344P Lenalidomide sustains CD4/CD8 T cell function in an exhaustion model

Background: Immune checkpoint inhibitors (ICIs) have limited efficacy in microsatellite-stable (MSS) or mismatch repair-proficient (pMMR) locally advanced rectal cancer (LARC).Combining ICIs with neoadjuvant chemoradiotherapy (nCRT) shows promise, but predictive biomarkers are lacking. Methods:We retrospectively analyzed 30 MSS/pMMR LARC patients treated with nCRT plus PD-1 blockade.Major pathological response (MPR) was defined as 10% residual tumor cells.Matched pre-and post-treatment samples underwent immunohistochemistry (IHC), multiplex immunofluorescence (mIF), bulk RNA sequencing, and single-cell RNA sequencing (scRNA-seq) to characterize immune and stromal changes.Results: Before treatment, the densities of CD8 + T cells and CD138 + plasma cells in both tumor parenchyma and stroma were higher in the MPR tumors compared with the non-MPR group.After treatment, MPR tumors showed increased CD3 + , CD8 + T cells, B cells, plasma cells, and NK cells in stroma, and elevated CD8 + T cells and plasma cells in tumor parenchyma.FOXP3 + regulatory T cells (Tregs) were significantly more abundant post-treatment in non-MPR tumors.Paired analysis revealed increased plasma cells and decreased Tregs post-treatment in MPR tumors, while non-MPR tumors had increased Tregs.Bulk RNA-seq showed upregulation of epithelial-mesenchymal transition (EMT) and cancer-associated fibroblast (CAF) genes in MPR tumors, while Wnt and TGF- signaling pathways were enriched in non-MPR tumors.scRNA-seq corroborated these findings, revealing expanded plasma cell populations and reduced Treg abundance in MPR tumors.In contrast, non-MPR tumors maintained FOXP3 + Tregs exhibiting an activated immunosuppressive phenotype co-expressing CTLA4 and TIGIT.Importantly, FOXP3 + Tregs significantly decreased after treatment only in MPR tumors. Conclusions:Response to nCRT plus PD-1 blockade in MSS/pMMR LARC is characterized by cytotoxic and humoral immune activation and Treg depletion.Resistance involves sustained immunosuppressive Tregs and Wnt/TGF- pathway activation.Targeting Tregs may improve treatment efficacy, and multiomic biomarkers can aid patient stratification.