Intrinsic immune dysfunction in CLL refers to the inherent alterations and impairments within the immune system that arise during the development and progression of the disease itself. These dysfunctions significantly contribute to the pathogenesis of CLL and its clinical manifestations, leading to a compromised immune response and an increased risk of infection and disease progression (19–23). For instance, leukemic B cells exhibit alterations that cause their accumulation in the blood and lymphoid tissues, which cause defects in their functional capabilities.

B cells naturally recognize specific antigens through the B-cell receptor (BCR), which consists of antigen-binding surface immunoglobulins and accessory molecules that interact with their corresponding antigens, leading to cell activation and antibody production. B-cell antibody production includes V(D)J recombination, somatic hypermutation, and class switching processes. In CLL, defects in antibody production and class switching often occur (24–26). The presence of dysfunctional B cells can reduce the ability to generate protective antibodies, leaving patients vulnerable to infections (27). BCR signaling is constitutively activated in CLL cells, whether this is attributable to self-antigens or environmental factors remains unclear (16–19). Nevertheless, signal transduction through the BCR activates pathways that promote the survival and growth of leukemic B cells, thereby facilitating disease progression.

Furthermore, patients with CLL have been shown to have an imbalance in the ratio of cluster of differentiation 4 (CD4)⁺ to CD8⁺ T cells (28). Although studies have documented a higher number of CD8⁺ T cells in CLL patients compared with age-matched healthy individuals, these cells often express exhaustion markers (i.e., programmed death cell protein 1[PD-1], cytotoxic T-lymphocyte associated protein 4 [CTLA-4], and lymphocyte-activation gene 3 [LAG-3]) and demonstrate reduced cytotoxic activity, which is perpetuated by improper immune synapse formation (29–31). Thus, CD8⁺ T cells often fail to release the cytotoxic granules necessary for eliminating leukemic cells. CLL patients often have a high frequency of CD4⁺ regulatory T cells (Treg). Treg cells are associated with hindered anti-tumor responses and immunosuppression, as they release immunosuppressive molecules (i.e., interleukin [IL]-10 and transforming growth factor beta [TGF-β]) that reduce CD8⁺ T-cell activation. Previous studies have demonstrated that removing Tregs leads to effective anti-tumor responses in animal models of CLL (32–34).

Natural killer (NK) cells, which are innate immune cells responsible for anti-tumor and anti-viral responses, are also present in the immune microenvironment of CLL. NK cell functions include degranulation, cytotoxicity, and the release of cytokines (35, 36). In CLL, NK cells’ cytolytic activity is impaired owing to defects in their cytotoxic machinery, and leukemic B cells employ immune evasion strategies to escape detection by NK cells (37).

Myeloid cells constitute a significant portion of the immune microenvironment in CLL. Nurse-like cells (NLCs) share lineage and functional similarities to tumor-associated macrophages (TAMs) and have been identified as CLL-specific TAMs (38). NLCs express stromal cell-derived factor 1 (SDF-1), which attaches to leukemic B cells and downregulates its receptor, C-X-C chemokine receptor type 4 (CXCR4), thereby protecting these cells from spontaneous apoptosis. NLCs also release B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), both of which contribute to the survival of CLL cells (39, 40). Another important myeloid cell component of the CLL TME are monocytes, which include CD14⁺⁺ CD16^- classical, CD14⁺ CD16⁺⁺ non-classical, and CD14⁺⁺ CD16⁺ intermediate subsets (41, 42). The recruitment of monocytes to the TME depends on CLL cells’ expression of C-C-chemokine receptor type 2 (CCR2) and several monocyte-attracting chemokines such as C-C-motif ligand 2 (CCL2) and CXCL10 (43, 44). When adoptively transferred into humanized MISTRG mice, monocyte subsets from CLL patients differentiate into TAMs (45). Monocytic myeloid-derived suppressor cells (M-MDSCs) are so named because of their common myeloid origin and immunoregulatory properties (46, 47). In CLL patients, an increase in M-MDSCs in the peripheral blood (PB) is associated with a poor prognosis (48, 49). Additionally, in CLL patients, M-MDSCs suppress T cell function and stimulate the production of Tregs. Lastly, CLL cells induce the transition of monocytes into M-MDSCs, suggesting cellular communication between CLL cells, M-MDSCs, and Tregs (50).

The intrinsic immune dysfunction observed in CLL results from a complex interplay of factors that collectively compromise the immune system’s ability to respond effectively to the disease. By understanding these intrinsic mechanisms, researchers and clinicians can develop more effective strategies to restore immune function and enhance therapeutic approaches.

Although chemotherapy, immunotherapy, and targeted therapy are vital for controlling CLL progression and improving outcomes, they often have significant side effects. A primary concern is these therapies’ ability to severely compromise the immune system and increase the risk of infection (9, 15, 17). Moreover, such therapy-induced immune dysfunction affects the immune system’s ability to perform essential surveillance, hindering its capability to detect and eliminate residual cancer cells, potentially causing relapse or disease progression.

Granulopoiesis, the process of neutrophil development, begins in the bone marrow where hematopoietic stem cells (HSC) differentiate into common myeloid progenitors [CMP (82)]. CMPs further differentiate into granulocyte-macrophage progenitors (GMP), which ultimately mature into neutrophils through a regulated process governed by various transcription factors. Recent research has identified unipotent cells within the neutrophil lineage; early neutrophil progenitors (eNePs) produce different types of neutrophils, categorized by function (83, 84). During the promyelocyte stage, neutrophils create primary granules such as neutrophil elastase (NE) and myeloperoxidase (MPO); during the myelocyte stage, they produce secondary granules. Once neutrophils develop into band cells, they form tertiary granules, such as arginase 1 (ARG1) and lysozyme. Fully differentiated neutrophils can leave the bone marrow in response to infection or inflammation (85–89). The bone marrow has a substantial reservoir of neutrophils. Granulocyte colony-stimulating factor (G-CSF) regulates granulopoiesis by inducing the proliferation of granulocytic precursors and influencing the release of mature neutrophils via the CXCR4-CXCL12 axis (88, 90).

Neutrophils are traditionally viewed as short-lived cells (88). However, recent research suggests that their lifespan in a disease condition is longer than previously believed (91). Specific transcription factors, such as GATA binding protein 1 (GATA1) and CCAAT/enhancer binding proteins (C/EBPs), regulate neutrophil production and their release into the bloodstream (92). The factors controlling circadian expression remain unknown. Signals from various cell types (i.e., bone marrow stromal cells, endothelial cells, and osteoblasts) collaborate to regulate the release or withholding of CXCL12 (92). Lastly, neutrophils can survive for extended periods following their activation by cytokines, inflammatory mediators, or microbial products (93). After completing their antimicrobial functions, neutrophils undergo apoptosis and are cleared by resident macrophages and dendritic cells (94).

Neutrophils rely on pre-existing effector molecules stored in intracellular granules to respond quickly to infection or inflammation (85). Degranulation is highly regulated and occurs hierarchically: secretory vesicles and tertiary granules are released first, followed by secondary and primary granules (95, 96). SNARE and Rab proteins regulate vesicle movement and degranulation (97).

Neutrophils produce ROS by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which generates superoxide (O₂-) and other ROS with potent antimicrobial activity (98). For instance, hydrogen peroxide (H₂O₂), in conjunction with MPO, produces hypochlorous acid (HOCl), a highly microbicidal substance (99). Some microbes can thwart ROS production by releasing toxins that prevent the assembly of the NADPH oxidase complex (100). Neutrophils have additional mechanisms to effectively combat pathogens.

Phagocytosis is facilitated by receptors that internalize particles into the phagosome, which is crucial for pathogen elimination (80). Neutrophils identify pathogens through pathogen-associated molecular patterns (PAMPs) or opsonins, which initiate signaling cascades that facilitate particle engulfment (101). The phagosome matures into a microbicidal vacuole known as the phagolysosome, which triggers ROS production by NADPH oxidase (102).

Neutrophils also form NETs to prevent the propagation of infection (81). During NETosis, a dynamic cell death program, neutrophils release chromatin fibers containing nuclear DNA along with antimicrobial proteins, which act as a framework to trap pathogens (103). However, unregulated NET formation can harm the host, contributing to noninfectious inflammatory diseases, such as cystic fibrosis and vasculitis. Excessive neutrophil recruitment or activation is linked to acute and chronic conditions such as myocardial infarction, stroke, systemic lupus erythematosus (SLE), and cancer (4), raising interest in neutrophils as a key player in various diseases.

Much of our understanding of neutrophils in cancer comes from studies of solid tumors in mouse models, and this information can act as a rationale for understanding the role neutrophils play in hematologic malignancies. Recent research has emphasized the crucial role of neutrophils in cancer, showing that the neutrophil-to-lymphocyte ratio (NLR) acts as a prognostic indicator in various solid tumors (104). For example, a high neutrophil count indicates a favorable prognosis in colon cancer patients, whereas lower neutrophil counts are associated with disease progression in breast cancer patients. This suggests that neutrophils can significantly influence the progression or regression of cancer (6, 105). NLR is emerging as a potentially useful prognostic indicator in hematologic malignancies, with studies suggesting that higher NLR values indicate increased inflammation and may be linked to poorer outcomes in patients (106). Neutrophils have been found to both combat and promote cancer by interacting with other immune cells within the TME and engaging in a range of functions, such as releasing substances that modulate the host response (107).

Neutrophils show remarkable adaptability in their role, either increasing or reducing inflammation. Their release of ROS is linked to DNA damage in lung cancer (108). In the TME, neutrophils are highly flexible, responding to signals that guide their activation and polarization, thereby influencing mechanisms that promote or hinder cancer development. It remains unclear whether neutrophils’ anti- or pro-tumor activities stem from specific activation under different microenvironmental conditions or if certain neutrophil subsets are predisposed to specific functions (3). Further research is needed to clarify the mechanisms behind the diverse functions of neutrophil subsets in both solid and hematologic cancers.

Patients with solid tumors and autoimmune diseases may have distinct subsets of mature neutrophils, including low-density granulocytes (LDGs (3)) and tumor-associated neutrophils (TANs) (5, 107). TANs are categorized as TAN1 and TAN2, which have opposing functions. TAN1 possesses anti-tumor properties; it releases cytokines such as IL-12 and tumor necrosis factor-alpha (TNF-α), which aid in the recruitment and activation of CD8⁺ T cells. In a murine model of lung carcinoma, TAN1 was associated with early tumor growth, whereas TAN2 emerged during tumor progression, influenced by the TME (109). In another study, the inhibition of TGF-β resulted in more cytotoxic and pro-inflammatory neutrophils, whose phenotype resembled that one of TAN1, whereas the presence of TGF-β leds to a pro-tumor phenotype. In human cancers, early-stage TANs acted as antigen-presenting cells (APCs), facilitating the priming, proliferation, and activation of anti-tumoral T cells (110).

LDGs, or low-density neutrophils (LDN), were first identified in autoimmune diseases as a pro-inflammatory subset linked to SLE progression. LDGs are thought to contribute to SLE pathogenesis by releasing NETs, which cause vascular damage and create autoantigens that induce autoimmunity (111). Increased LDGs are observed in SLE patients with a significant interferon (IFN) signature, a common marker of various autoimmune diseases, especially SLE (112, 113). In SLE, IFN- α production by plasmacytoid dendritic cells primes neutrophils to release NETs via Toll-like receptor 9 (TLR9) signaling (114, 115). NET levels positively correlate with lymphoma progression and childhood acute leukemia development (116, 117). In solid cancer, LDGs from tumor-bearing mice show reduced anti-tumor cytotoxicity, decreased phagocytosis, lower ROS production, increased anti-inflammatory activity, and tumor-promoting properties (118).

Further research clarifying the specific behaviors and roles of neutrophil subsets within the intricate and dynamic human TME would enhance our understanding of how these neutrophil subsets interact with the TME and how they might be therapeutically targeted.

The protein arginine deiminase (PAD) family consists of a group of enzymes that are responsible for the post-translational modification (PTM) of protein arginine residues through deamination or demethylation, leading to the formation of citrulline. PAD4 is the only member of the family with a nuclear localization sequence. PAD4 competes with other nuclear enzymes to modify multiple arginine residues in histone H3 and H4. The citrullination of histone H3 (CitH3) has been linked to NET formation, highlighting the potential significance of targeting PAD4 in pathological conditions associated with excessive NET formation, such as cancer (144, 145).

BMS-P5 was the first PAD4 inhibitor to be developed. Li et al. demonstrated that administering BMS-P5 to human multiple myeloma (MM) cell lines reduced NETosis. Furthermore, this compound inhibited NET formation in primary bone marrow cells obtained from MM patients. Subsequently, in vivo studies revealed a significant reduction in DNA and CitH3 in a BMS-P5-treated mouse model of MM (146).

JBI-589 is another promising selective PAD4 inhibitor, notable for its oral bioavailability. Studies have shown that JBI-589 can stimulate immune responses against lung tumors, resulting in a substantial delay in tumor progression in lung cancer. Like BMS-P5, JBI-589 exhibited a PAD4-mediated preventative effect against NET formation and citrullination in murine neutrophils and arthritis models (147). PAD4 inhibitors could offer a novel approach to treat CLL and restore the proper function of neutrophils by disrupting pro-survival signals that leukemic B cells receive from NETs in the TME.

Nanoparticles have emerged as a promising strategy for the targeted delivery of compounds to cells. Sivelestat was first used to target acute lung injury caused by systemic inflammatory response syndrome (SIRS (148)). This compound specifically inhibits the NE stored in neutrophil granules. Cruz et al. demonstrated the efficacy of nanoparticle-mediated sivelestat delivery in inhibiting NET formation (149). They noted a significant decrease in serum levels of NE and other proinflammatory cytokines; furthermore, treatment with nanoparticle-mediated sivelestat protected mice against endotoxic shock. Nanoparticle-mediated agents could serve as innovative therapeutic options able to reduce off-target effects, circumvent resistance mechanisms, and enhance drug effectiveness (150–152). These potential benefits could aid patients with CLL.

Prostaglandin E2 (PGE2) is an endogenous protein with oxytocic properties that is widely seen in various clinical settings, including gastrointestinal cancer (153, 154). Studies with patients who have received autologous and allogeneic hematopoietic stem cell transplants have shown that PGE2 can inhibit NET formation through the induction of protein kinase A and Epac (155). In neutrophils, PGE2 effectively regulates NET release via autophagy induction, highlighting this compound’s ability to regulate NET formation and release. Besides its role in NET formation, PGE2 is linked to several pro-tumor mechanisms, including suppressing antitumor immunity, regulating tumor immune evasion, and promoting tumor progression (156). The specific role of PGE2 in CLL remains unclear; therefore, further research is needed to determine if inhibiting PGE2 could be an effective strategy.

Disulfiram, a medication approved by the U.S. Food and Drug Administration for alcohol use disorder, inhibits aldehyde dehydrogenase. Studies have demonstrated its ability to block gasdermin D (GSDMD) in macrophages. In neutrophils, GSDMD is a pore-forming protein crucial for NET formation (157). Adrover et al. demonstrated that disulfiram effectively inhibited PMA-induced NET formation by neutrophils isolated from the peripheral blood of patients with COVID-19 (158); they also observed similar inhibitory effects in a mouse model of severe transfusion-related acute lung injury. In CLL, it is unexplored whether disulfiram can effectively reduce NET formation. A concern is its impact on macrophages, which may aggravate the immune dysfunction observed in patients. Overall, exploring alternative uses for FDA-approved drugs might present valuable opportunities for patients.

Neutrophils exhibit remarkable plasticity, capable of assuming different phenotypes based on their maturation and surrounding environment (i.e., TAN1 or TAN2). Ohms et al. pioneered the attempt to polarize human neutrophils into distinct subsets. They exposed human neutrophils to either a TAN1 cocktail (containing LPS, IFN-β, and IFN-γ) or a TAN2 cocktail (containing L-lactate, adenosine, TGF-β, IL-10, PGE2, and G-CSF) and demonstrated that each respective cocktail could polarize mature neutrophils into tumor-associated subsets (159). Their findings suggest that interventions designed to polarize tumor-promoting neutrophils into anti-tumor subsets can be used to treat cancer and eventually CLL. Integrating these therapeutic strategies into the current treatment landscape for CLL might have the potential to significantly reduce neutrophil-mediated immune dysfunction in patients. By specifically targeting the formation of NETs and modulating the immune response, these approaches may not only hinder tumor progression but also improve overall patient survival rates.