Local immunosuppressive microenvironment underlies reduced responsiveness to imiquimod treatment in recurrent or residual cervical HSIL

Objective: The topical immune modifier imiquimod is used as alternative treatment for cervical high-grade squamous intraepithelial lesions (cHSIL). While its clinical efficacy in primary cHSIL (pcHSIL) is approximately 60%, this is only 33% for recurrent or residual cHSIL (rrcHSIL) due to reasons that are not well understood. Since the immune microenvironment plays a key role in response to imiquimod in pcHSIL, we performed an in depth comparison of the pcHSIL to the rrcHSIL immune microenvironment. Methods: Transcriptome analysis was performed using the nCounter® Human PanCancer IO360™ panel on 6 pcHSIL and 6 rrcHSIL. Multispectral immunofluorescence (13 markers) was used to analyze T- and myeloid-cell composition of pcHSIL (n = 40) and rrcHSIL (n = 10). Differences in pre-treatment immune infiltrates of rrcHSIL were related to clinical response after imiquimod. Results: Transcriptomic analyses revealed lower expression of genes involved in leukocyte activation, immune cell recruitment, T cell engagement, lymphocyte and B cell infiltration in rrcHSIL compared to pcHSIL. Gene set enrichment analysis (GSEA) supported these findings, demonstrating decreased IL2-STAT 5 signaling in rrcHSIL. Immunofluorescence staining results corroborated the transcriptomic data, with rrcHSIL displaying lower intraepithelial infiltration with CD4+(Tbet + ) T cells, and (CD14 + )CD68 + CD163- M1-like macrophages, but higher numbers of CD8+(PD1 + ) T cells and CD68 + CD163 + M2-like macrophages. RrcHSIL non-responders to imiquimod exhibited an even more immunosuppressive microenvironment than complete responders, characterized by increased infiltration of CD4 + FoxP3 + regulatory T cells and (CD14 + )CD68 + CD163 + M2-like macrophages and CD14 + HLADR- monocytic myeloid derived suppressor cells. Conclusions: The limited efficacy of imiquimod in rrcHSIL may be explained by a strong immunosuppressive microenvironment.