CD69 on Tumor-Infiltrating Cells Correlates With Neuroblastoma Suppression by Simultaneous PD-1 and PD-L1 Blockade

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).

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.

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.

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.