Seung Hyuck Jeon¹, Minyong Kang^2,3,4, Minwoo Jeon¹, Youseung Chung¹, A Reum Kim¹, Yong Joon Lee¹, Eui-Soon Kim¹, Heejin Nam¹, Junsik Park⁵, Jung-Yun Lee⁵, Eui-Cheol Shin¹, Seong Il Seo², and Su-Hyung Park¹

¹Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. 
²Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 
³Samsung Genome Institute, Samsung Medical Center, Seoul, Republic of Korea. 
⁴Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea. 
⁵Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul, Republic of Korea.

S.H. Jeon and M. Kang contributed equally to this article.
Corresponding Authors: Su-Hyung Park; Seong Il Seo; and Eui-Cheol Shin

Purpose: Regulatory T cells (Tregs) exert immunosuppressive functions and hamper anti-tumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies.

Experimental design: From 42 patients with renal cell carcinoma and 5 patients with ovarian cancer, immune cells from tumor and peripheral blood were isolated. We performed multicolor flow cytometry and RNA-sequencing to characterize the phenotypes and heterogeneity of intratumoral Tregs. In vitro functional assays were performed to evaluate suppressive capacity of Tregs and effect of CEACAM1-mediated depletion. The CT26 tumor model was used to evaluate the association between intratumoral Tregs and tumor growth, and examine the in vivo role of CEACAM1+ intratumoral Tregs on anti-tumor immunity.

Results: We found that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was selectively expressed on intratumoral Tregs, while its expression on peripheral Tregs or other immune cells was low. The CEACAM1+ intratumoral Tregs accumulated with tumor progression, while the CEACAM1- subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of tumor-infiltrating T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1--mediated reinvigoration of exhausted CD8+ T cells.

Conclusions: CEACAM1 marks highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs. These results may inform future studies on CEACAM1-mediated depletion in cancer patients.