This work was supported by the National Science Foundation for Outstanding Young Scholars of China (No. 82122028 to LX), the National Natural Science Foundation of China (No. 82173094 to LX), Natural Science Foundation of Chong Qing (No. 2023NSCQ-BHX0087 to SW).

The C57BL/6J mice (referred to B6 mice) and naive P14 transgenic mice9,27 used were 6-10 weeks of age weighing 18-22 g. Both male and female were included without randomization or blinding. All animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Qingdao Agricultural University.
1. Preparation of medium and reagents
<ol>
	<li>Prepare B16F10-GP melanoma cell culture medium, named D10 (complete DMEM medium) by adding DMEM, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine with an additional 100 U/mL puromycin. Maintain a sterile D10 and store for up to 2 weeks at 2-4 &#176;C.</li>
	<li>Prepare R2 medium (for termination of red blood cell lysis) by adding RPMI-1640 with 2% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.</li>
	<li>Prepare fluorescence-activated cell sorting (FACS) buffer by adding 1x phosphate-buffered saline (PBS) with 2% FBS and 0.01% of sodium azide. The addition of sodium azide can prolong the storage time of FACS buffer, which can be stored for months at 2-4 &#176; C.</li>
	<li>Prepare red blood cell lysis (RBL) buffer by adding 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM ethylenediamine tetraacetic acid (EDTA) into double distilled water and adjust its pH to 7.3. Store RBL buffer at room temperature (RT) and it remains stable for up to 3 months.</li>
	<li>Prepare the anesthetic, 2,2,2-tribromoethanol, as described below.
	<ol>
		<li>Weigh out appropriate-amount of 2,2,2-tribromoethanol crystal in a 50 mL sterile conical tube wrapped with aluminum foil. Add appropriate amount of 2-methyl-2-butanol into the crystal to prepare the stock solution with the concentration of 500 mg/mL.</li>
		<li>Swirl it to mix and warm the tube in a 37 &#176;C water bath until the crystal is dissolved. Make sure all crystals are dissolved.</li>
		<li>Mix the solution thoroughly. Filter the stock solution with a 0.22 &#181;m filter into a sterile container. Store the stock solution frozen, shielded from light, at -20 &#176;C or dilute with PBS into a working solution at the concentration of 12.5 mg/mL and stored at 4 &#176;C. 
		&#8203;NOTE: It is best to use the prescribed concentration as higher concentrations of the liquid are irritating.</li>
	</ol>
	</li>
</ol>
2. Preparation of B16F10-GP cell suspension
<ol>
	<li>Thaw and culture a vial of 1 x 106 B16F10-GP cells with 5 mL of D10 in a cell culture incubator at 37 &#176;C and 5% CO2. Subculture cells when they reached 80% to 90% density as described below.
	<ol>
		<li>Discard the original culture medium by aspiration with a pipetting gun and wash the cells 2x with PBS. When cleaning adherent cells with PBS in a cell culture dish, move the PBS to the side wall or drop it into the dish.</li>
		<li>After aspiration of PBS, add 0.5-1 mL of 0.25% trypsin EDTA solution to the cell culture dish or flask. Gently shake to cover the entire cell surface. Place the cell culture dish or flask in an incubator at 37 &#176;C for about 1 min or at RT until the cells are rounded and separated. Observe exfoliation of cells with the help of an inverted microscope.</li>
		<li>Add equal volume of freshly formulated D10 to terminate trypsin digestion. Purge the lower surface of the cell culture flask with a pipetting gun to ensure that all cells were separated.</li>
		<li>Transfer the B16F10-GP cell suspension to a 15 mL centrifuge tube and centrifuge at 163 x g for 4 min at RT.</li>
		<li>Aspirate the supernatant and discard. Resuspend the cells in 1-2 mL of D10 for precipitation. Transfer B16F10-GP cell suspension to a new cell culture dish or flask containing 8-10 mL of D10 and then incubate in a cell incubator at 37 &#176;C at 5% CO2.</li>
	</ol>
	</li>
	<li>On the day of tumor implantation, collect B16F10-GP cells with a density of approximately 90% as described in steps 2.1.1 to 2.1.4. After centrifugation, aspirate the supernatant and discard. Resuspend the cell precipitate in 1 mL of PBS.</li>
	<li>Count viable cells using a 0.4% trypan blue hemocytometer. Add PBS to dilute the cell to 5 x 105 cells per 100 &#181;L. Place the cells on ice until use.</li>
</ol>
3. Ectopic inoculation of B16F10-GP cells in the bilateral&#160;inguinal region of mice
<ol>
	<li>Aspirate 100 &#181;L of prepared B16F10-GP cell suspension using a 1 mL syringe. Flick the tube wall to move the bubbles to the top and push the piston to move the top bubble out.</li>
	<li>Hold the mouse in a supine position, exposing its abdomen. Press the right hind limb of the mouse in the supine position with a finger so that the skin in the right inguinal region is fully exposed (Same with the left inguinal region).</li>
	<li>Remove the fur on the abdomen with depilatory cream, then clean the bilateral abdomen area with cotton containing 75% ethanol.</li>
	<li>Before inserting the needle, make sure that the skin in the inguinal region is tightened. Insert the needle at a 45&#176; angle in the subcutaneous space of the inguinal region of the upper thigh. Make sure the needle is beveled&#160;upward and the depth of the needle to 0.5-1 cm.</li>
	<li>Apply gentle suction before injection, if there is no blood, inject the cells slowly injected into the subcutaneous tissue. At the same time, observe a small bolus (formation of fluid pocket) in the subcutaneous region.</li>
	<li>After injection, remove the needle, put it in the sharps box, and return the mouse to its cage.</li>
</ol>
4. Adoptive transfer of P14 T cells into tumor-bearing mice
<ol>
	<li>Perform adoptive transfer in tumor-bearing mice 6-8 days after tumor implantation, when the tumors are palpable (approximately 3-5 mm in diameter). Intraperitoneally inject 4 mg cyclophosphamide the day before transfer28,29,30. 
	NOTE: The aim of CTX injection is to create space in the lymphatic compartment for adoptively-transferred T cells by transiently eliminating proliferating lymphocytes.</li>
	<li>Isolate lymphocytes from the spleen and LNs of na&#239;ve P14 transgenic mice as described below31,32 (6-10 weeks old, the same sex as tumor-bearing mice to avoid rejection issues). 
	NOTE: Perform the following procedures in a biosafety cabinet to ensure strictly sterile conditions.
	<ol>
		<li>Prepare two 6 cm dishes and add 3 mL of R2 medium to one of the dishes and 3 mL of RBL buffer to the other dish. Place a 70 &#181;m cell filter in the Petri dish containing RBL buffer.</li>
		<li>Euthanize P14 mice in airtight containers containing isoflurane followed by cervical dislocation. Adjust the number of P14 mice according to the number of recipient mice.</li>
		<li>Harvest the spleen, inguinal, and axillary lymph nodes from the euthanized mice and transfer them to 6 cm dishes containing 4 mL of R2 medium and placed on ice.</li>
		<li>Place the spleen in a strainer soaked with 3 mL of RBL buffer, grind the spleen with the putter inside of the 1 mL syringe. Incubate the cells at RT for 1-2 min, then terminate the reaction with 3 mL of cold R2 medium.</li>
		<li>Grind the LNs with the putter inside of the 1 mL syringe until only connective tissue remains in the strainer with a 70 &#181;m cell filter. Rinse the filter with cold R2 medium and transfer the cell suspension to a new 15 mL centrifuge tube. Centrifuge the samples at 500 x g for 6 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cells with 3 mL of PBS. Use a 70 &#181;m cell filter to remove flocculent material from the cell suspension.</li>
		<li>Centrifuge the cell suspension at 500 x g for 6 min at 4 &#176;C. Resuspend the cells with 3 mL of PBS and place the tube on ice. Take a small sample, mix with trypan blue, and count cells using a blood cell counting plate.</li>
	</ol>
	</li>
	<li>Determine the percentage of P14 (live/dead-CD45.1+CD8+V&#945;2+) cells by flow cytometry. Ensure the transferred donor cells exhibit distinct congenic marker with recipient mice (tumor-bearing B6 mice is CD45.2+). Perform staining prior to transfer to verify the correct phenotype of the transferred cells.
	<ol>
		<li>Add 5 x 104- 1 x 105 cells to a 1.5 mL centrifuge tube containing 1 mL of FACS buffer. Centrifuge the cell suspension at 500 x g at 4 &#176;C for 3 min.</li>
		<li>Discard the supernatant, flick the bottom of the tube to disperse the cells, and place the tube on ice.</li>
		<li>Prepare the following conjugated antibody mixture9,32 diluted in 100 &#181;L of FACS buffer: anti-CD8, 1:200; anti-TCR V&#945;2, 1:100; anti-CD45.1, 1:200; Live/dead stain, 1:400. (Table of Materials).</li>
		<li>Centrifuge the antibodies mixture at 15,000 x g for 3 min to aggregate the particles. Place the mixture on ice and shield it from light by wrapping it in foil. Only take the supernatant to avoid the aspiration of particles.</li>
		<li>Resuspend the cells in 100 &#181;L of the antibody mixture and thoroughly mix by flicking the tube wall. After wrapping in tin foil, incubate the tube for 30 min on ice.</li>
		<li>Wash the cells 2x with FACS buffer. Centrifuge the tube at 500 x g at 4 &#176;C for 3 min. Resuspend the cells with 200 &#181;L of FACS buffer and transfer the cell suspension to a flow tube.</li>
		<li>Run the flow tube with stained cells on a flow cytometer to determine the percentage of live/dead-CD45.1+CD8+V&#945;2+ cells. 
		NOTE: Generally, over 90% of CD8+ T cells from P14 transgenic mice harbored V&#945;2+TCRs, which are specific to H-2Db GP33-41 epitope of LCMV (lymphocytic choriomeningitis virus).</li>
	</ol>
	</li>
	<li>Calculate the absolute number of live/dead-CD45.1+CD8+V&#945;2+ cells by multiplying its percentage and the live cell number obtained in steps 4.2 and 4.3.</li>
	<li>Aspirate 200 &#181;L of P14 cell (5 x 105 cells) suspension with a 100 U insulin syringe and remove bubbles. The initial number of transferred naive P14 cells (5 x 103 - 5 x 105) did not affect the phenotype of transferred cells9.</li>
	<li>Place the mice in a cage and warm&#160;with an infrared lamp for 5-10 min to expand the tail vein. Hold in place with an appropriately sized mouse fixator, straighten the tail, and wipe it with a cotton ball containing 75% ethanol to make the veins visible.</li>
	<li>Insert the needle parallel to the tail vein and gently pull back the plunger. If there is blood flowing into the syringe, slowly push the cell suspension into the vein. 
	NOTE: If there is resistance or swelling of the tail during injection, the injection position needs to be adjusted. The injection site should start at the distal end.</li>
	<li>After the injection is completed, pull out the needle quickly and press the injection site gently with a cotton ball. Return the mouse to a new clean cage and closely observe it for several minutes for any adverse effects.</li>
</ol>
5. Resection of the primary tumor
NOTE: Ensure all surgical instruments are autoclaved before use. Sterilize the operating area inside the biosafety cabinet with 75% ethanol, followed by UV irradiation for at least 30 min. Wear clean gowns, hats, masks, and sterile gloves during surgery.
<ol>
	<li>When the tumor is palpable (approximately 5 mm in diameter), resect the primary tumor.</li>
	<li>Anesthetize the mice with intraperitoneal injection of Ketamine (75 mg/kg). Pinch foot pad to evaluate the degree of anesthesia, if there is no pain reflex, it indicates the right timing for surgery. If the hind limbs were withdrawn, provide another dose of 10-30 &#181;L. 
	NOTE: Alternatively, intraperitoneal medetomidine&#160;(1 mg/kg body weight; Domitor) is recommended to anesthetize mice.</li>
	<li>Apply the drying preventive ointment on mouse eyes. Remove the fur on the abdomen with depilatory cream to fully expose the surgical field.</li>
	<li>Place the mouse in a biosafety cabinet and place it on an anatomical board covered with clean absorbent paper in a supine position so that the longitudinal axis of the mouse is parallel to the experimenter. 
	NOTE: To maintain a strict sterile environment, the following procedures must be performed in a biosafety cabinet.</li>
	<li>Disinfect the abdomen of the mice with a cotton ball soaked in povidone-iodine. Incise the skin near the tumor-bearing site with a sterile scalpel or opthalmic scissors. Insert closed scissors tip into the incision to clearly expose the tumor. When incising the skin, make sure not to damage the inguinal lymph nodes.</li>
	<li>During tumor removal, keep the capsule as intact as possible. Carefully and gently remove the connective tissue adjacent to the tumor with sterile scissors. Remove the tumor in situ completely, otherwise it may recur.</li>
</ol>
6. Intranodal injection of B16F10-GP cells in the inguinal lymph node
NOTE: After bilateral tumor clearance, B16F10-GP cells were injected into unilateral inguinal lymph node and PBS was injected into&#160;the other side.
<ol>
	<li>Aspirate 20 &#181;L (5 x 104 cells) of B16F10-GP cell suspension with a 100 U insulin syringe, remove bubbles and then inject into one inguinal lymph node. Inject an equal volume of PBS into the inguinal lymph node on the other side.
	<ol>
		<li>During the injection, insert the needle from the distal end of the lymph node, and then slowly insert the needle to the center of the lymph node. At this time, if the fluid is accurately injected into the lymph node, the lymph node can be seen to swell significantly. 
		NOTE: When the needle is inserted from the distal end of the lymph node, make sure not to puncture the node.</li>
	</ol>
	</li>
	<li>Suture the incision with 2-3 stitches using a 3-0 suture. Disinfect the skin surrounding the wound with cotton impregnated with povidone iodine. Take care to bypass lymph nodes during suturing.</li>
	<li>Place the mouse in a clean cage and keep warm using infrared light in the lateral decubitus position. Monitor continuously until consciousness is recovered. Ensure that the mice that has undergone surgery is not returned to the company of other animals until fully recovered.</li>
	<li>Give buprenorphine every 4-6 h for 3 consecutive days after the operation to relieve postoperative pain (at a dose of 0.1-0.5 mg/kg, SQ or IP). Monitor the feeding, drinking, movement, and activity areas of the mice. Typically, mice recover from surgical trauma within 3 days. 
	NOTE: If mice are unable to resume normal feeding and activities and show any signs of infection, consult a veterinarian for intervention or euthanize it.</li>
	<li>Sacrifice mice at different time points: day 8 and day 18 after intranodal injection of tumor cells. Recover activated donor cells using flow cytometry analysis.</li>
</ol>

Evaluating Antigen-Specific CD8+ T Cells During Lymph Node Metastasis

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophosphamide-facilitated+adoptive+immunotherapy+of+an+established+tumor+depends+on+elimination+of+tumor-induced+suppressor+T+cells.">Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells.</a> J Exp Med. 155 (4), 1063-1074 (1982).</li><li>Maine, G. N., Mule, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+room+for+T+cells.">Making room for T cells.</a> J Clin Invest. 110 (2), 157-159 (2002).</li><li>Xue, G., 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=Adoptive+cell+therapy+with+tumor-specific+th9+cells+induces+viral+mimicry+to+eliminate+antigen-loss-variant+tumor+cells.">Adoptive cell therapy with tumor-specific th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells.</a> Cancer Cell. 39 (12), 1610.e9-1622.e9 (2021).</li><li>Prokhnevska, N., 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=CD8++T+cell+activation+in+cancer+comprises+an+initial+activation+phase+in+lymph+nodes+followed+by+effector+differentiation+within+the+tumor.">CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.</a> Immunity. 56 (1), 107.e5-124.e5 (2023).</li><li>Wang, 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=Tumor+transplantation+for+assessing+the+dynamics+of+tumor-infiltrating+CD8++T+cells+in+mice.">Tumor transplantation for assessing the dynamics of tumor-infiltrating CD8+ T cells in mice.</a> J Vis Exp. (172), e62442 (2021).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-specific+memory+cd8(+)+t+cells+are+strictly+resident+in+draining+lymph+nodes+during+tumorigenesis.">Tumor-specific memory cd8(+) t cells are strictly resident in draining lymph nodes during tumorigenesis.</a> Cell Mol Immunol. 20 (4), 423-426 (2023).</li><li>Fransen, M. F., 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=Tumor-draining+lymph+nodes+are+pivotal+in+pd-1/pd-l1+checkpoint+therapy.">Tumor-draining lymph nodes are pivotal in pd-1/pd-l1 checkpoint therapy.</a> JCI Insight. 3 (23), e124507 (2018).</li><li>Francis, D. 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=Blockade+of+immune+checkpoints+in+lymph+nodes+through+locoregional+delivery+augments+cancer+immunotherapy.">Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy.</a> Sci Transl Med. 12 (563), eaay3575 (2020).</li><li>Garner, H., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+crosstalk+in+cancer+progression+and+metastatic+spread:+A+complex+conversation.">Immune crosstalk in cancer progression and metastatic spread: A complex conversation.</a> Nat Rev Immunol. 20 (8), 483-497 (2020).</li></ol>

The authors declare no competing interests.

During tumorigenesis, antigen-presenting cells (APCs) engulf tumor antigens and migrate to TdLN where they prime CD8+ T cells. After priming and activation, CD8+ T cells leave the TdLN and infiltrate the tumor to kill tumor cells10. Through TdLN resection and the administration of FTY720 which block the exit of immune cells from the lymphoid organs, several studies have demonstrated the pivotal role of TdLN in ensuring the efficacy of PD-1/PD-L1 checkpoint therapy<sup class="...

Cancer immunotherapy, especially the immune checkpoint blockade (ICB), has revolutionized cancer therapy1. ICB blocks the coinhibitory immunoreceptors (such as PD-1, Tim-3, LAG-3, and TIGIT), which are highly expressed in exhausted CD8+ T cells in the tumor microenvironment (TME), leading to the reinvigoration of exhausted CD8+ T cells2. Considering the heterogeneity of exhausted CD8+ T cells, accumulating evidence revealed that tumor-specific CD8+ T cells derived from the periphery, including draining lymph node (dLN), but not in TME, mediate the efficacy of ICB3,4,5,6,7,8. Recently, TdLN derived TCF-1+TOX- tumor-specific memory CD8+ T cells (TdLN-TTSM) was confirmed to be the genuine responders to ICB which embody several functional properties of conventional memory T cells and could further expand and differentiate into progeny exhausted cells upon ICB treatment9. Altogether, these findings corroborated the importance of LN in mounting anti-tumor immunity.
Lymph node functions as a critical place in facilitating the priming and activation of tumor-specific CD8+ T cells by providing structural basis as well as biological signals10. Several types of cancer cells frequently seed sentinel lymph node (SLN, the first LN draining a primary tumor) before systematic dissemination11. The presence of SLN metastasis is linked with poor outcome in human cancer and preclinical models showed that tumor cells in TdLN could spread to distant organs through both the lymphatic vessels and blood vessels of the node12,13,14,15. SLN biopsy now represents a standard procedure to guide subsequent treatment decisions in many solid tumor types which could avoid unnecessary resection of uninvolved LN16,17. Even to the involved LN, it remains controversial whether and when surgical resection is needed as several studies have demonstrated that the removal of regional LN did not exhibit improved overall survival compared to those that received radiation or systemic therapy without regional LN resection18,19. One interpretation is that metastatic LN (mLN) with microscopic disease may retain some capacity to educate immune cells and provide some therapeutic benefits. So, it is critically important to elucidate how LN metastasis affects the anti-tumor immune response, especially the properties and functions of TdLN-TTSM.
Until now, both preclinical and clinical data have revealed some structural and cellular alterations in mLN20. However, the dynamic changes of tumor-specific CD8+ T cells during LN metastasis have not been delineated. Therefore, developing a compelling model of LN metastasis is needed for further investigation. Indeed, several studies have reported mLN mouse models through different ways14,21,22. For example, spontaneous metastasis in axillary LNs was conducted through the implantation of 4T1 breast cancer cells into the mammary fat pad22. In another study, Reticker-Flynn et al. generated melanoma cell lines with high incidence of spread from subcutaneous primary tumor to LNs through serial inoculation of tumor cells cultured from dissociated mLN tissues (nine rounds)14. Another commonly used model was prepared by the injection of tumor cells into the footpad and the metastatic loci would be formed in popliteal LN22. Notably, it is difficult to evaluate the precise timepoints of intervention because LN metastasis in these models is not always faithful.
In the present study, a murine LN metastatic model was established through the intranodal injection of B16F10-GP cells23,24, generated by CRISPR/Cas9-mediated insertion of LCMV virus glycoprotein (GP) gene sequence into the genome of B16F10 cell line9. Then, these mice were transferred with P14 cells which harbor transgenic T cell receptors (TCRs) specifically recognize the H-2Db GP33-41 epitope25,26 and the systemic and local dynamics of antigen-specific CD8+ T cells during LN metastasis could be investigated. Our experimental design provides a useful model for the study of immune responses, especially the antigen-specific CD8+ T cells during the LN metastasis which excludes the perturbation of bystander CD8+ T cells. These results would affect the clinical treatment options of whether to remove or retain the mLN and shed new light on the manipulation of mLN to achieve maximum therapeutic benefits.

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			<td>PerCP/Cy5.5 anti-mouse CD8a&#160;</td>
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			<td>Clone:53-6.7</td>
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			<td>RPMI-1640</td>
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			<td>S12003-09&#160;</td>
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			<td>F34004-30&#160;</td>
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			<td>trypsin-EDTA</td>
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			<td>T4049-100ml</td>
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draining lymph node metastasis model for assessing the dynamics of antigen-specific cd8+ t cells during tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

The schematic diagram of this experimental design is shown in Figure 1A. A total of 5 x 105 B16F10-GP cells in 100 &#181;L of PBS were subcutaneously (s.c.) implanted into the bilateral inguinal region of CD45.2 C57BL/6J mice. After 7 days, these tumor-bearing mice were intraperitoneally (i.p.) injected with 4 mg CTX, followed by the adoptive transfer of 5 x 105 CD45.1+P14 cells through tail intravenous (i.v.) injection. When tumors grew to approximately 3-5 ...

Watch this Scientific Journal Video about Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis at JoVE.com

Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. ...

draining-lymph-node-metastasis-model-for-assessing-dynamics-antigen

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Immunology and Infection

1.5K Views.  Qingdao Agricultural University. The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Video: Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...