Given the growing significance of immunotherapy in addressing the limitations of conventional acute myeloid leukemia (AML) treatments, this study aimed to elucidate the hotspot evolution and frontiers of immunotherapy in AML using bibliometric analysis. With a strict retrieval strategy applied in the Web of Science Core Collection, 2411 publications were obtained and exported. The temporal and geographical distributions of these publications and the countries, institutions, journals, and authors who contributed to the field were investigated. An in-depth content analysis was performed. The United States had various research institutions dedicated to AML immunotherapy. Frontiers in Immunology had the highest number of publications, but Blood had the highest H-index. Marion Subklewe was the most productive author. The current research hotspots of AML immunotherapy included chimeric antigen receptor-T-cell therapy, antibody-based immunotherapies, immune checkpoint blockade, and combination therapy, highlighting the key aspects of immunotherapy for AML treatment and providing comprehensive insights into the research status and advances in this field. Novel immunotherapies combined with chemotherapy may become the primary focus of AML treatment.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are randomly expressed by natural killer (NK) cell subsets and recognize motifs shared by HLA class-I (HLA-I) allotypes. Such interactions prevent NK cell autoreactivity while enhancing their response against cells lacking those HLA-I molecules (missing self), a situation defined in transplantation as iKIR-HLA-I mismatch (iKIR-MM), whose genotypic prediction has been associated with microvascular inflammation (MVI). Herein, we compared iKIR-MM in kidney transplant recipients with MVI ≥2 (n = 19) and controls with MVI ≤1 (n = 36). In parallel to genetic analysis of iKIR-MM, which was more frequent in MVI ≥2 patients, putative alloreactive iKIR-MM NK cells were defined by flow cytometry as NKG2A(-) cells bearing self-specific but lacking donor-specific iKIR. Although iKIR-MM NK cells were detected in both groups, their pretransplant numbers were higher in MVI ≥2 patients (median = 11.02, interquartile range = 0-58.31 vs median = 0, interquartile range = 0-9.46), especially in the presence of donor-specific antibodies or C4d, and correlated with MVI grade. Pretransplant, a subset of MVI ≥2 patients showed high proportions and numbers of oligoclonal iKIR-MM NK cells, which displayed an NKG2C(+) adaptive phenotype associated with cytomegalovirus infection. This pilot study provides a novel perspective on the contribution of iKIR-MM NK cells to MVI, with potential practical implications.

High-grade serous ovarian cancer (HGSOC) remains an urgent unmet clinical need, with more than 70% of patients presenting with metastatic disease. Many patients develop large volumes of ascites, which promotes metastasis and is associated with poor therapeutic response and survival. Immunotherapy trials have shown limited success, highlighting the need to better understand HGSOC immunology. Here, we analyzed cytotoxic lymphocytes [natural killer (NK), T, and innate T cells] from patients with HGSOC and observed widespread dysfunction across primary and metastatic sites. Although nutrient rich, ascites was immunosuppressive for all lymphocyte subsets. NK cell dysfunction was driven by uptake of polar lipids, with associated dysregulation in lipid storage. Phosphatidylcholine was a key immunosuppressive metabolite, disrupting NK cell membrane order and cytotoxicity. Blocking lipid uptake through SR-B1 protected NK cell antitumor functions in ascites. These findings offer insights into immune suppression in HGSOC and have important implications for the design of future immunotherapies.

Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials.

Advanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.

We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function in vitro and in vivo. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo.

The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

Natural Killer (NK) cells are an important population of the immune system, and NK cell-based therapy has shown great potential in the treatment of cancers. However, to apply NK cells clinically, producing a large number of cells with high cytotoxicity remains a challenge. Current strategies focus on employing different irradiated feeder cells to stimulate NK expansion, maturation, and cytotoxicity. While co-stimulatory signals play critical roles in promoting NK cell proliferation and activating their functions, the exploitation of these signals for expanding NK cells has not been fully explored. To identify the optimal engineered feeder cells for expanding umbilical cord blood-derived NK cells, we generated different feeder cells expressing the co-stimulatory molecules CD80, 4-1BBL, or membrane-bound IL-21 (mbIL21). We then evaluated the transduction efficacy of a chimeric antigen receptor (CAR) construct into expanded NK cells using various lentiviral vectors. Our results showed that CD80, in combination with 4-1BBL and mbIL21, induced the highest expansion of NK cells from cord blood. The expanded NK cells displayed higher cytotoxicity toward target cells compared to T cells following CAR transduction using BaEV lentivirus.

Prevention of graft-versus-host disease (GVHD) is critical to successful allogeneic hematopoietic cell transplantation (HCT), but for many years was difficult to achieve. Advances in the understanding of allogeneic HCT biology and immunology have paved the way for novel clinical approaches to GVHD prophylaxis, highlighted by the broad adoption of posttransplant cyclophosphamide and the approval of abatacept by the US Food and Drug Administration to prevent acute GVHD. Patients undergoing allogeneic HCT are now experiencing severe acute GVHD at historically low rates, and significant improvements in preventing chronic GVHD are also being achieved. This review highlights key pharmacological approaches and graft manipulation strategies being used or investigated for GVHD prophylaxis. Furthermore, we discuss the ongoing unmet needs in GVHD prevention and the challenges in addressing these areas in future clinical trials.

Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor with limited treatment options. Although natural killer (NK) cell-based immunotherapy is promising, its efficacy remains limited, necessitating strategies to enhance NK cell cytotoxicity. Histone deacetylase (HDAC) inhibition demonstrate potential to enhance NK-mediated killing. However, the combination of HDAC inhibitors and NK cell therapy for DMG remains unexplored.

Patient-derived DMG cell lines and orthotopic mouse models were used to evaluate the effects of the class I HDAC inhibitor MS-275 on cytotoxicity. NK cell-mediated lysis was measured using both luciferase and calcein AM-based assays. The downstream signaling pathways affected by MS-275 were investigated via RNA-seq, CUT&Tag assay, RT‒qPCR, and chromatin immunoprecipitation with qPCR.

Based on bioinformatic analysis, class I HDACs are identified as therapeutic targets in DMG. The corresponding HDAC inhibitor, MS-275 upregulated NK cell-mediated cytotoxicity pathway through GSEA analysis. Pretreating DMG cells with MS-275 elevated NK cell ligand gene expression and enhanced NK cell-induced lysis. In addition to NK-activating ligands, MS-275 elevated the NK-inhibitory ligand HLA-E, thereby enhancing the efficacy of immunotherapies targeting the NKG2A-HLA-E axis. Mechanistically, MS-275 increased HLA-E expression by promoting STAT3 acetylation at lysine 685. Combining MS-275 with NK cell therapy and blockade of the NKG2A-HLA-E axis extended overall survival in orthotopic mouse models.

This study is the first to demonstrate that HDAC inhibition enhances NK cell-mediated cytotoxicity in DMG. Combining HDAC inhibition with NK cell therapy represents a promising therapeutic strategy for treating DMG by targeting NKG2A-HLA-E axis.

Multiple myeloma (MM) is a haematological malignancy originating from terminally differentiated B cells, resulting in significant morbidity and mortality. Currently, MM is regarded as an incurable disease, often exhibiting a relapse-remitting pattern that necessitates multiple lines of therapy. It is now well-established that ineffective immunosurveillance plays a critical role in the progression of MM. Consequently, strategies that redirect immune effector cells against MM have emerged as effective treatment modalities, particularly in cases where standard care therapies fail. T cell-based immunotherapy has gained considerable attention in ongoing clinical trials; however, natural killer (NK) cells, known for their ability to execute cytotoxicity against infected and malignant cells with precision, may offer complementary therapeutic advantages over T cells and possess untapped therapeutic potential. This review seeks to introduce readers to the significance of NK cell-mediated immunosurveillance in the context of MM, explore the potential benefits of redirecting NK cells against MM, and illustrate how current treatment strategies are often reliant on the functionality of NK cells. Most importantly, new promising mechanisms of harnessing NK cell-based immunity against MM are reviewed and put into a clinical perspective to highlight their implications for patient treatment and outcomes.

Natural killer (NK) cells are emerging as a promising tool for cancer immunotherapy due to their innate ability to selectively recognize and eliminate cancer cells. Over the past 3 decades, strategies to harness NK cells have included cytokines, small molecules, antibodies, and the adoptive transfer of autologous or allogeneic NK cells, both unmodified and genetically engineered. Despite favorable safety profiles in clinical trials, challenges such as limited in vivo persistence, exhaustion, and the suppressive tumor microenvironment continue to hinder their efficacy and durability. This review categorizes NK cell-based therapies into 3 major approaches: (i) cellular therapies, including unmodified and chimeric antigen receptor-engineered NK cells; (ii) cytokine-based strategies such as interleukin-2 and interleukin-15 derivatives; and (iii) antibody-based therapies, including immune checkpoint inhibitors and NK cell engagers. We highlight these advancements, discuss current limitations, and propose strategies to optimize NK cell-based therapies for improved cancer treatment outcomes.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS.

Murine NK cells were activated and expanded ex vivo with superagonist interleukin (IL)-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, graft-versus-host disease and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray.

AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared with synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Combining CD155 and anti-DNAM-1 pretreated alloNK did not affect survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS.

These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT.

Chimeric antigen receptor (CAR) T and natural killer (NK) cell therapies represent a promising strategy for the treatment of cancers and other chronic diseases. Engineered CAR constructs endow immune cells with the ability to target desired antigens with high specificity, allowing for directed responses to antigen-expressing cells. CAR T and NK cells have shown marked success in the treatment of hematologic malignancies, although there remains a large population of patients with disease that fails to respond to CAR therapies, and their efficacy in solid tumors is still limited. In this review, we provide a broad overview of the development, design, and implementation of CAR therapies from bench to bedside. We discuss the building blocks of CAR constructs and how these can be manipulated to optimize CAR functionality, review the possible sources of T and NK cells for CAR therapies, and examine the limitations of both CAR T and CAR NK cells. Finally, we discuss recent breakthroughs in the CAR field and consider how these advances may affect the success of CAR therapies in the years to come.

Post-transplant cyclophosphamide (PT-CY) has been pivotal in controlling graft-versus-host disease (GvHD) following T-cell-replete haploidentical bone marrow transplantation (haplo-BMT). However, the widely adopted regimen is associated with high relapse rates, particularly in patients without GvHD. Our preclinical studies indicate that pre- or post-transplant bendamustine (PT-BEN) may reduce GvHD, enhance graft-versus-leukemia (GvL) effects, and induce significant alterations in the proportion, phenotype, and function of various immune cell subsets.

We initiated a Phase Ia/Ib, single-center trial with a standard 3 + 3 dose-escalation design, sequentially replacing post-transplant (PT)-CY with BEN (PT-CY/BEN). Multi-parameter flow cytometry and TCR β sequencing of genomic DNA was performed on isolated PBMCs on PT days +30, +60, +100, +180, and +365.

Overall, the PT-CY/BEN (n=14) regimen was associated with earlier neutrophil and platelet engraftment, reduced transfusion requirements, and comparable clinical outcomes to PT-CY (n=10), including survival and relapse rates. PT-CY/BEN patients exhibited distinct immune reconstitution patterns, characterized by earlier CD4+ T-cell recovery, impaired CD8+ T-cell engraftment, and reduced NK-cell counts. Notably there were no significant changes in B-cells, Tregs, or MDSCs. Enhanced T-cell repertoire diversity in the PT-CY/BEN cohort was associated with improved CMV control.

Our Phase Ia findings demonstrate the well-tolerability of PT-CY/BEN and its association with early engraftment, a more diverse T-cell repertoire, and earlier CD4+ T-cell reconstitution. Future studies are warranted to confirm our findings and investigate potential additional benefits of PT-CY/BEN over PT-CY alone.

Over the past few years, cellular immunotherapy has emerged as a promising treatment for certain hematologic cancers, with various CAR-T therapies now widely used in clinical settings. However, challenges related to the production of autologous cell products and the management of CAR-T cell toxicity highlight the need for new cell therapy options that are universal, safe, and effective. Natural killer (NK) cells, which are part of the innate immune system, offer unique advantages, including the potential for off-the-shelf therapy. A recent first-in-human trial of CD19-CAR-NK infusion in patients with relapsed/refractory lymphoid malignancies demonstrated safety and promising clinical activity. Building on these positive clinical outcomes, current research focuses on enhancing CAR-NK cell potency by increasing their in vivo persistence and addressing functional exhaustion. There is also growing interest in applying the successes seen in hematologic malignancies to solid tumors. This review discusses current trends and emerging concepts in the engineering of next-generation CAR- NK therapies. It will cover the process of constructing CAR-NK cells, potential targets for their manufacturing, and their role in various solid tumors. Additionally, it will examine the mechanisms of action and the research status of CAR-NK therapies in the treatment of solid tumors, along with their advantages, limitations, and future challenges. The insights provided may guide future investigations aimed at optimizing CAR-NK therapy for a broader range of malignancies.

The new era of cell therapeutics has started with autologous products to avoid immune rejection. However, therapeutics derived from allogeneic cells could be scaled and made available for a much larger patient population if immune rejection could reliably be overcome. In this review, we outline gene engineering concepts aimed at generating immune-evasive cells. First, we summarize the current state of allogeneic immune cell therapies, and second, we compile the still limited data for allogeneic cell replacement therapies. We emphasize the advances in this fast-developing field and provide an optimistic outlook for future allogeneic cell therapies.

Natural Killer (NK) cells have shown promising prospects in 'off-the-shelf' cell therapy, particularly the NK-92 cell line, which can serve as a foundation for the next generation of universal chimeric antigen receptor (CAR)-engineered NK products. A key strategy for generating universal cellular products is the elimination of the beta-2-microglobulin (B2M) gene, which encodes a component of MHC class I molecules (MHC-I) that plays a role in the presentation of foreign antigens and in the 'licensing' or 'education' of NK cells. To functionally study the impacts of MHC-I deficiency on NK-92, we generated a B2M knockout (KO) NK-92MI (B-92) cell line and compared the multidimensional properties of B2M KO and wild-type NK-92MI cells in terms of biological phenotypes, effector functions, and transcriptomic signatures. We observed a decrease in activating receptors, cytokine production, and cytotoxicity in B-92 cells. Further analysis of signalling events revealed that the upregulated expression and phosphorylation of SHP-1 in B-92 cells inhibited the phosphorylation levels of STAT3 and ERK, thereby affecting their killing function. By knocking out SHP-1 (PTPN6), we partially restored the cytotoxic function of B-92 cells. Notably, we also found that CAR modification can overcome the hyporesponsiveness of B-92 cells. These findings will facilitate further exploration in the development of NK cell-based products.

With the rapid development of the cell and gene therapy industry, there is an increasing demand for lentiviral vectors that can efficiently infect cells of different purposes. BaEV lentiviruses have been shown to efficiently infect hematopoietic stem cells, primary B cells, and NK cells, which traditional VSV-G lentiviruses cannot infect. However, there is a problem of low virus yield in the production of BaEV lentivirus. The formation of syncytium and apoptosis occur after plasmid transfection, and resulting in a reduction in virus production. In this study, the issue of low production of BaEV lentivirus was comprehensively improved. By increasing the cell density of inoculation, reducing the amount of BaEV plasmid, and adjusting the harvest time, the maximum titer of BaEV lentivirus reached 4.43E+ 06 IU/ml, representing an increase of 369 times compared to the unoptimized condition. Further, the purification method of lentivirus solution was optimized, and the infection titer of lentivirus reached 1.00E+ 08 IU/ml, which is 10300 times higher than pre-optimization levels.

Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR-Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4KO NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4KO NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4KO conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.

NK cell adoptive cell therapy (ACT) has emerged as a promising strategy for cancer immunotherapy, offering advantages in scalability, accessibility, efficacy, and safety. Ex vivo activation and expansion protocols, incorporating feeder cells and cytokine cocktails, have enabled the production of highly functional NK cells in clinically relevant quantities. Advances in NK cell engineering, including CRISPR-mediated gene editing and chimeric Ag receptor technologies, have further enhanced cytotoxicity, persistence, and tumor targeting. Cytokine support post-adoptive transfer, particularly with IL-2 and IL-15, remains critical for promoting NK cell survival, proliferation, and anti-tumor activity despite persistent challenges such as regulatory T cell expansion and cytokine-related toxicities. This review explores the evolving roles of IL-2 and IL-15 in NK cell-based ACT, evaluating their potential and limitations, and highlights strategies to optimize these cytokines for effective cancer immunotherapy.

Innate immune cells, including natural killer cells, macrophages and γδ T cells, are gaining prominence as promising candidates for cancer immunotherapy. Unlike conventional T cells, these cells possess attributes such as inherent antitumor activity, rapid immune responses, favorable safety profiles and the ability to target diverse malignancies without requiring prior antigen sensitization. In this Review, we examine the engineering strategies used to enhance their anticancer potential. We discuss challenges associated with each cell type and summarize insights from preclinical and clinical work. We propose strategies to address existing barriers, providing a perspective on the advancement of innate immune engineering as a powerful modality in anticancer treatment.

Peripheral blood stem cell (PBSC) donation is the primary procedure used to collect haemopoietic stem and progenitor cells (HSPCs) for haemopoietic stem cell transplants (HSCT), however there is a clinical need to reduce collection times and achieve sufficient HSPC doses for successful engraftment. Short bouts of interval cycling transiently enrich peripheral blood with HSPCs and cytolytic natural killer (CD56dim NK) cells, which predict engraftment success and prevent post-transplant complications respectively. Despite this, feasible protocols for use during PBSC collections (≈ 3 h) have yet to be evaluated.

In a randomised crossover design, 18 adults (9 young: 22.7 ± 3.2 years, 9 older: 65.2 ± 12.9 years) completed 3 × 3-h trials: high-intensity interval exercise (HIIE, 9 × 2-min cycling at 80-85% heart rate (HR)max/9 × 18 min rest), moderate-intensity interval exercise (MIIE, 9 × 4-min cycling at 65-70% HRmax/9 × 16 min rest) and REST (180 min). Immune cell subsets, including HSPCs and CD56dim NK concentrations (cells/µL) were determined across 18 timepoints and area under the curve (AUC, cells/µL x minutes) and total cell dose (cells/kg) were estimated.

By design, MIIE elicited lower average and peak HR and rating of perceived exertion than HIIE and was reported as more enjoyable. All cell subset concentrations increased following each interval of MIIE and HIIE. Across all participants, the estimated cell dose of total lymphocytes, monocytes, T cells, CD56bright and CD56dim NK was greater in MIIE and HIIE versus REST (p < 0.03), but there were no differences between MIIE and HIIE. The magnitude of change versus REST was greatest for CD56dim NK versus all cell subsets, and AUC was significantly greater in HIIE versus REST for this cell type only (p < 0.0001). There were no statistically significant differences in HSPC AUC (p = 0.77) or cell dose (p = 0.0732) in MIIE and HIIE versus REST. Age did not predict any changes across trials or timepoints for any cell type.

Persistent mobilisation of peripheral blood immune cells throughout 3 h of MIIE and HIIE evoked sustained numbers of CD56dim NK cells, but there was no reliable difference in HSPCs compared to a time-matched period of rest.

Chimeric antigen receptor (CAR) NK cell therapy has emerged as a promising alternative to CAR T cell therapy, offering significant advantages in terms of safety and versatility. Here we explore the current clinical landscape of CAR NK cells, and their application in hematologic malignancies and solid cancers, as well as their potential for treating autoimmune disorders. Our analysis draws from data collected from 120 clinical trials focused on CAR NK cells, and presents insights into the demographics and characteristics of these studies. We further outline the specific targets and diseases under investigation, along with the major cell sources, genetic modifications, combination strategies, preconditioning- and dosing regimens, and manufacturing strategies being utilized. Initial results from 16 of these clinical trials demonstrate promising efficacy of CAR NK cells, particularly in B cell malignancies, where response rates are comparable to those seen with CAR T cells but with lower rates of severe adverse effects, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and graft-versus-host disease (GvHD). However, challenges remain in solid tumor applications, where only modest efficacy has been observed to date. Our analysis reveals that research is increasingly focused on enhancing CAR NK cell persistence, broadening their therapeutic targets, and refining manufacturing processes to improve accessibility and scalability. With recent advancements in NK cell engineering and their increased clinical applications, CAR NK cells are predicted to become an integral component of next-generation immunotherapies, not only for cancer but potentially for immune-mediated diseases as well.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) remains a curative therapeutic option for patients with high-risk hematologic malignancies. When a fully matched donor is unavailable, haploidentical hematopoietic stem cell transplantation (haplo-HCT) provides a viable alternative. Over time, haplo-HCT procedures have significantly evolved, improving outcomes in treatment related mortality (TRM), especially in graft-versus-host disease (GvHD). However, challenges such as delayed immune reconstitution and disease relapse persist. Advances in in vivo graft manipulation techniques, such as post-transplant cyclophosphamide (PTCy) and ex vivo approaches, including TCRα/β and CD19 depletion, have shown promise in reducing the risk of severe GvHD without increasing the relapse rates. Innovative strategies, such as haploidentical donor lymphocyte infusions, "suicide-switch" mechanisms, ORCA-Q product infusions, and CAR based therapies offer potential to further optimize outcomes. This review examines the graft manipulation modalities in the haplo-HCT setting, highlighting their role in advancing cellular therapies and providing new hope in the fight against life-threatening diseases.

One of the most successful treatments in hematologic cancer is chimeric antigen receptor (CAR)-T cell-based immunotherapy. However, CAR-T therapy is not without challenges like the costly manufacturing process required to personalize each treatment for individual patients or graft-versus-host disease. Umbilical cord blood (UCB) has been most commonly used for hematopoietic cell transplant as it offers several advantages, including its rich source of hematopoietic stem cells, lower risk of graft-versus-host disease, and easier matching for recipients due to less stringent HLA requirements compared to bone marrow or peripheral blood stem cells. In this review, we have discussed the advantages and disadvantages of different CAR-T cell manufacturing strategies with the use of allogeneic and autologous peripheral blood cells. We compare them to the UCB approach and discuss ongoing pre-clinical and clinical trials in the field. Finally, we propose a cord blood bank as a readily available source of CAR-T cells.

Head and neck squamous cell carcinoma (HNSCC) presents a significant therapeutic challenge because of the limited effectiveness of current treatments including immunotherapy and chemotherapy. This study investigated the potential of a novel combination therapy using allogeneic natural killer (NK) cells and cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, to enhance anti-tumor efficacy in HNSCC. Allogeneic NK cells were tested against HNSCC cells in vitro and NOG (NOD/Shi-scid/IL-2Rγ null) xenograft mouse models for cytotoxicity. In vitro assays demonstrated enhanced cytotoxicity against HNSCC cells when NK cells were combined with cetuximab, a phenomenon attributed to antibody-dependent cellular cytotoxicity. In vivo, the combination therapy exhibited a significant anti-tumor effect compared to either monotherapy, with high NK cell infiltration and cytotoxic activity in the tumor microenvironment. Tumor infiltration by NK cells was confirmed using flow cytometry and immunohistochemistry, highlighting the increased presence of NK cells (CD3- CD56+). These findings suggest that combination allogeneic NK cells and cetuximab could be a potential therapeutic modality for HNSCC and provide a foundation for future clinical trials to improve patient outcomes.

Natural Killer (NK) cells hold significant promise as therapeutic agents in immuno-oncology due to their ability to target and eliminate cancerous and infected cells without causing graft-versus-host disease or cytokine release syndrome. However, the limited availability of robust, scalable methods for generating clinical-grade NK cells remains a limiting factor to broader clinical application.

Here we report the development of a novel feeder-cell-free culture system optimized for producing NK cells from cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs). Our method eliminates the need for feeder cells while achieving high yields of NK cells that exhibit unique marker expression and cytotoxic functions. Cord blood CD34+ HSPCs were cultured in our established hDLL 4 culture system and generated large numbers of human T lymphoid progenitors (ProTcells) in 7 days. ProTcells were further cultured in a hDLL4-free, feeder-cell-free system for NK cell differentiation and supplemented with cytokines. Following a 7- or 14-day culture, this method produced highly pure NK cell populations (>90% CD3-CD56+).

Flow and mass cytometric analysis confirmed the expression of activating receptors, transcription factors (ID2, T-bet) and cytotoxic molecules (perforin, granzyme A/B), all essential for ProT-NK cell functionality. These cells are in an immature state, indicated by the absence of maturation markers (CD16, KIRs). Functional assays demonstrated that these ProT-NK cells are capable of degranulation and cytokines production (TNFα) upon stimulation with K562 target cells and showed cytotoxicity against K562 cells superior to that of Peripheral Blood (PB)-NK. In NSG-Tg(hIL-15) mice, ProT-NK cells colonize bone marrow, the liver, and the spleen and persist and mature in bone marrow for at least 9 days post-injection. Compared to ProT-NK D21, ProT-NK D14 was superior in functional and homing potential. In vivo, an anti-tumor assay that uses a subcutaneous K562 model has demonstrated the anti-tumor potential of ProT-NK cells.

Our ex vivo culture process supports scalable ProT-NK cell production in high yields, reducing dependency on feeder cells and mitigating contamination risks. Our findings demonstrate the feasibility of generating large, functional NK cell populations from HSPCs isolated from readily available cord blood sources and offer an efficient alternative to PB-NK cell therapies.

Efforts to produce adoptive cell therapies in AML have been largely unfruitful, despite the success seen in lymphoid malignancies. Identifying targetable antigens on leukemic cells that are absent on normal progenitor cells remains a major obstacle, as is the hostile tumor microenvironment created by AML blasts. In this review, we summarize the challenges in the development of adoptive cell therapies such as CAR-T, CAR-NK, and TCR-T cells in AML, discussing both autologous and allogeneic therapies. We also discuss methods to address myelotoxicity associated with these therapies, including rapidly switchable CAR platforms and CRISPR-Cas9 genetic engineering of hematopoietic stem cells. Finally, we present the current clinical landscape in these areas, along with future directions in the field.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that carries a grim prognosis. Because of the dearth of treatment options available for treatment of GBM, Chimeric Antigen Receptor (CAR)-engineered T cell and Natural Killer (NK) therapy could provide alternative strategies to address the challenges in GBM treatment. In these approaches, CAR T and NK cells are engineered for cancer-specific immunotherapy by recognizing surface antigens independently of major histocompatibility complex (MHC) molecules. However, the efficacy of CAR T cells is hindered by GBM's downregulation of its targeted antigens. CAR NK cells face similar challenges, but, in contrast, they offer advantages as off-the-shelf allogeneic products, devoid of graft-versus-host disease (GVHD) risk as well as anti-cancer activity beyond CAR specificity, potentially reducing the risk of relapse or resistance. Despite CAR T cell therapies being extensively studied in clinical settings, the use of CAR-modified NK cells in GBM treatment remains largely in the preclinical stage. This review aims to discuss recent advancements in NK cell and CAR T cell therapies for GBM, including methods for introducing CARs into both NK cells and T cells, addressing manufacturing challenges, and providing evidence supporting the efficacy of these approaches from preclinical and early-phase clinical studies. The comprehensive evaluation of CAR-engineered NK cells and CAR T cells seeks to identify the optimal therapeutic approach for GBM, contributing to the development of effective immunotherapies for this devastating disease.

We present an easy-to-use, disposable, thermoplastic microwell chip designed to support screening and high-resolution imaging of single-cell behavior in two- and three-dimensional (2D and 3D) cell cultures. We show that the chip has excellent optical properties and provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly sized spheroids. We then demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells grown under different metabolic conditions to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on natural killer (NK)-cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.

Cutaneous melanoma is one of the most aggressive skin cancers originating from skin pigment cells. Patients with advanced melanoma suffer a poor prognosis and generally cannot benefit well from surgical resection and chemo/target therapy due to metastasis and drug resistance. Thus, adoptive cell therapy (ACT), employing immune cells with specific tumor-recognizing receptors, has emerged as a promising therapeutic approach to display on-tumor toxicity. This review discusses the application, efficacy, limitations, as well as future prospects of four commonly utilized approaches -including tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR) T cell, engineered T-cell receptor T cells, and chimeric antigen receptor NK cells- in the context of malignant melanoma.

Natural killer cells, an important component of the innate immune system, can directly recognize and lyse virally infected or transformed cells. However, NK cells fail to restrain the growth of malignancies, such as B-cell acute lymphoblastic leukemia (B-ALL). The molecular genetics of NK cells in the B-ALL bone marrow microenvironment and the mechanisms underlying the inhibited function of NK cells at the single-cell level remain largely elusive.

In this study, we studied the frequency and absolute number of NK cells in peripheral blood samples collected from 43 healthy volunteers and 104 pediatric B-ALL patients diagnosed at Hunan Children's Hospital. We also analyzed published single-cell RNA sequencing (scRNAseq) data from B-ALL and normal bone marrow samples using unsupervised clustering. Our findings were further validated using bulk transcriptomic data and clinical data from a cohort of 139 B-ALL bone marrow samples.

We found that the frequency and number of NK cells were significantly decreased in the bone marrow and peripheral blood of B-ALL patients. In-depth analysis of scRNAseq data identified 12 NK cell clusters. Among them, the C2 cluster, which is present in healthy bone marrow but reduced in B-ALL bone marrow, displays overexpression of a transcription factor KLF2 and a significant downregulation of the "leukocyte proliferation" pathway. Furthermore, we found that the expression of KLF2 in B-ALL at diagnosis was positively correlated with the percentage of leukemia cells and the positive rate of minimal residual disease (MRD), indicating that KLF2 is a marker of poor prognosis.

There are dramatic differences at the single-cell level in the transcriptomics of NK cells between healthy donors and B-ALL patients. A transcription factor, KLF2, which is enriched in the C2 cluster of NK cells, has been suggested to regulate the proliferation of NK cells and is associated with poor prognosis of pediatric B-ALL.

Increase in cancer cases and research driven by understanding its causes, facilitated development of novel targeted immunotherapeutic strategies to overcome nonspecific cytotoxicity associated with conventional chemotherapy and radiotherapy. These target specific immunotherapeutic regimens have been evaluated for their efficacy, including: (1) vaccines harnessing tumor specific/associated antigens, (2) checkpoint blockade therapy using monoclonal antibodies against PD1, CTLA-4 and others, and (3) adoptive cell transfer approaches viz. chimeric antigen receptor (CAR)-cell-based therapies. Here, we review recent advancements on these target specific translational immunotherapeutic strategies against cancer/s and concerned limitations.

Haploidentical donor transplantation (HIDT) or cord blood transplantation (CBT) are common alternatives for patients lacking human-leukocyte antigen (HLA)-matched donors. In addition to the donor source, NK cell alloreactivity due to HLA-mismatch setting may affect outcomes in alternative-donor hematopoietic cell transplantation (HCT). However, a limited number of studies have evaluated their impacts in adult acute lymphoblastic leukemia (ALL).

We aimed to assess the effects of donor source and KIRL-MM on outcomes of alternative-donor HCT, with a special focus on adult ALL.

We retrospectively compared clinical outcomes between HIDT (n=47) and double unit CBT (DCBT) (n=134). Patients received fludarabine and busulfan-based reduced toxicity conditioning before HIDT, and TBI-based myeloablative conditioning before DCBT. KIR ligands were determined using a web-based calculator. For DCBT, donor KIR ligand groups were defined by the dominant CB unit after engraftment.

After a median follow-up of 39.4 months, DCBT showed higher 3-year non-relapse mortality (NRM) (22.8% vs. 9.0%, p=0.038), whereas the cumulative incidence of relapse (CIR) was significantly higher in HIDT (47.9% vs. 18.9%, p<0.001). Estimated disease-free survival (DFS) was comparable (DCBT 58.5% vs. HIDT 44.3%, p=0.106). GVH direction KIRL-MM showed lower incidence of acute GVHD in both HIDT and DCBT. However, GVH direction KIRL-MM was associated with poorer DFS (37.2% vs. 66.0%, p=0.008) only in the DCBT subgroup, mostly due to specifically higher NRM rate (35.0% vs 18.4%, p=0.057).

Our study supports the usefulness of DCBT in the HIDT-dominant era and suggests potential ways to improve survival outcomes of DCBT.

Chimeric antigen receptor-natural killer (CAR-NK) cells represent a breakthrough in cancer immunotherapy, making this a highly popular research area. However, comprehensive analyses of this field using bibliometric methods are rare. To our knowledge, this study has collected highest number of publications (1,259) on CAR-NK therapy from January 1, 2004, to December 31, 2023, and utilized CiteSpace and VOSviewer to analyze regions, institutions, journals, authors, and keywords to forecast the latest trends in CAR-NK therapy research. The United States and China, contributing over 60% of publications, are the primary drivers in this field. The Helmholtz Association and Harvard University are the most active institutions, with most publications appearing in Frontiers in Immunology. Winfried S. Wels is the most prolific author, while EL Liu is the most frequently co-cited author. "Immunotherapy," "T-cells," and "Cancer" are the most extensively covered topics in CAR-NK therapy research. Our study reveals current CAR-NK research trends, identifies potential research hotspots, and visualizes references through bibliometric methods, providing valuable guidance for future research in this field.

Natural killer (NK) cell activity is influenced by cytokines and microenvironment factors, resulting in remarkably diverse functions, by contributing to inflammatory responses or serving as rheostats of adaptive immunity. Using single cell RNA sequencing (scRNA-seq), we identified a TGFβ1 highCD56brightNK cell population associated with hematopoietic stem cell transplant recipients protected from acute graft-versus-host disease (GVHD). We further define a role for the combination of interleukin-2 (IL-2) and transforming growth factor β1 (TGF-β1) in promoting a regulatory phenotype in NK cells. "Induced" regulatory NK cells produce high amounts of TGF-β1, inhibited T cells, could promote naive T cells differentiation into regulatory T cells, and exhibited a unique transcriptional program that includes expression of IKZF2 (HELIOS) and ZNF683 (HOBIT). This phenotype was not stable, and "induced" regulatory NK cells lost the ability to secrete TGF-β1 upon exposure to different cytokines. These findings define protective CD56brightNK cells post-hematopoietic stem cell transplantation, and demonstrate the combination of IL-2 and TGF-β1 promotes regulatory activity in NK cells.

Posttransplant cyclophosphamide (PTCy) has revolutionized the landscape of human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (haplo-HCT), providing a pivotal therapeutic option for patients with hematological malignancies who lack an HLA-matched donor.

In this retrospective analysis involving 54 adult patients undergoing PTCy-based haplo-HCT, we evaluated the impact of inhibitory killer immunoglobulin-like receptor (KIR)/HLA mismatch, alongside patient, donor, and transplant factors, on clinical outcomes within a homogeneous cohort characterized by a myeloablative conditioning regimen and bone marrow graft.

With a median follow-up of 73.2 months, our findings reveal promising outcomes: 6-year overall survival, relapse-free survival, and graft-versus-host disease (GVHD) and relapse-free survival rates were 63% (95% CI: 51-79), 58% (95% CI: 46-74), and 42% (95% CI: 30-58), respectively. Notably, the cumulative incidence rates of relapse and non-relapse mortality at 6 years post-haplo-HCT were 29% (95% CI: 19-45) and 12% (95% CI: 6-26), respectively. Acute GVHD at day 100 posttransplantation occurred with a cumulative incidence of 33% (95% CI: 22- 49) for grades II-IV and 9% (95% CI: 3-23) for grades III-IV. Furthermore, 41% of patients developed chronic GVHD within 1 year posttransplantation, distributed as follows: 28% mild, 9% moderate, and 4% severe.

Within our cohort, several variables were associated with outcomes following PTCy-based haplo-HCT. However, inhibitory KIR/HLA mismatch did not influence these outcomes.