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Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL. [ABSTRACT FROM AUTHOR]

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with chronic lymphocytic leukemia (CLL), the most frequent B cell malignancy in western countries, develop a progressive immunosuppression, leading to diminished anti-tumor immunity. Within the last years, immune checkpoint blockade has revolutionized anti-cancer therapies. Nonetheless, patients with CLL failed to achieve clinical benefits from therapies targeting widely-studied checkpoints such as PD-1/PD-L1 or CTLA-4. In this context, our results provide new insights about LAG-3 expression dysregulation in CLL and its role promoting tumor escape. Our data suggest that increased LAG-3 expression on leukemic cells correlates with shorter time to treatment and poor outcome in CLL. Moreover, treatment with relatlimab, a novel anti-LAG-3 blocking monoclonal antibody currently under clinical trial for different solid and hematological malignancies including CLL, restored, at least in part, NK and T cell-mediated anti-tumor responses. Altogether, our data provide the rationale to further investigate the role of LAG-3 in the pathogenesis of 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Chronic lymphocytic leukemia (CLL) represents the most frequent B cell malignancy in Western countries and still remains as an incurable disease. Despite recent advances in targeted therapies including ibrutinib, idelalisib or venetoclax, resistance mechanisms have been described and patients develop a progressive immunosuppression. Since immune checkpoint blockade has demonstrated to reinvigorate T and NK cell-mediated anti-tumor responses, the aim of this work was to elucidate whether this immunosuppression relies, at least in part, in BTLA/HVEM axis in patients with CLL. Our results demonstrate that BTLA and HVEM expression is deeply dysregulated on leukemic and NK cells and correlates with poor outcome. Moreover, soluble BTLA levels correlated with adverse cytogenetics and shorter time to treatment. BTLA blockade restored, at least in part, NK cell-mediated responses in patients with CLL. Altogether, our results provide the rationale to further investigate the role of BTLA/HVEM axis in the pathogenesis of CLL. 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. [ABSTRACT FROM AUTHOR]

The correlation between progression and the genetic characteristics of Binet stage A patients with chronic lymphocytic leukemia (CLL) detected by whole exome sequencing (WES) was analyzed in 55 patients. The median follow-up for the patients was 102 months. During the follow-up, 24 patients (43%) progressed. Univariate Cox analysis showed that the presence of driver mutations, the accumulation of two or more mutations, the presence of adverse mutations, immunoglobulin heavy chain genes (IGHV) mutation status and unfavorable single copy number abnormalities (SCNAs) were associated with a higher risk of progression. Particularly, the occurrence of an adverse mutation and unfavorable SCNAs increased the risk of progression nine-fold and five-fold, respectively. Nevertheless, only the occurrence of adverse mutations retained statistical significance in the multivariate analysis. All patients carrying an unfavorable mutation progressed with a median progression-free survival (PFS) of 29 months. The accumulation of two or more mutations also increased the risk of progression with a median PFS of 29 months. The median PFS of patients with unfavorable SCNAs was 38 months. Combining mutations and SCNAs, patients may be stratified into three groups with different prognostic outcomes: adverse (17% probability of five-year PFS), protective (86% probability of five-year PFS) and neither (62% probability of five-year PFS, p < 0.001). Overall, the analysis of the mutational status of patients with CLL at an early stage of the disease may allow the identification of patients with a high risk of progression. The feasibility of an early therapeutic intervention in these particular patients requires further investigation. [ABSTRACT FROM AUTHOR]