Deciphering tph cells as prognostic drivers of angioimmunoblastic T-cell lymphoma

Abstract INTRODUCTION Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive malignancy derived from follicular helper T (Tfh) cells, characterized by systemic immune dysregulation and heterogeneous clinical outcomes despite anthracycline-based chemotherapy 1-2. The prognosis of AITL remains poor, with limited efficacy of current treatments, and patient outcomes vary significantly even among those with similar clinical staging. Recent studies have highlighted the role of peripheral immune signatures in T-cell lymphoma, but comprehensive single-cell profiling of AITL's immune landscape is lacking 3-4. This study leverages CyTOF and single-cell RNA sequencing (scRNA-seq) to map the peripheral and tumor microenvironment (TME) immune heterogeneity in AITL, identifying T peripheral helper (Tph) cells as a novel prognostic subset and exploring their mechanistic role in disease progression. RESULTS Our CyTOF analysis of peripheral blood mononuclear cells (PBMCs) from 17 AITL patients firstly revealed significant heterogeneity in immune cell distribution, with Tph cells (CD3+, CD4+, CD278+, CD279+, CD185-) emerging as a distinct subset enriched in AITL compared to healthy controls and other T-cell lymphomas. Tph cells demonstrated strong prognostic value, with higher proportions correlating with poorer progression-free survival (PFS). Pseudotemporal analysis positioned Tph cells later than Tfh cells in differentiation trajectories, suggesting a terminal state linked to disease aggressiveness. Mechanistically, Tph cells drive immune dysregulation through ligand-receptor interactions, particularly CD70-mediated costimulation and IL-21 signaling, which promote B-cell activation and inflammatory responses. Multi-immunofluorescence (mIF) of tumor tissues confirmed Tph cell infiltration in patients with adverse outcomes, while scRNA-seq identified Tph-specific gene signatures and communication networks, including upregulated pro-inflammatory pathways and DNA repair mechanisms. METHOD We integrated CyTOF, scRNA-seq, and mIF to dissect AITL's immune landscape. PBMCs from AITL patients and controls were analyzed using a 42-marker CyTOF panel, followed by PhenoGraph clustering and t-SNE visualization. For scRNA-seq, 35,546 single cells from AITL lymph nodes were sequenced, annotated, and subjected to pseudotime and cell-cell communication analyses. Tumor tissues were stained with mIF to quantify Tph and Tfh spatial distribution. Statistical analyses included Spearman correlation, Kaplan-Meier survival curves, and ROC analysis to evaluate prognostic significance. The study adhered to ethical guidelines, with approval from Zhejiang Cancer Hospital's IRB. CONCLUSIONS This study establishes Tph cells as a key prognostic driver in AITL, bridging peripheral immune signatures with TME dysregulation. By combining CyTOF and scRNA-seq, we unveiled Tph's role in promoting immune evasion and inflammation via CD70/CD27 and IL-21/IL-21R axes, offering new targets for precision therapy. Our multi-omics framework provides a blueprint for investigating immune heterogeneity in malignancies with complex microenvironments, paving the way for improved stratification and targeted interventions in AITL. Future work will validate these findings in larger cohorts and explore therapeutic strategies to disrupt Tph-mediated pathways. CONTACT INFORMATION Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China Email: yanghy@zjcc.org.cn