Background The deletion of 13q14 (del(13q14)) is the most common genetic alteration in Chronic Lymphocytic Leukemia (CLL), found in ~55% of patients and associated with favorable prognosis. Homozygous deletion of the minimal deleted region (MDR), including DLEU2 and miR-15a/16-1, leads to CLL development in ~40% of mice by 15–18 months (Klein et al., 2010).

In patients, progression is marked by increasing T-cell exhaustion and dysfunction. BTK inhibition can reduce immunosuppression and restore T-cell function. We hypothesized that immune surveillance initially restrains CLL in MDR-deleted mice, but progressive dysfunction triggers disease in some mice. BTK inhibition before disease onset could modulate immune– CLL interactions, preventing immune escape and overt CLL.

Our aims were to define immune mechanisms driving CLL via longitudinal immune profiling, and to assess how early intervention with acalabrutinib impacts CLL development and immune trajectories in MDR-deleted mice.

Methods Two cohorts were established: one with 62 MDR-deleted and 10 wild-type (WT) mice, with monthly blood sampling from 12 months to endpoint; and another with 61 MDR-deleted mice randomized to acalabrutinib (n=28, 25 mg/kg daily) or vehicle (n=33) from 9 months of age (prior to CLL onset), with sampling starting at 8 months. Immune dynamics were assessed by spectral flow cytometry. Longitudinal changes in CLL and immune subsets (T, NK, monocytes) were analyzed using linear mixed models. Generalized Bayesian trajectory modeling (GBTM) clustered mice by immune trajectories. CLL was defined as >10% CD5⁺B220^low among CD19⁺ cells in PB. Spleens were paraffin-embedded and stained for PAX5 and CD5 to evaluate B cell distribution and CLL infiltration.

Results Untreated MDR-deleted mice had shorter survival than WT (median 20 vs. 25 months, p=0.01), although total T, NK, and monocyte levels overtime were comparable. Forty-three percent of MDR-deleted mice developed CLL (median onset: 18 months), with leukemic burden rising over time: 5% at 12 months, 23% at 20 months, and 48% at 25 months. Those developing CLL showed greater increases in regulatory T cells (Tregs: CD4⁺CD25⁺, CD4⁺CD39⁺), CD4⁺IL33R⁺, T follicular helper (Tfh: CD4⁺CXCR5⁺), and exhausted CD4⁺PD1⁺ T cells compared to non-diseased MDR-deleted mice. They also showed early CD4⁺ naïve reduction and EM expansion. CD8⁺PD1⁺ and CD8⁺CD244⁺ cells were higher, with CD8⁺ naïve decline and memory gain. All these differences were already present before disease onset.

GBTM was used to cluster mice by immune trajectories over time. Results identified >70% of CLL-developing mice followed trajectories with rising Tregs and a shift from naïve to EM CD8+ T cells (–55% naïve, +20% memory) by endpoint.

Acalabrutinib treatment before CLL onset did not extend survival but reduced CLL incidence (6% vs. 32% in vehicle). Mean CLL burden remained lower with acalabrutinib: 2% vs. 6% at 12 months; 2% vs. 11% at 19 months; and 3% vs. 13% at 24 months. Vehicle-treated mice showed increased Tregs and IL33R⁺ cells, and a marked rise in Tfh and PD1⁺CD4⁺ T cells (p<0.05 during >50% of follow-up), with greater CD4⁺ naïve decline and EM expansion (p<0.05 from 12–17 months). CD8⁺PD1⁺ cells were significantly higher in vehicle mice from 15-17 months, and naïve CD8⁺ T cells declined faster (p<0.05 from months 12-17), with memory CD8⁺ increase. These changes resembled those in MDR-deleted vs. WT mice.

Consistently, GBTM confirmed that 57% of vehicle-treated mice followed immune trajectories with regulatory/exhausted T-cell expansion and naïve CD8⁺ loss (–50%) and +20% EM gain. In contrast, >70% of acalabrutinib-treated mice showed only modest immune shifts and preserved naïve CD8⁺ cells (–15%).

Immunohistochemistry of spleens from MDR-deleted mice revealed PAX5⁺CD5dim regions, more pronounced in mice with high leukemic burden. Some showed dense infiltration and mitotic figures suggestive of early Richter-like evolution. In isolated cases, PAX5⁺CD5dim regions were detected without evidence of leukemia in circulation.

Conclusion CLL development is driven by progressive immune dysfunction, detectable before disease onset. Preemptively BTK inhibition with acalabrutinib reshapes immune trajectories, prevents CLL in most mice, and delays onset in others. These findings demonstrate that modulating immunity can block tumor-suppressor-driven CLL, highlighting the need for both genetic and immune alterations to enable overt disease.

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2025
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