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.
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.
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.
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
2. Preparation of B16F10-GP cell suspension
3. Ectopic inoculation of B16F10-GP cells in the bilateral inguinal region of mice
4. Adoptive transfer of P14 T cells into tumor-bearing mice
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.
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 the other side.
The schematic diagram of this experimental design is shown in Figure 1A. A total of 5 x 105 B16F10-GP cells in 100 µL 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 mm in diameter (about 7 days after P14 cells transfer), primary tumors were resected, and 5 x 104 B16F10-GP cells in 20 µL of PBS were directly injected into unilateral inguinal lymph node. The inguinal lymph node on the other side was injected with equal volumes of PBS. Representative hematoxylin and eosin (H&E, 100x) staining of the non-metastatic lymph node (nLN) and metastatic lymph node (mLN) at indicated time points are shown in Figure 1B. The structure of nLN was intact. At the early stage of LN metastasis (D8), mLN was partially occupied with tumor cells (black arrow), and there is still some remaining area with lymphocytes that have not been invaded by tumor cells (red arrow). While at the late stage of LN metastasis (D18), mLN is filled with tumor cells accompanied by tumor angiogenesis and little lymphocytes. Activated P14 cells recovered in TdLN produced high level of IFN-γ after GP33-41 peptide stimulation9,31. Here, the percentages of activated P14 cells were analyzed through flow cytometry at different time points and the gating strategy is shown in Figure 2. The frequency of antigen-specific CD8+ T cells in peripheral blood at the early stage (D8) and late stage (D18) is 2.81% and 1.48%, respectively (Figure 3A). Tumor-specific CD8+ T cells have been reported to strictly reside in dLN during tumorigenesis and non-draining LN recovered limited donor cells33. The percentage of antigen-specific P14 cells in nLN was stable during LN metastasis. Intriguingly, antigen-specific CD8+ T cells in mLN were transiently boosted at the early stage, evidenced by the higher frequency of P14 cells compared to nLN, while it sharply decreased at the late stage (Figure 3B).
Figure 1: Schematic diagram of the experimental design. (A) C57BL/6J mice (CD45.2+) are implanted with 5 x 105 B16F10-GP tumor cells on the bilateral inguinal region. After 7 days, these mice are intraperitoneally injected with 4 mg CTX, and followed by the adoptive transfer of different congenically marked (CD45.1+) P14 cells the next day. When tumors grow to approximately 3-5 mm in diameter (about 7 days after P14 cells transfer), primary tumors are resected, then 5 x 104 B16F10-GP cells in 20 µL of PBS are directly injected into unilateral inguinal lymph node, and the inguinal lymph node on the other side is injected with equal volumes of PBS. (B) Representative hematoxylin and eosin (H&E,100x) staining of the LNs. Abbreviations: s.c. = subcutaneous; CTX= cyclophosphamide; i.v. = intravenous; Sac = sacrifice; nLN = non-metastatic LN; mLN = metastatic LN. Please click here to view a larger version of this figure.
Figure 2: Gating strategy for flow cytometry analysis. Gating strategy used to identify donor-derived activated antigen-specific CD8+ T cells. Abbreviations: L/D = live/dead. Please click here to view a larger version of this figure.
Figure 3: Dynamics of antigen-specific CD8+ T cells during LN metastasis. The proportion of antigen-specific CD8+ T cells in (A) peripheral blood, (B) nLN and mLN at different time points. Abbreviations: nLN = non-metastatic LN; mLN = metastatic LN. Please click here to view a larger version of this figure.
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 therapy34,35. Consistent with this, we recently found that tumor-specific memory CD8+ T cells (TTSM) predominantly reside in TdLN, these TdLN-TTSM cells serve as bona fide responders to ICB9. Unfortunately, several types of cancer cells often spread to TdLNs from primary tumor sites, which lead to structural reconfirmation and immune cell dysfunction in TdLNs. The impaired quantity and quality of CD8+ T cells in mLNs has already been described20,36. However, the dynamic changes of CD8+ T cells, especially the tumor antigen-specific CD8+ T cells during the LN metastatic cascade have not been elucidated.
Here, we developed a convenient mouse model to monitor the dynamics of systemic and local antigen-specific CD8+ T cells during the process of LN metastasis. B16F10-GP melanoma cells were subcutaneously implanted into the bilateral inguinal region, followed by adoptively transferring with P14 cells which could be specifically activated by the GP-derived peptide GP33-41. At day 7 post cell transfer when P14 cells in inguinal LNs were fully activated, primary tumors in both sides were resected and B16F10-GP cells were directly injected into unilateral inguinal LN, sham operation on the other side was performed through the injection with equal volumes of PBS. This ingenious design enables the comparison of P14 cells in mLN and non-metastatic LN (nLN) within the same mice. Simultaneously, P14 cells in periphery blood within the same mice were detected at different time points during LN metastasis by bleeding the orbital vein. Apart from the frequencies of P14 cells in periphery blood and TdLNs at different stages during LN metastasis, the transcriptional and epigenetic properties of these antigen-specific CD8+ T cells could be further examined with other techniques.
Of note, several steps should be performed cautiously. Firstly, the primary tumors should be excised thoroughly, as the residual tumor cells would regrow rapidly. Secondly, the operation should be performed gently, as tumor tissues contain abundant new blood vessels which are usually inevitably broken during primary tumor resection and lead to the death of surgical mice due to massive hemorrhage. Therefore, the timing of primary tumor resection is critically important. Generally, it is relatively safe to remove it when the tumor grows to the size about 5 x 5 mm, and tumor cells should be precisely injected into LN rather than the bottom or adjacent tissues. Moreover, the volume of tumor cell suspensions should be controlled below 20 µL, otherwise spill would form new tumor outside the LN. Lastly, a limitation of this protocol is that the operation is traumatic, and mice may experience infection during healing process, which would affect the properties of antigen-specific CD8+ T cells. Therefore, it is critically important to maintain strict asepsis during the surgery and sham-operated PBS injection should be done to exclude the impact of injection-induced physical damage to the antigen-specific CD8+ T cells.
Overall, we provide a convenient model to investigate the antigen-specific CD8+ T cells during LN metastasis and the interactions between antigen-specific CD8+ T cells and other immune cells or stroma cells during LN metastasis could also be elucidated. In addition, it could be easily extended to several other tumor models. Collectively, this protocol offers a useful reproductive model for the study of cancer immunology.
The authors have nothing to disclose.
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).
1.5 mL centrifuge tube | KIRGEN | KG2211 | |
100 U insulin syringe | BD Biosciences | 320310 | |
15 mL conical tube | BEAVER | 43008 | |
2,2,2-Tribromoethanol (Avertin) | Sigma | T48402-25G | |
2-Methyl-2-butanol | Sigma | 240486-100ML | |
70 μm nylon cell strainer | BD Falcon | 352350 | |
APC anti-mouse CD45.1 | BioLegend | 110714 | Clone:A20 |
B16-GP cell line | Beijing Biocytogen Co.Ltd, China | Custom | |
BSA-V (bovine serum albumin) | Bioss | bs-0292P | |
cell culture dish | BEAVER | 43701/43702/43703 | |
centrifuge | Eppendorf | 5810R-A462/5424R | |
cyclophosphamide | Sigma | C0768-25G | |
Cyclophosphamide (CTX) | Sigma | PHR1404 | |
Dulbecco's Modified Eagle Medium | Gibco | C11995500BT | |
EDTA | Sigma | EDS-500g | |
FACS tubes | BD Falcon | 352052 | |
fetal bovine serum | Gibco | 10270-106 | |
flow cytometer | BD | FACSCanto II | |
hemocytometer | PorLab Scientific | HM330 | |
isoflurane | RWD life science | R510-22-16 | |
KHCO3 | Sangon Biotech | A501195-0500 | |
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation | Life Technologies | L10199 | |
needle carrier | RWD Life Science | F31034-14 | |
NH4Cl | Sangon Biotech | A501569-0500 | |
paraformaldehyde | Beyotime | P0099-500ml | |
PE anti-mouse TCR Vα2 | BioLegend | 127808 | Clone:B20.1 |
Pen Strep Glutamine (100x) | Gibco | 10378-016 | |
PerCP/Cy5.5 anti-mouse CD8a | BioLegend | 100734 | Clone:53-6.7 |
RPMI-1640 | Sigma | R8758-500ML | |
sodium azide | Sigma | S2002 | |
surgical forceps | RWD Life Science | F12005-10 | |
surgical scissors | RWD Life Science | S12003-09 | |
suture thread | RWD Life Science | F34004-30 | |
trypsin-EDTA | Sigma | T4049-100ml |