Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model.

BACKGROUND: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation. METHODS: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice. RESULTS: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer. CONCLUSION: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.

Fatty acid oxidation in immune function.

Cellular metabolism is a crucial determinant of immune cell fate and function. Extensive studies have demonstrated that metabolic decisions influence immune cell activation, differentiation, and cellular capacity, in the process impacting an organism's ability to stave off infection or recover from injury. Conversely, metabolic dysregulation can contribute to the severity of multiple disease conditions including autoimmunity, alloimmunity, and cancer. Emerging data also demonstrate that metabolic cues and profiles can influence the success or failure of adoptive cellular therapies. Importantly, immunometabolism is not one size fits all; and different immune cell types, and even subdivisions within distinct cell populations utilize different metabolic pathways to optimize function. Metabolic preference can also change depending on the microenvironment in which cells are activated. For this reason, understanding the metabolic requirements of different subsets of immune cells is critical to therapeutically modulating different disease states or maximizing cellular function for downstream applications. Fatty acid oxidation (FAO), in particular, plays multiple roles in immune cells, providing both pro- and anti-inflammatory effects. Herein, we review the major metabolic pathways available to immune cells, then focus more closely on the role of FAO in different immune cell subsets. Understanding how and why FAO is utilized by different immune cells will allow for the design of optimal therapeutic interventions targeting this pathway.

Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

Human effector CD8+ T cells with an activated and exhausted-like phenotype control tumour growth in vivo in a humanized tumour model.

BACKGROUND: Humanized tumour models could be particularly valuable for cancer immunotherapy research, as they may better reflect human-specific aspects of the interfaces between tumour and immune system of human cancer. However, endogenous antitumour immunity in humanized models is still largely undefined. METHODS: We established an autologous humanized mouse tumour model by using NSG mice reconstituted with human immune cells from hematopoietic progenitors and tumours generated from transformed autologous human B cells. We demonstrate growth of solid lymphoid tumours after subcutaneous implantation, infiltration by endogenous human immune cells and immunocompetence of the model. FINDINGS: We found human T cell subsets described in human cancer, including progenitor exhausted (Tpex), terminally exhausted (Tex-term) and tissue-resident (TRM) cells in tumour-bearing humanized mice with accumulation of Tex-term and TRM in the tumour. In addition, we identified tumour-reactive CD8+ T cells through expression of CD137. This subpopulation of de novo arising human CD137+ CD8+ T cells displayed a highly proliferative, fully activated effector and exhausted-like phenotype with enhanced expression of activation and exhaustion markers like PD-1, CD39, CD160, TIM-3, TIGIT and TOX, the senescence marker CD57 (B3GAT1) and cytolytic effector molecules such as PRF1, GZMH and NKG7. Moreover, these CD137+ CD8+ T cells exhibited tumour-specific clonal expansion and presented signature overlap with tumour-reactive CD8+ T cells described in human cancer. We demonstrate superior anticancer activity of this activated and exhausted-like human CD8+ T cell subset by adoptive transfer experiments using recipients bearing autologous human tumours. Mice adoptively transferred with CD137+ CD8+ T cells showed reduced tumour growth and higher CD8+ T cell tumour infiltration, correlating with control of human tumours. INTERPRETATION: We established an immunocompetent humanized tumour model, providing a tool for immunotherapy research and defined effective anticancer activity of human effector CD8+ T cells with an activated and exhausted-like phenotype, supporting clinical exploration of such cells in adoptive T cell therapies. FUNDING: Swiss Cancer Research foundation.

CXCR4 has a dual role in improving the efficacy of BCMA-redirected CAR-NK cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.

Differential impact of genetic deletion of TIGIT or PD-1 on melanoma-specific T-lymphocytes.

Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGITKO T-cells. Functional analyses showed that PD-1KO and TIGITKO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGITKO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.

A general pHLA-CD80 scaffold fusion protein to promote efficient antigen-specific T cell-based immunotherapy.

Inadequate antigen-specific T cells activation hampers immunotherapy due to complex antigen presentation. In addition, therapeutic in vivo T cell expansion is constrained by slow expansion rates and limited functionality. Herein, we introduce a model fusion protein termed antigen-presenting cell-mimic fusion protein (APC-mimic), designed to greatly mimicking the natural antigen presentation pattern of antigen-presenting cells and directly expand T cells both in vitro and in vivo. The APC-mimic comprises the cognate peptide-human leukocyte antigen (pHLA) complex and the co-stimulatory marker CD80, which are natural ligands on APCs. Following a single stimulation, APC-mimic leads to an approximately 400-fold increase in the polyclonal expansion of antigen-specific T cells compared with the untreated group in vitro without the requirement for specialized antigen-presenting cells. Through the combination of single-cell TCR sequencing (scTCR-seq) and single-cell RNA sequencing (scRNA-seq), we identify an approximately 600-fold monoclonal expansion clonotype among these polyclonal clonotypes. It also exhibits suitability for in vivo applications confirmed in the OT-1 mouse model. Furthermore, T cells expanded by APC-mimic effectively inhibits tumor growth in adoptive cell transfer (ACT) murine models. These findings pave the way for the versatile APC-mimic platform for personalized therapeutics, enabling direct expansion of polyfunctional antigen-specific T cell subsets in vitro and in vivo.

Generation of non-genetically modified, CAR-like, NK cells.

BACKGROUND: Natural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been disappointing. This is partially due to the lack of target specificity. Recently, CD19-specific chimeric antigen receptor (CAR)-NK cells have proven to be safe and potent in patients with B-cell tumors. However, the generation of CAR-NK cells is a complicated manufacturing process. We aim at developing a targeted NK cell therapy without the need for cellular genetic modifications. We took advantage of the natural expression of the IgG Fc receptor CD16a (FcγRIIIa) to induce strong antigen-specific effector functions through antibody-dependent cell-mediated cytotoxicity (ADCC). We have generated the new technology "Pin", which enables the arming of modified monoclonal antibodies (mAbs) onto the CD16a of ex vivo expanded NK (eNK) cells. Methods Ex vivo eNK were prepared from umbilical cord blood cells and expanded using interleukin (IL)-2/IL-15 and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid feeder cells. mAbs were engineered with four substitutions called Pin mutations to increase their affinity to CD16a. eNK were incubated with anti-CD20 or anti-CD19 Pin-mAbs to generate "armed" eNK and were used to assess effector functions in vitro on cancer cell lines, lymphoma patient cells and in vivo. RESULTS: CD16a/Pin-mAb interaction is stable for several days and Pin-mAb eNK inherit the mAb specificity and exclusively induce ADCC against targets expressing the cognate antigen. Hence, Pin-mAbs confer long-term selectivity to eNK, which allows specific elimination of the target cells in several in vivo mouse models. Finally, we showed that it is possible to arm eNK with at least two Pin-mAbs simultaneously, to increase efficacy against heterogenous cancer cell populations. CONCLUSIONS: The Pin technology provides an off-the-shelf NK cell therapy platform to generate CAR-like NK cells, without genetic modifications, that easily target multiple tumor antigens.

Proteomic and phenotypic characteristics of memory-like natural killer cells for cancer immunotherapy.

BACKGROUND: Human and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell lines. The resulting cells retain enhanced lytic ability for up to 7 days as well as after cryopreservation, and memory-like NK cells (mlNK) have been shown to induce complete remissions in patients with hematological malignancies. No single phenotype has been described for mlNK and the physiological changes induced by the short-term cytokine or tumor-priming which are responsible for these enhanced functions have not been fully characterized. Here, we have generated mlNK by cytokine and tumor-priming to find commonalities to better define the nature of NK cell "memory" in vitro and, for the first time, in vivo. METHODS: We initiated mlNK in vitro from healthy donors with cytokines (initiated cytokine-induced memory-like (iCIML)-NK) and by tumor priming (TpNK) overnight and compared them by high-dimensional flow cytometry, proteomic and metabolomic profiling. As a potential mechanism of enhanced cytolytic function, we analyzed the avidity of binding of the mlNK to NK-resistant tumors (z-Movi). We generated TpNK from healthy donors and from cancer patients to determine whether mlNK generated by interaction with a single tumor type could enhance lytic activity. Finally, we used a replication-incompetent tumor cell line (INKmune) to treat patients with myeloid leukaemias to potentiate NK cell function in vivo. RESULTS: Tumor-primed mlNK from healthy donors and patients with cancer showed increased cytotoxicity against multiple tumor cell lines in vitro, analogous to iCIML-NK cells. Multidimensional cytometry identified distinct memory-like profiles of subsets of cells with memory-like characteristics; upregulation of CD57, CD69, CD25 and ICAM1. Proteomic profiling identified 41 proteins restricted to mlNK cells and we identified candidate molecules for the basis of NK memory which can explain how mlNK overcome inhibition by resistant tumors. Finally, of five patients with myelodysplastic syndrome or refractory acute myeloid leukemia treated with INKmune, three responded to treatment with measurable increases in NK lytic function and systemic cytokines. CONCLUSIONS: NK cell "memory" is a physiological state associated with resistance to MHC-mediated inhibition, increased metabolic function, mitochondrial fitness and avidity to NK-resistant target cells.

A strategy to reconstitute immunity without GVHD via adoptive allogeneic Tscm therapy.

Introduction: Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD. Methods: The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence. Results: Our protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the "foreign" antigen without causing GVHD after they were transferred into the recipients. Discussion: Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.

Lipid-based nanosystems: the next generation of cancer immune therapy.

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy.

Potentials of bone marrow cells-derived from naïve or diabetic mice in autoimmune type 1 diabetes: immunomodulatory, anti-inflammatory, anti hyperglycemic, and antioxidative.

BACKGROUND: The scarcity of transplanted human islet tissue and the requirement for immunosuppressive drugs to prevent the rejection of allogeneic grafts have hindered the treatment of autoimmune type 1 diabetes mellitus (T1DM) through islet transplantation. However, there is hope in adoptively transferred bone marrow cells (BMCs) therapy, which has emerged as a propitious pathway for forthcoming medications. BMCs have the potential to significantly impact both replacement and regenerative therapies for a range of disorders, including diabetes mellitus, and have demonstrated anti-diabetic effects. AIM: The main goal of this study is to evaluate the effectiveness of adoptively transferred bone marrow cells derived from either naïve mice (nBMCs) or diabetic mice (dBMCs) in treating a T1DM mice model. METHODS: Male Swiss albino mice were starved for 16 h and then injected with streptozotocin (STZ) at a dose of 40 mg/kg body weight for 5 consecutive days to induce T1DM. After 14 days, the diabetic mice were distributed into four groups. The first group served as a diabetic control treated with sodium citrate buffer, while the other three groups were treated for two weeks, respectively, with insulin (subcutaneously at a dose of 8 U/kg/day), nBMCs (intravenously at a dose of 1 × 106 cells/mouse/once), and dBMCs (intravenously at a dose of 1 × 106 cells/mouse/once). RESULTS: It is worth noting that administering adoptively transferred nBMCs or adoptively transferred dBMCs to STZ-induced T1DM mice resulted in a significant amelioration in glycemic condition, accompanied by a considerable reduction in the level of blood glucose and glycosylated hemoglobin % (HbA1C %), ultimately restoring serum insulin levels to their initial state in control mice. Administering nBMCs or dBMCs to STZ-induced T1DM mice led to a remarkable decrease in levels of inflammatory cytokine markers in the serum, including interferon-γ (INF-γ), tumor necrosis factor- α (TNF-α), tumor growth factor-ß (TGF-ß), interleukin-1 ß (L-1ß), interlekin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10). Additionally, STZ-induced T1DM mice, when treated with nBMCs or dBMCs, experienced a notable rise in total immunoglobulin (Ig) level. Furthermore, there was a significant reduction in the levels of islet cell autoantibodies (ICA) and insulin autoantibodies (IAA). Furthermore, the serum of STZ-induced T1DM mice showed a significant increase in Zinc transporter 8 antigen protein (ZnT8), islet antigen 2 protein (IA-2), and glutamic acid decarboxylase antigen protein (GAD) levels. Interestingly, the administration of nBMCs or dBMCs resulted in a heightened expression of IA-2 protein in STZ-induced T1DM mice treated with nBMCs or dBMCs. Furthermore, the level of malondialdehyde (MDA) was increased, while the levels of catalase (CAT) and superoxide dismutase (SOD) were decreased in non-treated STZ-induced T1DM mice. However, when nBMCs or dBMCs were administered to STZ-induced T1DM mice, it had a significant impact on reducing oxidative stress. This was accomplished by reducing the levels of MDA in the serum and enhancing the activities of enzymatic antioxidants like CAT and SOD. STZ-induced T1DM mice displayed a significant elevation in the levels of liver enzymes ALT and AST, as well as heightened levels of creatinine and urea. Considering the crucial roles of the liver and kidney in metabolism and excretion, this research further examined the effects of administering nBMCs or dBMCs to STZ-induced T1DM mice. Notably, the administration of these cells alleviated the observed effects. CONCLUSION: The present study suggests that utilizing adoptively transferred nBMCs or adoptively transferred dBMCs in the treatment of T1DM led to noteworthy decreases in blood glucose levels, possibly attributed to their capacity to enhance insulin secretion and improve the performance of pancreatic islets. Additionally, BMCs may exert their beneficial effects on the pancreatic islets of diabetic mice through their immunomodulatory, antioxidant, anti-inflammatory, and anti-oxidative stress properties.

Lipid Nanoparticle-mRNA Engineered Dendritic Cell Based Adoptive Cell Therapy Enhances Cancer Immune Response.

Lipid nanoparticles encapsulating mRNA (LNP-mRNA) revolutionized medicine over the past several years. While clinically approved indications currently focus on infectious disease vaccination, LNP-mRNA based treatments also hold promise for cancer immunotherapy. However, the route of dosing may impact treatment efficacy, safety, and dose. To minimize adverse effects, it is hypothesized that LNP-mRNA can be used to activate and engineer dendritic cells (DC) ex vivo before re-administration of these cells. Here, it is shown that LNP-mRNA engineered DCs can indeed vaccinate recipient mice. Vaccinated mice showed strong anti-tumor T cell responses, rejected tumor challenge, and displayed no evidence of toxicity. Further, it is found that DC specific ablation of the immune activating kinase NFkB inducing kinase (NIK) abrogated vaccination efficacy, demonstrating that adoptively transferred DCs can be functionally modified in addition to their antigen presentation capacity. Collectively, these studies show that ex vivo LNP-mRNA engineering of DCs is a feasible and robust therapeutic strategy for cancer.

Deciphering Natural Killer Cell Cytotoxicity Against Medulloblastoma in vitro and in vivo: Implications for Immunotherapy.

Purpose: Medulloblastoma (MB) is the most prevalent paediatric brain tumour. Despite improvements in patient survival with current treatment strategies, the quality of life of these patients remains poor owing to the sequelae and relapse risk. An alternative, or, in addition to the current standard treatment, could be considered immunotherapy, such as Natural Killer cells (NK). NK cells are cytotoxic innate lymphoid cells that play a major role in cancer immunosurveillance. To date, the mechanism of cytotoxicity of NK cells, especially regarding the steps of adhesion, conjugation, cytotoxic granule polarisation in the cell contact area, perforin and granzyme release in two and three dimensions, and therapeutic efficacy in vivo have not been precisely described. Materials and Methods: Each step of NK cytotoxicity against the three MB cell lines was explored using confocal microscopy for conjugation, Elispot for degranulation, flow cytometry, and luminescence assays for target cell necrosis and lysis and mediators released by cytokine array, and then confirmed in a 3D spheroid model. Medulloblastoma-xenografted mice were treated with NK cells. Their persistence was evaluated by flow cytometry, and their efficacy in tumour growth and survival was determined. In addition, their effects on the tumour transcriptome were evaluated. Results: NK cells showed variable affinities for conjugation with MB target cells depending on their subgroup and cytokine activation. Chemokines secreted during NK and MB cell co-culture are mainly associated with angiogenesis and immune cell recruitment. NK cell cytotoxicity induces MB cell death in both 2D and 3D co-culture models. NK cells initiated an inflammatory response in a human MB murine model by modulating the MB cell transcriptome. Conclusion: Our study confirmed that NK cells possess both in vitro and in vivo cytotoxic activity against MB cells and are of interest for the development of immunotherapy.

Evaluation of the safety and efficiency of cytotoxic T cell therapy sensitized by tumor antigens original from T-ALL-iPSC in vivo.

Background: Since RNA sequencing has shown that induced pluripotent stem cells (iPSCs) share a common antigen profile with tumor cells, cancer vaccines that focus on iPSCs have made promising progress in recent years. Previously, we showed that iPSCs derived from leukemic cells of patients with primary T cell acute lymphoblastic leukemia (T-ALL) have a gene expression profile similar to that of T-ALL cell lines. Methods: Mice with T-ALL were treated with dendritic and T (DC-T) cells loaded with intact and complete antigens from T-ALL-derived iPSCs (T-ALL-iPSCs). We evaluated the safety and antitumor efficiency of autologous tumor-derived iPSC antigens by flow cytometry, cytokine release assay, acute toxicity experiments, long-term toxicity experiments, and other methods. Results: Our results indicate that complete tumor antigens from T-ALL-iPSCs could inhibit the growth of inoculated tumors in immunocompromised mice without causing acute and long-term toxicity. Conclusion: T-ALL-iPSC-based treatment is safe and can be used as a potential strategy for leukemia immunotherapy.

A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Immunomodulatory Effects of Yu-Ping-Feng Formula on Primary Sjögren's Syndrome: Interrogating the T Cell Response.

Ethnopharmacological treatments have shown beneficial effects in the clinical practice of autoimmune disorders. However, the underlying mechanism of immunomodulatory effects remains challenging, given the complicate composition of herbal medicines. Here, we developed an immunological approach to interrogate the T helper cell response. Through data mining we hypothesized that Chinese medicine formula, Yu-Ping-Feng (YPF) might be a promising candidate for treating primary Sjögren's syndrome (pSS), a common autoimmune disease manifested by exocrine gland dysfunction. We took advantage of a mouse model of experimental Sjögren's syndrome (ESS) that we previously established for YPF formula treatment. YPF therapy ameliorated the ESS pathology in mice with active disease, showing improved salivary function and decreased serum levels of autoantibodies. Phenotypic analysis suggested that both effector T and B cells were significantly suppressed. Using co-culture assay and adoptive transfer models, we demonstrated that YPF formula directly restrained effector/memory T cell expansion and differentiation into Th17 and T follicular helper (Tfh) cells, the key subsets in ESS pathogenesis. Importantly, we recruited 20 pSS patients and conducted a pilot study of 8-week therapy of YPF formula. YPF treatment effectively improved fatigue symptoms, exocrine gland functions and reduced serum IgG/IgA levels, while effector T and B cell subsets were significantly decreased. There was a trend of reduction on disease activity, but not statistically significant. Together, our findings suggested a novel approach to assess the immunomodulatory effects of YPF formula, which may be favorable for patients with autoimmune disorders.

Collaborative adsorption and photocatalytic degradation of high concentration pharmaceutical pollutants in water using a novel dendritic fibrous nano-silica modified with chitosan and UiO-66.

This study presents a novel hybrid mesoporous material for degrading drug pollutants in water. The hybrid materials, derived from UiO-66 metal-organic framework and chitosan, coated on nano-silica, showed excellent drug adsorption through hydrogen-bonding interactions and efficient photodegradation of antibiotics. The hybrid material's enhanced conductivity and reduced band gap significantly improved pollution reduction by minimising electron-hole recombination. This allows for more efficient charge transport and better light absorption, boosting the material's ability to break down pollutants. Structural and morphological analyses were conducted using various techniques, including scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, Brunauer-Emmett-Teller analysis, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Optimising the adsorption-photodegradation process involved investigating pH, catalyst dose, and radiation time. Non-linear optimisation revealed an efficiency exceeding 85 % for 400 mg/L tetracycline and doxycycline, the model antibiotics. The optimal parameters for maximal elimination were determined as pH = 4.3, hybrid mesosphere dose = 4.0 mg/mL, and radiation time = 10 min. Kinetic studies favored pseudo-second-order diffusion models over pseudo-first-order models. The hybrid mesosphere showed sustained efficiency after three cycles and performed well in real aqueous samples, removing over 80 % of each antibiotic. This study demonstrates the potential of the hybrid mesoporous material for removing pharmaceutical pollutants in water systems.

Off-the-shelf CAR-NK cells targeting immunogenic cell death marker ERp57 execute robust antitumor activity and have a synergistic effect with ICD inducer oxaliplatin.

BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and poor infiltration of engineered NK cells. Here, we explore whether immunogenic cell death (ICD) marker ERp57 translocated from endoplasmic reticulum to cell surface after drug treatment could be used as a target for CAR-NK therapy. METHODS: To target ERp57, a VHH phage display library was used for screening ERp57-targeted nanobodies (Nbs). A candidate Nb with high binding affinity to both human and mouse ERp57 was used for constructing CAR-NK cells. Various in vitro and in vivo studies were performed to assess the antitumor efficacy of the constructed CAR-NK cells. RESULTS: We demonstrate that the translocation of ERp57 can not only be induced by low-dose oxaliplatin (OXP) treatment but also is spontaneously expressed on the surface of various types of tumor cell lines. Our results show that G6-CAR-NK92 cells can effectively kill various tumor cell lines in vitro on which ERp57 is induced or intrinsically expressed, and also exhibit potent antitumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the antitumor activity of G6-CAR-NK92 cells is synergistically enhanced by the low-dose ICD-inducible drug OXP. CONCLUSION: Collectively, our findings suggest that ERp57 can be leveraged as a new tumor antigen for CAR-NK targeting, and the resultant CAR-NK cells have the potential to be applied as a broad-spectrum immune cell therapy for various cancers by combining with ICD inducer drugs.

CAR-NKT Cells in Asthma: Use of NKT as a Promising Cell for CAR Therapy.

NKT cells, unique lymphocytes bridging innate and adaptive immunity, offer significant potential for managing inflammatory disorders like asthma. Activating iNKT induces increasing IFN-γ, TGF-ß, IL-2, and IL-10 potentially suppressing allergic asthma. However, their immunomodulatory effects, including granzyme-perforin-mediated cytotoxicity, and expression of TIM-3 and TRAIL warrant careful consideration and targeted approaches. Although CAR-T cell therapy has achieved remarkable success in treating certain cancers, its limitations necessitate exploring alternative approaches. In this context, CAR-NKT cells emerge as a promising approach for overcoming these challenges, potentially achieving safer and more effective immunotherapies. Strategies involve targeting distinct IgE-receptors and their interactions with CAR-NKT cells, potentially disrupting allergen-mast cell/basophil interactions and preventing inflammatory cytokine release. Additionally, targeting immune checkpoints like PDL-2, inducible ICOS, FASL, CTLA-4, and CD137 or dectin-1 for fungal asthma could further modulate immune responses. Furthermore, artificial intelligence and machine learning hold immense promise for revolutionizing NKT cell-based asthma therapy. AI can optimize CAR-NKT cell functionalities, design personalized treatment strategies, and unlock a future of precise and effective care. This review discusses various approaches to enhancing CAR-NKT cell efficacy and longevity, along with the challenges and opportunities they present in the treatment of allergic asthma.