The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.

The inclusion of monoclonal antibodies targeting immune checkpoints such PD-1/PD-L1 or CTLA-4 has revolutionized the landscape of anti-cancer therapy. However, PD-1 and CTLA-4 blockade failed to achieve clinical benefit in CLL, thus attention has been focused on emerging checkpoints in this malignancy. LAG-3 is an immune checkpoint receptor that negatively regulates T cell-mediated responses by inducing an hyporesponsive state, thus promoting tumor escape. Patients with chronic lymphocytic leukemia (CLL) develop a profound immune suppression that leads to lessened immunosurveillance and increased risk of developing a secondary neoplasia. In the study herein, we report the profound dysregulation of LAG-3 on leukemic cells in CLL. Likewise, natural killer (NK) and T cells showed increased LAG-3 expression, hence suggesting a role for this checkpoint in CLL-associated immunosuppression. High LAG-3 expression, as well as high levels of soluble LAG-3 (sLAG-3), correlated with adverse cytogenetics and poor outcome in patients with CLL, highlighting the clinical relevance of this immune checkpoint. Treatment of peripheral blood mononuclear cells (PBMCs) from patients with CLL with relatlimab, a new anti-LAG-3 blocking antibody currently evaluated in numerous clinical trials, depleted leukemic cells and restored NK cell- and T cell-mediated responses. Moreover, combination of LAG-3 with the immunomodulatory drug (IMiD) lenalidomide significantly increased IL-2 production by T cells and antibody-dependent cytotoxicity (ADCC) mediated by NK cells. Altogether, these data provide new insights into the potential anti-leukemic effects of relatlimab, currently in clinical trials in CLL, and provides the rationale to further investigate its combination with IMiDs for the management of hematological malignancies.

One of the cardinal features of chronic lymphocytic leukemia (CLL) is its association with a profound immunosuppression. NK cell function is markedly impaired in CLL patients, who show a significant dysregulation of the expression of activating and inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2, also termed LIR-1, LILRB1) in the regulation of NK cells in CLL. Our results show that ILT2 expression was significantly decreased on leukemic cells and increased on NK cells of CLL patients, particularly in those with advanced disease and with bad prognostic features, such as those carrying chromosome del(11q). The immunomodulatory drug lenalidomide may regulate the expression of ILT2 and its ligands in CLL since it significantly increased the expression of ILT2 and partially reestablished the expression of its ligands on leukemic cells. Furthermore, lenalidomide significantly increased the activation and proliferation of NK cells, which was strongly enhanced by ILT2 blockade. Combining ILT2 blockade and lenalidomide activated NK cell cytotoxicity resulting in increased elimination of leukemic cells from CLL patients. Overall, we describe herein the role of an inhibitory receptor involved in the suppression of NK cell activity in CLL, which is restored by ILT2 blockade in combination with lenalidomide, suggesting that it may be an interesting therapeutic strategy to be explored in this disease.

Chronic lymphocytic leukemia (CLL) is characterized by progressive immunosuppression and diminished cancer immunosurveillance. Immune checkpoint blockade (ICB)-based therapies, a major breakthrough against cancer, have emerged as a powerful tool to reinvigorate antitumor responses. Herein, we analyzed the role of the novel inhibitory checkpoint BTLA and its ligand, HVEM, in the regulation of leukemic and natural killer (NK) cells in CLL. Flow cytometry analyses showed that BTLA expression is upregulated on leukemic cells and NK cells from patients with CLL, whereas HVEM is downregulated only in leukemic cells, especially in patients with advanced Rai-Binet stage. In silico analysis revealed that increased HVEM, but not BTLA, mRNA expression in leukemic cells correlated with diminished overall survival. Further, soluble BTLA (sBTLA) was found to be increased in the sera of patients with CLL and highly correlated with poor prognostic markers and shorter time to treatment. BTLA blockade with an anti-BTLA monoclonal antibody depleted leukemic cells and boosted NK cell-mediated responses ex vivo by increasing their IFN-γ production, cytotoxic capability, and antibody-dependent cytotoxicity (ADCC). In agreement with an inhibitory role of BTLA in NK cells, surface BTLA expression on NK cells was associated with poor outcome in patients with CLL. Overall, this study is the first to bring to light a role of BTLA/HVEM in the suppression of NK cell-mediated immune responses in CLL and its impact on patient’s prognosis, suggesting that BTLA/HVEM axis may be a potential therapeutic target in this disease.