Single-Cell Multi-Tissue T Cell Clonal Dynamics Reveal Distinct Immune Coercion Landscapes in MSI and MSS Colorectal Cancer

The efficacy of immunotherapy in colorectal cancer (CRC) has long been considered to be closely associated with microsatellite instability (MSI) status. Patients with microsatellite stable (MSS) tumors typically exhibit poor responses to PD-1/PD-L1 inhibitors and a poor prognosis, often being categorized as immunologically ‘cold’ tumors. However, some MSS patients can still achieve favorable therapeutic responses, sometimes even surpassing those of certain MSI patients. Immune-cold and immune-hot tumor phenotypes are largely determined by the abundance, clonal expansion, and functional states of tumor-infiltrating T cells. This suggests that immunotherapy responses are driven by dynamic remodeling of T-cell clonality rather than by MSI status alone. To elucidate the underlying T cell clonal dynamics, integrated single-cell transcriptome (scRNA-seq) and T cell receptor sequencing (scTCR-seq) data analyses from 43 blood and tissue samples of MSI and MSS colorectal cancer patients before and after anti-PD-1 therapy were performed. Using our developed TCR reconstruction pipeline (TORBiT), we systematically analyzed the clonal architecture of the TCR repertoire, inter-tissue migration, and its association with T-cell functional state transitions. From a TCR clonal kinetic perspective, we revealed two distinct modes of immune Coercion that may further affect the immune response: a “high-fluctuation, deep-exhaustion” pattern in MSI tumors and a “high-baseline, strong-suppression” pattern in MSS tumors. These findings provide a novel theoretical foundation and research perspective for understanding the responsiveness and resistance mechanisms to immune checkpoint inhibitors.