Lymphoma is a general category of hematological malignancies that arise from aberrant lymphocytes, which are part of the immune system. Lymphomas are typically divided into Hodgkin’s lymphoma (HL) and non-Hodgkin lymphoma (NHL), the latter being much more common than the former. Among the subtypes of NHL, the most common are B-cell lymphomas, which include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and mantle cell lymphoma. In contrast, T-cell lymphomas and HL are less common but have important clinical subtypes with unique pathologic and clinical features [1]. Despite advances in chemotherapy and targeted therapy regimens, the outlook for the vast majority of patients with relapsed or refractory B-cell lymphoma remains unfavorable. Standard first-line treatment regimens, including R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) have dramatically increased survival rates in B-cell lymphomas, particularly diffuse large B-cell lymphoma (DLBCL). However, these regimens have limited long-term benefits in patients with high-risk or advanced disease. In addition, autologous stem cell transplantation, although beneficial for some, is not an option in all cases. Similarly, resistance to treatment and relapse are often seen in other types of lymphoma, such as some T-cell lymphomas and HLs [2]. These limitations highlight the need for more robust and individualized treatment approaches across the spectrum of lymphatic malignancies.

Cell-based immunotherapy has revolutionized cancer treatment by leveraging immune system cells to identify and eradicate tumor cells. Among the diverse cell-based immunotherapeutic strategies, the utilization of natural killer (NK) cells has emerged as a promising therapeutic approach in lymphoma. As innate immune effectors, NK cells possess distinctive properties that render them attractive candidates for immunotherapy, notably their capacity to recognize and eliminate tumor cells without prior sensitization. NK cells play a critical role in the immune surveillance against tumor development and viral infections. They possess a delicate balance of activating and inhibitory receptors that allows them to distinguish between healthy cells and those that exhibit altered self-characteristics, such as infected or malignant cells [3].

However, in the context of lymphoma, aberrations within the tumor microenvironment can suppress NK cell activity, compromising their ability to effectively target and eliminate tumor cells. Thus, strategies to enhance NK cell function and overcome immunosuppressive mechanisms are being explored to unleash the full potential of these cells in lymphoma treatment. These strategies include targeting immunosuppressive factors, modulating cytokine levels, and overcoming NK cell exhaustion. By understanding the complex interplay between NK cells and the lymphoma microenvironment, novel immunotherapeutic strategies can be developed to improve anti-tumor responses and enhance NK cell-mediated immunity in lymphoma.

Despite the importance of NK cells in the immune response against cancer cells, lymphomas employ various mechanisms to evade or suppress NK cell activity. The most common suppressive factors are tumor-derived soluble factors, which are released by lymphoma cells and have immunosuppressive effects on NK cells. These factors include cytokines, chemokines, and growth factors, such as TGF-β and IL-10, which inhibit the activity of NK cells and impair their cytotoxic functions [11]. The interaction between inhibitory receptors expressed on NK cells and their corresponding ligands on lymphoma cells represents another mechanism that suppresses NK cell activity. Inhibitory receptors recognize specific ligands on target cells, and lymphoma cells can upregulate the expression of ligands for these inhibitory receptors, such as Major Histocompatibility Complex class I molecules. These ligands engage with inhibitory receptors like KIRs and NKG2A, transmitting inhibitory signals to the NK cells and dampening their cytotoxicity against lymphoma cells [12]. In addition, immunoregulatory cells, such as Tregs and MDSCs, that often exist in the lymphoma microenvironment also suppress NK cell function. These cells produce immunosuppressive molecules and inhibit NK cell activity, preventing effective anti-tumor responses [13]. Furthermore, extracellular matrix components and stromal cells in the lymphoma microenvironment, such as cancer-associated fibroblasts (CAFs), secrete factors and remodel the extracellular matrix, creating a physical barrier that hampers NK cell infiltration into the tumor site. This limited access to malignant lymphocytes imposes spatial constraints that impede NK cell recognition and subsequent mediated cytotoxicity [14]. Metabolic reprogramming derived from lymphoma cells also leads to alterations in the tumor microenvironment that affect NK cell function. For example, increased consumption of nutrients and competition for resources within the microenvironment impair the NK cell metabolism and compromise their effector functions [15]. Immune checkpoint molecules, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), expressed in NK cells are upregulated in the lymphoma microenvironment [16]. Engagement of these checkpoints by their ligands on lymphoma cells leads to NK cell exhaustion and functional suppression. These immune checkpoint molecules are negative regulators of T-cell and NK cell function that limit the amplitude of T cell and NK cell activation. The blockade of immune checkpoints, such as PD-1 and CTLA-4, has shown promising results in enhancing antitumor immunity and producing durable clinical responses [17]. Moreover, genetic and epigenetic changes within lymphoma cells contribute to NK cell suppression. These alterations affect the expression of molecules involved in NK cell recognition and activation, impairing their ability to effectively target and eliminate lymphoma cells. It has been observed that promoter methylation/hypermethylation of tumor suppressor genes, miRNA expression or methylation level, and Long noncoding RNA (LncRNA) expression were associated with lymphoma disease progression [18].

The treatment of lymphoma relies heavily on medication, which varies depending on the type and stage of lymphoma, as well as individual patient factors. Common types of medication used in lymphoma treatment include chemotherapy, monoclonal antibodies, immunomodulatory drugs, and targeted therapies [19].

NK cells are crucial in host immunity against cancer, but cancers develop mechanisms to escape NK cell attack or induce defective NK cells. NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. These approaches include expanded allogeneic NK cells, stable allogeneic NK cell lines, and genetic modification of fresh NK cells or NK cell lines. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells, or even induced pluripotent stem cells (iPSCs) [31]. A variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. In the first approach, expanded allogeneic NK cells are not inhibited by self-histocompatibility antigens like autologous NK cells, making them suitable for adoptive cellular immunotherapy. In the second approach, stable allogeneic NK cell lines are more practical for quality control and large-scale production. Thirdly, genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors, and/or chimeric tumor-antigen receptors is another approach [31]. In the context of lymphoma, the objective of NK cell therapy revolves around exploiting the inherent anti-tumor attributes possessed by these cells, with the specific aim of selectively targeting and eradicating lymphoma cells. The implementation of this therapy entails the introduction of ex vivo expanded and activated autologous (derived from the patient themselves) or allogeneic (derived from a donor) NK cells into the patient's system [32], as illustrated in Table 1. The ultimate goal lies in bolstering the immune response against lymphoma cells, thereby promoting regression of the tumor. Clinical trials investigating allogeneic NK cell therapies in patients with relapsed or refractory hematologic malignancies, including those with B-cell lymphoma, have supported the significant anti-tumor activity of these cells. These therapies may provide a superior safety profile, as they are associated with a lower incidence of cytokine release syndrome (CRS) and neurologic toxicity compared to T-cell therapies. Nevertheless, the use of allogeneic NK cell therapies is hampered by several limitations, such as the scarcity of appropriate donors, the relatively short in vivo persistence, and the manufacturing constraints that hinder the consistent administration of multiple doses [33].

NK cell therapy for lymphoma can be administered either as a standalone treatment or in conjunction with other therapeutic modalities such as chemotherapy, radiation therapy, or targeted therapies. The intention behind combination approaches is to amplify the effectiveness of standard treatments while concurrently enhancing the immune response against lymphoma [34]. Table 2 outlines selected clinical studies using allogeneic NK cells in lymphoma, while Table 3 provides preclinical data supporting their potential efficacy.

Chimeric Antigen Receptors (CARs) are genetically engineered to recognize and bind to antigens on the cell surface. Receptors allow cells to recognize specific targets, for example, tumor cells, and trigger potent cytotoxic activity against them. CARs are usually designed to bind to particular antigens on the surface of cancer cells and therefore provide targeted therapies against tumors. While the earliest cancer treatments were mainly based on CAR-T cell therapy, recent studies have also proposed CAR-NK cell therapy (CAR-engineered NK cells) as a promising candidate for tumor treatment [35].

These treatments make use of NK cells, which already have the innate ability to recognize and eradicate cancer cells. By using CAR technology, the cells are engineered to recognize and eliminate specific tumor antigens. These cells, due to their unique features, i.e., not requiring HLA matching and possessing allogeneic potential, can effectively target cancer cells. Furthermore, the ability of CAR-NK cells to exert potent antitumor effects without inducing severe toxicities (specifically CRS, neurotoxicity, or graft-versus-host disease [GvHD]) establishes this modality as a safe and efficacious alternative to traditional cellular therapies [36].

The efficacy of CAR-NK cells in treating lymphoma and other hematologic malignancies is being investigated in several studies. A pivotal study by Liu et al., published in The New England Journal of Medicine, demonstrated that cord blood-derived CAR-NK cells targeting CD19 induced high response rates without CRS, neurotoxicity, or GvHD; this represents a significant milestone in NK cell therapy development (Clinical Trial: NCT03056339) [37].

Building upon this foundational work, Rezvani et al. reported the successful application of off-the-shelf allogeneic CAR-NK cells with sustained efficacy and favorable safety profiles in patients with relapsed or refractory lymphoid malignancies, including lymphoma. These findings further highlighted the feasibility and clinical relevance of CAR-NK cells as a scalable, low-toxicity alternative to CAR-T cell therapies [38]. One of the most promising clinical trials was conducted by Fate Therapeutics, in which iPSC-derived CAR-NK cells (FT596) were administered to treat relapsed and refractory B-cell lymphomas. FT596 was designed to target three antigens, CD19, CD16, and IL-15 in order to increase anti-tumor activity and persistence. The trial enrolled 86 patients across nine US research sites. The outcomes revealed that 50 % of the patients who received a high dose of FT596 (900 million cells) and 33 % of the patients receiving lower doses achieved a complete response. Side effects were low, primarily including mild CRS that resolved without intensive care. Notably, no cases of immune effector cell-associated neurotoxicity syndrome (ICANS) or GvHD were observed (Clinical Trial: NCT04245722) [39]. Further advancements were made by Nkarta Therapeutics, who conducted trials using their allogeneic CAR-NK product, NKX019, targeting CD19 antigens. This trial demonstrated an impressive overall response rate (ORR) of approximately 80 % and a complete response (CR) of 70 %. Most importantly, no CRS or neurotoxicity occurred, confirming both the efficacy and safety (Clinical Trial: NCT05020678) [40]. Similarly, the TAK-007 (ARC-TCR) trial employed umbilical cord blood-derived CAR-NK cells for the treatment of indolent NHL (iNHL) and large B-cell lymphoma (LBCL). Preliminary results indicated an ORR of 78 % in iNHL and 50 % in LBCL, again without major side effects such as CRS and neurotoxicity (Clinical Trial: NCT05020015) [41]. A more recent approach by Allogene Therapeutics involved the use of allogeneic CAR-NK cells (ALLO-316) specifically engineered to target CD19 antigens in refractory B-cell lymphomas. This study reported a 60 % ORR, with no high-grade toxicities like CRS or neurotoxicity, positioning ALLO-316 as a promising novel therapy in the management of difficult-to-treat lymphomas (Clinical Trial: NCT04263594) [42]. Key clinical trials evaluating CAR-NK cell therapy in B-cell lymphoma are summarized in Table 4.

Presently, ongoing research endeavors and clinical trials are dedicated to refining these aforementioned aspects of NK cell therapy with a primary focus on evaluating the safety, efficacy, and long-term outcomes in lymphoma patients. Affimed (AFMD), a clinical-stage immuno-oncology company, focuses on developing targeted immuno-oncology therapies. Affimed has three programs (AFM13, AFM24, and AFM26). AFM13 (acimtamig) is a first-in-class tetravalent bispecific innate cell engager, engineered as a tandem antibody (TandAb) construct to treat CD30-expressing malignancies. It facilitates targeted tumor lysis by providing bivalent binding to CD30 on malignant cells and CD16A on NK cells and macrophages. It recruits NK (NK) cells by binding to CD16A as immune effector cells [31]. AFM13 induces NK cell-mediated cytotoxicity, resulting in tumor cell lysis. Results of a Phase 1 dose-escalation study conducted on 28 patients with heavily pretreated relapsed or refractory HL show that AFM13 is well-tolerated and active, with an objective response rate of 50 %. Hence, AFM13 represents a new, feasible, targeted immunotherapy for heavily pretreated patients with HL. The dose regimen of AFM13 has to be optimized and the treatment duration has to be prolonged in order to increase the clinical efficacy [43]. The second study is an ongoing Phase II clinical trial that aims to evaluate the antitumor activity and safety of AFM13 in patients with CD30-positive peripheral T-cell lymphoma (PTCL) or transformed mycosis fungoides (tMF). The trial is investigating the efficacy of AFM13 as monotherapy in patients with relapsed or refractory CD30-positive T-cell lymphoma. The results of the trial are not yet available [44]. Peripheral T-cell lymphomas are highly aggressive and one of the most difficult-to-treat forms of lymphoma. Affimed, after underwhelming monotherapy data, has decided to focus on testing AFM13 in combination with the AB-101 NK cell product of Artiva. AB-101 NK is an allogeneic, non-genetically modified cord blood-derived NK cell. It is a cryopreserved NK cell therapy that has been optimized for combination with monoclonal antibodies to enhance ADCC and anti-tumor responses. The combination of AFM13 and AB-101 NK cells has the potential to provide a more durable and effective response compared to monotherapy with AFM13 [45].

AFM13 is a tetravalent bispecific antibody engineered to engage simultaneously CD30 on cancer cells and CD16A on NK cells, thereby enhancing ADCC. To further improve its efficacy, more recent studies have focused on combining AFM13 with umbilical cord blood-derived NK cells, particularly those activated with cytokines to enhance their persistence and cytotoxic activity. Kerbauy et al. demonstrated in preclinical models that cytokine-induced memory-like NK cells derived from cord blood, which were precomplexed with AFM13, exhibited enhanced antitumor activity against CD30-positive lymphoma cells. The NK cells were hyperactivated and cytotoxic despite the presence of immunosuppressive cytokines such as TGF-β and IL-10, which are typically upregulated within the tumor microenvironment [46]. Thereafter, a Phase I/II first-in-human trial by Nieto et al. at MD Anderson Cancer Center evaluated the safety and efficacy of this combination in relapsed or refractory CD30-positive lymphomas. The trial consisted of ex vivo AFM13 precomplexing with cytokine-preactivated cord blood-derived NK cells before infusion. Preliminary outcomes report an ORR of 93 % and CR of 66 % with favorable safety outcomes [47]. These findings underscore the effectiveness of AFM13 combined with NK cells derived from umbilical cord blood as a potent method in the treatment of CD30-positive lymphomas, particularly in relapsed or refractory disease, and implicate the utility of innate immune effectors in next-generation cancer immunotherapies.

One of the most important advances in NK cell-based immunotherapy is the generation of cytokine-induced memory-like NK (CIML-NK) cells. The cells are generated by a temporary pre-activation with IL-12, IL-15, and IL-18, which are capable of inducing memory-like functional properties. CIML-NK cells exhibit increased IFN-γ production, increased cytotoxicity, and increased proliferative capacity upon secondary stimulation. Phenotypically, they are CD56brightCD16dim with strong expression of activating receptors (NKG2D, NKp30, and NKp46) and transcription factors (T-bet and Eomes) [48]. Epigenetic reprogramming following cytokine stimulation is a hallmark of CIML-NK cells, leading to lasting changes in phenotype and function [49]. The epigenetic modifications involve demethylation of regulatory elements in the IFNG locus (e.g., CNS1) and regulation of master genes such as PRDM1/BLIMP1 and ZBTB32, which together allow long-term IFN-γ expression in progeny cells as well as enhanced cytotoxic activity. This long-term reprogramming makes CIML-NK cells attractive candidates for cancer immunotherapy, particularly for treating refractory and relapsed lymphomas [50].

Despite their tremendous potential, there are currently few clinical trials evaluating CIML-NK cells in the context of lymphoma, and no specific studies have been formally published yet. Most clinical data come from a Phase I study (Clinical Trial: NCT01898793) that focuses primarily on patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In this study, NK cells were preactivated with IL-12, IL-15, and IL-18, and infused into patients to assess safety and preliminary efficacy. Although lymphoma was not the primary target, the study illustrated the broad potential of CIML-NK cells for the treatment of refractory hematological malignancies [43].

Preclinical research has further proved the efficacy of CIML-NK cells against lymphoma and melanoma models. In such experimental models, CIML-NK cells, which had been activated with IL-12, IL-15, and IL-18, were infused into mice with established tumors. The results demonstrated that CIML-NK cells effectively controlled tumor growth and increased survival rates, thus confirming their notable therapeutic value in treating tumor resistance to NK cells [51]. However, specialized clinical trials are needed to confirm these results in lymphoma patients.

Various combinatorial approaches have been established to further improve the activity of CIML-NK cell therapy. A combination of CIML-NK cells with monoclonal antibodies like rituximab (anti-CD20) has augmented ADCC and clinical responses considerably [52]. Moreover, strategic targeting of the immune checkpoint pathways suppressing NK cell function, specifically NKG2A and PD-1/PD-L1, is a novel approach in reconstituting CIML-NK cytotoxicity for the immunosuppressive tumor microenvironment [53]. Cytokine support strategies, specifically using IL-15 superagonists such as ALT-803 (n - 803), have been found to augment the in vivo stability, expansion, and activation of CIML-NK cells, resulting in durable antitumor activity [54].

Moreover, genetic modification of CIML-NK cells through the use of CARs has significantly enhanced their therapeutic efficacy. A recent preclinical study demonstrated that CIML-NK cells transduced with CD19-targeting CAR exhibited potent antitumor activity against CD19-expressing malignancies. In comparison to the conventional CAR-NK cells, CAR-CIML-NK cells produced more IFN-γ and exhibited greater degranulation against CD19+ target cells, resulting in greater cytotoxicity against leukemic and lymphomatous cells. Furthermore, the IL-12, IL-15, and IL-18 induced memory-like characteristics enhanced the in vivo persistence of CAR-CIML-NK cells and effectively inhibited tumor growth in CD19+ lymphoma mouse models, thereby resulting in extended survival [55]. For a succinct overview of the role of NK cells as a therapeutic intervention in lymphoma, pertinent studies and evidence are summarized in Table 2.

Various preclinical and clinical studies have demonstrated that improving the clinical efficacy of NK cell therapy for hematopoietic malignancies, such as lymphoma and leukemia, requires overcoming significant challenges related to optimal proliferation, long-term persistence, and effector function.

Appropriate selection of cell sources, advances in expansion methods and genetic modification techniques (CAR, Bispecific Killer Cell Engagers [BiKEs], high-affinity, non-cleavable CD16 [hnCD16]), cytokine adjuvants, antibody design and stem cell biology have been effective in overcoming these limitations.

Recently, induced pluripotent stem cells (iPSCs) and the NK-92 cell line have emerged as promising substrates for CAR engineering, owing to their inherent homogeneity and virtually unlimited proliferative capacity. However, due to the lymphoma origin of NK-92 cell lines, there are some limitations. Furthermore, technical complexities inherent to iPSC differentiation have hindered the large-scale manufacturing of NK cells from this progenitor source. However, regarding the potential and ability of NK 92 lines and iPSCs as an off-the-shelf therapy, advances in the use of these cells, expansion methods, and specific targeting employing genetic engineering have been made and are being investigated. These advances promise to overcome many challenges, thus bringing the application of NK 92 lines and iPSC-derived NK cells closer as clinically approved options for the treatment of hematologic and solid malignancies. Until now, the NK92 cell line has not been used in any registered trials, however, iPSC-derived NK cells have been used in Phase I clinical trials of lymphoma and other malignancies (Clinical Trials: NCT04245722; NCT04023071; NCT04630769; NCT0455188; NCT03841110). In recent years, engineered iPSC-derived NK cells have demonstrated promising results in preclinical lymphoma models. Specifically, FT516 was engineered to express a high-affinity CD16 receptor for targeting both CD19 and CD20 antigens. This platform was further optimized in FT596 as a triple-gene-engineered product incorporating these modifications alongside a membrane-bound 15/IL-15Rα fusion protein. Subsequent iterations have utilized transgenic overexpression of ARID5B or CRISPR-mediated knockout of CD38 to enhance in vivo persistence and effector function.

Regarding expansion methods, it was observed that the ex vivo incubation of NK cells with IL-15 and nicotinamide (NAM) exhibited improved proliferation, persistence, cytotoxicity, and inflammatory cytokine production (e.g., Gamida cell; GDA-201 - Clinical Trial: NCT05296525) [56]. In addition to cytokine-based approaches, feeder cell-based expansion has shown significant promise. For example, irradiated K562 feeder cells genetically engineered to express membrane-bound IL-15 (mbIL-15), IL-21 (mbIL-21), and 4–1BBL have been extensively used to promote robust NK cell expansion. These feeder-based protocols can achieve over 1000-fold expansion within two to three weeks and improve NK cell cytotoxicity and IFN-γ secretion. Feeder-supported methods have been used in good manufacturing practice-compliant protocols for clinical-grade NK cell production and are currently under evaluation in clinical trials (e.g., NCT00995137, NCT02028407) [57]. Nevertheless, safety concerns regarding the tumor origin of feeder cell lines have led to parallel development of feeder-free systems and artificial antigen-presenting cells (aAPCs).

In recent years, CAR- and BiKE-NK cells have been used as alternative safer cells compared to CAR-T cells, for treating hematological malignancies. NK-92 cell lines represent one of the most widely utilized sources for CAR-NK cells; however, due to their limited in vivo expansion, umbilical cord blood (UCB) has emerged as a viable alternative. UCB-derived NK cells exhibit a higher proliferative rate and enhanced in vivo persistence compared to cell line-based models. Recently, several CAR-NK cell products have entered Phase I/II clinical trials for solid tumors and hematologic malignancies (Clinical Trials: NCT05654038, NCT05528341). Furthermore, promising Phase II trials are currently underway for patients with relapsed or refractory lymphoma who have previously failed brentuximab vedotin and PD-1 inhibitors. These studies, including LuminICE-203 (Clinical Trial: NCT05883449) and AFM13–104 (Clinical Trial: NCT04074746), utilize allogeneic cord blood-derived NK cells pre-complexed with AFM13, followed by multiple infusions of AFM13 monotherapy. These trials assess dose-escalation of pre-complexed, off-the-shelf NK cells, followed by expansion cohorts in Phase I/II for relapsed or refractory lymphoma, particularly CD30+ peripheral T-cell lymphoma.

Recent studies on the treatment of relapsed and refractory lymphoma suggest that, in addition to selecting optimal NK cell sources and enhancing in vivo persistence, combinatorial strategies, including the use of anti-lymphoma monoclonal antibodies, BiKEs, or checkpoint inhibitors, yield superior efficacy compared to monotherapeutic cell therapy. These improved outcomes are primarily mediated through more precise tumor targeting and the blockade of inhibitory regulators.

Preclinical trials using new NK products with improved expansion, demonstrate increased anti-lymphoma activity due to enhanced persistence and potency of transferred NK cells. The findings from these trials will impact the design of future clinical protocols; when integrated with multimodal strategies and the optimization of dosage and administration schedules, these advancements may lead to superior therapeutic outcomes in the treatment of lymphoma.

NK cells are garnering significant attention in lymphoma therapy due to their unique properties, most notably their ability to exert rapid and potent antitumor responses without prior sensitization. However, the complex interplay between NK cells and the lymphoma microenvironment, coupled with immune escape mechanisms and the suppressive effects of certain medications, can impair NK cell function. Integrating NK cell therapy with chemotherapy or immune checkpoint inhibitors holds significant potential for synergistic effects and may overcome these hurdles. While limitations regarding optimal proliferation, long-term persistence, and effector function remain, recent advances are addressing these challenges. Specifically, the selection of appropriate cell sources, progress in expansion protocols, and genetic modifications, including CAR, BiKEs, and hnCD16, alongside cytokine adjuvants and stem cell biology, are proving effective in enhancing the therapeutic efficacy of NK cells.

This project was supported by Hematopoietic Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

The data that support the findings of this study are available from the corresponding author upon reasonable request.