IFN-γ-induced CD4+ CADM1+ T cells emerged as malignancy-progressive biomarkers in EBV NK/T lymphoproliferative disorders and potential drivers of malignant expansion in AITL Free

Epstein-Barr virus (EBV) infection of T and NK cells leads to EBV-associated NK/T-cell lymphoproliferative disorders (EBV NK/T LPDs), a spectrum of aggressive and poorly understood diseases that range from chronic inflammation to fulminant hemophagocytic lymphohistiocytosis. Accurate diagnosis remains challenging due to disease heterogeneity and technical barriers. Although immunostaining and PCR of biopsies are commonly used, obtaining biopsies is invasive and risky, and PCR of sorted cells often yields ambiguous results. Compared to EBV-associated B-cell neoplasms, which benefit from targeted therapies such as rituximab, EBV NK/T LPDs lack effective molecular therapies and respond poorly to chemotherapy. These diagnostic and therapeutic challenges are compounded by our limited understanding of pathogenesis and molecular mechanisms underlying the EBV NK/T LPDs. This study aims to characterize the immune and molecular landscape of EBV NK/T LPDs and to identify novel diagnostic biomarkers and therapeutic targets to improve patient outcomes.

Methods We collected and analyzed single-cell RNA-sequencing (scRNA-seq) and TCR-sequencing (scTCR-seq) data of peripheral blood mononuclear cells (PBMCs) from 15 patients: 2 with infectious mononucleosis (IM), 8 with chronic active EBV (CAEBV) at various grades, 2 with EBV (+) angioimmunoblastic T-cell lymphoma (AITL), 2 with EBV (+) peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), and 1 with EBV (+) nodular-type NK/T-cell lymphoma. EBER determined EBV positivity in situ hybridization on pathological tissue sections: EBV (+) AITL cases showed scattered EBER (+) cells, while EBV (+) PTCL-NOS cases exhibited diffuse EBER (+). Flow cytometry, multiplex immunofluorescence (mIF), and cytometric bead array (CBA) were employed to profile immune cell populations and cytokine levels. In vitro functional assays were conducted to investigate transcriptional regulation and phenotypic plasticity of candidate cell types.

Results Using scRNA-seq data, we first identified a population of CD4⁺ CADM1⁺ T cells as a malignancy-progressive signature in EBV NK/T LPDs, their frequency increased significantly with disease progression, peaking in CAEBV grade 3 and EBV (+) NK/T non-Hodgkin lymphomas (NK/T NHL) (R² = 0.48, p = 0.0058). Combining with published scRNA-seq datasets, we also found that these cells were scarcely detected in healthy controls or EBV (−) NK/T NHL. Flow cytometry further verified that CD4⁺ CADM1⁺ T cells were significantly more abundant in EBV (+) NK/T NHL (n=5) compared to EBV (−) NK/T NHL (n=6, p = 0.0015), and also significantly elevated in CAEBV (n=4, p = 0.0166) relative to EBV (−) NK/T NHL. Notably, the EBV (−) NK/T NHL group included AITL cases with EBER (-) (n = 2), PTCL-NOS cases with EBER (-) (n = 3), and one PTCL-NOS case with scattered EBER (+) (n = 1). Moreover, mIF also demonstrated significantly greater infiltration of CD4⁺ CADM1⁺ T cells in tissue sections from EBV (+) NK/T-NHL compared to EBV (−) cases. We further observed significantly elevated plasma IFN-γ levels in EBV NK/T LPDs (n=13) compared to healthy controls via CBA assay (n=20, p = 0.0165), suggesting a cytokine milieu consistent with Th1-type inflammation. IFN-γ aligns with the emergence of CD4⁺ CADM1⁺ T cells, which exhibit a Th1-like transcriptional profile and may contribute to disease pathogenesis. Functional experiments also revealed that IFN-γ upregulates CADM1 expression in H9 cells via IRF2, promoting a Th1-like phenotypic shift. In AITL, CD4⁺ CADM1⁺ T cells exhibited clonal TCR expansion, suggesting malignant potential. Notably, mIF analysis of AITL tissues revealed substantial spatial colocalization between CADM1 and PD-1, an established marker of neoplastic T follicular helper cells. This histopathological finding provides strong evidence that CD4⁺ CADM1⁺ T cells represent malignant components within AITL lesions.

Conclusions Our study reveals CD4⁺ CADM1⁺ T cells as a distinct immunophenotypic and molecular hallmark of EBV NK/T LPDs, correlating with disease progression. These cells may serve as a minimally invasive diagnostic biomarker and a potential therapeutic target. These insights enhance our understanding of EBV NK/T LPDs and may facilitate the development of novel strategies for early detection and treatment of this aggressive disease group.