Pertussis toxin-induced lymphocytosis shifts the tumor microenvironment toward immune activation via CXCL9 signaling

A decrease in peripheral lymphocyte count in cancer patients predicts poorer prognosis and can be a useful discriminator in response to anti-tumor immunotherapy. However, therapeutic interventions to induce lymphocytosis and improve the efficacy of anti-tumor immunotherapy have not been investigated. In this study, we used pertussis toxin (PTX) to induce lymphocytosis; toxicity was minimal at controlled doses. Using an in vivo squamous cell carcinoma (SCC) model, the effects of PTX on anti-PD1 treatment efficacy were evaluated. To characterize the underlying changes associated with PTX pretreatment, we conducted immunohistochemistry, transcriptomic analysis, flow cytometry, and chemokine array analyses. PTX increased lymphocyte counts in SCC-tumor-bearing mice and shifted tumors from low to high immune scores. PTX-induced elevation of CXCL9 led to lymphocytosis and increased immune cell infiltration in tumor tissues. Combining PTX with an anti-PD1 antibody significantly suppressed in vivo SCC tumor growth compared with anti-PD1 treatment alone. The combination treatment induced a greater proportion of T cells in the tumor microenvironment, and these findings were associated with increased terminally differentiated CD8+ T cells and higher Ki-67 expression. In addition, tumor-specific IFN-γ production was augmented in animals treated with anti-PD1 antibody and combination treatments, but not with PTX alone. PTX can induce high lymphocyte infiltration into the tumor microenvironment, creating a favorable immune environment for immune checkpoint inhibitors. These PTX actions could enhance the efficacy of anti-PD1 treatment in an in vivo SCC model.