Tregs in malignant ascites from advanced EOC patients
Regulatory T cells (Tregs) have been reported to play an important role in the immune inhibitory network of EOC [15]. Subtype-specific biology of Tregs has been observed in some types of cancer but not in EOC [16] . Therefore, we investigated the prevalence of Tregs and the subtypes, based on the expression patterns of FoxP3 and CD45RA on CD4+ T cells, in malignant ascites from 41patients with advanced EOC. Fig. 1A shows the representative analysis pipeline for Tregs in ascites cells from advanced EOC. We examined the frequency of Tregs and their subtypes (CD45RA+FoxP3lo, naïve Tregs, CD45RA-FoxP3hi effector-type Tregs, and CD45RA- FoxP3lo as non-Tregs) among all CD4+ T cells in the ascites from each patient. As illustrated in Fig.1B, Tregs comprised 4.2% (range,0.2-14.5%) of all CD4+ T cells in ascites, and 0.2% (0-0.8%), 2.0% (0-11.4%) and 1.5% (0.1-6.3%) of CD4+ T cells were naïve Tregs, effector-type Tregs and non-Tregs, respectively.
Relationship between effector-type Tregs and clinicopathological factors in patients with advanced EOC
Recent reports have described the importance in evaluating effector-type Tregs rather than total Tregs [11] [16]. We queried whether there were any correlations between effector-type Tregs and clinical factors. Table1 summarizes the relationship between clinicopathological features and the proportion of effector-type Tregs among all CD4+ T cells in the ascites. We defined the patients who exhibited high or low frequency of effector-type Tregs based on the median value (2.0%). The effector-type Tregs were observed significantly higher frequency in patients with high-grade serous carcinoma (HGSC) compared with those in non‑serous histological types (P=0.042, Student's t‑test). No significant correlation was found between the patients with high frequency of effector-type Tregs and other clinicopathological variables.
Effector-type Tregs and outcomes in advanced EOC patients
We analyzed the potential association between the effector-type Tregs and outcomes in advanced EOC patients. Of the 41 patients included in the analysis, the median follow-up for all patients was 20 months. Based on the median value of effector-type Tregs among all CD4+ T cells, the patients with higher frequency for effector-type Tregs showed a trend for increased progression-free survival (PFS). However, there was survival difference in neither PFS (p=0.18; Fig.2A) nor overall survival (OS) (p=0.36; Fig.2B) in all EOC patients. Next, we analyzed in a subgroup of patients with HGSC because they had a higher frequency of effector-type Tregs than other histotypes (Table.I). No correlation was observed between the outcomes and effector-type Tregs in patients with HGSC, but with a trend toward better PFS for effector-type Tregs. (Fig.2C and D)
CCR4 expression on effector-type Tregs from advanced EOC patients
C-C chemokine receptor 4 (CCR4) has been reported to be predominantly expressed on the cell surface of effector-type Tregs, in both cancer tissues and peripheral blood from patients [11]. We investigated whether or not effector-type Tregs in malignant ascites from advanced EOC patients express CCR4. CCR4 expression was observed in effector-type Tregs (Fig. 3).
Association between the expression of immune checkpoint molecules on CD8+ T cells and effector-type Tregs in ascites
To elucidate the multiple immune-inhibitory networks in malignant ascites of EOC, we investigated the association between the expression of immune checkpoint molecules on CD8+ T cells and the frequency of Tregs in ascites. As shown in Fig.4, the expression of PD-1 (p=0.048) and TIM-3 on CD8+ T cells (p=0.0095) were significantly greater in patients with higher frequency of effector-type Tregs than in those with fewer effector-type Tregs, based on the median value. We next investigated the associations between the expression of each of the four immune checkpoint molecules (PD-1, TIM-3, LAG-3 and BTLA) and the frequency of effector-type Tregs (Supplementary Table I). We observed that 57.1% of the advanced EOC patients exhibiting high PD-1expression on the CD8+ T cells also showed a high frequency of effector-type Tregs in ascites, suggesting multiple immune-inhibitory mechanism in malignant ascites of advance EOC patients.