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

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

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

종양 형성 중 항원 특이적 CD8+ T 세포의 역학을 평가하기 위한 배액 림프절 전이 모델

마우스의 서혜부 림프절에서 B16F10-GP 세포의 결절 내 주입

100-U 인슐린 주사기를 사용하여 B16F10-GP 흑색종 세포 현탁액 20마이크로리터를 흡입하는 것으로 시작합니다. 주사기에서 기포를 제거합니다. 다음으로, 종양 절제 수술을 받은 마우스에서 세포 현탁액을 하나의 서혜부 림프절에 주입합니다.림프절의 말단부에서 바늘을 삽입하고 림프절 중앙으로 천천히 움직입니다. 림프절이 크게 부풀어 오르는 것을 관찰하여 체액이 정확하게 주입되었음을 알 수 있습니다. 반대쪽의 서혜부 림프절에 동일한 양의 PBS를 주입합니다.3-0 봉합사를 사용하여 절개 부위를 봉합합니다. 포비돈 요오드로 상처 주위의 피부를 소독한 다음 마우스를 깨끗한 케이지에 넣고 적외선을 사용하여 따뜻함을 유지합니다. 마우스를 측면 욕창 자세로 유지하고 의식이 회복될 때까지 지속적으로 모니터링합니다.B16F10-GP 세포에 이식한 후, 헤마톡실린 및 에오신 염색에 의해 림프절 전이가 관찰되었습니다. 초기 단계에서 전이성 림프절은 종양 세포에 의해 부분적으로 점유되어 있었고 일부 영역에는 여전히 영향을 받지 않은 림프구가 포함되어 있었습니다. 말기에 이르러 전이성 림프절은 종양 혈관신생을 동반한 종양 세포로 채워졌습니다.또한, 말초혈액 내 항원 특이적 CD8 양성 T세포의 빈도는 초기에 2.81%였으나 말기에는 1.48%로 떨어졌다. 흥미롭게도, CD8 양성 T 세포의 비율은 비전이성 림프절에서 안정적으로 유지되었지만, 초기에는 전이성 림프절에서 일시적으로 증가했으나 말기에는 급격히 감소했습니다.

Research

JoVE Journal

Immunology and Infection

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

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. ...

연구

면역학 및 감염병학

Ectopic Inoculation of B16F10-GP Melanoma Cells in Inguinal Region of Mice

마우스의 서혜부 영역에서 B16F10-GP 흑색종 세포의 이소성 접종

Adoptive Transfer of P14 T-Cells into Tumor-Bearing Mice

P14 T-세포를 종양이 있는 마우스로 입양 이식

Resection of the Primary Tumor From Mice

생쥐의 원발성 종양 절제