eating or immobility), abnormal appearance (lack of grooming) or posture (pilo-erection or hunched posture) for the time the bandage is in place (1 week).</li>
</ol>
5. Bandage Removal (6-7 Days Post-transplantation)
<ol>
	<li>Anesthetize mice by inhalation of a 3% Isofluorane solution in a transparent induction box (see above).</li>
	<li>Cut bandage and plaster with artery scissors and carefully remove them. Note: avoid stretching of the transplant site.</li>
	<li>Monitor the transplanted mice for clinical signs (see 4.3) and score for rejection of skin grafts (Table 1). Note: The graft score system takes advantage of the different hair textures on the tail-skin compared to the dorsal-skin. The appearance of the transplant and its rejection is categorized in 3 grades. Allogeneic skin (MHC II mismatched, e.g., I-Abm12) grafts of C57BL/6 immunocompetent mice are usually rejected within 12-15 days post transplantation (own data and 15-20).</li>
</ol>

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H., Singer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+prolongation+of+MHC+class+II+disparate+skin+allografts+by+in+vivo+administration+of+anti-IFN-gamma+monoclonal+antibody.">Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody.</a> Journal of immunology. 144 (12), 4648-4650 (1950).</li><li>Goes, N., Sims, T., Urmson, J., Vincent, D., Ramassar, V., Halloran, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+MHC+regulation+in+the+IFN-gamma+knockout+mouse.+Evidence+for+three+states+of+MHC+expression+with+distinct+roles+for+IFN-gamma.">Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma.</a> Journal of immunology. 155 (10), 4559-4566 (1995).</li><li>Surquin, M., 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=IL-4+deficiency+prevents+eosinophilic+rejection+and+uncovers+a+role+for+neutrophils+in+the+rejection+of+MHC+class+II+disparate+skin+grafts.">IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts.</a> Transplantation. 80 (10), 1485-1492 (2005).</li><li>Gaylo, A. E., Laux, K. S., Batzel, E. J., Berg, M. E., Field, K. 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The authors declare that they have no competing financial interests.

Skin transplantation is a widely used method for studying rejection and tolerance dependent on T cells. Since the skin transplantation model was established, several adaptations and changes have been applied. In the described procedure, I-Abm12 tail-skin transplantation is performed using anesthetic gas (isofluorane). The use of gas anesthesia decreases the time of execution and mice recovery, which diminishes the stress on transplanted mice. The procedure employs tissue adhesive to fix the tail-skin, which tends to curl more than ear or dorsal-skin tissue. Moreover it is important to flatten the tail-skin before cutting and trimming the edges as this reduces curling and improves positioning of the tail-skin on the dorsal excision site.
To assess graft rejection or acceptance it is required to prepare the tail-skin with the black stripe (e.g., in C57BL/6 mice) in the middle of the graft to facilitate scoring of transplants based on hair texture differences.
The mutation of the MHC Class II I-Abm12 molecule exclusively activates CD4+ T cells in immunocompetent and immunodeficient mice (of the same genetic background). On the one hand this facilitates the interpretation of the results, but on the other hand it limits the method to CD4+ T cells studies. To extend T cell activation to other T cell subsets, tail-skin from other donors, e.g., same genetic background including I-Abm1 mice and different genetic background including Balb/c or DBA mice can be transplanted.
In the acute rejection studies, we benefit from the naturally occurring mutation in the MHC class II groove and the activation of allogeneic CD4+ T cells to study organ rejection in mice. C57BL/6 mice rejected I-Abm12 skin grafts within 14 days after transplantation, which is consistent with most studies using I-Abm12 skin transplants15-20. In addition the acute rejection model is widely used to test the allogeneic potential of CD4+ T cells from gene-deficient mice. Indeed, IFN-&#947;-/- mice showed delayed I-Abm12 skin graft rejection (unpublished data and16). Blocking IFN-&#947; by administration of blocking antibody similarly delayed rejection15,17,18. This indicates a key role of IFN-&#947; in initiating MHC class II expression on Ag-presenting and endothelial cells (rev. in 12). Since perforin-deficient mice did not delay I-Abm12 skin graft rejection21, the role of cytokines seems to be more important for rejection. Indeed, IL-6 administration resulted in prolonged graft survival, possibly by downregulating IFN-&#947;-production15. In addition, neutrophils seem to play a major role in the rejection of I-Abm12 skin grafts in IL-4-deficient mice19 indicating a fine-tuning of innate and adaptive immune cell derived cytokines. This hypothesis is strengthened by the observation that ABT-100 blocks cytokine secretion and delays I-Abm12 graft rejection in mice20. Administration of anti-CD3 antibody delays I-Abm12 skin graft rejection in immunocompetent mice (unpublished data). Moreover, we showed that T cell deficient CD3&#949;-/- and Rag2-/- mice accept the allogeneic skin grafts for up to 100 days. Tolerance in these mice can be easily reverted/broken by adoptive transfer of CD4+ T cells from immunocompetent C57BL/6 mice. Transfer of 2 x 104 CD4+ T cells or antigen-specific ABM cells is sufficient to induce I-Abm12 graft rejection around day 14. Isolated V&#945;2V&#946;8-positive ABM cells showed extensive proliferation and IFN-&#947; production in the lymph nodes before rejection, however rejection was dependent on I-Abm12 expression in the skin graft.
In summary, this mouse model is an excellent way to study activation of low T cell numbers during rejection or tolerance. The tail-skin transplantation is easy to perform and can be applied to mice with different genetic backgrounds to study the allogeneic responses of T cells without incurring a severe physiological effect. Moreover, this model is ideal for interventions with chemical and biological substances that induce or break skin graft tolerance.

Transplantation of solid organs such as skin, heart and kidneys is now a standard procedure in medical practice worldwide1. Successfully transplanted organs can be rejected by activation of the recipient immune system, which recognizes the major histocompatibility antigens of the donor. Therefore transplanted patients need treatment with immunosuppressive drugs2. Allogeneic skin transplantation in mice was established by Medawar and colleagues in 1955 and was helpful for identifying the targeted molecules later described as major histocompatibility complex (MHC) class I and II. Since then, the skin transplantation model has been continuously modified and adapted to study the role of T cell subsets and the relevance of chemical and biological intervention in suppressing graft rejection2-4. Skin from the ear and trunk are more difficult to prepare and are more susceptible to hypoxia and necrosis than tail-skin5; however, the transplantation procedure is similar. In addition the monitoring of tail-skin transplants is easy due to the characteristic hair texture of the skin.
This article provides a detailed procedure for MHC class II mismatch tail-skin transplantation that allows for the study of different aspects of CD4+ T cell-mediated allograft rejection and tolerance in mice. The natural three-point mutation in the MHC class II molecule I-Ab (called I-Abm12)6-9 is sufficient to induce rejection of skin allografts in C57BL/6 mice8. The I-Abm12 molecule activates CD4+ T cells with various &#945;&#946;-T cell receptor (TcR) chains from C57BL/6 mice, among which V&#945;2V&#946;8-TcR-specific T cells were identified in order to generate a TcR-transgenic mouse10. The adoptive transfer of V&#945;2V&#946;8-TcR-specific T cells has been used to establish a rejection model in immunodeficient C57BL/6 Rag2-/- mice transplanted with I-Abm12 skin.
Genetic differences between donor and recipient impact the outcome of transplant acceptance and rejection. There are different types of transplants: autografts are transplants from the recipient individual itself; syngrafts and allografts are transplants from genetically identical and genetically unrelated individuals respectively. Acceptance of different allogeneic organ transplants has been demonstrated by chemical and biological intervention in patients and mouse models11,3,4. In a basic approach, anti-CD3 antibody-treated C57BL/6 mice showed prolonged survival of I-Abm12 tail-skin (unpublished data). Depletion of CD4+ and CD8+ T cells before transplantation in recipient mice resulted in acceptance of MHC class I and II mismatched grafts (rev. in 12). Interestingly, rejection of skin grafts depends on the presence of CD4+ T cells (rev. in 12). In this model, targeting specific interactions between different immune cells by blocking costimulatory molecules with antibodies or suppression with regulatory T cells might induce tolerance (unpublished data). Indeed, blocking both CD40 and CD28 led to long-term skin allograft tolerance13,14.
Tail-skin transplantation is easy to perform and easy to monitor compared to transplantation of other organs. In addition, tail-skin transplants are easy to prepare and are less susceptible to ischemia than other skin tissues. In contrast to injected anesthetics, the use of anesthetic gas (isofluorane) during transplantation shortens both the procedure and recipient recovery time. Curling of the tail-skin transplant, which may lead to incomplete wound healing and graft rejection, is prevented by application of tissue adhesive. Furthermore, the I-Abm12 tail-skin transplantation model exclusively activates CD4+ T cells in both immunocompetent and immunodeficient mice (of the same genetic background) facilitating the interpretation of the results.
This protocol describes a reliable, reproducible and easily monitored mouse model that allows for chemical and biological intervention. The model is intended for investigating rejection and tolerance induction of tail-skin transplants.

<table><tbody><tr><td>Betadine standard solution</td><td>Mundipharma</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Carl Roth GmbH</td><td>31025060</td><td></td></tr><tr><td>Dafalgan , UPSA</td><td>Bristol Myers Squibb UPSA</td><td></td><td></td></tr><tr><td>Hansaplast finger strips</td><td>Beierdorf AG</td><td>REF.76861</td><td></td></tr><tr><td>Histoacryl tissue adhesive</td><td>Braun</td><td>REF.1050052</td><td></td></tr><tr><td>Leukotape classic , 2 cm x 10 m</td><td>BSN Medical SAS</td><td>REF.02204-00</td><td></td></tr><tr><td>PBS, Phosphate Buffered Saline, pH 7.4</td><td>Invitrogen</td><td>10010015</td><td>GIBCO</td></tr><tr><td>Sterile gauze, 5 x 5 cm, 8 ply</td><td>MaiMed GmbH</td><td>21010</td><td></td></tr><tr><td>Narrow pattern forceps</td><td>FST</td><td>11003-12</td><td></td></tr><tr><td>Fine iris scissor curved</td><td>FST</td><td>14095-11</td><td></td></tr><tr><td>Fine iris scissor</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Mayo scissors</td><td>FST</td><td>14010-15</td><td></td></tr><tr><td>Artery scissors ball tip 11.5 cm</td><td>FST</td><td>14080-11</td><td></td></tr><tr><td>Tissue forceps</td><td>FST</td><td>11021-14</td><td></td></tr><tr><td>Surgical blade No. 20</td><td>Swann-Morton LTD</td><td>3006</td><td>Carbon steel</td></tr><tr><td>Surgical blade handles</td><td>Swann-Morton LTD</td><td></td><td></td></tr><tr><td>Syringe, 1 ml</td><td>ARTSANA</td><td></td><td>Disposable</td></tr><tr><td>Temgesic, buprenorphine</td><td>ESSEX Chemie AG</td><td></td><td>0.3 mg/ml</td></tr><tr><td>Tissue Culture dishes 10 cm, 60.1 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>TPP</td><td></td><td></td></tr><tr><td>Vaseline</td><td>Vifor SA</td><td></td><td></td></tr><tr><td>Warm pad</td><td>Solis</td><td></td><td>Type 223</td></tr></tbody></table>

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.

In a first approach C57BL/6 mice were transplanted with I-Abm12 allografts and I-Ab syngrafts. After bandage removal, grafts manifest signs of wound healing without closure of the contact zone in C57BL/6 mice (Figure 1A). After bandage removal, CD4+ T cell-mediated inflammation led to the appearance of necrotic areas (red spots) and rejection of I-Abm12 allografts in C57BL/6 mice within 13 days after transplantation (Figure 1B-C, filled symbols), while syngrafts were tolerated for up to 100 days (Figure 1B-C, open symbols). These data show the progression of acute rejection of I-Abm12 grafts in immunocompetent C57BL/6 mice.
Various studies demonstrate that rejection of MHC class II mismatched tail-skin depends on the presence of allogeneic CD4+ T cells (rev. in12 ). Indeed, T cell deficient CD3&#949;-/- mice and T- and B-cells deficient Rag2-/- mice showed complete wound healing and tolerated the I-Abm12 grafts up to 100 days (Figure 2), whereas C57BL/6 mice rejected grafts within 13 days (Figure 2). The acceptance of I-Abm12 mismatched tail-skin allows for the study of allogeneic CD4+ T cells and their effector functions in a mouse model without severe disease pathologies.
To break the established tolerance in CD3&#949;-/- and Rag2-/- mice, CD4+ T cells were adoptively transferred: 2 x 104 polyclonal C57BL/6 CD4+ T cells were sufficient to induce rejection of I-Abm12 grafts within 14 days (data not shown). Transfer of 50,000 and 100,000 C57BL/6 CD4+ T cells did not accelerate the graft rejection process in CD3&#949;-/- and Rag2-/- mice. These data underline the role of allogeneic CD4+ T cells in the acute skin graft rejection model.
The advantage of this tail-skin transplantation model is that functional analysis of CD4+ T cells can be easily done by adoptive transfer of antigen-specific CD4+ T cells. The transgenic ABM mouse9 is considered an ideal tool to delineate the activation and the phenotypic behavior of the population without the influence of non-specific CD4+ T cells. The transfer of 2 x 104 antigen-specific ABM cells was sufficient to induce rejection of I-Abm12 grafts in Rag2-/- mice within 13 days (Figure 3A-B). In addition, ABM cells are easy to isolate and can be tracked using the expression of the V&#945;2V&#946;8-TcR chains (Figure 3C). ABM cell proliferation and production of cytokines in I-Abm12 transplanted Rag2-/- mice were investigated. ABM cells proliferated extensively as determined by eFluor670 dilution assay (Figure 3D) and up to 30% of the ABM cells produced IFN-&#947; upon PMA-stimulation (Figure 3E).
Taken together, these data show that relatively few allogeneic CD4+ T cells are required to induce rejection of I-Abm12 skin grafts, rendering this model ideal for the analysis of allogeneic T cells with minimal severe pathology.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51724/51724fig1highres.jpg" width="500" /> 
Figure 1. Transplantation of mice with allogeneic and syngeneic tail-skin. (A) C57BL/6 mice were transplanted with allogeneic (I-Abm12) and syngeneic (I-Ab) tail-skin (n = 5 per group). Pictures illustrate allografts (left) and syngrafts (right) after bandage removal on day 7. (B) Scoring and (C) survival of skin grafts from day 7 until day of rejection or final monitoring (day 100). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51724/51724fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/51724/51724fig2highres.jpg" width="400" /> 
Figure 2. Allografts are tolerated in the absence of T cells in mice. Survival of Rag2-/-- I-Abm12- mouse skin grafts transplanted in C57BL/6, CD3&#949;-/- and Rag2-/- mice (n = 4-6 per group) and illustration of rejected and accepted grafts. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51724/51724fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51724/51724fig3highres.jpg" width="500" /> 
Figure 3. Anti-I-Abm12 specific CD4+ T cells proliferate and produce IFN-&#947; during graft rejection. (A) Scoring and (B) survival of I-Abm12 tail-skin grafts in Rag2-/- mice (n = 5) adoptively transferred with 2 x 104 antigen-specific ABM cells. (C) V&#945;2V&#946;8-staining of ABM cells isolated from lymph nodes of transgenic ABM mice. (D) eFluor670 dilution of proliferating ABM cells and (E) IFN-&#947; production of PMA-stimulated ABM cells isolated from lymph nodes of I-Abm12 skin grafted Rag2-/- mice on day 9 after adoptive ABM cell transfer (n = 5 mice). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51724/51724fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:84px;">Graft Score</td>
			<td style="width:485px;">Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3+</td>
			<td>No red areas and presence of a shiny black stripe&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2+</td>
			<td>Appearance of small red areas, loss of a shiny black stripe and dryness&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1+</td>
			<td>Appearance of large red areas, no black stripe and shrinkage&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0</td>
			<td>Graft rejection defined as 80% necrosis&#160;</td>
		</tr>
	</tbody>
</table>
Table 1. Graft score system. The appearance of the transplant and its rejection is categorized in 3 grades, taking advantage of the different hair textures on the tail- and dorsal-skin.

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

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

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.4K Views.  University of Basel and University Hospital Basel. 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.

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

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Biology

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 ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Cancer Research

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.

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.

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

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 ...

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 ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Medicine

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

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

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

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

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

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.
The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.
In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Rag&#947;c mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10504/10504fig1.jpg" /> 
Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Rag&#947;c mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10504/10504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Summary

Explore More Videos

Chapters in this video

Murine Skin Transplantation

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

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8⁺ T Cells in Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Murine Full-thickness Skin Transplantation

Orthotopic Hind Limb Transplantation in the Mouse

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

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

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS