NK Cell Activity Research Articles

MyCAF-derived exosomal PWAR6 accelerates CRC liver metastasis via altering glutamine availability and NK cell function in the tumor microenvironment

BackgroundLiver metastasis from colorectal cancer (CRC) is a major clinical challenge that severely affects patient survival. myofibroblastic cancer-associated fibroblasts (myCAFs) are a major component of the CRC tumor microenvironment, where they contribute to tumor progression and metastasis through exosomes.MethodsSingle-cell analysis highlighted a notable increase in myCAFs in colorectal cancer liver metastases (CRLM). Exosomal sequencing identified PWAR6 as the most significantly elevated lncRNA in these metastatic tissues. In vivo and in vitro assays confirmed PWAR6's roles in CRC cell stemness, migration, and glutamine uptake. RNA pulldown, RIP, and Co-IP assays investigated the molecular mechanisms of the PWAR6/NRF2/SLC38A2 signaling axis in CRC progression, flow cytometry was used to assess NK cell activity and cytotoxicity.ResultsClinically, higher PWAR6 expression levels are strongly associated with increased 68Ga FAPI-PET/CT SUVmax values, particularly in CRLM patients, where expression significantly exceeds that of non-LM cases and normal colon tissues. Regression analysis and survival data further support PWAR6 as a negative prognostic marker, with elevated levels correlating with worse patient outcomes. Mechanistically, PWAR6 promotes immune evasion by inhibiting NRF2 degradation through competitive binding with Keap1, thereby upregulating SLC38A2 expression, which enhances glutamine uptake in CRC cells and depletes glutamine availability for NK cells.ConclusionmyCAFs derived exosomes PWAR6 represents a pivotal marker for CRC liver metastasis, and its targeted inhibition with ASO-PWAR6, in combination with FAPI treatment, effectively curtails metastasis in preclinical models, offering promising therapeutic potential for clinical management.Graphical abstract

Hypoimmunogenic HLA-E Single Chain Inhibits Alloreactive Immune Responses.

Chimeric Ag receptor T cells derived from universal donors are susceptible to recipient immunologic rejection, which may limit their invivo persistence and compromise treatment efficacy. In this study, we generated HLA class I-deficient T cells by disrupting β2-microglobulin to evade recognition by HLA-mismatched CD8+ T cells, and then restored NK cell tolerance by forced expression of an HLA-E single-chain receptor. We specifically report on an optimized hypoimmunogenic disulfide trap HLA-E4 (dtHLA-E4) molecule that exhibited increased surface expression, enhanced NK cell inhibitory potential, and abrogated CD8-dependent T cell recognition. Our dtHLA-E4 molecule comprised the CD4 (4) transmembrane domain and truncated cytoplasmic region, as well as disulfide trap mutations to anchor an HLA class I signal sequence-derived peptide. Functional comparison of dtHLA-E4 molecules fused to different VL9 epitopes showed that peptides derived from HLA-A and HLA-C allotypes maximized NK cell inhibition and minimized NKG2C+ NK cell activation. Furthermore, incorporation of mutations into the α3 domain of HLA-E diminished the immunogenicity of dtHLA-E4 by reducing CD8+ T cell recognition, but crucially, these mutations left NK cell inhibitory function intact. These findings demonstrate the systematic construction of a hypoimmunogenic dtHLA-E4 molecule, which promises to facilitate persistence of allogeneic HLA class I-deficient chimeric Ag receptor T cells by overcoming NK cell missing-self recognition.

Interferon gamma-mediated prevention of tumor progression in a mouse model of multiple myeloma.

Malignant plasma cells in multiple myeloma patients reside in the bone marrow and continuously interact with local immune cells. Progression and therapy response are influenced by this immune environment, highlighting the need for a detailed understanding of endogenous immune responses to malignant plasma cells. Here we used the 5TGM1 murine transfer model of multiple myeloma to dissect early immune responses to myeloma cells. We modeled stable and progressive disease by transferring 5TGM1 murine myeloma cells into C57Bl/6 mice and KaLwRij mice, respectively. We used flow cytometry and single-cell and bulk transcriptomic analyses to characterize differential immune responses in stable and progressive disease. Transfer of 5TGM1 cells in C57Bl/6 mice led to stable disease with low tumor burden in a subset of animals. Stable disease was associated with sustained activation and expansion of NK cells, ILC1, and CD8+ T cells, a response that was lost upon disease progression. Single-cell RNA-sequencing of immune cells and bulk RNA sequencing of immune and mesenchymal stromal cells implicated the activation of interferon responses as a central immune pathway during stable disease. Experimentally, neutralization of IFNγ significantly increased myeloma development and progression in C57Bl/6 mice, testifying to the importance of this pathway in early disease control. In conclusion, we provide a framework for studying immune responses to multiple myeloma progression in immunocompetent and genetically modifiable mice and highlight the importance of bone marrow immunity in tumor control.

Immunostimulatory Effects of Red Beet Ferment Extract on Cyclophosphamide-Induced Immunosuppression in Wistar Rats

Red beet extract contains a variety of bioactive compounds, including antioxidant and immune-stimulating properties. Red beets are rich in nutrients and have been reported to help mitigate cardiovascular disease, while also possessing potent antioxidant capacity. In this study, we sought to determine the immune-enhancing effects of red beet fermented extract. The immunosuppressive drug cyclophosphamide (Cy) and red beet fermented extract were orally administered to Wistar rats once daily for four weeks. Blood analysis was performed to measure the expression of immune cytokines. We also examined the immunostimulatory effects of red beet fermented extract by measuring the activity and viability of spleen cells and the expression of immune cytokines. The expression of immune cytokines in the blood and spleen cells was increased in the red beet fermented extract treatment group compared to that in the negative control group, which induced immunosuppression of cytokines. It also increased the activity of NK cells and improved the viability of spleen cells. In conclusion, red beet fermented extract increased the expression of immune cytokines and increased the activity of adipocytes and NK cells, confirming its immunostimulatory effect. Therefore, red beet fermented extract has the potential to be utilized as a nutraceutical ingredient to enhance immunity.

Maimendong decoction inhibits lung cancer metastasis by increasing the proportion and killing activity of NK cells

Ethnopharmacological relevanceIn China, Maimendong Decoction (MMDD) is a classic Chinese medicine prescription for treating lung diseases, such as cough, upper respiratory tract infection, bronchitis, asthma, pulmonary fibrosis and so on. However, the mechanism by which MMDD inhibits lung cancer metastasis remains unclear. Aim of the studyThis study seeks to examine the effect of MMDD on suppressing lung cancer metastasis and to uncover its mechanism through the regulation of tumor immunity. Materials and methodsThe impact of MMDD on lung cancer cell growth and metastasis was assessed via cell apoptosis, proliferation, and migration assays, alongside lung metastasis models in mice. Hematoxylin-eosin (H&E) staining, immunohistochemistry, and immunofluorescence were used to examine the tissue morphology of metastatic tumors, Ki-67 protein expression, and NK cell infiltration. The potential anti-lung cancer mechanism of MMDD was explored through network pharmacology. Immune cell levels and NK cell function in mouse peripheral blood were analyzed using flow cytometry. Additionally, co-culture and lactate dehydrogenase (LDH) assays were performed to assess NK cell cytotoxicity. ResultsMMDD significantly reduced the number of metastatic tumors and the neoplastic burden in the lungs of mice. While MMDD did not affect the proliferation, apoptosis, or migration of lung cancer cells at 1 mg/ml concentration in vitro, it substantially increased NK cell proportion in peripheral blood and metastatic tumor tissues. Moreover, MMDD significantly boosted NK cell cytotoxicity and enhanced their killing effect on lung cancer cells. Importantly, depletion of NK cells abolished the survival-prolonging effect of MMDD in mice. ConclusionThis study demonstrates that MMDD restrains lung cancer cell metastasis primarily by increasing NK cell levels and enhancing their cytotoxic activity. These results offer experimental support for the use of MMDD in treating lung cancer metastasis.

Plant-produced SARS-CoV-2 antibody engineered towards enhanced potency and in vivo efficacy.

Prevention of severe COVID-19 disease by SARS-CoV-2 in high-risk patients, such as immuno-compromised individuals, can be achieved by administration of antibody prophylaxis, but producing antibodies can be costly. Plant expression platforms allow substantial lower production costs compared to traditional bio-manufacturing platforms depending on mammalian cells in bioreactors. In this study, we describe the expression, production and purification of the originally human COVA2-15 antibody in plants. Our plant-produced mAbs demonstrated comparable neutralizing activity with COVA2-15 produced in mammalian cells. Furthermore, they exhibited similar capacity to prevent SARS-CoV-2 infection in a hamster model. To further enhance these biosimilars, we performed three glyco- and protein engineering techniques. First, to increase antibody half-life, we introduced YTE-mutation in the Fc tail; second, optimization of N-linked glycosylation by the addition of a C-terminal ER-retention motif (HDEL), and finally; production of mAb in plant production lines lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO). These engineered biosimilars exhibited optimized glycosylation, enhanced phagocytosis and NK cell activation capacity compared to conventional plant-produced S15 and M15 biosimilars, in some cases outperforming mammalian cell produced COVA2-15. These engineered antibodies hold great potential for enhancing in vivo efficacy of mAb treatment against COVID-19 and provide a platform for the development of antibodies against other emerging viruses in a cost-effective manner.

Ashwagandha (Withania somnifera) in Cancer Therapy: Anticancer Activities and Their Mechanisms

Cancers are characterized by rapid development of abnormal cells that multiply uncontrollably, leading to invasive tumors. By 2040, new cancer cases worldwide are expected to reach 30.2 million. In response, research focuses on the anti-cancer properties of ashwagandha (Withania somnifera), used in traditional Indian medicine. Ashwagandha exhibits anti-inflammatory effects by modulating pathways like NF-kB, Nrf2/HO-1, JAK-STAT, MAPK, and NLRP3, reducing cancer-related inflammation. It also has strong antioxidant properties due to compounds like withanolides, increasing antioxidant enzyme activity and reducing oxidative stress, potentially lowering cancer risk. Ashwagandha extract shows cytotoxic effects against cancer cells, inducing apoptosis and arresting cells in the resting phase, leading to DNA damage in cancer cells. It inhibits angiogenesis, the process of forming new blood vessels that supply tumors, through mechanisms such as VEGF inhibition and reduced macrophage infiltration. Additionally, ashwagandha modulates T lymphocytes and cytokine production, enhancing the immune response against cancer, and stimulates NK cell activity, crucial for controlling tumor growth and development. Supplementation with ashwagandha reduces cortisol levels and improves cognitive functions and quality of life in people experiencing stress, potentially lowering cancer risk. Research suggests ashwagandha may be a valuable addition to anti-cancer therapy, acting on various levels of cancer pathogenesis. However, further clinical studies are needed to fully understand its mechanisms of action and to evaluate its efficacy and safety in cancer treatment.

EXTH-85. ASSESSMENT OF ANTI-CD69 ANTIBODY THERAPY ALONE OR IN COMBINATION WITH ANTI-PD-1 IN MURINE GBM

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressive central nervous system malignancy with a dismal prognosis. There is continued interest in trying multiple immunotherapy modalities for this intractable cancer. Cluster of differentiation 69 (CD69), is an early marker of T and NK cell activation. CD69 play multiple roles in T cell-mediated immunoregulation with immune inhibitory role in antitumor immunity. Previous preclinical work in non-CNS malignancies has demonstrated that targeting CD69 can improve clinical outcome and anti-tumor immunity. We aimed to assess anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. METHODS We explored transcriptomic data from 163 GBM patients using the TCGA database. We then tested the therapeutic value of anti-CD69 immunotherapy in a preclinical model of GBM and assessed the ideal timing of treatment with anti-CD69 Ab by evaluating survival and immune tumor-microenvironment by flow cytometry. Two treatment-timings with anti-CD69 Ab were further evaluated either alone, or in combination with anti-PD1 Abs and compared to control and to anti-PD1 only treatment. Survival data was collected. RESULTS CD69 expression was significantly increased in GBM patients tissues as compared with normal brains and increased CD69 expression was associated with worse progression-free survival. Treatment-timing with single dose anti-CD69 Ab at day 1 or three doses at days 4, 8 and 12 post tumor-injection minimally improved the median survival time of animals. In addition, effective CD69 blockage and significantly elevated PD-1 expression on NK cells was found in these timings, making them good candidates to evaluate anti-CD69 Ab therapy alone or in combination with anti-PD-1 Ab. However, when assessing these treatment-timings with anti-CD69 Ab alone or in combination with anti-PD1 no survival benefit was found. CONCLUSION This is the first study assessing anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. While some effect was found on the cellular level, this didn’t translate to survival benefit and results are overall negative.

CTNI-81. PROPOSAL FOR A PHASE IB PILOT CLINICAL TRIAL TO STUDY SAFETY AND IMPROVING ANTI-NEOPLASTIC ADVERSE EFFECTS BY ADDITION OF NATURAL MARINE PRODUCT FUCOIDAN TO THE STANDARD THERAPY FOR PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (GBM)

Abstract INTRODUCTION GBM is the most aggressive primary brain tumor. Despite standard of care (SOC) with the Stupp protocol and tumor treating fields (TTFs), survival remains poor. In addition, SOC treatment is associated with 96% any adverse effects (AE) and 25% severe AEs (Chinot et al, NEJM 2014), which affects quality of life (QOL) and can lead to early treatment termination. Therefore, novel therapies are needed and minimization of chemoradiation-related AEs is crucial. Marine natural product, fucoidan, may have an important role in oncological management as tumor-directed therapy and as a protectant against chemoradiation-related AEs. Fucoidan is a seaweed-derived sulfated polysaccharide. Mounting evidence garnered from randomized clinical trials for colon, head and neck, and lung cancer patients suggests that fucoidan can improve certain AEs associated with chemoradiation. Furthermore, data gleaned from multiple studies have shown that fucoidan is tolerable with a good safety profile. HYPOTHESIS Fucoidan is safe in combination with chemoradiation and chemotherapy and improves treatment related AE profile. Fucoidan may improve survival outcome in GBM patients by augmenting cytotoxic effects, inhibiting angiogenesis, and improving the tolerability of SOC therapy. METHOD 12 newly diagnosed GBM patients will be enrolled. A Fucoidan-based product will be administered orally at the established dose for cancer patients daily with concurrent chemoradiation therapy and adjuvant chemotherapy over 6 months. Patients will be evaluated weekly to monthly for AEs and QOL scoring; laboratory and brain MRI monitoring will be performed per SOC guidelines. NK cell activity, proinflammatory cytokines, including interleukin (IL)-1β, will be measured. POTENTIAL OUTCOME This pilot study may establish a foundation for future larger randomized clinical trials to treat brain cancer more effectively by adding nature products.

TLR3 activation enhances antitumor effects of sorafenib in hepatocellular carcinoma by activating NK cell functions through ERK and NF-κB pathways

Background Sorafenib is a standard therapeutic agent for advanced hepatocellular carcinoma (HCC). However, its efficacy is moderate, as the survival of patients is prolonged for only a few months, and the response rate is low. The mechanism of low efficacy remains unclear. In this study, we investigated the effect of Toll-like receptor 3 (TLR3) on the effects of sorafenib on HCC. Methods Polyinosinic-polycytidylic acid [poly(I: C)] was used as a double-stranded RNA analog and TLR3 agonist in subsequent experiments. After orthotopic implantation of HCC tumors in BALBc nu/nu or C57BL/6 mice, survival time, tumor growth, and metastasis in the abdomen and lungs were analyzed. Flow cytometry and cytotoxicity assays were used to analyze NK cells isolated from the spleen or peripheral blood. ELISA was used to detect the expression of plasma interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1. In addition, the expression of phosphorylated-extracellular regulated kinase 1/2 (pERK1/2), phosphorylated-protein kinase B (pAKT), ERK1/2 and AKT was analyzed by Western blotting. Results Sorafenib reduced the number and activity of NK cells in tumor-bearing mice and simultaneously decreased the levels of MCP-1 and IFN-γ in the plasma. The combination of sorafenib and poly(I: C) synergistically inhibited tumor growth and metastasis in tumor xenograft mice and prolonged survival. Poly(I: C) not only exerts a direct inhibitory effect on tumor growth and metastasis by targeting the TLR3 receptor on tumor cells but also facilitates the proliferation and activation of NK cells, indirectly impeding tumor progression. Mechanistically, poly(I: C) decreased the sorafenib-induced inhibition of ERK phosphorylation and increased the phosphorylation of IκB in NK cells, thereby enhancing NK cell function. Conclusion Activation of TLR3 can enhance the antitumor effect of sorafenib on HCC. The combination of a TLR3 activator and sorafenib may be a new strategy for the treatment of HCC.

A phase 1 study of interleukin-15 in combination with mogamulizumab in relapsed and refractory T-cell malignancies

IntroductionRecombinant human interleukin-15 (rhIL-15) is an immunotherapeutic which enhances NK-cells to augment the antibody directed cellular cytotoxicity (ADCC) of monoclonal antibodies. Mogamulizumab is a CCR4 directed monoclonal antibodies that exerts cytotoxicity through ADCC and depletes regulatory T-cells within the tumor microenvironment. MethodsWe conducted a phase 1 clinical trial of rhIL-15 combined with mogamulizumab. Patients with relapsed or refractory adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF) and Sezary syndrome (SS) received fixed dose mogamulizumab combined with escalating doses of rhIL-15 to identify the maximum tolerated dose (MTD). ResultsSix patients were enrolled, 4 with ATLL and 2 with MF/SS. The most common adverse events were rash, infection and fever (67%, in all). Two patients (33%) had grade 4 acute kidney injury and 25% of cycles had grade ≥3 anemia. The MTD was dose level 1. One patient with ATLL had a partial response despite receiving only 4 cycles due to grade 4 myositis. Circulating NK cells were increased in all patients during the first cycle and a rapid reduction in tumor cells within the peripheral circulation was noted. Ex vivo assessment demonstrated increased NK cell activation and increased cell lysis in the presence of monoclonal antibodies after only 5 days. DiscussionOur small study suggests that rhIL-15 combined with mogamulizumab leads to effector NK cell activation and regulatory T-cell depletion but has an unfavorable safety profile. Future development of combinations of immunotherapy that target the microenvironment in relapsed or refractory T-cell lymphomas remain rational. NCT04185220

Interleukin-12 decorated nanosized semiflexible Immunofilaments enable directed targeting and augmented IFNγ responses of natural killer cells

Immunotherapies are a powerful strategy to treat cancer by modulating the immune system to raise an anti-tumor immune response. A prime example of immunotherapies are cytokines - small immunomodulatory molecules that are widely used to stimulate immune cells. Undirected administration of cytokines, however, can cause severe side effects, preventing the use of potent cytokines, such as Interleukin (IL)-12, which induces IFNγ responses by cytotoxic effector lymphocytes, including NK cells. Biomaterials, like nanoparticles, can encapsulate IL-12 and accumulate at the tumor site to alleviate side effects. Yet, the released IL-12 might not be directly targeted to extracellular IL-12 receptors on the specific effector cells, thereby potentially compromising the cytokine's therapeutic efficacy. Here, we develop a polymer-based platform to target NK cells, which we call immunofilaments. Immunofilaments are nanosized linear polymers that present an anti-CD16 antibody and IL-12 effectively to NK cells and lead to synergistic NK cell activation as highlighted by an increase in TNFα and IFNγ production and upregulation of multiple activation markers, including CD25, CD69, and degranulation marker CD107a. NK cell proliferation is enhanced in the presence of both anti-CD16 antibody and IL-12 compared to giving IL-12 separately. Finally, we demonstrate that the IF platform is suitable for in vivo applications, as immunofilaments readily activate human NK cells upon administration to mice. Statement of SignificanceIL-12 is a potent cytokine that stimulates IFNγ responses in NK cells, which supports an anti-tumor immune response. Due to its high potency, the delivery of IL-12 needs to be highly controlled to prevent severe adverse side effects, which can be achieved by using biomaterials. This study shows that nanosized polymers termed Immunofilaments can be used to immobilize IL-12 and effectively target and activate NK cells by co-conjugation of anti-CD16 antibodies. This work is a prime example of careful engineering of innovative biomaterials to improve immunotherapy.

Asciminib stands out as the superior tyrosine kinase inhibitor to combine with anti-CD20 monoclonal antibodies for the treatment of CD20+ Philadelphia-positive B-cell precursor acute lymphoblastic leukemia in preclinical models.

Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCP-ALL) is a high-risk subtype of acute lymphoblastic leukemia characterized by the presence of the BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKI) combined with chemotherapy are established as the first-line treatment. Additionally, rituximab, an anti-CD20 monoclonal antibody is administered to adult BCP-ALL patients with ≥20% CD20+ blasts. In this study, we observed a marked prevalence of CD20 expression in patients diagnosed with Ph+ BCP-ALL, indicating a potential widespread clinical application of rituximab in combination with TKI. Consequently, we examined the influence of TKI on the antitumor effectiveness of anti-CD20 monoclonal antibodies by evaluating levels of CD20 on the cell surface and conducting in vitro functional assays. All tested TKI were found to uniformly downregulate CD20 on leukemic cells, diminishing the efficacy of rituximab-mediated complement- dependent cytotoxicity. Interestingly, these TKI displayed varied effects on natural killer (NK) cell-mediated antibody- dependent cytotoxicity and macrophage phagocytic function. While asciminib demonstrated no inhibition of effector cell functions, dasatinib notably suppressed the anti-CD20-monoclonal antibody-mediated NK cell cytotoxicity and macrophage phagocytosis of BCP-ALL cells. Dasatinib and ponatinib also decreased NK cell degranulation in vitro. Importantly, oral administration of dasatinib, but not asciminib, compromised NK cell activity in patients' blood, as determined by an ex vivo degranulation assay. Our results indicate that asciminib might be preferred over other TKI for combination therapy with anti-CD20 monoclonal antibodies.

Multi-omics revealed that ELAVL3 regulates MYCN in neuroblastoma via immunogenic cell death: Risk stratification and experimental research

Neuroblastoma (NB), a common and highly lethal malignant disease in pediatrics, still lacks an effective therapeutic approach that addresses all conditions. Immunogenic Cell Death (ICD) plays a crucial role in tumor cell death and triggers a potent anti-tumor immune response. In this study, we report an ICD-related index (ICDR-Index) in NB through various machine learning methodologies, utilizing bulk transcriptome data from 1244 NB samples and 16 scRNA-seq datasets. Our results showed that the ICDR-Index could accurately identify different risk subtypes of patients with NB and provide predictive value for prognosis. Importantly, we found that high-risk patients with NB exhibited significantly poor overall survival (OS) rates, adverse clinical phenotypes, poor immune cell infiltration, and low sensitivity to immunotherapy. Furthermore, we identified ELAVL3 as a key gene within the ICDR-Index, where high expression levels were associated with malignancy and poor OS in NB. Additionally, targeted silencing of ELAVL3 down-regulated MYCN gene expression and reduced the malignancy of NB cells. Notably, the si-ELAVL3-transfected NB cells enhanced the anti-tumor activity of NK cells. Collectively, this study offers avenues for predicting the risk stratification of patients with NB and reveals a potential mechanism by which ELAVL3 regulates NB cell death.

Low-Intensity Focused Ultrasound-Responsive Phase-Transitional Liposomes Loaded with STING Agonist Enhances Immune Activation for Breast Cancer Immunotherapy.

Background: Pharmacologically targeting the STING pathway offers a novel approach to cancer immunotherapy. However, small-molecule STING agonists face challenges such as poor tumor accumulation, rapid clearance, and short-lived effects within the tumor microenvironment, thus limiting their therapeutic potential. To address the challenges of poor specificity and inadequate targeting of STING in breast cancer treatment, herein, we report the design and development of a targeted liposomal delivery system modified with the tumor-targeting peptide iRGD (iRGD-STING-PFP@liposomes). With LIFU irradiation, the liposomal system exploits acoustic cavitation, where gas nuclei form and collapse within the hydrophobic region of the liposome lipid bilayer (transient pore formation), which leads to significantly enhanced drug release. Methods: Transmission electron microscopy (TEM) was used to investigate the physicochemical properties of the targeted liposomes. Encapsulation efficiency and in vitro release were assessed using the dialysis bag method, while the effects of iRGD on liposome targeting were evaluated through laser confocal microscopy. The CCK-8 assay was used to investigate the toxicity and cell growth effects of this system on 4T1 breast cancer cells and HUVEC vascular endothelial cells. A subcutaneous breast cancer tumor model was established to evaluate the tumor-killing effects and therapeutic mechanism of the newly developed liposomes. Results: The liposome carrier exhibited a regular morphology, with a particle size of 232.16 ± 19.82 nm, as indicated by dynamic light scattering (DLS), and demonstrated low toxicity to both HUVEC and 4T1 cells. With an encapsulation efficiency of 41.82 ± 5.67%, the carrier exhibited a slow release pattern in vitro after STING loading. Targeting results indicated that iRGD modification enhanced the system's ability to target 4T1 cells. The iRGD-STING-PFP@liposomes group demonstrated significant tumor growth inhibition in the subcutaneous breast cancer mouse model with effective activation of the immune system, resulting in the highest populations of matured dendritic cells (71.2 ± 5.4%), increased presentation of tumor-related antigens, promoted CD8+ T cell infiltration at the tumor site, and enhanced NK cell activity. Conclusions: The iRGD-STING-PFP@liposomes targeted drug delivery system effectively targets breast cancer cells, providing a new strategy for breast cancer immunotherapy. These findings indicate that iRGD-STING-PFP@liposomes could successfully deliver STING agonists to tumor tissue, trigger the innate immune response, and may serve as a potential platform for targeted immunotherapy.

Natural killer cell engagers: From bi-specific to tri-specific and tetra-specific engagers for enhanced cancer immunotherapy.

Innovative NKCEs: NK cell engagers (NKCEs) represent a promising new class of immunotherapeutics targeting tumours by activating NK cells. Multi-specific formats: The transition from bi-specific to multi-specific NKCEs enhances their versatility and therapeutic efficacy. NKCEs have the potential to improve NK cell activation by engaging activating receptors and incorporating cytokines. Current clinical trials demonstrate the safety and efficacy of various NKCEs across different cancer types. Future research directions: Optimising NKCE designs and exploring combination therapies are essential for overcoming challenges in cancer treatment.

Impact of Vitamin D deficiency on immunological and metabolic responses in women with recurrent pregnancy loss: focus on VDBP/HLA-G1/CTLA-4/ENTPD1/adenosine-fetal-maternal conflict crosstalk

Background and aimRecurrent pregnancy loss (RPL), also known as recurrent implantation failure (RIF), is a distressing condition affecting women characterized by two or more consecutive miscarriages or the inability to carry a pregnancy beyond 20 weeks. Immunological factors and genetic variations, particularly in Vit D Binding Protein (VDBP), have gained attention as potential contributors to RPL. This study aimed to provide insight into the immunological, genetic, and metabolic networks underlying RPL, placing a particular emphasis on the interactions between VDBP, HLA-G1, CTLA-4, ENTPD1, and adenosine-fetal-maternal conflict crosstalk.MethodsA retrospective study included 198 women with three or more consecutive spontaneous abortions. Exclusion criteria comprised uterine abnormalities, endocrine disorders, parental chromosomal abnormalities, infectious factors, autoimmune diseases, or connective tissue diseases. Immunological interplay was investigated in 162 female participants, divided into two groups based on their Vit D levels: normal Vit D-RPL and low Vit D-RPL. Various laboratory techniques were employed, including LC/MS/MS for Vit D measurement, ELISA for protein detection, flow cytometry for immune function analysis, and molecular docking for protein–ligand interaction assessment.ResultsGeneral characteristics between groups were significant regarding Vit D and glucose levels. Low Vit D levels were associated with decreased NK cell activity and downregulation of HLA-G1 and HLA-G5 proteins, while CTLA-4 revealed upregulation. VDBP was significantly downregulated in the low Vit D group. Our findings highlight the intricate relationship between Vit D status and adenosine metabolism by the downregulation of SGLT1, and NT5E, key components of adenosine metabolism, suggests that Vit D deficiency may disrupt the regulation of adenosine levels, leading to an impaired reproductive outcome. HNF1β, a negative regulator of VDBP, was upregulated, while HNF1α, a positive regulator, was downregulated in low Vit D women after RPL. Molecular docking analysis revealed crucial residues involved in the interaction between Vit D and HNF1β.ConclusionCollectively, these findings underscore the importance of Vit D in modulating immune function and molecular pathways relevant to pregnancy maintenance, highlighting the need for further research to elucidate the mechanisms and potential therapeutic interventions for improving pregnancy outcomes in individuals with Vit D deficiency and RPL.

Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity.

Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors' expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated. CD56⁺ CD3- NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10× magnification for 24 h. Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation. Our findings point to the role of Siglec-sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies.

Reduced T and NK Cell Activity in Glioblastoma Patients Correlates with TIM-3 and BAT3 Dysregulation.

Inhibitory receptors are critical for regulating immune cell function. In cancer, these receptors are often over-expressed on the cell surface of T and NK cells, leading to reduced anti-tumor activity. Here, through the analysis of 11 commonly studied checkpoint and inhibitory receptors, we discern that only HAVCR2 (TIM3) and ENTPD1 (CD39) display significantly greater gene expression in glioblastoma compared to normal brain and lower grade glioma. Cell surface TIM-3, but not ENTPD1, was also elevated on activated CD4+ and CD8+ T cells, as well as on NK cells from glioblastoma patients compared to healthy donor T and NK cells. A subsequent analysis of molecules known to co-ordinate TIM-3 function and regulation was performed, which revealed that BAT3 expression was significantly reduced in CD4+ and CD8+ T cells, as well as NK cells from glioblastoma patients compared to counterparts from healthy donors. These pro-inhibitory changes are also correlated with reduced levels of the activation marker CD69 and the pro-inflammatory cytokine IFNγ in CD4+ and CD8+ T cells, as well as NK cells from glioblastoma patients. Collectively, these data reveal that glioblastoma-mediated CD4+ and CD8+ T cell and NK cell suppression is due, at least in part, to dysregulated TIM-3 and BAT3 expression and the associated downstream immunoregulatory and dysfunctional effects.

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy

The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.