A Single Cell Transcriptomic Atlas of CD8+ Cytokine-Induced Killer Cells Generated from Metastatic Colorectal Cancer Patients

Abstract Cytokine-induced killer (CIK) cell therapy is an adoptive cell immunotherapy under investigation for malignancies including metastatic colorectal cancer (mCRC). Clinical activity has been attributed primarily to CD3⁺CD56⁺ NK-like T cells, with CD8⁺ cells considered a key effector subset. However, the transcriptional and clonal states underlying CD8⁺ CIK-cell expansion and function remain poorly undefined. We combined single-cell RNA and paired T-cell receptor sequencing to profile 43,047 cells from day-14 CIK cell products generated from five patients with mCRC. Among 14,973 CD8⁺ CIK cells, ten transcriptional states were identified, comprising six conventional and four NK-like states (C3, C5, C7 and C9). NK-like T-cell frequency increased during CIK expansion in all patients. The ten most expanded CD8⁺ clonotypes preferentially occupied NK-like states in four patients, while memory-like states predominated in one, revealing inter-patient heterogeneity. Despite this, NK-like states showed significant clonal overlap (permutation test, Benjamini–Hochberg-adjusted P < 0.05), consistent with phenotypic plasticity, whereas memory-like states were largely clonally distinct. Trajectory analysis supported a branching model in which intermediate C5 was associated with trajectories toward highly cytotoxic Temra-like C9 or cytotoxic C3, with a further trajectory from C3 toward dysfunctional-like C7. Regulon analysis identified state-specific programs, including KLF2 in C5, GFI1 and IKZF2 in C9, IKZF1 in C7 and FOXM1 in proliferative C8. Together, these findings identify an apparent branch point within NK-like CD8⁺ CIK cells that may shape cytotoxic and dysfunctional phenotypes. This suggests that targeting pathways governing this transition may improve CIK-cell manufacturing and lead to more effective therapeutic outcomes in mCRC.