CCR8 positive Tregs and their correlation with immunotherapy response in advanced non-small cell lung cancer (NSCLC).

2635 Background: Regulatory T cells (Tregs) expressing the chemokine receptor CCR8 are pivotal modulators of the tumor immune microenvironment. CCR8 has recently emerged as a promising therapeutic target due to its selective expression on activated Tregs in the tumor microenvironment and its role in promoting immunosuppression. This study investigates the prognostic and therapeutic implications of CCR8-positive Tregs in non-small cell lung cancer (NSCLC), focusing on their impact in relation to tertiary lymphoid structure (TLS) status. Methods: A validated 6-plex multiplex immunofluorescence (mIF) panel was used to analyze tumor samples from NSCLC patients treated with immune checkpoint blockers (ICB) in the BIP precision medicine study (ClinicalTrials.gov: NCT04389143). Markers included CD4, CD8, CD20, FoxP3, PanCK, and CCR8, alongside DAPI staining to assess immune contexture (infiltrated, excluded, desert), TLS status, and CCR8/FOXP3 double-positive Tregs. Clinical outcomes, including progression-free survival (PFS) and objective response rate (ORR), were analyzed in 50 responders and 50 non-responders. Findings were validated using transcriptomic data from the POPLAR (NCT01903993) and OAK (NCT02008227) studies, which evaluated atezolizumab versus docetaxel in advanced NSCLC. Kaplan-Meier curves, hazard ratios, and Cox regression models were used for survival analyses. Results: CCR8-expressing Tregs were significantly enriched in infiltrated tumors, showing a 1.5-fold increase compared to excluded tumors (p = 0.057), a 3.3-fold increase compared to desert tumors (p = 0.001), and a 1.8-fold increase in TLS-positive tumors compared to TLS-negative tumors (p = 0.003). These findings highlight that activated Tregs co-infiltrate with CD8 T cells and other immune cell types. This enrichment was confirmed in samples from the POPLAR and OAK studies using transcriptomic analyses (3-fold increase, p = 2e-16). Due to their correlation with overall immune cell infiltration, the presence of CCR8-positive Tregs was significantly associated with better survival (HR 0.45, p < 0.001) across the entire patient cohort. However, when stratified for TLS-positive tumors, the presence of activated Tregs was associated with diminished objective response rate and progression-free survival suggesting a negative impact of these immunosuppressive cells on response to ICB. Conclusions: This study provides the first evidence linking CCR8-positive Tregs with immunotherapy resistance in NSCLC, particularly in TLS-positive tumors. These findings parallel observations in TLS-positive sarcomas, where Treg abundance predicted poor outcomes (Italiano et al., Nature Medicine , 2022). This study supports the exploration of CCR8-targeted therapies to deplete immunosuppressive Tregs and enhance the efficacy of immunotherapy in TLS-positive NSCLC.