HPV16 E7-Containing Exosomes May Contribute to PD-L1 Expression in Tumor-Infiltrating Lymphocytes of Cervical Cancer.

BACKGROUND

Human papillomavirus 16 (HPV16) plays a major role in the development of cervical cancer (CC). Programmed cell death ligand 1 (PD-L1) expression in tumor-infiltrating lymphocytes (TILs) is an important feature of the tumor immune microenvironment and may affect responses to immune checkpoint blockade. However, the relationship between the HPV16 E7 oncoprotein and PD-L1 expression in TILs remains unclear.

AIM: To investigate the presence of HPV16 E7 protein in exosomes from CC tissues and plasma, determine the association between exosomal E7 and PD-L1 expression in TILs, and explore whether E7-rich tumor-derived exosomes contribute to increased PD-L1 expression in TILs.

STUDY

DESIGN

This study combined clinical observational analyses and in vitro experiments. Three CC single-cell RNA sequencing datasets, a 30-patient HPV16-positive cohort, and a 104-patient tissue microarray cohort were analyzed. In vitro experiments were conducted to further examine the potential relationship between exosomal E7 and PD-L1 expression in TILs.

METHODS

Blood and pathological samples from 30 patients with HPV16-positive CC, as well as culture fluid from the C33A and SiHa CC cell lines, were collected. Exosomes were isolated, and E7 protein expression was assessed. Bioinformatics analyses of CC single-cell RNA sequencing datasets were performed to evaluate CD274 mRNA expression in TILs. Immunohistochemistry (IHC) was used to assess PD-L1 protein expression in TILs in the 30-patient cohort, while multiplex immunofluorescence was used to evaluate E7 and PD-L1 expression in TILs in a 104-patient tissue microarray. Exosome uptake by TILs was assessed using confocal microscopy. TILs were co-cultured with exosomes, and HPV16 E7 and PD-L1 expression was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting in the TIL blank control, co-culture blank control, co-culture empty vector, and co-culture E7-overexpression groups.

RESULTS

E7 protein was detected in exosomes from the peripheral blood of 28 of 30 patients (93.3%) and in exosomes from SiHa cell culture. PD-L1 was expressed in TILs, with a positive rate of 43.3% (13/30). In the tissue microarray cohort (n = 103; 28 events), higher CD3+PD-L1+/CD3+ expression was associated with poorer survival (HR = 2.97, p = 0.009). CD3+E7+/CD3+ expression showed a borderline association with poorer survival in the dichotomized analysis (HR = 2.15, p = 0.052) but was significantly associated with survival when modeled as a continuous variable (per 10%: HR = 1.28, p = 0.002). In the 30-patient cohort, exosomal E7 protein content was significantly associated with PD-L1 expression in TILs (Cramér's V = 0.728, p = 0.0001). qRT-PCR and western blotting demonstrated increased PD-L1 mRNA and protein expression in TILs exposed to exosomes from E7-overexpressing cells.

CONCLUSION

HPV16 E7 protein is present in exosomes from patients with CC and HPV16-positive CC cells. Higher exosomal E7 levels are associated with increased PD-L1 expression in TILs, and exposure to E7-rich exosomes is associated with upregulated PD-L1 expression in TILs. These findings suggest that exosomal E7 may contribute to PD-L1 regulation in TILs and may be associated with poorer prognosis in patients with CC. Further studies are needed to determine whether E7 directly mediates this effect and to clarify its functional and clinical significance.