Sanghee Shin ^a,1, Inseong Jung ^a,1, Dokyung Jung ^c, Christine Seulki Kim ^a, Sung-Min Kang ^c, Suyeon Ryu ^c, Sung-Jin Choi ^a, Soojeong Noh ^a, Jongwon Jeong ^a, Beom Yong Lee ^a, Jun-Kook Park ^a, Jiwon Shin ^a, Hanchae Cho ^c, Jong-Ik Heo ^a, Youngtae Jeong ^a, Sun Ha Choi ^d,e, Shin Yup Lee ^d,e, Moon-Chang Baek ^c,*, Kyungmoo Yea ^a,b,*

^a Department of New Biology, DGIST, Daegu, 42988, Republic of Korea ^b New Biology Research Center, DGIST, Daegu, 43024, Republic of Korea ^c Department of Molecular Medicine, CMRI, Exosome Convergence Research Center (ECRC), School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea ^d Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea ^e Lung Cancer Center, Kyungpook National University Chilgok Hospital, Daegu, 41404, Republic of Korea

^* Corresponding author.
¹ S. Shin and I. Jung contributed equally to this work.

Extracellular vesicles (EVs) mediate cell-cell crosstalk by carrying bioactive molecules derived from cells. Recently, immune cell-derived EVs have been reported to regulate key biological functions such as tumor progression. CD4⁺ T cells orchestrate overall immunity; however, the biological role of their EVs is unclear. This study reveals that EVs derived from CD4⁺ T cells increase the antitumor response of CD8⁺ T cells by enhancing their proliferation and activity without affecting regulatory T cells (Tregs). Moreover, EVs derived from interleukin-2 (IL2)-stimulated CD4⁺ T cells induce a more enhanced antitumor response of CD8⁺ T cells compared with that of IL2-unstimulated CD4⁺ T cell-derived EVs. Mechanistically, miR-25-3p, miR-155-5p, miR-215-5p, and miR-375 within CD4⁺ T cell-derived EVs are responsible for the induction of CD8⁺ T cell-mediated antitumor responses. In a melanoma mouse model, the EVs potently suppress tumor growth through CD8⁺ T cell activation. This study demonstrates that the EVs, in addition to IL2, are important mediators between CD4⁺ and CD8⁺ T cells. Furthermore, unlike IL2, clinically used as an antitumor agent, CD4⁺ T cell-derived EVs stimulate CD8⁺ T cells without activating Tregs. Therefore, CD4⁺ T cell-derived EVs may provide a novel direction for cancer immunotherapy by inducing a CD8⁺ T cell-mediated antitumor response.