Abstract
[Introduction] Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy that develops decades after HTLV-1 infection. ATL cells show characteristic “flower cell” morphology and express CD4⁺CD25⁺ Treg-like markers. Aggressive ATL is characterized by uncontrolled proliferation, tissue infiltration, and poor prognosis. In ATL, constitutive expression of the HTLV-1 bZIP factor (HBZ) and acquired mutations in signaling pathways have been observed. Notably, mutations in TCR signaling molecules (e.g., PLCG1, PRKCB, and CARD11) occur in ~90% of cases. Additionally, NOTCH1 PEST domain mutations are found in ~20% of cases, frequently coexisting with TCR pathway mutations. In our previous mouse study, CD4⁺ T-cell–restricted expression of HBZ combined with a Card11(E626K) mutation (Card11(E626K)CD4-Cre) was insufficient to fully recapitulate the aggressive ATL phenotype. Transcriptomic comparisons between aggressive ATL patient samples and Card11;HBZ double-mutant mice (Card11;HBZ-Tg) suggested that the additional activation of NOTCH signaling is necessary to recapitulate aggressive ATL pathology. In this study, we investigated whether the triple genetic alterations—HBZ expression, Card11 mutation, and Notch1 mutation—drive aggressive ATL in a mouse transplantation model. In addition, we examined whether HBZ-expressing hematopoietic cells support the proliferation of T cells harboring oncogenic mutations, as suggested by the human disease context in which ATL arises from a pool of HTLV-1–infected, non-malignant T cells.
[Methods] CD4⁺ T cells were purified from the spleens of donor Card11;HBZ-Tg mice using MACS-based magnetic separation. These cells were transduced with a lentiviral vector encoding a Notch1 mutant (PEST domain deletion) or an empty control vector (EGFP). For the primary transplantation, to assess the oncogenic potential of triple-mutant CD4⁺ T cells (expressing HBZ, mutant Card11, and mutant Notch1) in a double-mutant hematopoietic background, 1 × 10⁶ splenocytes—including the transduced CD4⁺ T cells—were co-transplanted with Card11;HBZ-Tg whole bone marrow (WBM) cells into lethally irradiated wild-type (WT) recipient mice. For the secondary transplantation, to evaluate the influence of the hematopoietic background—particularly the presence of HBZ-expressing hematopoietic cells—on the proliferation of mutant cells, 1 × 10⁶ splenocytes from primary recipients—containing either Card11;HBZ;Notch1 or Card11;HBZ;EGFP cells—were transplanted into non-irradiated secondary WT or HBZ-Tg recipient mice. Peripheral blood parameters, organ infiltration, cellular distributions, morphology, histopathology, or survival outcomes (overall survival [OS] and median survival time [MST]) were assessed.
[Results] In the primary transplantation, recipients receiving Card11;HBZ;Notch1 CD4⁺ T cells (n = 10) developed a lethal lymphoproliferative disease (LPD) and succumbed primarily to lung lesions, whereas control recipients receiving Card11;HBZ;EGFP CD4⁺ T cells (n = 16) did not develop a lethal LPD (median survival time [MST], 9.8 months vs. not reached; p = 0.016). Histopathological analysis of lung tissue revealed that infiltrating T cells exhibited irregular nuclear morphology and were frequently positive for CD25⁺, FOXP3⁺, and Ki-67⁺. These results suggest that, at least in the original hematopoietic context characterized by HBZ expression and Card11 mutation, a limited number of triple-mutant CD4⁺ T cells may possess oncogenic potential.
To further assess the role of the hematopoietic environment in supporting oncogenesis, we performed secondary transplantation experiments. Non-irradiated HBZ-Tg recipients receiving splenocytes containing triple-mutant CD4⁺ T cells (n = 5) developed aggressive LPD, whereas non-irradiated WT recipients (n = 17) did not develop LPD (MST, 2.8 months vs. not reached, OS 100% at 6 months; p < 0.001). Histopathological analysis revealed severe infiltration of proliferating T cells in multiple organs in the HBZ-Tg recipients, indicating that an HBZ-expressing hematopoietic environment promotes the expansion of triple-mutant CD4⁺ T cells and drives the development of aggressive ATL.
[Conclusion] The combination of HBZ expression, Card11, and Notch1 mutations in CD4⁺ T cells within an HBZ-expressing hematopoietic environment drives aggressive ATL-like LPD in mice. Further dissection of tumor–microenvironment interactions may uncover new strategies for disease control.
