Abstract 5613: PD-1 and TIGIT are major immune checkpoint receptors expressed in breast cancer-infiltrating T cells
Authors: Kim Jee Ye , Kwon Minsuk , Lim Sung Mook , Kim Joo Heung , Park Hyung Seok , Park Seho , Kim Seung Il , Cho Young Up , Paik Soonmyung , Shin Eui-Cheol 
Published: July 2017

Abstract
Immune checkpoint blockers, which target co-inhibitory receptors of T cells, have provided promising responses against various tumors. However, the expression of immune checkpoint receptors (ICRs) in breast cancer remains poorly understood. We aimed to investigate the expression pattern of multiple ICRs in tumor-infiltrating and peripheral blood (PB) lymphocytes in human breast cancer.
We isolated lymphocytes from fresh breast tumor and paired PB from 21 patients who underwent surgery between July 2016 and November 2016. Multi-color flow cytometry was performed primarily focusing on expression of multiple ICRs in CD8+ T cells and regulatory T cells (Tregs).
In CD8+ T cell subsets, PD-1+ or TIGIT+ cells were more frequently observed in tumor-infiltrating CD8+ T cells than in PB CD8+ T cells (p&lt;0.0001 and p=0.02, respectively). Around half of the tumor infiltrating CD8+ T cells expressed PD-1 (median 58.20%) and TIGIT (45.95 %). In contrast, LAG3 and TIM3 were hardly detected in both tumor-infiltrating and PB CD8+ T cells. Intriguingly, about 40% of tumor-infiltrating CD8+ T cells were PD-1+TIGIT+, and &gt;70% of PD-1+ tumor-infiltrating CD8+ T cells co-expressed TIGIT, indicating that functional exhaustion of breast tumor-infiltrating CD8+ T cells might be mediated not by PD-1 alone but by multiple receptors including TIGIT. However, the expression of PD-1 and TIGIT showed different patterns in detail. PD-1 was frequently expressed by CCR7-CD45RA- effector memory T cells (TEM) (p=0.001) whereas TIGIT was frequently expressed by CCR7-CD45RA+ effector memory RA T cells (TEMRA) (p=0.03), suggesting that TIGIT is expressed during terminal differentiation of CD8+ T cells.
Next, we examined breast tumor-infiltrating Tregs. The frequency of CD25+FoxP3+ Tregs among CD4+ T cells were higher in tumor than in PB (16.60% vs. 7.86%; p=0.002). In particular, CD45RA-FoxP3hi activated suppressive Tregs account for 77.6% of tumor-infiltrating Tregs (vs. PB 32.2%; p=0.005). Tumor-infiltrating Tregs showed higher expression of CD39 (p=0.005), a marker of the suppressive activity of Tregs. We also examined the expression of ICRs on Tregs as upregulation of these receptors is associated with enhanced suppressive activity of Tregs. CD4+CD25+FoxP3+ Tregs in tumors showed higher expression levels of PD-1, TIGIT and CTLA-4 compared to PB Tregs (p&lt;0.05).
There were no noteworthy correlations between ICR expression and clinical features, such as age, stage and subtype.
We show that PD-1 and TIGIT are major ICRs expressed in breast tumor-infiltrating CD8+ T cells. Moreover, we found that CD4+CD25+FoxP3+ Tregs are abundant in breast tumors and overexpress PD-1, TIGIT and CTLA-4. Our data provide an understanding of comprehensive phenotypes of immune checkpoint expression on T cells in breast cancer. Functional changes of CD8+ T cells and Tregs by blocking of single or multiple ICRs are being investigated.
Citation Format: Jee Ye Kim, Minsuk Kwon, Sung Mook Lim, Joo Heung Kim, Hyung Seok Park, Seho Park, Seung Il Kim, Young Up Cho, Soonmyung Paik, Eui-Cheol Shin. PD-1 and TIGIT are major immune checkpoint receptors expressed in breast cancer-infiltrating T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5613. doi:10.1158/1538-7445.AM2017-5613