Venetoclax-based therapy remodels T-cell and NK-cell immunity in patients with chronic lymphocytic leukemia

Abstract

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is characterized by profound immune dysregulations. Venetoclax (Ven), a selective BCL-2 protein inhibitor, has shown robust clinical activity in both treatment-naïve and relapsed/refractory (R/R) CLL. In Ven-treated patients with CLL, a reduction in the number of non-malignant lymphocytes and a decline in tumor-supportive T-cell subsets were reported. However, the effects of Ven treatment on the patients' immunological profile remain incompletely characterized. Aims: This study aims to characterize the immunological effects of Ven-based therapies in CLL by tracking longitudinal changes in T-cell and NK-cell phenotypic and functional profiles. Methods:In this multicenter, observational study 68 patients with R/R CLL, eligible for therapy with Ven-based regimens, were enrolled. Forty-six patients received Ven + rituximab and 22 Ven monotherapy. Peripheral blood (PB) samples were collected at the time of treatment start (n=68), and after 1 (n=60), 6 (n=56) and 12 (n=57) months of therapy. The percentage and the phenotype of T and NK cells were assessed by multicolor flow cytometry. Results: Data of 58 enrolled patients have been analyzed to date. In parallel with the reduction of circulating leukemic cells, Ven treatment induced a significant decline in absolute CD3⁺, CD4⁺ and CD8⁺ T-cell counts, which was already evident after one month and persisted at the 6- and 12-month timepoints. During treatment, in the CD4⁺ T-cell compartment, the frequency of naïve (TN) and central memory (TCM) subsets remained stable over time, while within the cytotoxic subsets we observed a decrease in the effector memory (TEM) T cells and a parallel, significant increase of the terminally differentiated (TEMRA) T cells compared to the pre-treatment distribution. In the CD8+ T-cell compartment we observed a similar rise in the proportion of TEMRA, which was paralleled by a decrease in the proportion of TCM and TEM subsets. The evaluation of immune checkpoint expression on CD4⁺ T cells revealed a downmodulation of PD-1 that was already evident after one month of treatment and a reduction of BTLA, TIM-3 and LAG-3 levels that became evident at the later 6- and 12-month timings. Interestingly, a more detailed analysis performed in a subgroup of patients showed that the observed reduction in immune checkpoint expression was mostly restricted to the less differentiated (i.e. TN, TCM) CD4+ subpopulations. Concerning the CD8⁺ T-cell compartment, Ven treatment significantly lowered BTLA expression - particularly within the TN and TCM subsets - across all timepoints, whereas the levels of the other immune checkpoints remained largely unaltered. Of note, an expansion of the immunosenescent CD57⁺PD-1⁻ subset was persistently detected within the CD8⁺ TEMRA subset already after one month of treatment and within the CD4⁺ TEM and TEMRA subsets at the later 12-month timepoint. T-cell homing capacity was also evaluated, based on CXCR3 and CD62L expression. Interestingly, both CD4⁺ and CD8⁺ T cells showed increased CD62L expression after 12 months of treatment, indicating enhanced migration capacity to lymph nodes. In the context of innate immunity, Ven administration resulted in an overall decrease in the absolute number of NK cells, detectable as early as one month after treatment initiation. Notably, the frequency of the CD56dimCD16⁺ NK-cell subset, mostly responsible for cytotoxic activity and ADCC, increased significantly at the 6- and 12-month timepoints. By contrast, the proportion of the less represented CD56bright NK-cell subset remained stable in the PB of Ven-treated patients. We did not detect any relevant difference in the modulation of T-cell and NK-cell phenotype between patients treated with Ven plus rituximab and those receiving Ven monotherapy. Conclusion: This study provides a longitudinal evaluation of the immunomodulatory effects of Ven treatment in the largest cohort of patients with CLL analyzed to date. Our results indicate that Ven therapy reshapes both the T-cell and NK-cell compartments by alleviating features of exhaustion, enhancing lymph node homing potential and promoting the expansion of cytotoxic subsets. At the time of data presentation, conclusive results from additional phenotypic analyses and functional assays will be available. These immunological modulations may be harnessed to elucidate Ven off-tumor effects and to refine optimal therapeutic sequencing strategies.