Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Summary

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.

Abstract

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 GP_33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2D^b. 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.

Introduction

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 ICB

Protocol

The C57BL/6J mice (referred to B6 mice) and naive P14 transgenic mice^9,27 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

1. Prepare B16F10-GP melan.......

Discussion

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 therapy

Materials

Name	Company	Catalog Number	Comments
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