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Updated: Jul 31, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
CD69 on Tumor-Infiltrating Cells Correlates With Neuroblastoma Suppression by Simultaneous PD-1 and PD-L1 Blockade
Seiichiro Inoue1, Yuta Takeuchi1, Yutaka Horiuchi2
1Department of Hepato-Biliary-Pancreatic and Pediatric Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.
Insights
Immune checkpoint inhibitors targeting PD-1/PD-L1 suppressed neuroblastoma growth only in mice with T cells. Increased CD8+ tumor-infiltrating lymphocytes (TILs) correlated with reduced tumor weight, suggesting their importance in treatment efficacy.
Area of Science:
- Immunology
- Oncology
- Cancer Research
Background:
- Tumor-infiltrating cells, particularly lymphocytes (TILs), are crucial in antitumor immunity.
- Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are vital in cancer treatment.
Purpose of the Study:
- To investigate the role of T lymphocytes in immune checkpoint inhibition for mouse neuroblastoma.
- To analyze immune cells within the tumor microenvironment during PD-1/PD-L1 blockade.
Main Methods:
- Neuroblastoma cells (Neuro-2a) were injected into immune-deficient nude mice and immunocompetent A/J mice.
- Mice received anti-PD-1 and anti-PD-L1 antibodies, and tumor growth was monitored.
- Immune cells in tumors and spleens were analyzed via flow cytometry.
Main Results:
- Antibody treatment suppressed tumor growth in A/J mice but not in nude mice.
- Activated CD8+ TIL infiltration increased in smaller tumors (<300 mg) and correlated negatively with tumor weight.
- No significant changes were observed in regulatory T cells or activated CD4+ and CD8+ lymphocytes in spleen tissue.
Conclusions:
- Lymphocytes are essential for the antitumor immune response mediated by PD-1/PD-L1 blockade.
- Enhancing CD8+ TIL infiltration into tumors may represent a promising therapeutic strategy for neuroblastoma.
Introduction:
Tumor-infiltrating cells play an important role in tumor immunology, and tumor-infiltrating lymphocytes (TILs) are critical in antitumor reaction related to immune checkpoint inhibition targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1).
Methods:
In nude mice, which are immune deficient because they lack T cells, and inbred A/J mice, which are syngeneic to neuroblastoma cells (Neuro-2a) and have normal T cell function, we investigated the importance of T lymphocytes in immune checkpoint inhibition in mouse neuroblastoma and analyzed the immune cells in the tumor microenvironment. Then, we subcutaneously injected mouse Neuro-2ainto nude mice and A/J mice, administered anti-PD-1 and anti-PD-L1 antibodies by intraperitoneal injection, and evaluated tumor growth. At 16 d after Neuro-2a cells injection, mice were euthanized, tumors and spleens were harvested, and immune cells were analyzed by flow cytometry.
Results:
The antibodies suppressed tumor growth in A/J but not in nude mice. The co-administration of antibodies did not affect regulatory T cells (culster of differentiation [CD]4+CD25+FoxP3+ cells) or activated CD4+ lymphocytes (expressing CD69). No changes in activated CD8+ lymphocytes (expressing CD69) were observed in spleen tissue. However, increased infiltration of activated CD8+ TILs was seen in tumors weighing less than 300 mg, and the amount of activated CD8+ TILs was negatively correlated with tumor weight.
Conclusions:
Our study confirms that lymphocytes are essential for the antitumor immune reaction induced by blocking PD-1/PD-L1 and raises the possibility that promoting the infiltration of activated CD8+ TIL into tumors may be an effective treatment for neuroblastoma.
