intranodal injection of b16f10-gp cells in the inguinal lymph node of mice

评估淋巴结转移过程中抗原特异性 CD8+ T 细胞

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.

作者聚焦：研究 LN 转移期间 CD8+ T 细胞全身和局部动力学的模型

用于评估肿瘤发生过程中抗原特异性 CD8+ T 细胞动力学的引流淋巴结转移模型

This protocol provides a useful reproductive model for the studies of antigen specific CD8 T-cells. They are lymph node metastasis, which excludes the participation of bystander CD8 T-cells. We try to reveal the systemic and local dynamics of antigen specific CD8 T cells They are lymph node metastasis.Recent accumulating evidence revealed that tumor specific CD8 T cells derived from the periphery, especially in tumor draining lymph node, but not in tumor microenvironment met the efficacy of immune checkpoint blockage therapy. Recently we identified this in one positive talks negative tumor sub 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 blockage. It is difficult to evaluate the precise time points of intubation because lymph nodes metastasis through other techniques is not always feasible.This protocol has provided an approach to precisely investigate the antigen specific CD8 T-cells immune responses. The lymph node metastasis, which excludes the participation of bystander CD8 T cells. Our experimental design provides a useful model to investigate the antigen specific CD8 T-cells, the lymph node metastasis and interactions between antigen specific CD8 T cells and other immune cells or stroma cells during lymph node metastasis.We'll try to elucidate how lymph node metastasis affects the anti-tumor immune response, especially the properties and the functions of TDR and TTSM cells. This results would affect the clinical treatment options of whether to remove or retain the MLN and show the new light on the manipulation of MLN to achieve maximum therapeutic benefits.

在小鼠腹股沟淋巴结中结内注射B16F10-GP细胞

首先使用 100-U 胰岛素注射器吸出 20 微升 B16F10-GP 黑色素瘤细胞悬液。从注射器中去除气泡。接下来，在接受肿瘤切除手术的小鼠中，将细胞悬浮液注射到一个腹股沟淋巴结中。从淋巴结远端插入针头，然后缓慢移动到淋巴结的中心。观察淋巴结的明显肿胀，表明液体被准确注射。将等体积的PBS注射到另一侧的腹股沟淋巴结中。使用 3-0 缝合线缝合切口。用聚维酮碘对伤口周围的皮肤进行消毒，然后将小鼠放在干净的笼子中，并使用红外线保持温暖。将鼠标保持在侧卧位并持续监测，直到意识恢复。B16F10-GP细胞植入后，通过苏木精和伊红染色观察淋巴结转移。在早期，转移性淋巴结显示部分被肿瘤细胞占据，部分区域仍含有未受影响的淋巴细胞。到了晚期，转移性淋巴结充满了肿瘤细胞，并伴有肿瘤血管生成。此外，外周血中抗原特异性CD8阳性T细胞的频率在早期为2.81%，在后期下降到1.48%。有趣的是，虽然CD8阳性T细胞的百分比在非转移性淋巴结中保持稳定，但在早期转移性淋巴结中短暂增强，但在晚期急剧下降。

intranodal-injection-b16f10-gp-cells-inguinal-lymph-node

Research

JoVE Journal

Immunology and Infection

Intranodal Injection of B16F10-GP Cells in the Inguinal Lymph Node of Mice

167 次观看.  Qingdao Agricultural University. 首先使用 100-U 胰岛素注射器吸出 20 微升 B16F10-GP 黑色素瘤细胞悬液。从注射器中去除气泡。接下来，在接受肿瘤切除手术的小鼠中，将细胞悬浮液注射到一个腹股沟淋巴结中。从淋巴结远端插入针头，然后缓慢移动到淋巴结的中心。观察淋巴结的明显肿胀，表明液体被准确注射。将等体积的PBS注射到另一侧的腹股沟淋巴结中。使用 3-0 缝合线缝合切口。用聚维酮碘...

视频: 在小鼠腹股沟淋巴结中结内注射B16F10-GP细胞

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 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. ...