Introduction/Background The mechanisms of innate immune response and the immune checkpoint (IC) system-governed mechanisms are the two interconnected areas currently becoming the focus of world research in view of the prospects for therapeutic reactivation of antitumor immunity. This may be especially relevant to virus-associated cancers (as, for example, most cases of cervical cancer), which are known to develop as a result of persistent infection, long-term antigen exposure and are frequently characterized by chronically inflamed environment and local and/or systemic immunosuppression. Studying the immune changes that accompany the earliest stages of tumor progression may provide insight into the mechanisms driving the onset of metastasis; and, in this respect, systemic changes observed in peripheral blood (PB) may possibly reflect local dysfunctions at the tumor site. In case of cervical cancer, these assumptions need more extensive research evidence.

Methodology To characterize the changes in the local microenvironment, we compared the transcriptomes of the early invasive squamous cell cervical cancer and its precursor high-grade lesions by performing RNA-sequencing and bioinformatics analysis on a panel of 12 fresh tissue samples comprising HPV(+) cervical intraepithelial neoplasia 3 (CIN3) and carcinoma at FIGO IA1-IIB stages, plus normal epithelium. PB samples were obtained from women with CIN3 and stage IA cancer immediately prior to treatment; PB from healthy women was used as control. Subsets of PB lymphocytes were phenotyped using multicolor flow cytometry.

Results Among the differentially expressed genes identified, there were a considerable number of genes that, according to Gene Ontology, are responsible for inflammatory and innate immune responses (including interferon type I and II pathways) and belong to the system of self/non-self DNA/RNA recognition, with multiple anti-inflammatory factors found to be down-regulated, while a spectrum of interferon-stimulated genes, anti-viral/anti-microbial factors, pro-inflammatory cytokines, as well as markers of immune suppression were found up-regulated in invasive cancer. Accordingly, ‘Influenza A’ and ‘Pyrimidine metabolism’ KEGG pathways appeared to be significantly enriched. SPEED enrichment analysis revealed the TLR-, TNF alpha-, and IL1-dependent signalings among the top pathways lying behind the alterations of gene expression patterns observed at the initial stage of invasion. PPI network analysis confirmed close interrelation of differently expressed genes encoding molecular components of inflammatory response and virus recognition system. Among the circulating lymphocyte functional markers that may mirror the described immune alterations, the expression of CD161 in iNKT/NK-like T/NK cells (defined by CD3/CD56, Va24Ja18/Vb11-TCR, and CD4/CD8), the level of CD27 and delta2/delta1 ratio in Tgammadelta subpopulation (defined by CD3/TCRgd), and co-expression pattern of PD1/PDL1/LAG3/TIM3 in 4 subsets of CD4/CD8 T cells defined by the level of CD25/CD127, as well as in NK/NKT cells, were measured, and specific correlated differences between the control and cancer groups and between different lymphocyte populations were detected.

Conclusion The findings suggest deep involvement of the inflammation-associated and IC-mediated mechanisms and coordinate contribution of various T cell subsets with innate-like properties in initiation and promotion of invasive growth of cervical carcinoma both at local and systemic levels.

Disclosures The study was supported by the state assignment of the Ministry of Science and Higher Education, project No.0752-2020-0007 (AAAA-A20-120070290151-6). The authors declare no conflicts of interest.