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

Модель метастазирования дренирующих лимфатических узлов для оценки динамики антиген-специфических CD8+ Т-клеток в процессе онкогенеза

Внутриузловое введение клеток B16F10-GP в паховый лимфатический узел мышей

Начните с аспирации 20 микролитров суспензии клеток меланомы B16F10-GP с помощью инсулинового шприца 100-U. Удалите пузырьки из шприца. Далее мыши, перенесшей операцию по резекции опухоли, вводят клеточную суспензию в один паховый лимфатический узел.Введите иглу из дистального конца лимфатического узла и медленно переместите ее к центру лимфатического узла. Наблюдают значительное увеличение лимфатического узла, показывающее, что жидкость введена точно. Введите равный объем PBS в паховый лимфатический узел с другой стороны.Зашить разрез шовным материалом 3-0. Продезинфицируйте кожу вокруг раны повидон-йодом, затем поместите мышь в чистую клетку и поддерживайте тепло с помощью инфракрасного света. Держите мышь в положении бокового пролежня и непрерывно наблюдайте до тех пор, пока сознание не восстановится.После имплантации клетками B16F10-GP метастазирование в лимфатические узлы наблюдалось по окрашиванию гематоксилином и эозином. На ранней стадии метастатический лимфатический узел был частично занят опухолевыми клетками, при этом некоторые участки все еще содержали непораженные лимфоциты. К поздней стадии метастатический лимфатический узел был заполнен опухолевыми клетками, что сопровождалось опухолевым ангиогенезом.Кроме того, частота антиген-специфических CD8-положительных Т-клеток в периферической крови составила 2,81% на ранней стадии, которая снизилась до 1,48% на поздней стадии. Интересно, что в то время как процент CD8-положительных Т-клеток оставался стабильным в неметастатических лимфатических узлах, он временно повышался в метастатическом лимфатическом узле на ранней стадии, но резко снижался на поздней стадии.

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. ...

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

Эктопическая инокуляция клеток меланомы B16F10-GP в паховой области мышей

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

Адоптивный перенос P14 Т-клеток мышам-носителям с опухолями

Resection of the Primary Tumor From Mice

Резекция первичной опухоли у мышей