Integrative profiling of t-follicular helper cells heterogeneity and tumor microenvironment signatures in angioimmunoblastic T cell lymphoma

Abstract Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of mature T-cell lymphoma (TCL) that arises from T follicular helper (TFH) cells and is characterized by distinct clinicopathologic features, poor prognosis, and resistance to current treatments. A hallmark of AITL is its complex lymphoma microenvironment (LME), in which neoplastic TFH cells typically comprise only 10–30% of the infiltrate, complicating diagnosis. The heterogeneous cellular composition of the LME, along with subtle morphological features and frequent overlap with other reactive lymphoid processes or classic Hodgkin lymphoma, often results in delayed diagnosis or misdiagnosis. This persistent diagnostic challenge, together with poor therapeutic responses, underscores the need to further define the transcriptional and spatial programs of neoplastic TFH cells and their environment. To address this, we performed single-cell RNA sequencing on five AITL lymph node specimens. After quality filtering, cells expressing 200–10,000 genes with fewer than 10% mitochondrial transcripts were retained. Seurat-based integration was used to mitigate batch effects and cluster cells by transcriptional similarity. Malignant TFH cells were identified by co-expression of ten canonical TFH genes (CD3E, CD4, BCL6, ICOS, PDCD1, CXCR5, CXCL13, IL21, CTLA4 and CD40LG) alongside monoclonal T-cell receptor expansions via scRepertoire. Gene set enrichment revealed consistent upregulation of oxidative phosphorylation signatures, implicating altered mitochondrial metabolism, and expression of central memory-associated transcripts. Notably, a subset of malignant TFH cells expressed FOXP3, a hallmark of regulatory T cells, suggesting phenotypic plasticity and context-dependent adoption of regulatory programs. Immunofluorescence validation confirmed FOXP3 expression in neoplastic cells. To study the LME, we performed sequential forty-plex immunofluorescence (COMET Lunaphore) in 14 AITL and 3 reactive lymph nodes. In AITL we identified two distinct microenvironmental archetypes. Both niches exhibited marked CD40 upregulation relative to controls, but with distinct cellular localizations: in the M1-skewed archetype CD40 was confined primarily to macrophages, whereas in the M2-skewed niche it was predominantly B-cells. CD40 single immunostaining was conducted in 200 additional cases, including other T-cell lymphomas and autoimmune disorders, and confirmed that while CD40-driven immune activation is a hallmark of AITL, elevated CD40 expression was also observed in a subset of other T-cell lymphomas. In our AITL PDX models using NSG mice, CD40 expression persisted in tumor-infiltrating B cells (patient derived), reinforcing the potential role of the CD40-CD40L axis in tumor–microenvironment crosstalk (PMID: 35921527). Altogether, our data indicates that neoplastic TFH cells in AITL possess phenotypic plasticity, adopting both Treg-like and central memory expression programs. These cells reside within two distinguishable microenvironmental archetypes which may underlie aspects of diagnostic complexity and variable clinical behavior. The consistent overexpression of CD40 across these niches suggests it could serve as a biomarker for stratifying microenvironmental contexts and monitoring therapeutic responses. While these findings provide preliminary insight into the cellular and spatial heterogeneity of AITL, further studies in larger patient cohorts are necessary to confirm these observations and to explore the feasibility of targeting the CD40-CD40L axis or related pathways as part of future therapeutic strategies.