This work was funded in part by sponsored research agreements from TRex Bio and grants from the NIH (1R01AR075864-01A1). JMM is supported by the Cancer Research Society (grant 26005). We acknowledge the Parnassus Flow Cytometry Core supported in part by grants NIH P30 DK063720, S10 1S10OD021822-01, and S10 1S10OD018040-01.

The present study was approved and performed in compliance with UCSF IACUC (AN191105-01H) and IRB (13-11307) protocols. Skin samples, discarded as part of routine elective surgical procedures, such as hernia repair, were used for the present research. The skin samples are either de-identified and certified as Not Human Subjects Research or, if clinically identifying information is required for downstream analyses, patients provided written consent under IRB protocol 13-11307. No other inclusion or exclusion criteria were utilized. NSG mice of either sex, 8-10 weeks of age, were employed in the study. The mice were obtained from commercial sources (see Table of Materials).
1. Processing of donor human skin sample
NOTE: The human skin sample used in this transplant was a large sample collected from the abdomen of a healthy patient. The sample must be at least 15 cm x 7.5 cm. Size limitations may affect the number of mice for which skin is available and the choice of graft size.
<ol>
	<li>Maintain the skin sample at cold temperatures (on ice; 4&#176;C) before preparation and grafting. Keep the sample moist in a closed specimen collection cup with the gauze soaked in phosphate-buffered saline (PBS). 
	NOTE: Storing the skin sample at 4 &#176;C for longer than 2 days is not recommended. However, reports exist where the skin samples are stored for a longer time12. 
	CAUTION: Treat all human tissue with standard biohazard precautions.</li>
	<li>Prepare to dermatome the human skin sample in a sterilized negative pressure tissue culture hood on a sterilized dissection board.</li>
	<li>Place the skin sample, epidermis side up, on the dissection board. Wipe the epidermis with a sterile alcohol prep pad and then with PBS.</li>
	<li>Pin the closer edge of the skin in place with a 1.5 in dissecting T-pin (see Table of Materials).</li>
	<li>Dermatome the skin specimen at a 400 &#181;m thickness, applying steady pressure while cutting forward at a 30&#176;-45&#176; angle. Follow all instrument-specific instructions and safety measures (see Table of Materials). 
	NOTE: For details on the dermatome technique, please see a previously published report13.</li>
	<li>Prepare a 100 mm x 20 mm Petri dish by placing a sterile gauze soaked in sterile PBS at the bottom of the dish. Place the skin, epidermis side up, onto the wet gauze.</li>
	<li>Seal and cover the plate edges with a semitransparent sealing film (see Table of Materials) to ensure the sample is not contaminated. Store the sample at 4&#176; C prior to grafting.</li>
</ol>
2. Pre-surgery conditioning and preparation
<ol>
	<li>Prepare the sterile instruments and a sterile surgical station for grafting. Use autoclaved paper towels as sterile surfaces for instrument and mouse placement. 
	NOTE: Mice may be grafted after weaning but are preferably grafted between 8-10 weeks of age. Mice of either sex may be grafted.</li>
	<li>Perform the surgical preparation, such as hair removal, in an area physically separated from the surgical station.</li>
	<li>Prepare the anti-GR1 (see Table of Materials) by diluting it to 1 mg/mL in sterile saline. Dose each mouse with 100 &#181;g/100 &#181;L of the anti-GR1 solution intraperitoneally following anesthesia induction.</li>
	<li>Anesthetize the mice, one at a time, with isoflurane or other institutionally approved anesthetics. 
	NOTE: Isoflurane needs to be given at a 3%-5% concentration during induction. Once the mouse is immobile, lower the isoflurane concentration to 1%-3% to effect for the duration of the surgery.
	<ol>
		<li>Monitor the mouse for appropriate depth of anesthesia by observing respiratory rate, absence of toe-pinch response, and appropriate pink coloration of ears and mouth. 
		CAUTION: Use appropriate anesthetic machinery and scavenging methods, and avoid exposure to isoflurane vapors.</li>
	</ol>
	</li>
	<li>Transfer the mouse to a heating pad or another heat source (see Table of Materials).</li>
	<li>Administer the ophthalmic ointment by dabbing a small drop of ointment on the eye with a gloved finger.</li>
	<li>Administer analgesics Buprenorphine (0.08 mg/kg) and Carprofen (5 mg/kg) (see Table of Materials) subcutaneously by pinching the skin and injecting at an angle parallel to the body. 
	NOTE: Prepare the pre-treatment analgesia following institutional protocols. Follow institutional guidelines for the selection and administration of analgesics. The method of analgesia used in this study is outlined in step 2.7 and Supplementary Figure 1.</li>
	<li>Administer the anti-GR1 (prepared in step 2.4) intraperitoneally by slightly lifting the mouse by the tail, exposing the abdomen, and injecting at a 30&#176; angle using an insulin 1 mL (12.7 mm) syringe.</li>
	<li>Use animal-safe electric clippers (see Table of Materials) to shave the middle and upper portions of the dorsal side of the mouse.</li>
	<li>Clear all the hair and apply a generous amount of hair removal ointment onto the shaved skin for 30 s to 1 min.</li>
	<li>Completely wipe away hair removal ointment with a paper towel and PBS.</li>
</ol>
3. Transplantation procedure
<ol>
	<li>Transfer the mouse to a secondary surgical location, away from the hair removal station.</li>
	<li>Sterilize the surgical site with the iodine swab stick in a circular motion, starting in the middle and working out toward the edge of the depilated area.</li>
	<li>Place a piece of sterile plastic wrap over the mouse and cut a window in the plastic slightly larger than the size of the area to be grafted.</li>
	<li>Cut a rectangle-shaped 10 mm x 10 mm portion of donor skin to be grafted with a scalpel. Do this by firmly holding the donor skin in place with the backside of the forceps and cutting alongside the forceps with the scalpel.</li>
	<li>Using the surgical scissors, snip a rectangular area of mouse skin matching the size of the donor skin piece, creating a graft bed. Use&#160;forceps to pull the skin away from the body, and cut the skin with the scissors angled away from the body to avoid cutting deeply into the facia.</li>
	<li>Place the donor skin piece, epidermis side up, onto the prepared graft bed.</li>
	<li>Using the back of the forceps, manipulate the skin, sliding back and forth until the donor skin lies completely flat against the graft bed.</li>
	<li>Add drops of surgical glue tissue adhesive (see Table of Materials) where the donor skin meets the mouse skin and hold the mouse and donor skin together with forceps for 1-2 s so that the glue adheres to the tissues. Completely seal the edge of the graft and allow for the glue to dry fully.</li>
	<li>Bandage the mice (Figure 1) following the steps below.
	<ol>
		<li>Cut a piece of petrolatum gauze (see Table of Materials) large enough to cover the graft area completely.</li>
		<li>Cover the graft with the petrolatum gauze, and lightly press the gauze against the skin using forceps.</li>
		<li>Cut a strip of a transparent film dressing lengthwise so that the width is large enough to cover the mouse&#39;s wound.</li>
		<li>Firmly press the transparent film dressing, adhesive side down, over the gauze. Quickly roll the mouse to wrap the dressing completely around the torso, ensuring it fits tightly without impeding respiration and all limbs are free for movement.</li>
		<li>Place the mouse in a recovery cage and monitor it until it is alert and moving around. Provide a heat source on part of the cage for at least 15 min following recovery. 
		NOTE: The animals are expected to recover within 1-5 min after placing them in the recovery cage.</li>
	</ol>
	</li>
	<li>Singly house the mice following grafting.</li>
	<li>Administer post-surgical analgesia as required by institutional protocols. 
	&#8203;NOTE: Buprenorphine (0.08 mg/kg) was administered subcutaneously 4-6 h later during the present study.</li>
</ol>
4. Post-surgical procedures
<ol>
	<li>Inject 100 &#181;L (100 &#181;g) of anti-GR1 intra-peritoneally at 4 days, 7 days, and 11 days post-grafting to prevent graft rejection (Supplementary Figure 1).</li>
	<li>Replace bandages as necessary and if removed by mice.</li>
	<li>Rebandage all the mice on day 7.</li>
	<li>Remove the bandages on day 14.</li>
	<li>Monitor the mice for signs of graft rejection and systemic inflammation (weight loss, hair loss, extreme lethargy).</li>
	<li>Harvest the mice between 3 and 6 weeks post-graft.
	<ol>
		<li>Euthanize the mice via regulator-controlled carbon dioxide (CO2) administration followed by cervical dislocation. 
		&#8203;NOTE: Follow institutional guidelines for euthanasia.</li>
		<li>Dissect the skin grafts from the mice14. Place a portion of the graft in 10% formalin for 24 h before paraffin embedding and sectioning for histology staining9.</li>
		<li>Mince the skin grafts with scissors and digest enzymatically with 250 IU/mL of Collagenase IV and 0.02 mg/mL of DNase in cell culture media overnight. Stain the cells for surface and intracellular markers following the previously described procedure14.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+skin+organoids:+potential+opportunities+and+challenges.">Generation of skin organoids: potential opportunities and challenges.</a> Frontiers in Cell and Developmental Biology. 9, 3176 (2021).</li><li>Crist&#243;bal, 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=Mouse+models+for+human+skin+transplantation:+a+systematic+review.">Mouse models for human skin transplantation: a systematic review.</a> Cells Tissues Organs. 210 (4), 250-259 (2021).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Ito, 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=NOD/SCID/&#947;cnull+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/&#947;cnull mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Meraz, I. 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=An+improved+patient-derived+xenograft+humanized+mouse+model+for+evaluation+of+lung+cancer+immune+responses.">An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses.</a> Cancer Immunology Research. 7 (8), 1267-1279 (2019).</li><li>Racki, W. J., 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=NOD-scid+IL2rgamma(null)+mouse+model+of+human+skin+transplantation+and+allograft+rejection.">NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.</a> Transplantation. 89 (5), 527-536 (2010).</li><li>Meehan, G. 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MDR is a founder of TRex Bio and Sitryx. MDR and MML receive research funding from Sitryx, Q32, and TRex Bio.

The mouse xenograft skin transplant model is a key technique to mechanistically dissect human skin immune responses in an in vivo setting14. Successful skin xenograft transplants rely upon appropriate preparation of mice and&#160;skin specimens and mice and adherence to aseptic rodent surgery methods15. Rapid cooling and proper storage of skin samples at cold temperatures in media (such as sterile saline) are important to ensure continued tissue health prior to transplant12. Collection of split-thickness samples, using an instrument such as a dermatome, allows consistency in sample depth between mice and increases the provision of nutrients from the host to improve graft survival10. Loss of graft integrity with epidermal sloughing may be observed, particularly in full-thickness grafts with disrupted blood supply16. Mouse survival through and after surgery is aided by sterile technique, provision of heat during and after surgery, and techniques that minimize surgical time, such as utilizing surgical glue15. Administration of anti-GR1 following transplant allows for suppression of the remaining mouse immune responses in the immunodeficient host to promote graft survival10. These methods increase the likelihood of mouse and graft survival during the xenograft transplant model and improve experimental numbers and reproducibility from one experiment to the next.
Although extremely useful, there are some key limitations to the xenograft skin transplant model, as demonstrated. First, the graft exists in an inflammatory environment, most likely secondary to transient ischemia and infiltration of human grafted tissue with murine myeloid cells. Thus, it does not reflect human skin at a steady state and may not fully reflect all human inflammatory skin diseases10,14. Studying human inflammatory disorders by grafting clinical biopsies from lesional skin is an alternative strategy, but limitations in the numbers of biopsies available and inconsistent control of grafting depth make this option less favorable. Additionally, xenograft transplants do not recapitulate the human peripheral immune architecture. Reconstituting the mouse periphery with human cells may also bear caveats, as the NSG mouse lymphoid structure is atrophic prior to human cell engraftment; this may lead to preferential cell selection in some settings10,17. Additionally, skin engraftment and immune cell recovery from skin specimens may vary from mouse to mouse, even in the absence of treatment. At least 10 replicates per condition are recommended for this model to observe consistent differences between groups. The appropriate duration of the assay may need to be adjusted for the experimental question, as graft healing and re-vascularization occur over the initial weeks following transplant, and the effect of interventions may depend upon the stage of engraftment.
Despite these drawbacks, the human xenograft skin transplant model remains one of the few assays to study human skin immune responses in the intact organ in vivo. While transgenic mouse models may allow for mechanistic pathway dissection, differences in anatomy, immune cell composition, and dominant immune pathways limit translation to human disease1. Ex vivo culture of human-derived cells, skin organoid models, and human tissue explant studies may be alternatively used but are also subject to limitations, including disruption of immune cell networks and egress of immune cells from the intact tissue18,19. Therefore, the xenograft skin transplant models remain a key method for studying therapeutic candidates and human skin responses to inflammation in a preclinical in vivo setting.

Mouse models are frequently used to make inferences about human biology and disease, partly due to their experimental reproducibility and capacity for genetic manipulation. However, mouse physiology does not completely recapitulate human organ systems, particularly skin, and therefore has limitations for use as a preclinical model in drug development1. Anatomical differences between mouse and human skin include differences in epithelial thicknesses and architecture, lack of murine eccrine sweat glands, and variations in hair cycling2. Furthermore, both the innate and adaptive arms of the immune system are divergent between the two species3. Mouse skin contains a unique immune population of dendritic epidermal T cells (DETCs), has a higher abundance of dermal &#947;&#948; T cells, and varies in immune cell subset localization in comparison to human tissue4. Therefore, experimental findings regarding human skin biology and inflammation benefit from validation with human tissue. While in vitro and organoid culture systems are widely utilized tools to study human tissue, these systems are limited by absent or incomplete immune reconstitution and a lack of connection to peripheral vasculature5. The humanized xenograft skin transplant model aims to allow for therapeutic or biological manipulation of immune and non-immune pathways in human tissues in vivo.
The human skin xenograft model has been utilized to study skin physiology and pharmacology, analyze immune rejection and responses, dissect human skin cancer mechanisms, and understand skin diseases and wound healing6. While applicable to multiple fields of skin research, the xenograft model has lower throughput than in vitro studies and lacks the ease of genetic manipulation employed in mouse models. Time points within this model may range from weeks to months, and successful grafting requires appropriate facilities and equipment to perform these surgeries. However, the xenograft model supplies biological and physiological context to experiments, while organoid culture systems, such as tissue explants, often require replicating a myriad of moving parts, such as exogenous signals, at specific time intervals7. Therefore, this model is best utilized to further validate findings observed in vitro and within mouse models, or for work that is not otherwise biologically feasible. Appropriate use of the xenograft model provides a unique opportunity to study and manipulate intact human tissue in vivo.
Optimization of the xenograft skin transplant model has relied on decades of research to preserve graft integrity over time. Critical to this process is utilizing the non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mouse, which lacks B and T adaptive immune cells, functional NK cells, and has deficiencies in macrophage and dendritic cells8. The immunodeficient nature of these NSG hosts allows for the transplant of human hematopoietic cells, patient-derived cancers, and skin8,9,10. Despite this immunosuppressive host environment, additional suppression of mouse neutrophilic immune responses by anti-GR1 administration is necessary for graft success10. The major roadblocks in transplanting intact tissue are infection, rejection, and difficulty in re-establishing blood flow to the graft, sometimes leading to loss of dermal and epidermal integrity11. Techniques including administration of anti-FR1 and use of appropriate graft depth improve graft survival10. Meticulous optimization makes it possible to perform human xenograft skin transplants on NSG mice with high efficiency and survival rates, ranging from 90%-100%.

<table><tbody><tr><td>10% Neutral Buffered Formalin</td><td>Fisher</td><td>SF100-20</td><td>Fixative for histology</td></tr><tr><td>3M Vetbond Tissue Adhesive</td><td>3M</td><td>1469SB</td><td>surgical glue</td></tr><tr><td>Alexa 700 CD45 monoclonal antibody (Clone 30F11)</td><td>Thermo Fischer</td><td>56-0451-82</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Anti-GR1 clone RB6-8C5</td><td>BioXcell</td><td>BE0075</td><td>Anti-rejection</td></tr><tr><td>APC mouse anti-human CD25&amp;nbsp; (Clone 2A3)</td><td>BD Biosciences</td><td>340939</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>APC-eFluor 780 anti-human HLA-DR (Clone LN3)</td><td>eBioscience</td><td>47-9956-42</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Autoclave pouches</td><td>VWR&amp;nbsp;</td><td>89140-800</td><td>For autoclaving tools and paper towels</td></tr><tr><td>Brilliant Violet 60 anti-human CD4 antibody (Clone OKT4</td><td>Biolegend</td><td>317438</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Brilliant Violet 65 anti-human CD8a antibody (Clone RPA-T8)</td><td>Biolegend</td><td>301042</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Brilliant Violet 711 anti-human CD3 antibody (Clone OKT3)</td><td>Biolegend</td><td>317328</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Buprenex 0.3 mg/mL</td><td>Covetrus</td><td>059122</td><td>Analgesia</td></tr><tr><td>Carprofen 50 mg/mL</td><td>Zoetis</td><td>NADA # 141-199</td><td>Analgesia</td></tr><tr><td>Collagenase Type IV</td><td>Worthington</td><td>4188</td><td>Skin digestion</td></tr><tr><td>D42 Dermatome blade</td><td>Humeca</td><td>5.D42BL10</td><td>dermatome (1 blade per sample)</td></tr><tr><td>Dermatome D42</td><td>Humeca</td><td>4.D42</td><td>dermatome</td></tr><tr><td>Disposable Scalpel</td><td>Bard-Parker</td><td>371610</td><td>skin preparation</td></tr><tr><td>Dissecting T-Pins; 1-1/2 inch, 1000/CS 1.5</td><td>Cole-Parmer</td><td>UX-10915-03</td><td>To pin skin specimen for dermatome</td></tr><tr><td>Dissection scissors</td><td>medicon</td><td>02.04.10</td><td>sample preparation and mouse dissection</td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25-1G</td><td>Skin digestion</td></tr><tr><td>eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent</td><td>eBioscience</td><td>00-5521-00</td><td>Flow cytometry analysis: Cell Fixation and Permeabilization</td></tr><tr><td>eFluor-450 FOXP3 monoclonal antibody (Clone PCH101)</td><td>eBioscience</td><td>48-4776-42</td><td>Flow cytometry analysis: Intracellular protein staining</td></tr><tr><td>Electric clippers</td><td>Kent</td><td>CL8787-KIT</td><td>hair removal</td></tr><tr><td>Epredia&amp;nbsp;Shandon Instant Eosin</td><td>Fisher Scientific</td><td>6765040</td><td>H&amp;amp;E</td></tr><tr><td>Epredia&amp;nbsp;Shandon Instant Hematoxylin</td><td>Fisher Scientific</td><td>6765015</td><td>H&amp;amp;E</td></tr><tr><td>FITC anti-human CD45 (Clone HI30)</td><td>Tonbo Biosciences</td><td>35-0459-T100</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>Forceps&amp;nbsp;</td><td>medicon</td><td>07.60.07</td><td>sample preparation and mouse dissection</td></tr><tr><td>Gauze</td><td>Fisherbrand</td><td>22-362-178</td><td>Sample preparation</td></tr><tr><td>Heating lamp</td><td>Morganville Scientific</td><td>HL0100</td><td>Post-surgical care</td></tr><tr><td>Heating pads 4&quot; x 10&quot;</td><td>Pristech</td><td>20415</td><td>Surgical heat supply</td></tr><tr><td>Insulin 1cc 12.7 mm syringes</td><td>BD</td><td>329410</td><td>drug administration</td></tr><tr><td>Isoflurane</td><td>United States Pharmacopeia (USP)&amp;nbsp;</td><td>NDC 66794-013-25</td><td>Anesthesia&amp;nbsp;</td></tr><tr><td>Isoflurane machine</td><td>VetEquip</td><td>911103</td><td>Anesthesia</td></tr><tr><td>Nair for Men</td><td>Nair</td><td>&amp;lrm; 10022600588556</td><td>hair removal</td></tr><tr><td>Neomycin and Polymyxin Bisulfates and Bacitracin Zinc Ophthalmic ointment</td><td>Dechra</td><td>&amp;nbsp;NDC 17478-235-35</td><td>eye ointment to prevent drying</td></tr><tr><td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td><td>The Jackson Laboratory</td><td>005557</td><td>Mice</td></tr><tr><td>Paper towels</td><td>Kleenex</td><td>100848</td><td>May be autoclaved for sterile surfaces</td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td>Semitransparent sealing film</td></tr><tr><td>PE mouse anti-human CD127 (Clone HIL-7R-M21)</td><td>BD Biosciences</td><td>557938</td><td>Flow cytometry analysis: Surface protein staining</td></tr><tr><td>PE-Cy-7 mouse anti-Ki-67 (Clone B56)</td><td>BD Biosciences</td><td>561283</td><td>Flow cytometry analysis: Intracellular protein staining</td></tr><tr><td>PerCP-eFluor-710 CD152 (CTLA-4) monoclonal antibody (Clone 14D3)</td><td>eBioscience</td><td>46-1529-42</td><td>Flow cytometry analysis: Intracellular protein staining</td></tr><tr><td>Permeabilization Buffer 10x</td><td>eBioscience</td><td>00-8333-56</td><td>Flow cytometry analysis: Intracellular protein staining buffer</td></tr><tr><td>Petri Dish 150 mm</td><td>Corning</td><td>430597</td><td>Sample storage</td></tr><tr><td>Plastic Wrap</td><td>Fisherbrand</td><td>22-305-654</td><td>Site preparation</td></tr><tr><td>Providone-Iodine Swab stick</td><td>PDI</td><td>S41350</td><td>Site sterilization</td></tr><tr><td>Soft-Feed and Oral Hydration (Napa Nectar)</td><td>Se Lab Group Inc</td><td>NC9066511&amp;nbsp;</td><td>For supplementing poorly recovering mice post-surgery</td></tr><tr><td>Specimen Collection Cups</td><td>Fisher Scientific</td><td>22-150-266</td><td>sample storage</td></tr><tr><td>Sterile alcohol prep pad</td><td>Fisherbrand</td><td>22-363-750</td><td>skin preparation</td></tr><tr><td>Sterile PBS</td><td>Gibco</td><td>14190-144</td><td>Media for sample storage</td></tr><tr><td>Sterile saline</td><td>Hospira</td><td>NDC 0409-4888-02</td><td>For drug dilution</td></tr><tr><td>Tegaderm Film 4&amp;rdquo; x 4&lt;sup&gt;3/4&lt;/sup&gt;&amp;rdquo;&amp;nbsp;</td><td>3M</td><td>1626</td><td>transparent film wound dressing</td></tr><tr><td>Vaseline Petrolatum Gauze 3&amp;rdquo; x 8&amp;rdquo;&amp;nbsp;</td><td>Kendall</td><td>414600</td><td>wound dressing</td></tr><tr><td>Violet 510 Ghost Dye&amp;nbsp;</td><td>Tonbo Biosciences</td><td>13-0870-T100</td><td>Flow cytometry analysis: Viability dye</td></tr></tbody></table>

xenograft skin model to manipulate human immune responses in vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Human skin xenografts were performed on NSG mice inside a super-barrier animal facility. Success was defined by the prolonged graft and mouse survival and behavioral health of mice post-transplant. Poor survival during the week following surgery was initially observed as the biggest barrier to experimental success, with up to 50% of mice requiring euthanasia. Improving sterile technique and better support of mouse body temperatures during and immediately after surgery increased surgical survival consistently to over 80% and often to 90%-100% survival. While the addition of antibiotics in the mouse drinking water was trialed, it was not determined to improve outcomes and was discontinued as a potential confounder of results. Successfully grafted mice should appear active and healthy in the days following the transplant. Mice were moving around their cage, exploring, building nests, and eating and drinking as usual. An unwell mouse may seem lazy, non-reactive, and may not be eating or drinking enough to maintain its weight. Supplementation of lethargic mice with soft-feed and oral hydration options may assist post-surgical recovery.
A xenograft that is healing correctly will first scab and then recover within about 50 days of surgery. Grafts may contract over time but remain adherent around the edges where the donor skin meets the mouse&#39;s skin (Figure 2). While graft scabbing should subside, grafts will remain thicker and more inflamed than healthy human skin (Figure 2). Excessive contraction of grafts can occur, reducing available tissue for endpoint analysis. Utilization of consistently sized grafts and appropriate graft placement is expected to mitigate such issues. All grafts throughout five independent experiments survived to the time of harvest, up to 50 days post-transplant.
Tissue analysis may include immunohistochemistry and single-cell analyses following enzymatic digestion. A portion of skin was processed by overnight digestion in 250 IU/mL of Collagenase Type IV and 0.02 mg/mL of DNase in cell culture media (see Table of Materials), and stained for surface and intracellular markers as previously described14. Analysis by flow cytometry revealed the sustained presence of human immune cells within most grafts but counts varied between xenografts (Figure 3A). Figure 3B shows representative results from a successful graft (left) and one that has failed to maintain human immune cells (right). Analysis of hematoxylin and eosin-stained (H&#38;E) sections taken from the center of the graft revealed intact, non-devitalized skin composed of human dermis and epidermis for both 35 and 50 day time points (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64040/64040fig01.jpg" /> 
Figure 1: Method for bandaging mouse after surgery. The mouse is wrapped tightly in petrolatum gauze and transparent film dressing while under anesthesia. (A) Petrolatum gauze is placed over an area slightly larger than the surgical wound. (B) The transparent film dressing is prepared: a long strip is cut slightly wider than the petrolatum gauze. The backing covering the adhesive side of the transparent film dressing is partially removed. (C) The adhesive side of the film is firmly pressed against the back of the mouse, leaving an inch of space on the front end of the film. (D) The mouse is turned on its back. The overhanging front portion of the film is pressed onto the mouse, and the backing is removed. (E) The mouse is tightly wrapped in the film, and the remaining support is removed as the mouse is wrapped. (F) The mouse is successfully wrapped in the dressing, and its arms and legs are unconstrained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64040/64040fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64040/64040fig02.jpg" /> 
Figure 2: Representative images from transplanted animals from Day 10 to 21 post-transplant. (A-C) Three different mice with optimal transplants at day 10. (D-E)&#160;Two different mice with transplants on day 21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64040/64040fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64040/64040fig03.jpg" /> 
Figure 3: Immune cell chimerism in grafted skin. Flow cytometry data of human immune cell recovery from xenografts. Data is gated on the events of singlet, live, human CD45+ mouse CD45-. (A) Number of cells (Live Human CD45+ Events) recovered from two independent experiments. (B) Representative plots of human and mouse CD45+ immune cell staining in a successful graft (left) compared to graft with unsuccessful maintenance of the human immune compartment (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64040/64040fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64040/64040fig04.jpg" /> 
Figure 4: Histology of grafted human skin at day 35 and 50. Grafted skin was harvested from mice on day 35 (A) or 50 (B), preserved in 10% formalin, and paraffin-embedded and stained for Hematoxylin and Eosin (H&#38;E). (A) The epidermis shows moderate hyperplasia (acanthosis), slight hyper-granulosis, and compact orthokeratosis. In the papillary dermis, there are occasional dilated and congested capillaries. Melanocytes and melanin pigment are present in the basal layer of the epidermis. The dermis is moderately cellular and is composed of plump oval fibroblasts with pale syncytial cytoplasm and scattered lymphocytes. (B) The epidermis comprises stratified squamous epithelium, basket-weave orthokeratosis in the cornified layer, and is of normal thickness. Melanocytes and melanin pigment are present in the basal layer of the epidermis. The dermis shows oval fibroblasts and slightly enhanced extracellular matrix deposition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64040/64040fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Timeline of the protocol adopted for the xenograft study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64040/Supplemental Figure 1 JoVE xenograft PDFv2.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

xenograft-skin-model-to-manipulate-human-immune-responses-in-vivo

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.8K Views.  UCSF. The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report. 

Video: Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

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

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Cancer Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medicine

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Developmental Biology

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

Technika przeszczepiania ludzkiej skóry w modelu mysim

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

Source: Li, Z., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59679">A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to generate a humanized xenograft mouse model. An immunocompromised mouse is injected with cyclophosphamide to cause immune cell depletion. Patient-derived tumor cells mixed with human peripheral blood mononuclear cells (PBMCs) in a hydrogel are subcutaneously injected into the mouse, forming a tumor with infiltrating T cells.

Technika generowania ludzkiego, humanizowanego modelu myszy ksenoprzeszczepu wszczepionego w PBMC

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

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Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

Murine Skin Transplantation

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34⁺ Cells Injection, and Cytokine Delivery

Technika przeszczepiania ludzkiej skóry w modelu mysim

Technika generowania ludzkiego, humanizowanego modelu myszy ksenoprzeszczepu wszczepionego w PBMC