Introduction Tumour-infiltrating lymphocytes (TIL) are an important prognostic indicator in high-grade serous ovarian carcinoma (HGSC). Certain types of TIL (in particular CD8+ effector T cells) predict better outcomes, whereas others (most notably CD4+CD25+FoxP3+ regulatory T cells; Tregs) predict worse outcomes. An unconventional subset of CD8+FoxP3+ T cells has been reported to be involved in autoimmunity and in immune response to several cancers. While the functional significance of CD8+FoxP3+ TIL remains poorly understood, they were associated with effective anti-tumour responses in a murine tumour model. Hypothesis CD8+FoxP3+ TIL are present in a subset of cases of HGSC and correlate with a strong immune response and increased patient survival. Experimental Design Multi-colour immunohistochemistry (IHC) was performed on a cohort of 44 primary HGSC specimens to enumerate and locate CD8+FoxP3+ TIL in comparison to CD8+FoxP3- and CD8-FoxP3+ TIL. Triple-colour IHC methodology was developed to further assess the phenotype of CD8+FoxP3+ TIL, including the measurement of additional markers CD4 and CD25 (classical markers of Tregs), Ki-67 (a marker of proliferation), and TIA-1 (a marker of cytotoxic potential). Intraepithelial versus stromal location was determined by staining adjacent sections for the epithelial marker pan-cytokeratin. Survival analysis was performed using a cohort of 188 cases of HGSC. Multi-colour staining was resolved using the Nuance™ multispectral imaging system in conjunction with Metamorph™ software. Survival analysis was performed using Kaplan-Meier and log rank tests. Results CD8+FoxP3+ cells were found in 60% of 44 cases of HGSC, in variable proportions ranging from 0.2 - 7.9% of CD8+ TIL and 0.5 – 12.7% of FoxP3+ TIL. CD8+FoxP3+ TIL were found to be either CD4+ (38.8%) or CD4- (61.2%). The majority of CD8+FoxP3+ TIL were also found to be CD25-TIA-1+Ki-67-, more closely resembling their CD8+FoxP3- counterparts. CD8+FoxP3+ TIL were found mainly in intraepithelial regions and were positively associated with patient survival (progression free survival; P = 0.0396). Conclusions CD8+FoxP3+ TIL are a component of the host immune response to HGSC. They appear to have a non-proliferative effector phenotype, consistent with an active role in the anti-tumour response. CD8+FoxP3+ TIL are associated with increased patient survival. An improved understanding of this new TIL subset may inform future immunotherapeutic strategies for this challenging malignancy. / Graduate / 0982 / sarakost@hotmail.com