Integrin-deficient T cell leukemia accumulates in the central nervous system

Reviewer #1 (Public review):

The manuscript by Lux et al. addresses how T-cell acute lymphoblastic leukemia (T-ALL) cells migrate into the central nervous system (leptomeninges), specifically through VLA-4 and LFA-1 integrins. VLA-4 and LFA-1 are important regulators of normal T-cell migration into the CNS, so the authors tested whether they also mediate T-ALL infiltration. They generated an intracellular NOTCH1 T-ALL mouse model and then used CRISPR/Cas9 gene targeting to delete VLA-4 and LFA-1. They show that integrin-deficient T-ALL cells accumulate in the CNS compared to control T-ALL cells. The authors performed a time course experiment and found that although WT T-ALL cells accumulated in the CNS before DKO T-ALL cells, over time, DKO T-ALL cells outgrew the WT T-ALL cells. Subsequently, they performed bulk RNA-sequencing and revealed that Integrin beta 7 (Itgb7) was upregulated in the DKO T-ALL cells. To test whether Itgb7 was compensating for the loss of VLA-4 and LFA-1, the authors generated a triple KO (TKO). The TKO T-ALL cells migrated to the CNS; however, CNS accumulation between the TKO and the DKO was not significantly different. To evaluate if there is reduced exit of T-ALL DKO cells from the meninges, they inhibited T-ALL exit via the dorsal meningeal lymphatics by generating an AAV VEGF-trap encoding the binding domain of VEGFR3, and then co-injected WT: DKO cells weeks later. There was no effect on the WT:DKO T-ALL ratio or on the overall number of T-ALL cells in the CNS with meningeal lymphatics regression, suggesting that the DKO does not preferentially accumulate in the CNS, or that delayed exit results in DKO T-ALL accumulation in the CNS.

Additionally, the authors tested whether DKO affected immune surveillance by injecting DKO:WT T-ALL cells into NRG mice. DKO T-ALL cells localized in the dura mater and were spread throughout the tissue, whereas WT T-ALL cells clustered near blood vessels. These observations lead the authors to hypothesize that differential access to nutrients or other signals may influence leukemic cell proliferation. However, EdU labeling revealed no differences, leading the authors to hypothesize that the unique stromal cell layer in the meninges supports the DKO proliferative advantage. Finally, the authors tested whether integrin blockade and chemotherapy might chemosensitize T-ALL cells in the CNS. After a single treatment with 5FU, DKO cells were depleted faster than the WT cells; however, a single treatment with integrin blockade was toxic. After combining 5FU with the integrin antibodies, the authors showed that T-ALL cells in the CNS were significantly more depleted than in treatment with either single therapy.

These data highlight how challenging it is to identify regulators of T-ALL migration and adherence. This study highlights the importance of these experiments and the clinical need to identify the molecules that influence leukemic infiltration into the CNS.

Overall, this study was well performed with appropriate statistical power to implicate integrins in T-ALL CNS infiltration and proliferation.

Reviewer #2 (Public review):

Summary:

In this study, the authors set out to understand how T cell leukemia cells enter and persist in the CNS, with a particular focus on the role of adhesion molecules known to regulate normal immune cell trafficking. Contrary to expectations, they find that loss of two key adhesion molecules does not impair CNS entry but instead leads to increased accumulation of leukemia cells, which is associated with enhanced cell proliferation in this environment. These findings challenge prevailing assumptions about how leukemia cells interact with tissue niches and suggest a potential therapeutic strategy combining adhesion blockade with chemotherapy.

Strengths:

The study addresses an important and longstanding question in leukemia biology using well-designed in vivo models and multiple complementary approaches. The key observation is robust and consistently supported across genetic models and experimental systems. The authors systematically test alternative explanations, including altered entry, exit, and immune evasion, which strengthens the interpretation that proliferation differences underlie the phenotype. The work has potential translational relevance, particularly in highlighting a possible strategy to enhance the efficacy of anti-proliferative therapies in the CNS.

Weaknesses:

While the central phenotype is clear, the mechanistic basis remains incompletely defined. Addressing the following points would strengthen the manuscript.

Major critiques:

(1) The central claim that integrin loss enhances CNS accumulation via increased proliferation is not mechanistically resolved; current data are correlative (EdU incorporation, distribution patterns) and do not establish that integrin-mediated signaling directly restrains cell cycle progression in the CNS niche. The authors should perform functional perturbation of candidate pathways identified (e.g., TGF-β) using pharmacologic inhibitors or genetic approaches (dominant-negative receptor or CRISPR knockdown) in vivo or in ex vivo CNS-derived T-ALL co-culture systems to test whether blocking this pathway rescues the WT proliferation phenotype; if not feasible, the mechanistic claims should be toned down and clearly presented as hypotheses.

(2) The relationship between altered spatial distribution and proliferation is suggestive but not directly demonstrated. The imaging data indicate differences in localization, but these observations are not quantitatively linked to cell cycle status. The authors could strengthen this point by incorporating spatially resolved proliferation analyses, such as combining EdU labeling with imaging or quantifying proximity to stromal or vascular niches, or alternatively by providing additional quantitative analysis of the existing imaging data.

(3) The conclusion that CNS accumulation is not due to altered trafficking (entry/exit) is suggestive but not definitive, as early seeding dynamics are not directly assessed. Authors should perform short-term homing or early time-point competitive trafficking assays (e.g., CNS quantification at 6-48h post-transfer) to rigorously exclude differences in entry kinetics; if such experiments are not feasible, this limitation should be explicitly acknowledged in the discussion.

(4) The therapeutic claim that integrin blockade synergizes with chemotherapy is promising but underdeveloped, as it lacks survival outcomes and a broader translational context. The authors should include survival analyses and, if possible, test combination treatment in a more clinically relevant setting (e.g., delayed intervention or alternative standard-of-care agents), or otherwise temper translational conclusions and discuss risks such as inducing proliferation in the absence of chemotherapy.