We are indebted to Alice Gaughan and Jiao-Jing Wang whose outstanding technical support, intellectual input, and moral support were instrumental in moving these studies ahead. These studies were supported by the NIH grant AI036532 to GAH.

1. Lethal Irradiation
<ol>
 <li>Place up to 10 recipient mice into a microisolator cage compatible with the irradiator to be used.</li>
 <li>Irradiate in 2 equal doses summing total dose (total dose = 9 cGy for BALB.B recipients). Second irradiation should be 3 hr after first. Injection should occur between 4-6 hr after final irradiation. Irradiate mice in either Cs137 source or RS2,000, irradiator.</li>
 <li>Following the second radiation dose, store mice in microisolator cage with acidified water until the time of transplant.</li>
</ol>
2. Splenocyte Preparation
<ol>
 <li>Euthanize one donor mouse according to institutional guidelines. Each donor generally yields 75x106 - 125x106 splenocytes. Using CD8 purification kits, each mouse generally yields 6x106 - 12x106 CD8 T cells.</li>
 <li>Remove the spleen by first making a vertical 2 cm incision 2 cm right of the midline directly under the rib cage. Cut through the fur, skin, and visceral membrane.</li>
 <li>Remove spleen and create single cell suspension by placing spleen in 40 &mu;l mesh screen in a Petri dish with 1640 RPMI with 5% FBS. Use a syringe plunger to break apart the spleen until the entire spleen has been passed through mesh screen.</li>
 <li>Repeat this process for each WT and L2G85.B6 donor. Collect all the WT single cell suspensions in 1-50 ml conical tube, and collect all the L2G85.B6 single cell suspensions in a separate 50 ml conical tube.</li>
 <li>Centrifuge cells at 1,200 rpm for 10 min at 4 &deg;C. Resuspend pellets in 1640 RPMI with 5% FBS and count cells.</li>
</ol>
3. Bone Marrow Preparation
<ol>
 <li>Remove skin from one hind limb of a donor mouse.</li>
 <li>Carefully cut away as much muscle tissue as possible from femur and tibia/fibula.</li>
 <li>Remove hind limb by cutting femur away at the hip joint. Cut away rear paw just below tibia/fibula intersection. All bone cuts should be made using sturdy scissors.</li>
 <li>Carefully remove any remaining muscle tissue. Cut off fibula-relatively little bone marrow is found in fibula and not worth the effort.</li>
 <li>Place hind limb in cold 1640 RPMI 5% FBS and repeat process with the second hind limb. Each mouse should yield between 20x106 - 40x106 bone marrow cells.</li>
</ol>
4. Bone Marrow Removal
<ol>
 <li>Remove one hind limb from media and place in large Petri dish with a small amount of cold media (~1 ml).</li>
 <li>Cut away the knee joint. Using a syringe (volume &gt; 5 ml), insert subcutaneous needle into tibia and depress syringe until all red material is removed from interior of tibia. Repeat process with femur. Discard remaining bones devoid of bone marrow. Repeat process with second hind limb.</li>
 <li>Create single cell suspension by pipetting media containing bone marrow into large Petri dish and use syringe plunger and 40 &mu;l mesh screen. Pipette single cell suspension into 50 ml conical tube and keep on ice.</li>
</ol>
5. CD3 Depletion
There are a variety of ways to deplete CD3+ cells from the bone marrow. Our lab uses a kit made by Miltenyi Biotec (CD3-biotin - 130-093-021). Deplete CD3+ cells from the bone marrow following manufacturer's protocol. The buffer for Miltenyi kits will henceforth be called MACS Buffer (2 mM EDTA, 0.5% BSA in PBS, pH 7.2).
<ol>
 <li>Wash cells by centrifuging splenocytes and bone marrow cells at 1200 rpm for 10 min at 4 &deg;C and count cells.</li>
 <li>Remove ALL supernatant. Resuspend bone marrow cells in 100 &mu;l per 10 million bone marrow cells in MACS Buffer.</li>
 <li>Continue with CD3 depletion using manufactures' protocol.</li>
 <li>Wash CD3 depleted bone marrow cells 3 times in sterile PBS. Count CD3 depleted bone marrow cells and resuspend at an appropriate volume to inject 107 cells.</li>
</ol>
6. L2G85.B6 CD8+ T Cell Purification
There are a variety of ways to purify CD8 T cells from L2G85.B6 mice. Also, there are several ways to deplete CD8 T cells from WT splenocyte donors. Our lab uses kits from Miltenyi Biotec (CD8 depletion - 130-049-401, CD8 purification - 130-095-236).
<ol>
 <li>Resuspend the WT pellet in 90 &mu;l MACS Buffer per 107 cells. Continue with CD8 T cell depletion according to manufactures' protocol (CD8 depletion - 130-049-401).</li>
 <li>Resuspend the L2G85.B6 pellet in 40 &mu;l MACS Buffer per 107 cells. Continue with CD8 T cell purification according to manufactures' protocol (CD8 purification - 130-095-236).</li>
 <li>Count each cell population and wash each population 3 times with sterile PBS. Resuspend each population in appropriate volume to inject 18x106 WT (CD8 depleted) splenocytes and 2x106 L2G85.B6 purified CD8 T cells.</li>
</ol>
7. Injection Preparation
<ol>
 <li>Combine 107 CD3 depleted bone marrow cells, 18x106 WT (CD8 depleted) splenocytes, and 2x106 L2G85.B6 purified CD8 T cells in a microcentrifuge tube. Wash with sterile PBS and resuspend in 300 &mu;l.</li>
 <li>Inject cell preparation into the tail vein of recipient mice. Injections should be done using 28 gauge needles.</li>
 <li>Store mice in microisolator cage with acidified water. Score mice daily using scoring GVHD scoring system developed by Cooke et al1.</li>
</ol>
8. Bioluminescent Imaging
<ol>
 <li>Six days post transplant, inject recipient mice with 4 mg D-luciferin. Allow 5 min for luciferin to react with luciferase.</li>
 <li>Anesthetize mouse in isoflurane chamber of bioluminescence imager and image recipients for 5 min with small binning. This will create a high resolution image while gathering as many events as possible.</li>
 <li>Analyze data using Living Image software. The scale of the pseudo-color image can be changed to yield the best results. However, it is imperative that the same scale be used across experiments.</li>
 <li>Regions of interest can be created using Living Image software and light emittance can be quantified by calculating the flux (photons/sec) being emitted from each region of interest.</li>
</ol>
9. Representative Results
Approximately 7-10 days post transplant, mice begin showing clinical signs of GVHD. Mice appear scruffy due to lack of grooming. Recipients will also begin to lose weight between 7-10 days post transplant. Activity and posture of recipient mice will remain relatively normal until approximately day 12-14 post transplant. Cumulative GVHD scores will steadily increase through the first 2-3 weeks post transplant (Figure 1A). Disease course is quite variable between mice; however, recipients should uniformly succumb to GVHD by 30-40 days post transplant (Figure 1B).
Figure 2 shows recipient mice that were imaged 6 days post transplant. Pseudo-colored scale shows varying light emittance throughout the body with the highest light intensity being emitted in the spleen and the gut. CD8 T cell accumulation in the gut is consistent with previous findings6. Recipient mice can be placed back in microisolator cage to be imaged at a later time point or euthanized for ex vivo imaging.
<table border="1">
 <tbody>
 <tr>
 <td>Criteria</td>
 <td>Grade 0</td>
 <td>Grade 1</td>
 <td>Grade 2</td>
 </tr>
 <tr>
 <td>Weight loss (wkly.)</td>
 <td>&lt; 10%</td>
 <td>&gt; 10% - &lt; 25%</td>
 <td>&gt;25%</td>
 </tr>
 <tr>
 <td>Posture</td>
 <td>Normal</td>
 <td>Hunching only at rest</td>
 <td>Several hunching; impairs movement</td>
 </tr>
 <tr>
 <td>Activity</td>
 <td>Normal</td>
 <td>Mild to moderate decrease in activity</td>
 <td>Stationary unless stimulated</td>
 </tr>
 <tr>
 <td>Fur texture</td>
 <td>Normal</td>
 <td>Mild to moderate ruffling</td>
 <td>Severe ruffling/poor grooming</td>
 </tr>
 <tr>
 <td>Skin texture</td>
 <td>Normal</td>
 <td>Scaling of paws/tail</td>
 <td>Obvious areas of denuded skin</td>
 </tr>
 </tbody>
</table>
Table 1. Cooke et al developed this scoring system in 19961. Mice should be scored daily on each of the criteria on the left. Each mouse is given a score of 0-2 for each criteria and the total score is a sum of all the individual scores.
<img alt="Figure 1" src="/files/ftp_upload/3697/3697fig1.jpg" /> 
Figure 1. Lethally irradiated BALB.B were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. A) Clinical score data of recipients of bone marrow alone or with CD8 T cell depleted WT splenocytes and purified L2G85.B6 CD8 T cells. B) Survival data of recipients of bone marrow alone or with CD8 T cell depleted WT splenocytes and purified L2G85.B6 CD8 T cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3697/3697fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3697/3697fig2.jpg" /> 
Figure 2. Lethally irradiated BALB.B mice were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. Recipients were injected with 4 mg D-luciferin via intraperitoneal injection and were imaged using Xenogen IVIS for 5 min at small binning. Pseudo-colored images are shown where purple represents low intensity and red represents high intensity Regions of interest were drawn around the entire mouse and total flux (photons/sec) were quantified.
<img alt="Figure 3" src="/files/ftp_upload/3697/3697fig3.jpg" /> 
Figure 3. Lethally irradiated BALB.B mice were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. Recipients were injected with 4 mg D-luciferin via intraperitoneal injection and were imaged using Xenogen IVIS for 5 min at small binning. Pseudo-colored images are shown where purple represents low intensity and red represents high intensity. Recipients were imaged on A) day 4, B) day 6, and C) day 8 post transfer.

<ol>
	<li>Cooke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Berger, M. <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+subsets+involved+in+lethal+graft-versus-host+disease+directed+to+immunodominant+minor+histocompatibility+antigens.">T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens.</a> Transplantation. 57, 1095-1102 (1994).</li><li>Nimer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+CD8++cells+for+prevention+of+graft-versus-host+disease+after+bone+marrowtransplantation.+A+randomized+controlled+trial.">Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial.</a> Transplantation. 57, 82-87 (1994).</li><li>Korngold, R., 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=Surface+markers+of+T+cells+causing+lethal+graft-vs-host+disease+to+class+I+vs+class+II+H-2+differences.">Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences.</a> Journal of Immunology. 135, 3004-3010 (1985).</li><li>Cao, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+using+labeled+donor+tissues+reveals+patterns+of+engraftment,+rejection,+and+survival+in+transplantation.">Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.</a> Transplantation. 80, 134-139 (2005).</li><li>Asady, R. E. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}-dependent+CD103+expression+by+CD8(+)+T+cells+promotes+selective+destruction+of+the+host+intestinal+epithelium+during+graft-versus-host+disease.">TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.</a> J. Exp. Med. 201, 1647-1657 (2005).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol Rev. 114, 181-217 (1990).</li><li>Karecla, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+E-cadherin+on+epithelial+cells+by+the+mucosal+T+cell+integrin+alpha+M290+beta+7+(alpha+E+beta+7).">Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).</a> Eur. J. Immunol. 25, 852-856 (1995).</li><li>Cepek, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+epithelial+cells+and+T+lymphocytes+mediated+by+E-cadherin+and+the+alpha+E+beta+7+integrin.">Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.</a> Nature. 372, 190-193 (1994).</li><li>Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+functional+characterization+of+a+dominant+minor+H+antigen,+H60.">The molecular and functional characterization of a dominant minor H antigen, H60.</a> J. Immunol. 161, 3501-3509 (1998).</li></ol>

No conflicts of interest declared.

The protocol for inducing GVHD in mice presented here represents a clinically relevant model of murine GVHD. Originally established by Berger et al. in 1994, the C57Bl/6 into BALB.B strain combination is MHC-matched, with GVHD mortality mediated by CD4 dependent, CD8 T effectors2, highly similar to the most common clinical scenario3. It is known that transplanting CD8 T cells alone does not cause GVHD in this model; however, disease progression is significantly worse when both CD4 and CD8 T cells are present as compared to CD4 T cells alone. Furthermore, there has been extensive work showing the immunodominant hierarchy of the immune response to minor histocompatibility disparities in this model10. However, there remains some ambiguity regarding the roles for T cell subsets during different phases of GVHD4. Unpublished studies from our laboratory suggest that CD4 T cells play a dominant role during early GVHD events, while CD8 T cells dominate at later time points. 
The ability to track T-cell accumulation patterns in vivo is a powerful tool that has the potential to test hypotheses regarding CD4 and CD8 T cell contributions during GVHD. Mice transgenic for firefly luciferase were initially developed in the laboratory of Dr. Robert Negrin5. Because of their constitutive luciferase expression, cells from these donor mice can be tracked in vivo using a non-invasive imaging technique5. Another advantage of this model is that individual mice can be monitored over time. However, despite repeat imaging of recipient mice, there remains experimental variability between recipients. As shown in Figure 2, the range of total body flux is 2.4 million and the the mouse with the lowest flux has less than 60% of total body flux than the mouse with the greatest flux. Because a single luciferase transgene is adequate for T-cell detection, the luciferase positive strain can be easily bred to create double transgenic mice. Studies in our lab have shown a role for the integrin CD103 in promoting GVHD6. CD103 is an integrin expressed on CD8 T cells7 and the ligand is E-cadherin, which is selectively expressed on epithelial cells8,9. Creating a CD103-/- mouse that is able to express luciferase will allow for experiments to test further hypotheses regarding the role of CD103 in GVHD. This experimental technique is limited to assessing accumulation patterns between organs. Alternative techniques must be utilized to elucidate cellular trafficking patterns within individual organs. Coupling a clinically relevant GVHD model with bioluminescent imaging will help shed light on the kinetics of T cell trafficking and relative contributions of CD4 and CD8 T cells following bone marrow transplantation.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>12633-012</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>Invitrogen</td>
 <td>10439016 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;M Cell Strainer</td>
 <td>BD Biosciences</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD3e-Biotin </td>
 <td>Miltenyi Biotech</td>
 <td>130-093-021</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-Biotin Microbeads</td>
 <td>Miltenyi Biotech</td>
 <td>130-091-147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD8a Microbeads</td>
 <td>Miltenyi Biotech</td>
 <td>130-049-401</td>
 <td>Used to deplete CD8 T cells from spleen.</td>
 </tr>
 <tr>
 <td>CD8a Purification Antibody Cocktail</td>
 <td>Miltenyi Biotech</td>
 <td>130-095-236</td>
 <td>Used to purify CD8 T cells from spleen.</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td>Caliper Life Sciences</td>
 <td>122796</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

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.

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 ...

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Immunology and Infection

15.6K Views.  The Ohio State University Medical Center. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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 ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

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

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

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

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

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...