CLL treatments have improved, but high-risk disease and drug resistance remain serious problems. This review asks whether natural killer cells can provide a more flexible way to attack leukemia. Chronic lymphocytic leukemia, or CLL, is one of the most common hematological diseases in western countries, with an annual incidence of forty-two per one hundred thousand.

Conventional chemotherapy and targeted therapeutic drugs have limitations in prognosis or efficiency in high-risk patients, while immunotherapy has the potential for better effect and prognosis. Natural killer cells can mediate anti-tumor activity by expressing activating and inhibiting receptors and recognizing specific ligands on tumor cells.

In CLL immunotherapy, NK cells are used through antibody-dependent cytotoxicity, allogeneic NK-cell therapy, and chimeric antigen receptor natural-killer, or CAR-NK, cell therapy. Targeted therapies, including B-cell Lymphoma 2 protein inhibitors and B-cell receptor signal inhibitors, have greatly improved treatment options and prognosis in CLL.

Even so, these drugs show potential limitations, including poor treatment efficiency in high-risk patients and drug resistance. That makes introducing novel therapies to achieve better treatment response and reduce drug resistance a necessity; immunotherapy is one of the most effective therapeutic approaches.

Immune checkpoint blocking and chimeric antigen receptor transduction have shown significant therapeutic effects in several lymphoid malignancies, but results in CLL have not met expectations. The review therefore turns to other immune cells, including natural killer cells, whose activated and inhibited receptors bind specific ligands on tumor cells and mediate anti-tumor activity.

Antibody-dependent cytotoxicity is an important cancer-killing mechanism for NK cells, and it is mediated by CD16 receptors. NK cells are an important part of the natural immune system and play key roles in microbial infection and tumor-cell recognition. They are generally classified by CD fifty-six density into CD3-negative, CD56-bright, CD16-negative NK cells and CD3-negative, CD56-dim, CD16-positive NK cells.

CD56-dim, CD16-positive NK cells mainly exist in peripheral blood and are associated with cytolytic activity through granzymes and perforin. CD56-bright, CD16-negative NK cells generally exist in secondary lymphoid tissues, where they release cytokines; CD56-bright cells may differentiate into CD56-dim cells after stimulation by peripheral-tissue fibroblasts.

NK-cell activity is controlled by a balance between activation, inhibition, and cytokine receptors that bind specific ligands on immune cells, host cells, and tumor cells. When NK cells contact normal immune or host-tissue cells, inhibitory signaling and MHC-one-specific ITIM-bearing receptors keep NK-cell toxic activity in a resting state.

When NK cells sense cancer cells or microbial infection, activated receptor-ligand signaling leads them to release cytokines and lytic granules that damage target cells. Because multiple interactions are needed to activate key apoptosis-related cytokines, NK cells do not normally injure healthy tissues on a large scale.

In tumor cells, NK cells mainly perform two functions: releasing cytokines to transmit signals to circulating immune cells, and inducing tumor-cell lysis and apoptosis by degranulation. Their cytolytic killing includes direct killing through perforin and granzyme release, as well as antibody-dependent cytotoxicity.

In antibody-dependent cytotoxicity, Fc receptors on NK cells bind the Fc portion of IgG one or IgG three whose Fab portion is bound to tumor cells, activating the NK cells. The activated NK cells can then release lytic granules to kill targeted tumor cells without pre-activation.

Figure three presents two routes by which NK cells kill target cells. In panel A, adhesion through LFA-one and ICAM-one, alongside talin and actin, helps organize cytotoxic particles for release of perforin and granzyme. Panels B and C show receptor-mediated apoptosis: FasL binds CD95 and signals through DISC to caspases eight and ten, while stimulated NK cells use TRAIL to engage death receptors.

Tumor cells take measures to avoid the killing effect of NK cells, and the review links this immune escape to decreased NK-cell killing of cancer cells. Clinical studies show that NK-cell dysfunction is prevalent in a variety of hematological malignancies and solid tumors.

Tumor cells can up-regulate inhibitory NK-receptor ligands such as KIR2DL4, ILT2, ILT4, and HLA-G, allowing cancer cells to escape NK-cell killing. In patients with CLL, blocking HLA-G on tumor cells is considered an effective way to sensitize NK-cell immunotherapy.

Figure four summarizes how a CLL cell can evade NK-cell immune surveillance by reshaping both sides of the interaction. On the NK-cell side, inhibitory signals involving KIR2DL4, ILT2, ILT4, and HLA-G are enhanced, while activating signals through IL-2R, NKP30, NKG2D, and TRAIL are impaired.

The CLL cell also displays factors linked to ligand shedding and death-receptor escape, including ERp5, ADAM10, ADAM17, BAG6, ULBP1-to-3, and TRAILR, helping explain reduced NK-cell killing. In the early nineteen eighties, NK cells in patients with CLL were found to have impaired cytolytic activity, believed to result from intrinsic NK-cell defects.

Later findings suggested that immune-escape mechanisms of CLL cells also caused impaired NK-cell cytolytic activity, in addition to intrinsic defects. Other studies found that peripheral-blood NK cells in patients with CLL retained degranulation, cytokine production, and antibody-dependent cytotoxicity.

The reported activity could be restored with adequate activating signals from certain interleukins, suggesting that NK-cell cytolytic impairment can be reversed in certain conditions. Low-level expression of HLA-class one molecules in CLL cells of most patients may also lead to NK-cell-mediated tumor killing when those molecules are missing.

Some studies found more peripheral NK cells in patients with CLL than in healthy people, and linked the increased amount of NK cells with better prognosis. Other studies did not support a correlation between an increased amount of NK cells and CLL prognosis.

One factor contributing to these different results is CMV infection, which increases mature NK cells expressing the activating receptor CD94 slash NKG2C and changes the NK-cell phenotype. Differences may also come from study design, subjects, inclusion and exclusion criteria, experimental methods, and signaling substances used to stimulate NK-cell receptors.

The review therefore concludes that insufficient studies have examined NK-cell mechanism and functionality in patients with CLL. In CLL immunotherapy, NK cells are used through stimulated or restored activity in patients’ own NK cells, or through administering NK cells with activity to patients.

Adoptive immunotherapy refers to the in vitro induction and culture of patient, healthy-donor, autologous, or allogeneic NK cells to directly or indirectly kill tumor cells. The source of NK cells is an important factor in determining treatment efficacy, and patient-derived peripheral-blood NK cells are difficult to expand in vitro as expected.

In one study, NK cells reached forty-seven-hundred-and-twenty-fold amplification in vitro and expressed NKG2D and CD16 strongly, but no clinical response was observed. One approach to NK-cell immunotherapy in CLL intensifies NK-cell-mediated antibody-dependent cytotoxicity using tumor-specific monoclonal antibodies, or bispecific and trispecific killer engagers.

The review points to Table one as the summary of tumor-specific antibody therapies used in this approach. Table 1 summarizes tumor-specific antibody therapies that intensify NK cell-mediated antibody-dependent cellular cytotoxicity in chronic lymphocytic leukemia.

For CD20, it lists rituximab’s effects, including complement-dependent cytotoxicity, direct target-cell apoptosis, antibody-dependent phagocytosis, and ADCC, alongside limitations such as CD20 loss and impaired NK-cell activity. It also highlights strategies including glycoengineering, afucosylation, Fc-engineering, and BiKEs or TriKEs to address immune evasion and improve tumor-cell recognition.

Tumor-specific monoclonal antibodies stimulate NK-cell-mediated antibody-dependent cytotoxicity and recognize ligands on CLL cells, including CD20, CD19, and CD37. The first monoclonal antibodies used in CLL immunotherapy targeted CD20, and the first approved for clinical use was rituximab.

Rituximab’s anti-tumor effects include complement-dependent cytotoxicity, direct target-cell apoptosis, antibody-dependent phagocytosis, and antibody-dependent cytotoxicity. Rituximab had low efficacy alone, while combining it with fludarabine and cyclophosphamide in FCR added to efficacy.

Limitations of anti-CD20 monoclonal antibody alone include loss of CD20 antigen, weak binding of Fc gamma receptor three A to rituximab, and increased release of inhibitory substances that weaken NK-cell-mediated antibody-dependent cytotoxicity. Figure five summarizes how anti-CD20 monoclonal antibodies, including rituximab, obinutuzumab, ublituximab, and ofatumumab, act against tumor cells.

The central diagram shows CD20-targeted antibodies engaging NK cells to produce antibody-dependent cellular cytotoxicity, or ADCC, while also depicting direct tumor-cell apoptosis and antibody phagocytosis. It also links these mechanisms with treatment effects, including recurrent-tumor control, and notes that rituximab efficacy can be limited by reduced CD20 antigen, Fc-gamma receptor expression, inhibitory substances, and NK-cell activity.

Bispecific and trispecific killer engagers trigger human NK-cell effector function through CD16 signaling and induce cytotoxicity and cytokine production. Bispecific engagers contain one single-chain variable fragment recognizing CD16 and another recognizing tumor antigens, allowing signal transfer through immunological synapses.

Trispecific engagers recognize two different tumor antigens, increasing the possibility of identifying tumor cells when one antigen is missing and reducing immune evasion compared with bispecific engagers. Studies found that a CD16-and-CD19 bispecific engager induced antibody-dependent cytotoxicity against primary CLL cells in vitro, while CD16, CD19, and CD22 trispecific engagers enhanced NK-cell cytotoxicity and interferon gamma production.

A trispecific engager recognizing CD16, CD19, and interleukin fifteen activated NK cells from healthy donors and caused killing of CLL cells, showing promising efficacy in refractory CLL. In haploidentical hematopoietic stem-cell transplantation for high-risk lymphoid leukemia, donor NK-cell KIRs do not bind HLA on patient leukemia cells, so the NK cells receive no inhibitory KIR-HLA signal and become activated.

Allogeneic donor NK cells are therefore important in the graft-versus-leukemia effect, which has been adequately studied and proved. In vitro studies also found that allogeneic NK cells can kill CLL cells without relying on KIR-HLA mismatches.

In contrast, the graft-versus-leukemia effect in allogeneic transplantation for CLL relies primarily on T cells, and T-cell-mediated effects may lead to graft-versus-host disease. Allogeneic NK cells may be a safer immunotherapy option because they do not directly mediate the graft-versus-leukemia effect.

Table two surveys six current NK-cell immunotherapy trials for CLL, listing each identifier, the NK-cell source, development phase, and treatment design. The approaches include allogeneic, cord-blood-derived, NK92, AT19, and non-HLA-matched donor cells, with combinations such as CAR-NK-CD19, conditioning chemotherapy, rituximab, mogamulizumab, or ALT803.

This matters because the table makes visible how clinical translation is exploring potentially off-the-shelf NK-cell therapies across different cellular sources and engineering strategies. CAR-NK cell therapy has become an emerging field of adoptive immunotherapy, with an increasing number of preclinical studies conducted on it.

The review presents CAR-NK cell therapy as a potential option comparable to, and possibly superior to, CAR T-cell therapy. CAR-NK cells are being explored for blood and solid tumors, with broad prospects, but factors still affect their clinical transformation. Unlike T cells, arming NK cells with CAR is challenging because peripheral-blood NK cells have a low transfection rate and short survival time in vivo.

Researchers are exploring ways to improve primary-NK-cell transfection efficiency, but no significant progress has been made. CAR-NK-cell activation is affected by the location of the CAR-binding epitope and by the distance between the CAR-NK-cell surface and that epitope.

Contamination of allogeneic NK cells by T cells can induce graft-versus-host disease or lymphoproliferative disorders. Figure six illustrates how anti-CD19 monoclonal antibodies recruit natural killer cells to attack CD19-positive tumor cells through antibody-dependent cellular cytotoxicity, or ADCC.

It highlights two antibody-enhancing strategies: afucosylation, represented by inebilizumab, which strengthens interaction with Fc gamma receptor three A on NK cells, and Fc-engineering, which also supports NK-cell activation. The figure matters because these modifications are presented as ways to intensify NK-cell-mediated antitumor activity in chronic lymphocytic leukemia.

The review summarizes NK-cell-based immunotherapy as a valid alternative treatment for patients with CLL according to current evidence. NK cells are presented as a promising approach for CLL immunotherapy, using mechanisms against leukemia cells that differ from earlier treatments.

NK-cell immunotherapy can provide a second choice for leukemia resistant to routine treatment and may reduce immune escape by motivating the potential of the patient’s own immune system. Its efficacy is being evaluated by more and more clinical trials, while research on receptors such as 2B4, CS1, and LLT1 and their ligands may clarify leukemia mechanisms and improve therapies.

The proliferation and persistence of NK cells in vivo may be enhanced by combining them with cytokines such as interleukin twelve, interleukin fifteen, and interleukin twenty-one. Although few studies have designed CAR structures based on NK-cell characteristics, CAR can powerfully increase NK-cell killing activity.

Most CAR-NK cells simply follow CAR-T-cell design without accounting for NK-cell-specific characteristics. Designing optimized CAR structures suitable for NK cells and transfecting them into memory-like NK cells or specific NK-cell subsets is presented as a promising research direction.

The review presents NK-cell immunotherapy as a promising alternative for CLL, spanning antibody-assisted killing, donor NK cells, and CAR-NK cells—but it also shows that dysfunction, immune escape, manufacturing, and clinical validation remain major barriers.