Single-cell atlas of peripheral blood immune landscape in pMMR/MSS rectal cancer treated with neoadjuvant chemoradiotherapy and PD-1 blockade.

Abstract

3562 Background: Most colorectal cancer (CRC) patients exhibit normal mismatch repair function/microsatellite stability (pMMR/MSS), presenting a poor response to immune checkpoint inhibitor monotherapy. Neoadjuvant chemoradiotherapy (nCRT) combined with PD-1 inhibitors can enhance the efficacy of immunotherapy. However, the peripheral immune dynamics underlying this combination remain poorly understood. Methods: In this study, we selected 15 patients from the CHOICE I trial (ChiCTR2100042785) who received nCRT combined with PD-1 inhibitors. A total of 55 peripheral blood mononuclear cell (PBMC) samples were collected at three critical time points: pre-treatment baseline, during radiotherapy, and during immunotherapy. All samples underwent single-cell RNA sequencing (scRNA-seq) analysis, complemented by paired TCR/BCR profiling, to comprehensively map the dynamic evolution of the peripheral immune landscape during combined therapy. Results: We analysed 223,363 high-quality single cells and classified them into 11 distinct immune phenotypes using classical marker genes, revealing treatment-related immune dynamics. TCR clonal tracking revealed divergent dynamics that CD8+ T cells exhibited high clonal persistence (Jaccard index 0.1–0.23), significantly greater in responders than non-responders, underscoring their pivotal role in antitumor immunity. In contrast, CD4+ T cells (particularly T follicular helper cells) showed minimal clonal overlap ( < 0.03), consistent with rapid turnover. Monocyte analysis uncovered fundamental differences. Monocytes from non-responders displayed enhanced yet dysfunctional antigen presentation alongside immunosuppressive features, including specific expansion of the pro-inflammatory Mono_CD14_IL1B subset characterized by high immunomodulatory molecule expression. Conversely, monocytes in responders exhibited robust type I interferon signaling. B-cell analyses revealed that responders preferentially underwent plasma-cell differentiation, whereas B cells from NR retained an antigen-presenting–like phenotype. NK-cell analysis revealed an expanded, activated cytotoxic pool in responders (featuring CD38+, KIR3DL1+ subsets), whereas non-responders showed dominance of KLRC2+ NK cells, suggesting a dysfunctional state. These efficacy-linked changes across immune populations collectively map the systemic immune landscape under combination therapy. Conclusions: This study provides a comprehensive single-cell atlas of peripheral immune remodeling during nCRT combined with PD-1 inhibitors in pMMR/MSS rectal cancer, offering crucial insights for future patient stratification and personalized combination strategies.