Induction of protective immune responses at respiratory mucosal sites.

Many pathogens enter the host through mucosal sites. Thus, interfering with pathogen entry through local neutralization at mucosal sites therefore is an effective strategy for preventing disease. Mucosally administered vaccines have the potential to induce protective immune responses at mucosal sites. This manuscript delves into some of the latest developments in mucosal vaccination, particularly focusing on advancements in adjuvant technologies and the role of these adjuvants in enhancing vaccine efficacy against respiratory pathogens. It highlights the anatomical and immunological complexities of the respiratory mucosal immune system, emphasizing the significance of mucosal secretory IgA and tissue-resident memory T cells in local immune responses. We further discuss the differences between immune responses induced through traditional parenteral vaccination approaches vs. mucosal administration strategies, and explore the protective advantages offered by immunization through mucosal routes.

Cytokines drive the formation of memory-like NK cell subsets via epigenetic rewiring and transcriptional regulation.

Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56bright or CD56dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

Expression of a single inhibitory Ly49 receptor is sufficient to license NK cells for effector functions.

Natural killer (NK) cells recognize target cells through germline-encoded activation and inhibitory receptors enabling effective immunity against viruses and cancer. The Ly49 receptor family in the mouse and killer immunoglobin-like receptor family in humans play a central role in NK cell immunity through recognition of MHC class I and related molecules. Functionally, these receptor families are involved in licensing and rejection of MHC-I-deficient cells through missing-self. The Ly49 family is highly polymorphic, making it challenging to detail the contributions of individual Ly49 receptors to NK cell function. Herein, we showed mice lacking expression of all Ly49s were unable to reject missing-self target cells in vivo, were defective in NK cell licensing, and displayed lower KLRG1 on the surface of NK cells. Expression of Ly49A alone on a H-2Dd background restored missing-self target cell rejection, NK cell licensing, and NK cell KLRG1 expression. Thus, a single inhibitory Ly49 receptor is sufficient to license NK cells and mediate missing-self in vivo.

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation.

The surface receptor CD8α is present on 20-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses for leukemia patients in prior studies, thus we hypothesized that CD8α may impact critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models, compared to CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These 'induced' CD8α+ ('iCD8α+') NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity, compared to those that sustained existing CD8α expression ('sustained CD8α+) or those that remained CD8α- ('persistent CD8α-'). These iCD8α+ cells originated from an IL-15Rß high NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identifies human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion and exhibits a suppressive effect on NK cell activating receptors.

Memory-like differentiation enhances NK cell responses against colorectal cancer.

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Cancer-associated fibroblast-derived Dickkopf-1 suppresses NK cell cytotoxicity in breast cancer.

Breast cancer is poorly immunogenic, hence able to evade T cell recognition and respond poorly to immune checkpoint blockade. Breast cancer cells can also evade NK cell-mediated immune surveillance, but the mechanism remains enigmatic. Dickkopf-1 (DKK1) is a Wnt/b-catenin inhibitor, whose levels are increased in breast cancer patients and correlate with reduced overall survival. DKK1 is expressed by cancer-associated fibroblasts (CAFs) in orthotopic breast tumors and patient samples, and at higher levels by bone cells. While bone-derived DKK1 contributes to the systemic elevation of DKK1 in tumor-bearing mice, CAFs represent the primary source of DKK1 at the tumor site. Systemic or bone-specific DKK1 targeting reduces primary tumor growth. Intriguingly, specific deletion of CAF-derived DKK1 also limits breast cancer progression, regardless of its elevated levels in circulation and in the bone. DKK1 does not support tumor proliferation directly but rather suppresses the activation and tumoricidal activity of NK cells. Importantly, increased DKK1 levels and reduced number of cytotoxic NK cells are detected in breast cancer patients with progressive bone metastases compared to those with stable disease. Our findings indicate that DKK1 creates a tumor-supporting environment through the suppression of NK cells in breast cancer.

Enhanced mucosal B- and T-cell responses against SARS-CoV-2 after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant.

Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as SARS-CoV-2, by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection to SARS-CoV-2.

Gender Affirming Hormones Do Not Affect the Exposure and Efficacy of F/TDF or F/TAF for HIV Preexposure Prophylaxis: A Subgroup Analysis from the DISCOVER Trial.

Purpose: Transgender women are disproportionately affected by HIV and are underutilizing preexposure prophylaxis (PrEP). The lower uptake of PrEP by transgender women may be, in part, owing to the perception that taking PrEP may lower the efficacy of gender-affirming hormone therapy (GAHT) or to provider concerns that GAHT may lower the efficacy of PrEP. Methods: DISCOVER was a randomized, double-blind, noninferiority trial comparing emtricitabine (FTC, F) and tenofovir alafenamide (F/TAF) versus emtricitabine and tenofovir disoproxil fumarate (F/TDF) as PrEP among transgender women and cisgender men who have sex with men (MSM). This nested substudy of the DISCOVER trial compared the exposure of the active intracellular metabolites of FTC and tenofovir (TFV), FTC triphosphate (FTC-TP) and TFV diphosphate (TFV-DP), in peripheral blood mononuclear cells (PBMC) among transgender women receiving GAHT versus MSM within the F/TAF and F/TDF groups. Results: Our results demonstrate that TFV-DP and FTC-TP levels in PBMC were comparable between transgender women on GAHT and MSM receiving F/TAF, and between transgender women on GAHT and MSM receiving F/TDF. TFV-DP concentrations remained above the EC90 of 40 fmol/106 cells across all groups. No clinically significant drug-drug interactions of GAHT were observed with either F/TAF or F/TDF in this subanalysis. Conclusions: These findings are consistent with the clinical pharmacology of GAHT, FTC, TDF, and TAF reported in previous studies, and support the continued use of F/TAF and F/TDF for PrEP in transgender women.Clinicaltrials.gov registration number: NCT02842086.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. EXPERIMENTAL DESIGN: We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. RESULTS: Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. CONCLUSIONS: These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines currently provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a decrease in immunogenicity with age. Robust antigen-specific IFN-γ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

T-BET and EOMES sustain mature human NK cell identity and antitumor function.

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

Wild-type SARS-CoV-2 neutralizing immunity decreases across variants and over time but correlates well with diagnostic testing.

Importance: The degree of immune protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants provided by infection versus vaccination with wild-type virus remains unresolved, which could influence future vaccine strategies. The gold-standard for assessing immune protection is viral neutralization; however, few studies involve a large-scale analysis of viral neutralization against the Omicron variant by sera from individuals infected with wild-type virus. Objectives: 1) To define the degree to which infection versus vaccination with wild-type SARS-CoV-2 induced neutralizing antibodies against Delta and Omicron variants.2) To determine whether clinically available data, such as infection/vaccination timing or antibody status, can predict variant neutralization. Methods: We examined a longitudinal cohort of 653 subjects with sera collected three times at 3-to-6-month intervals from April 2020 to June 2021. Individuals were categorized according to SARS-CoV-2 infection and vaccination status. Spike and nucleocapsid antibodies were detected via ADVIA Centaur® (Siemens) and Elecsys® (Roche) assays, respectively. The Healgen Scientific® lateral flow assay was used to detect IgG and IgM spike antibody responses. Pseudoviral neutralization assays were performed on all samples using human ACE2 receptor-expressing HEK-293T cells infected with SARS-CoV-2 spike protein pseudotyped lentiviral particles for wild-type (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants. Results: Vaccination after infection led to the highest neutralization titers at all timepoints for all variants. Neutralization was also more durable in the setting of prior infection versus vaccination alone. Spike antibody clinical testing effectively predicted neutralization for wild-type and Delta. However, nucleocapsid antibody presence was the best independent predictor of Omicron neutralization. Neutralization of Omicron was lower than neutralization of either wild-type or Delta virus across all groups and timepoints, with significant activity only present in patients that were first infected and later immunized. Conclusions: Participants having both infection and vaccination with wild-type virus had the highest neutralizing antibody levels against all variants and had persistence of activity. Neutralization of WT and Delta virus correlated with spike antibody levels against wild-type and Delta variants, but Omicron neutralization was better correlated with evidence of prior infection. These data help explain why 'breakthrough' Omicron infections occurred in previously vaccinated individuals and suggest better protection is observed in those with both vaccination and previous infection. This study also supports the concept of future SARS-CoV-2 Omicron-specific vaccine boosters.

A multicomponent intranasal adjuvant drives durable humoral, cellular, and mucosal immune responses to SARS-CoV-2 in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a marked decrease in immunogenicity with age. Robust antigen-specific IFNγ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

A comparative study of two chemical models for creating subsurface caries lesions on aprismatic and prismatic enamel.

PURPOSE: To investigate the mineral density and lesion depth of artificial caries lesions on aprismatic enamel and prismatic enamel created by lactic acid and acetic acid buffers. METHODS: Forty bovine enamel blocks were allocated to: aprismatic enamel (Group A) and prismatic enamel (Group C) in acetic acid buffer for 192 h and aprismatic enamel (Group B) and prismatic enamel (Group D) in lactic acid buffer for 96 h. The mineral loss and lesion depth were measured using micro-computed tomography. RESULTS: A significant difference (P = 0.01) was observed in the mineral loss (%) in the lesions on aprismatic enamel and prismatic enamel treated with lactic acid buffer while no significant difference (P = 0.51) was observed in the mineral loss (%) in the lesions on aprismatic enamel and prismatic enamel treated with acetic acid buffer. No significant difference was noted in the mean lesion depth of lesions on aprismatic enamel and prismatic enamel treated with acetic acid and lactic acid buffers (P > 0.05). CONCLUSION: Aprismatic enamel and prismatic enamel have similar mineral loss in acetic acid while prismatic enamel showed more mineral loss compared to aprismatic enamel in lactic acid.

The unfulfilled potential of mucosal immunization.

Recent events involving the global coronavirus pandemic have focused attention on vaccination strategies. Although tremendous advances have been made in subcutaneous and intramuscular vaccines during this time, one area that has lagged in implementation is mucosal immunization. Mucosal immunization provides several potential advantages over subcutaneous and intramuscular routes, including protection from localized infection at the site of entry, clearance of organisms on mucosal surfaces, induction of long-term immunity through establishment of central and tissue-resident memory cells, and the ability to shape regulatory responses. Despite these advantages, significant barriers remain to achieving effective mucosal immunization. The epithelium itself provides many obstacles to immunization, and the activation of immune recognition and effector pathways that leads to mucosal immunity has been difficult to achieve. This review will highlight the potential advantages of mucosal immunity, define the barriers to mucosal immunization, examine the immune mechanisms that need to be activated on mucosal surfaces, and finally address recent developments in methods for mucosal vaccination that have shown promise in generating immunity on mucosal surfaces in human trials.

Consenso peruano para el uso de la resonancia magnética en el diagnóstico y seguimiento de pacientes con esclerosis múltiple / Peruvian consensus for the use of Magnetic Resonance Imaging in the diagnosis and follow-up of patients with Multiple Sclerosis

RESUMEN El uso de la resonancia magnética (RM) en el diagnóstico y seguimiento de pacientes con esclerosis múltiple (EM) ha optimizado el cuidado de los pacientes afectados. Diversos grupos internacionales de trabajo han intentado clarificar y normatizar el uso global de la RM pero, en muchas ocasiones, se extrapolan datos de otras regiones que no contemplan la realidad de cada lugar o son difíciles de implementar. Objetivo: Consensuar aspectos relacionados con el uso de RM en el diagnóstico y seguimiento de pacientes con EM en el Perú. Material y Métodos: Un grupo de expertos peruanos, conformado por neurólogos y radiólogos, condujo la elaboración del consenso mediante metodología de ronda de encuestas a la distancia. Resultados: Las recomendaciones, basadas en la evidencia publicada y en el criterio de los expertos, enfocaron tanto el rol de las técnicas convencionales de RM como el de la medición de la atrofia cerebral en pacientes con EM al momento del diagnóstico y durante el periodo de seguimiento. Conclusiones: Las recomendaciones del consenso podrán potencialmente homogenizar y optimizar el cuidado y seguimiento de pacientes con EM en nuestro país.

SUMMARY The use of Magnetic Resonance Imaging (MRI) in the diagnosis and follow-up of patients with Multiple Sclerosis (MS) has optimized the care of the affected patients. Several international working groups have tried to clarify and standardize the global use of MRI but, on many occasions, data are extrapolated from other regions, do not contemplate local realities or are difficult to implement. Objective: To reach a consensus on aspects related to the use of MRI in the diagnosis and follow-up of patients with MS in Peru. Material and Methods: A group of Peruvian experts (neurologists and radiologists) worked on the elaboration of the consensus using a remote survey round methodology. Results: The recommendations, established on the basis of published evidence and on the experts' criteria, focused on the role of both, the conventional MRI techniques and the measurement of brain atrophy in MS patients both at the time of diagnosis and during the follow-up period Conclusions: The consensual recommendations could potentially assist in the standardization and optimization of the care and follow-up of patients with MS in our country.

A novel fusion protein scaffold 18/12/TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy.

Hematopoietic cell transplantation donor-derived memory-like NK cells functionally persist after transfer into patients with leukemia.

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.