Myeong Joon Kim  ^1,2,8, Kyungsoo Kim^2,3,8, Hyo Jin Park¹, Gil-Ran Kim^4,5, Kyeong Hee Hong^1,2, Ji Hoon Oh^1,2, Jimin Son^1,2, Dong Jin Park^1,2, Dahae Kim^1,2, Je-Min Choi^4,5,6,7, Insuk Lee  ^2,3 & Sang-Jun Ha  ^1,2

¹Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea. 
²Brain Korea 21 (BK21) FOUR Program, Yonsei Education & Research Center for Biosystems, Yonsei University, Seoul, Republic of Korea. 
³Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea. 
⁴Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Republic of Korea. 
⁵Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea. 
⁶Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea. 
⁷Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, Republic of Korea. 
⁸These authors contributed equally: Myeong Joon Kim, Kyungsoo Kim.  

Corresponding authors: Correspondence to Insuk Lee or Sang-Jun Ha.

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP−Cre-ERT2(+/−) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.