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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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.

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 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

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

Representative Results

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 .......

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

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
1.5 mL centrifuge tubeKIRGENKG2211
100 U insulin syringeBD Biosciences 320310
15 mL conical tube BEAVER 43008
2,2,2-Tribromoethanol (Avertin) Sigma T48402-25G 
2-Methyl-2-butanolSigma240486-100ML 
70 μm nylon cell strainerBD Falcon 352350
APC anti-mouse CD45.1 BioLegend 110714Clone:A20 
B16-GP cell lineBeijing Biocytogen Co.Ltd, ChinaCustom
BSA-V (bovine serum albumin) Biossbs-0292P
cell culture dishBEAVER 43701/43702/43703 
centrifugeEppendorf5810R-A462/5424R 
cyclophosphamideSigma C0768-25G 
Cyclophosphamide (CTX)SigmaPHR1404
Dulbecco's Modified Eagle Medium Gibco C11995500BT 
EDTASigmaEDS-500g 
FACS tubesBD Falcon352052
fetal bovine serum Gibco10270-106
flow cytometerBDFACSCanto II
hemocytometerPorLab ScientificHM330
isofluraneRWD 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 BiotechA501569-0500 
paraformaldehydeBeyotimeP0099-500ml 
PE anti-mouse TCR Vα2BioLegend127808Clone:B20.1 
Pen Strep Glutamine (100x)Gibco10378-016
PerCP/Cy5.5 anti-mouse CD8a BioLegend100734Clone:53-6.7
RPMI-1640SigmaR8758-500ML
sodium azideSigmaS2002 
surgical forcepsRWD Life Science F12005-10
surgical scissorsRWD Life Science S12003-09 
suture threadRWD Life ScienceF34004-30 
trypsin-EDTASigmaT4049-100ml

References

  1. Morad, G., Helmink, B. A., Sharma, P., Wargo, J. A. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 184 (21), 5309-5337 (2021).
  2. Korman, A. J., Garrett-Thomson, S. C., Lonberg, N.

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