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

This protocol provides a useful reproductive model for the studies of antigen-specific CD8+T cells during lymph node metastasis, which excludes the perturbation of bystander CD8+T cells. We tried to reveal the systemic and local dynamics of antigen-specific CD8+T cells during lymph node metastasis. Recent accumulating evidence revealed that tumor-specific TD8+cells derived from the periphery, especially in tumor draining lymph node but not in tumor microenvironment mediated the efficacy of immune checkpoint blockade therapy.

Recently, we identified TCF-1+TOX-tumor-specific memory T cells in tumor draining lymph nodes of both mice tumor models and human hepatocellular carcinoma patients, which serve as general responders to immune checkpoint blockade. It is difficult to evaluate the precise time points of intervention because lymph node metastasis through other techniques is not always feasible. This protocol has provided an approach to precisely investigate the antigen-specific CD8+T cell immune responses during lymph node metastasis, which excludes the perturbation of bystander CD8+T cells.

Our experimental design provides a useful model to investigate the antigen-specific CD8+T cells during lymph node metastasis and their interactions between antigen-specific CD8+T cells and other immune cells or stroma cells during lymph node metastasis. We will try to elucidate how lymph metastasis affects the anti-tumor immune response, especially the properties and the functions of TdLN-TTSM 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.

To begin, prepare a B16F10-GP melanoma cell suspension. Before aspirating the cell suspension, flick the tube wall to move the bubbles to the top. Using a 1 mL syringe, aspirate 100 L of the cell suspension and push the piston to move the top bubble out.

Next, hold the mouse in a supine position, exposing its abdomen. Press the left hind limb of the mouse with a finger to fully expose the skin in the left inguinal region. Shave the left lower abdomen of the mouse and clean it with cotton containing 75%ethanol.

Then, maintaining the angle of needle insertion at approximately 45 degrees with an upward incline, insert the needle in the subcutaneous tissue of the inguinal region of the upper left thigh at a depth of 0.5 to 1 cm. Insert the needle gently. At the same time, inject the cells slowly into the subcutaneous tissue.

Observe a small bolus, signifying the formation of a fluid pocket in the subcutaneous region. After injection, buckle the protective sleeve, dispose of it in the sharps box, and return the mouse to its cage. To perform adoptive transfer, check the tumor-bearing mice six to eight days after tumor implantation when the tumors are palpable.

On the day before the transfer, intraperitoneally inject 4 mg of cyclophosphamide. On the day of transfer, aspirate 200 L of P14 cell suspension using a 100-U insulin syringe. Remove bubbles from the syringe.

Next, place the mouse in a cage and irradiate it with an infrared lamp for 5 to 10 minutes to expand the tail vein. Then use a mouse fixator to hold the mouse in place. Straighten the tail and wipe it with 75%ethanol.

Insert the needle parallel to the tail vein and gently pull back the plunger. If there is no blood flowing into the syringe, slowly push the cell suspension into the vein. After the injection is completed, quickly pull out the needle.

Press the injection site gently with a cotton ball and return the mouse to a new clean cage. Closely observe the mouse for several minutes for any adverse effects. To begin, palpate the primary tumor and determine if the tumor is approximately 5 mm in diameter.

To perform tumor resection, place the mouse in a biosafety cabinet and position it on an anatomical board covered with clean absorbent paper. Orient the mouse in a supine position, so that its longitudinal axis is parallel to the experimenter. Disinfect the abdomen of the mouse using a cotton ball soaked in povidone iodine.

Next, using a sterile scalpel, make an incision in the skin near the tumor-bearing site. Insert the closed tip of sterile forceps into the incision to clearly expose the tumor. Remove the tumor, ensuring the capsule remains as intact as possible.

Carefully and gently remove the connective tissue adjacent to the tumor using sterile scissors. Begin by aspirating 20 L of B16F10-GP melanoma cell suspension using a 100-U insulin syringe. Remove bubbles from the syringe.

Next, in a mouse that underwent tumor resection surgery, inject the cell suspension into one inguinal lymph node. Insert the needle from the distal end of the lymph node and slowly move it to the center of the lymph node. Observe significant swelling of the lymph node, showing that the fluid is accurately injected.

Inject an equal volume of PBS into the inguinal lymph node on the other side. Suture the incision using a 3-0 suture. Disinfect the skin surrounding the wound with povidone iodine.

Then, place the mouse in a clean cage and maintain warmth using infrared light. Keep the mouse in the lateral decubitus position and monitor continuously until consciousness is recovered. After implantation with B16F10-GP cells, lymph node metastasis was observed by hematoxylin and eosin staining.

In the early stage, the metastatic lymph node showed partial occupation by tumor cells with some areas still containing unaffected lymphocytes. By the late stage, the metastatic lymph node was filled with tumor cells accompanied by tumor angiogenesis. Further, the frequency of antigen-specific CD8+T cells in peripheral blood was 2.81%at the early stage, which dropped to 1.48%at the late stage.

Interestingly, while the percentage of CD8+T cells remained stable in the non-metastatic lymph nodes, it was transiently boosted in the metastatic lymph node at the early stage, but sharply decreased at the late stage.