Papillary Thyroid Carcinoma with Terminal Immune Exhaustion Phenotype Correlates with Increased Risk of Lymph Node...

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, with lymph node metastasis (LNM) being a key predictor of recurrence and poor prognosis. Preoperative detection of LNM remains challenging due to the limitations of imaging modalities, leading to inadequate surgical resection in 20-30% of patients. While immune checkpoint molecules have been implicated in PTC progression, the heterogeneity of CD8⁺ T cell exhaustion subsets and their specific association with LNM remain poorly defined. In this study, we aimed to perform an exploratory characterization of the distinct immune landscape of PTC prone to LNM, with a focus on terminal immune exhaustion, in order to generate hypotheses for improved risk stratification and therapeutic strategies.

Fresh PTC tissues from 40 patients (22 LNM-positive and 18 LNM-negative) were analyzed via flow cytometry (FCM) to quantify immune cell subsets, inflammatory cytokines, and chemokines. Immunohistochemistry (IHC) validated CD45⁺ immune cell infiltration. Transcriptomic and clinical data from 448 PTC patients in The Cancer Genome Atlas (TCGA-PTC) cohort were used for bioinformatic analysis consistent with the observed phenotype, including Gene Set Variation Analysis (GSVA) of terminal exhaustion gene signatures.

LNM-positive PTC exhibited a unique inflammatory milieu with significantly elevated IL-6, IL-1ra, CCL5, and IL-9 levels (all p < 0.05) in tumor interstitial fluid. FCM analysis revealed that LNM-positive PTC had increased infiltration of total CD45⁺ immune cells, CD3⁺ T cells, and CD3⁺CD8⁺ T cells (all p < 0.05). Critically, terminally exhausted PD-1^hiTIM-3⁺ CD8⁺ T cells were significantly enriched in LNM-positive PTC (p = 0.022) and positively correlated with extrathyroidal extension (p = 0.044). Additionally, LNM risk was associated with increased CD4⁺ regulatory T (Treg) cell frequency (p = 0.023) and elevated CTLA-4 expression on CD4⁺ T cells (p = 0.047). In TCGA-PTC validation, the terminal exhaustion gene signature was predominantly enriched in LNM-positive (p < 0.0001) and advanced-stage PTC (p < 0.001) and strongly correlated with BRAF mutation (predominantly V600E) (p < 0.0001)-the most common oncogenic driver in aggressive PTC.

Our findings suggest a terminal immune exhaustion phenotype (characterized by PD-1^hiTIM-3⁺ CD8⁺ T cells and Treg enrichment) as a potential key feature associated with LNM-prone PTC. This phenotype shows consistency across clinical samples and TCGA datasets, linking BRAF mutation (predominantly V600E) to immune suppression and metastatic potential. These insights provide a novel exploratory immune-based biomarker for LNM risk stratification and support the potential of combining anti-PD-1/TIM-3 therapy with BRAF inhibitors for high-risk PTC, which should be confirmed in future studies.