Published: June 12th, 2021
ERRATUM NOTICEImportant: There has been an erratum issued for this article. Read more …
Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.
T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.
CD8+ T cell-mediated immune response plays a pivotal role in controlling tumor growth. During tumorigenesis, naive CD8+ T cells get activated upon antigen recognition in an MHC class I-restricted manner and subsequently differentiate into effector cells and infiltrate into tumor mass1,2. However, within the tumor microenvironment (TME), prolonged antigen exposure, as well as immunosuppressive factors, drive infiltrated tumor-specific CD8+ T cells into a hyporesponsive state known as "exhaustion"3. Exhausted T cells (Tex) are distinct from effecto....
All mouse experiments were performed in compliance with the guidelines of the Institutional Animal Care and Use Committees of the Third Military Medical University. Use 6-8-week-old C57BL/6 mice and naïve OT-I transgenic mice weighing 18-22 g. Use both male and female without randomization or "blinding."
1. Preparation of medium and reagents
The schematic of this protocol is shown in Figure 1. Eight days after tumor inoculation, CD45.1+ and CD45.1+CD45.2+ OT-I cells were injected into B16F10-OVA tumor-bearing C57BL/6 mice. The tumor was surgically dissected from CD45.1+ OT-I cell-implanted mice (donor) on day 8 post-transfer and transplanted into tumor-matched CD45.1+CD45.2+ OT-I cell-implanted mice (recipient) in the dorsal flank on the same side as the implanted.......
T cell-mediated immunity plays a crucial role in immune responses against tumors, with CTLs playing the leading role in eradicating cancerous cells. However, the origins of tumor antigen-specific CTLs within TME have not been elucidated30. The use of this tumor transplantation protocol has provided an important clue that intratumoral antigen-specific CD8+ T cells may not persist for a long time, despite the existence of stem-like TCF1+ progenitor CD8+ T cells. Nota.......
This study was supported by grants from the National Natural Science Fund for Distinguished Young Scholars (No. 31825011 to LY) and the National Natural Science Foundation of China (No. 31900643 to QH, No. 31900656 to ZW).....
|0.22 μm filter
|1 mL tuberculin syringe
|1.5 mL centrifuge tube
|100 U insulin syringe
|15 mL conical tube
|70 μm nylon cell strainer
|APC anti-mouse CD45.1
|B16F10-OVA cell line
|BSA-V (bovine serum albumin)
|BV421 Mouse Anti-Mouse CD45.2
|cell culture dish
|Dulbecco's Modified Eagle Medium
|EasySep Mouse CD8+ T Cell Isolation Kit
|fetal bovine serum
|RWD life science
|LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation
|RWD Life Science
|PE anti-mouse TCR Vα2
|Pen Strep Glutamine (100x)
|PerCP/Cy5.5 anti-mouse CD8a
|RWD Life Science
|RWD Life Science
|RWD Life Science
ErratumErratum: Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
An erratum was issued for: Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice. The Protocol was updated.
Step 6.10 of the Protocol was updated from:
Administer penicillin every 8-12 h after the surgery for 3 days. Monitor the mouse's eating, drinking, moving, and the area operated on. Return the transplant recipient to the company of other animals only after it has fully recovered.
NOTE: The administration of buprenorphine is suggested to prevent post-surgical pain [delete sentence]. The mouse typically recovers from the trauma of the surgery within 3 days. If the mouse is not back to normal feeding and mobility and shows any manifestations of infection, consult a veterinarian for interventions or euthanize it.
Administer buprenorphine subcutaneously at a dose of 0.1 mg/kg body weight every 8 h three times after surgery to alleviate the pain. Monitor the mouse's eating, drinking, moving, and the area operated on. Return the transplant recipient to the company of other animals only after it has fully recovered.
NOTE: The mouse typically recovers from the trauma of the surgery within 3 days. If the mouse is not back to normal feeding and mobility and shows any manifestations of infection, consult a veterinarian for interventions or euthanize it.
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