Immune cell analysis in equine penile papilloma, in situ squamous cell carcinoma and invasive squamous cell carcinoma: FoxP3+ T regulatory lymphocytes differ according to equine papillomavirus 2 status

Equine penile tumors are common in horses and are often related to infection with equine papillomavirus type 2 (EcPV2). This study investigated the immune cell infiltrate (ICI) of these tumors in horses, focusing on the role of EcPV2. Using multiplex immunohistochemistry (mIHC) for CD3, CD20, and IBA-1 and immunohistochemistry (IHC) for FoxP3, 27 horses with papillomas (5/27), in situ carcinomas (CISs) (3/27), and squamous cell carcinomas (SCCs) (19/27) were evaluated. Eighteen cases tested positive for EcPV2 by either or both in situ hybridization (ISH) and polymerase chain reaction (PCR) (18/27 by PCR, of which 16 were ISH+). The ICIs were more abundant in EcPV2-positive tumors, although differences were not statistically significant. The number of FoxP3+ regulatory T-cells was significantly higher in EcPV2+ tumors, both in intraepithelial and stromal compartments. There were higher IBA-1+ macrophage densities in SCCs than in papillomas or CISs. p53 IHC was performed, and non-basal positivity was associated with malignancy. The <i>TP53</i> mutational analysis with next-generation sequencing revealed that 13/21 cases had a wild-type <i>TP53</i>, while <i>TP53</i> variants were detected in 4/21 cases. The ICIs did not vary according to <i>TP53</i> status. Tumor proliferation was also assessed with Ki67, which indicated progressively higher proliferation from benign to malignant tumors. In conclusion, although the number and distribution of B-cells, T-cells, and macrophages did not vary according to EcPV2 status, FoxP3 regulatory T-cells were observed in significantly higher numbers in EcPV2+ neoplasms, indicating a different immune landscape compared to EcPV2-negative tumors.