Activation Of Effector Cells Research Articles

Abstract 4005: HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC

Abstract Approximately 3% of non-small cell lung cancer (NSCLC) cases harbor ERBB2/HER2 mutations. Notably, the majority of actionable HER2 mutations in NSCLC are in-frame insertions in exon 20. Current HER2-targeting approaches, including antibody-drug conjugates (ADCs) such as trastuzumab deruxtecan, have shown promising response rates. To explore novel strategies for targeting HER2-positive NSCLC, we investigated chimeric antigen receptor (CAR)-based therapies to enhance the anti-tumor activity of immune effector cells. We engineered a series of HER2 CAR constructs utilizing scFvs derived from trastuzumab, pertuzumab, and FRP5, recognizing distinct domains of HER2. These scFvs were integrated into second-generation BB3z (with 4-1BB and CD3z intracellular domains) and third-generation 28BB3z (with CD28, 4-1BB, and CD3z intracellular domains) CAR backbones, which were subsequently transduced into activated T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. High transduction efficiencies were achieved, with over 80% in CAR-T cells and over 50% in CAR-NK cells. We next evaluated their activity against a panel of NSCLC cell lines expressing varying levels of HER2 in vitro using firefly luciferase assays. All HER2 CAR-T cells demonstrated more than 90% killing against H2170 (HER2 high) and H322 (HER2 medium) at an effector:target (E:T) ratio of 10:1, whereas HER2 CAR-T cells yielded a killing effect of 35% against A549 (HER2 low) cells. HER2-targeting CAR-NK cells also displayed potent cytotoxicity against HER2-expressing NSCLC cells. Moreover, we observed that treatment of HER2 mutant and HER2 amplified NSCLC cells with low doses of HER2 tyrosine kinase inhibitors (TKIs) such as poziotinib enhanced the expression of HER2 on the cell surface. This upregulation resulted in enhanced activation of HER2 CAR-T and CAR-NK cells in coculture experiments as measured by CD107a expression on effector cells. Additionally, treatment with the HER2 TKI poziotinib potentiated the killing activity mediated by HER2 CAR-T and CAR-NK cells. Using a HER2 YVMA duplication patient-derived xenograft (PDX) NSCLC model, we observed that the in vivo combination of poziotinib with HER2 CAR-NK cells exhibited superior anti-tumor activity as compared to poziotinib treatment alone. In conclusion, HER2 CAR-T and CAR-NK cells effectively target HER2-mutant and HER2-positive NSCLC, and their combination with HER2 TKIs enhances vulnerability to HER2-targeting strategies. Citation Format: Yan Yang, Monique B. Nilsson, Alissa Poteete, Junqin He, Qian Huang, John V. Heymach. HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4005.

Abstract 1281: Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota

Abstract The lung has been traditionally regarded as a sterile environment. Emerging evidence has overturned this belief unveiling the existence of a specific lung microbiota that plays a pivotal role in modulating lung immune tolerance. Recent research also sheds light on how the tissue microbiota is significantly altered by the presence of a growing tumor. Here, we investigated the compositional changes in lung-colonizing bacterial populations associated with the growth of LLC1 lung carcinoma cells in C57BL/6 mice, by analyzing the lung 16S rRNA profiling. While overall microbial diversity and structure were similar in tumor-bearing and healthy lungs, abundance analysis of amplicon sequence variants (ASVs) revealed that 48 ASVs were differentially modulated at significant level (FDR ≤ 0.05). Notably, local treatment with aerosolized vancomycin/neomycin antibiotics starting 4 days after tumor injection caused a different representation of 47 ASVs (FDR ≤ 0.05), mostly reducing those ASVs specifically emerging in the lung by the tumor growth, belonging to Bacterioidales, Clostridiales, Lactobacillales and Enterobacteriales. A different microbial composition was also suggested by the results of Partial Least Squares Discriminant Analysis (PLS-DA) that clustered lung tumor samples as saline or antibiotic-aerosolized. Based on the reported ability of some bacteria belonging to these orders to induce immunosuppressive cells in the gut, we examined the impact of antibiotic aerosol on tumor-associated Tregs in the lung. The presence of the tumor promoted the appearance of Tregs expressing CD69 and PD-1 activation markers, but vancomycin/neomycin aerosol treatment strongly dropped Treg frequency in tumor-bearing lungs, compared to saline. In a therapeutic setting, aerosolization of antibiotics starting 4 days after LLC1 tumor intravenous injection significantly reduced the number of lung metastases (p=0.0116). This effect was associated with an increased cytotoxic activity of immune infiltrating effector cells suggesting that the modulation of the lung microbiota impairs tumor growth and enhances antitumor immune response. Some cultivable bacterial strains, that were in agreement with metataxonomic profile results, were isolated from tumor-bearing lungs, but not from healthy lungs. Ongoing experiments suggest an effect of their in vivo transfer in the lung by aerosol on tumor growth and the ability to induce Treg expansion in vitro. Overall, this study unveils that the growth of the murine LLC1 tumor shapes the lung microbial composition by favoring the expansion of microbes with potential immunosuppressive activity eventually affecting the tumor progression. Moreover, our findings unravel the therapeutic potential of localized antibiotics to revert the immunosuppressive microenvironment by targeting tumor-associated bacteria. Citation Format: Giancarla Bernardo, Valentino Le Noci, Elena Montanari, Emerenziana Ottaviano, Giorgio Gargari, Elisa Borghi, Simone Guglielmetti, Federico Ambrogi, Tiziana Triulzi, Elda Tagliabue, Serenella M. Pupa, Michele Sommariva, Lucia Sfondrini. Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1281.

Impact of donor NKG2D and MICA gene polymorphism on clinical outcomes of adult and paediatric allogeneic cord blood transplantation for malignant diseases.

NKG2D is an activating receptor expressed by natural killer (NK) and CD8+ T cells and activation intensity varies by NKG2D expression level or nature of its ligand. An NKG2D gene polymorphism determines high (HNK1) or low (LNK1) expression. MICA is the most polymorphic NKG2D ligand and stronger effector cell activation associates with methionine rather than valine at residue 129. We investigated correlation between cord blood (CB) NKG2D and MICA genotypes and haematopoietic stem cell (HSC) transplant outcome. We retrospectively studied 267 CB HSC recipients (178 adult and 87 paediatric) who underwent transplant for malignant disease between 2007 and 2018, analysing CB graft DNA for NKG2D and MICA polymorphisms using Sanger sequencing. Multivariate analysis was used to correlate these results with transplant outcomes. In adult patients, LNK1 homozygous CB significantly improved 60-day neutrophil engraftment (hazard ratio (HR) 0.6; 95% confidence interval (CI) 0.4-0.9; p = .003). In paediatrics, HNK1 homozygous CB improved 60-day engraftment (HR 0.4; 95% CI 0.2-0.7; p = .003), as did MICA-129 methionine+ CB grafts (HR 1.7 95% CI 1.1-2.6; p = .02). CB NKG2D and MICA genotypes potentially improve CB HSC engraftment. However, results contrast between adult and paediatric recipients and may reflect transplant procedure disparities between cohorts.

Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy

BackgroundClinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified monoclonal antibodies (mAb) to Ara h 2 and structurally characterized their epitopes. ObjectiveWe investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. MethodsWe developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis (PCA) in mice with humanized FcεRI receptors. ResultsDiverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of two neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to two non-neutralizing mAbs (non-nAbs). Post-OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine PCA after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. ConclusionsSerum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.

Nanomedicines for an Enhanced Immunogenic Cell Death-Based In Situ Cancer Vaccination Response.

Cancer vaccines have shown tremendous potential in preventing and treating cancer by providing immunogenic antigens to initiate specific tumor immune responses. An in situ vaccine prepared from an autologous tumor can mobilize a patient's own tumor cell lysate as a reservoir of specific antigens, thus triggering a broad immune response and diverse antitumor immunity in an individually tailored manner. Its efficacy is much better than that of conventional vaccines with a limited number of epitopes. Several conventional therapies, including radiotherapy (RT), chemotherapeutics, photodynamic therapy (PDT), and photothermal therapy (PTT) can activate an anticancer in situ vaccine response by inducing immunogenic cell death (ICD), triggering the exposure of tumor-associated antigens (TAAs), cancerous testis antigens, neoantigens, and danger-associated molecular patterns (DAMPs) with low cost. However, the immunogenicity of dying tumor cells is low, making released antigens and DAMPs insufficient to initiate a robust immune response against malignant cancer. Moreover, the immunosuppressive tumor microenvironment (TME) severely hinders the infiltration and sensitization of effector immune cells, causing tolerogenic immunological effects.Herein, we mainly focus on the research in developing nanoplatforms to surmount the major challenges met by ICD-based in situ vaccines. We first summarized a variety of nanotechnologies that enable enhanced immunogenicity of dying cancer cells by enhancing antigenicity and adjuvanticity. The robust antigenicity was obtained via regulating the tumor cells death mode or the dying state to amplify the recognition of tumor debris by professional antigen-presenting cells (APCs). The adjuvanticity was potentiated by raising the level or intensifying the activity of endogenous adjuvants or promoting the intelligent delivery of exogenous immunostimulants to activate immune cell recruitment and promote antigen presentation. Additionally, versatile approaches to reverse immunosuppressive TME to boost the in situ tumor vaccination response are also highlighted in detail. On one hand, by modulating the cell metabolism in TME, the expansion and activity of effector versus immunosuppressive cells can be optimized to improve the efficiency of in situ vaccines. On the other hand, regulating cellular components in TME, such as reversing adverse immune cell phenotypes or inhibiting the activity of interstitial cells, can also significantly enhance the ICD-based antitumor immunotherapy effect. Finally, our viewpoint on the future challenges and opportunities in this hopeful area is presented. We expect that this Account can offer much more insight into the design, planning, and development of cutting-edge in situ tumor vaccine platforms, promoting more attention and academic-industry collaborations, accelerating the advanced progress of in situ tumor vaccine-based immunotherapy in the clinic.

Non–IgE-Mediated Immediate Drug-Induced Hypersensitivity Reactions

Immediate drug-induced hypersensitivity reactions (IDHSRs) have conventionally been attributed to an immunoglobulin E (IgE)-mediated mechanism. Nevertheless, it has now been acknowledged that IDHSRs can also occur independently of IgE involvement. Non-IgE-mediated IDHSRs encompass the activation of effector cells, both mast cell-dependent and -independent and the initiation of inflammatory pathways through immunogenic and nonimmunogenic mechanisms. The IDHSRs involve inflammatory mediators beyond histamine, including the platelet-activating factor, which activates multiple cell types, including smooth muscle, endothelium, and MC, and evidence supports its importance in IgE-mediated reactions in humans. Clinically, distinguishing IgE from non-IgE mechanisms is crucial for future treatment strategies, including drug(s) restriction, readministration approaches, and pretreatment considerations. However, this presents significant challenges because certain drugs can trigger both mechanisms, and their presentations can appear similarly, ranging from mild to life-threatening symptoms. Thus, history alone is often inadequate for differentiation, and skin tests lack a standardized approach. Moreover, drug-specific IgE immunoassays have favorable specificity but low sensitivity, and the usefulness of the basophil activation test remains debatable. Lastly, no biomarker reliably differentiates between both mechanisms. Whereas non-IgE-mediated mechanisms likely predominate in IDHSRs, reclassifying most drug-related IDHSRs as non-IgE-mediated, with suggested prevention through dose administration adjustments, is premature and risky. Therefore, continued research and validated diagnostic tests are crucial to improving our capacity to distinguish between these mechanisms, ultimately enhancing patient care.

Enhancing CAR Macrophage Efferocytosis Via Surface Engineered Lipid Nanoparticles Targeting LXR Signaling.

The removal of dying cells, or efferocytosis, is an indispensable part of resolving inflammation. However, the inflammatory microenvironment of the atherosclerotic plaque frequently affects the biology of both apoptotic cells and resident phagocytes, rendering efferocytosis dysfunctional. To overcome this problem, a chimeric antigen receptor (CAR) macrophage that can target and engulf phagocytosis-resistant apoptotic cells expressing CD47 is developed. In both normal and inflammatory circumstances, CAR macrophages exhibit activity equivalent to antibody blockage. The surface of CAR macrophages is modified with reactive oxygen species (ROS)-responsive therapeutic nanoparticles targeting the liver X receptor pathway to improve their cell effector activities. The combination of CAR and nanoparticle engineering activated lipid efflux pumps enhances cell debris clearance and reduces inflammation. It is further suggested that the undifferentiated CAR-Ms can transmigrate within a mico-fabricated vessel system. It is also shown that our CAR macrophage can act as a chimeric switch receptor (CSR) to withstand the immunosuppressive inflammatory environment. The developed platform has the potential to contribute to the advancement of next-generation cardiovascular disease therapies and further studies include in vivo experiments.

Endothelial cells in tumor microenvironment: insights and perspectives.

The tumor microenvironment is a highly complex and dynamic mixture of cell types, including tumor, immune and endothelial cells (ECs), soluble factors (cytokines, chemokines, and growth factors), blood vessels and extracellular matrix. Within this complex network, ECs are not only relevant for controlling blood fluidity and permeability, and orchestrating tumor angiogenesis but also for regulating the antitumor immune response. Lining the luminal side of vessels, ECs check the passage of molecules into the tumor compartment, regulate cellular transmigration, and interact with both circulating pathogens and innate and adaptive immune cells. Thus, they represent a first-line defense system that participates in immune responses. Tumor-associated ECs are involved in T cell priming, activation, and proliferation by acting as semi-professional antigen presenting cells. Thus, targeting ECs may assist in improving antitumor immune cell functions. Moreover, tumor-associated ECs contribute to the development at the tumor site of tertiary lymphoid structures, which have recently been associated with enhanced response to immune checkpoint inhibitors (ICI). When compared to normal ECs, tumor-associated ECs are abnormal in terms of phenotype, genetic expression profile, and functions. They are characterized by high proliferative potential and the ability to activate immunosuppressive mechanisms that support tumor progression and metastatic dissemination. A complete phenotypic and functional characterization of tumor-associated ECs could be helpful to clarify their complex role within the tumor microenvironment and to identify EC specific drug targets to improve cancer therapy. The emerging therapeutic strategies based on the combination of anti-angiogenic treatments with immunotherapy strategies, including ICI, CAR T cells and bispecific antibodies aim to impact both ECs and immune cells to block angiogenesis and at the same time to increase recruitment and activation of effector cells within the tumor.

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Epigenetic reprogramming of T cells: unlocking new avenues for cancer immunotherapy.

T cells, a key component of cancer immunotherapy, undergo a variety of histone modifications and DNA methylation changes since their bone marrow progenitor stages before developing into CD8+ and CD4+ T cells. These T cell types can be categorized into distinct subtypes based on their functionality and properties, such as cytotoxic T cells (Tc), helper T cells (Th), and regulatory T cells (Treg) as subtypes for CD8+ and CD4+ T cells. Among these, the CD4+ CD25+ Tregs potentially contribute to cancer development and progression by lowering T effector (Teff) cell activity under the influence of the tumor microenvironment(TME). This contributes to the development of therapeutic resistance in patients with cancer. Subsequently, these individuals become resistant to monoclonal antibody therapy as well as clinically established immunotherapies. In this review, we delineate the different epigenetic mechanisms in cancer immune response and its involvement in therapeutic resistance. Furthermore, the possibility of epi-immunotherapeutic methods based on histone deacetylase inhibitors and histone methyltransferase inhibitors are under investigation. In this review we highlight EZH2 as the principal driver of cancer cell immunoediting and an immune escape regulator. We have addressed in detail how understanding T cell epigenetic regulation might bring unique inventive strategies to overcome drug resistance and increase the efficacy of cancer immunotherapy.

CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.

CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.

Engineered Nanomaterials for Immunomodulation: A Review.

The immune system usually provides a defense against invading pathogenic microorganisms and any other particulate contaminants. Nonetheless, it has been recently reported that nanomaterials can evade the immune system and modulate immunological responses due to their unique physicochemical characteristics. Consequently, nanomaterial-based activation of immune components, i.e., neutrophils, macrophages, and other effector cells, may induce inflammation and alter the immune response. Here, it is essential to distinguish the acute and chronic modulations triggered by nanomaterials to determine the possible risks to human health. Nanomaterials size, shape, composition, surface charge, and deformability are factors controlling their uptake by immune cells and the resulting immune responses. The exterior corona of molecules adsorbed over nanomaterials surfaces also influences their immunological effects. Here, we review current nanoengineering trends for targeted immunomodulation with an emphasis on the design, safety, and potential toxicity of nanomaterials. First, we describe the characteristics of engineered nanomaterials that trigger immune responses. Then, the biocompatibility and immunotoxicity of nanoengineered particles are debated, because these factors influence applications. Finally, future nanomaterial developments in terms of surface modifications, synergistic approaches, and biomimetics are discussed.

A microrheological examination of insulin-secreting β-cells in healthy and diabetic-like conditions.

Pancreatic β-cells regulate glucose homeostasis through glucose-stimulated insulin secretion, which is hindered in type-2 diabetes. Transport of the insulin vesicles is expected to be affected by changes in the viscoelastic and transport properties of the cytoplasm. These are evaluated in situ through particle-tracking measurements using a rat insulinoma β-cell line. The use of inert probes assists in decoupling the material properties of the cytoplasm from the active transport through cellular processes. The effect of glucose-stimulated insulin secretion is examined, and the subsequent remodeling of the cytoskeleton, at constant effects of cell activity, is shown to result in reduced mobility of the tracer particles. Induction of diabetic-like conditions is identified to alter the mean-squared displacement of the passive particles in the cytoplasm and diminish its reaction to glucose stimulation.

Unique Peripheral Immune Profile in Young Women with Metastatic Breast Cancer

Abstract Background: The occurrence of breast cancer in young women (BCYW), i.e., under 40 years old (<40 years), is increasing, and tumors in this group often exhibit different biology and prognosis compared to those in older counterparts. Aim and Study Design: This study aimed to access the peripheral immune cell populations in metastatic BCYW at diagnosis and compare them with older (≥40) metastatic breast cancer (BC) patients. Blood samples were collected when the metastatic disease was diagnosed and before treatment began. Materials and Methods: A total of 90 patients were included, with a median age of 52.5 years at baseline (27–88 years). Among them, 17 patients were under 40 years old, and 73 were 40 years old or older. We isolated peripheral blood mononuclear cells using Histopaque-1077 Hybri-Max to access immune cells by flow cytometry. For comparisons between two groups, the Mann-Whitney-Wilcoxon test was used, and Dunn’s test was used for comparisons between more than two groups. Results: The presence of metastatic BCYW was characterized by a lower proportion of regulatory T-cells (Treg) and central memory (CM) CD8+ T-cells, as well as a higher proportion of total effector T-cells (Teff). In young patients with widespread metastasis (plurimetastatic), there was a higher proportion of Teff cells, Vδ2+ T-cells, and effector Vδ1+ T-cells, with fewer Treg cells. Conversely, older plurimetastatic patients had more Treg cells and fewer effector T-cells. Furthermore, young oligometastatic patients had a lower proportion of CM CD8+ T-cells. Conclusions: BCYW presents a distinct immunologic profile with higher effector cell activity and lower levels of regulatory and memory cells, regardless of metastatic burden and menopausal status. This finding suggests that targeting immune pathways may offer new therapeutic strategies to improve outcomes for young women with BC, potentially leading to more personalized and effective treatments.

The Role of NKG2D and Its Ligands in Autoimmune Diseases: New Targets for Immunotherapy.

Natural killer (NK) cells and CD8+ T cells can clear infected and transformed cells and generate tolerance to themselves, which also prevents autoimmune diseases. Natural killer group 2 member D (NKG2D) is an important activating immune receptor that is expressed on NK cells, CD8+ T cells, γδ T cells, and a very small percentage of CD4+ T cells. In contrast, the NKG2D ligand (NKG2D-L) is generally not expressed on normal cells but is overexpressed under stress. Thus, the inappropriate expression of NKG2D-L leads to the activation of self-reactive effector cells, which can trigger or exacerbate autoimmunity. In this review, we discuss the role of NKG2D and NKG2D-L in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), type I diabetes (T1DM), inflammatory bowel disease (IBD), and celiac disease (CeD). The data suggest that NKG2D and NKG2D-L play a pathogenic role in some autoimmune diseases. Therefore, the development of strategies to block the interaction of NKG2D and NKG2D-L may have therapeutic effects in some autoimmune diseases.

Tumor-derived extracellular vesicles in the immune microenvironment of head and neck squamous cell carcinoma: Foe or future?

Head and neck squamous cell carcinoma (HNSCC) is considered a “cold tumor” due to its suppressive immune microenvironment, and is associated with a poor prognosis. Tumor-derived extracellular vesicles (EVs) play an essential role in the tumor microenvironment and mediate intercellular communications. EVs have been proven to be key immune regulators involved in antitumor immune responses and escape from immune surveillance. Tumor-derived EVs favor the formation of an immunosuppressive tumor microenvironment by regulating the differentiation, proliferation and activation of innate and adaptive immune effector cells, as well as myeloid cells, acting as a “foe” in the microenvironment. However, EVs are also valuable for predicting and improving the prognosis of HNSCC, and represent hope for future treatments. In this review, we summarize the impact of HNSCC-derived EVs on the immune microenvironment, describe their roles as biomarkers and for drug delivery in disease monitoring and treatment. We provide insights into important areas for future research and identify potential therapeutic targets for HNSCC treatment.

Abstract A071: Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma

Abstract Background Metastatic pancreas adenocarcinoma (PDAC) is highly lethal and minimally responsive to immunotherapy. Gamma delta T cells (gDTs) are a unique T cell subset found within the PDAC microenvironment. Animal models have shown that gDTs promote oncogenic progression and ablating gDT delays cancer progression and improves survival. Understanding the activity of immune effector cells against cancer organoids provides a scalable platform to understand the impact of activation and small molecule modulation to redirect gDT killing in PDAC. Methods Patient-derived organoids (PDOs) were generated from fresh tissue sampling and exapanded using a defined, serum-free media. Upon passaging, media was collected from PDAC PDOs. Allogeneic gDTs isolated from healthy donors were cultured in PDAC PDO conditioned media from 3 separate PDOs, in 3 separate biological replicates, and then subjected to bulk RNAseq. Allogeneic gDTs were isolated and cultured with PDAC PDOs at ratio of 5:1 (gDTs:PDAC PDOs) for 48 hours. After 48 hours, staining was performed for induction of apoptosis (cleaved caspase-3/7FITC). Images were acquired by high content imaging (Cytation 5, BioTek) and quantified for response across organoid populations. Based on RNAseq nominated candidates, low density lipoprotein (LDL) was added (10 ug/mL) as well as a small molecule inverse agonist of the Liver X Receptor (LXR) beta transcription factor (SR9243) to assess the ability of these factors to modulate gDT mediated PDAC killing. Results Exposure of gDTs to PDAC PDO secreted factors results in differential regulation of cholesterol handling, including upregulation of cholesterol efflux, and downregulation of cholesterol uptake and biosynthesis related genes in conditioned T cell populations. gDTs cause marked apoptosis in PDAC PDOs, including 40% staining of % organoid area versus negligible staining in control populations of 1.9% organoid area (effect size = 9.5, p<0.001). This response was directly compared to standard chemotherapy at the time of clinical resistance to FOLFIRINOX which conferred only 2% staining of mean organoid area (effect size = 0.11, NS). Given RNAseq data demonstrating cholesterol handling changes after exposure to PDAC secreted factors, we investigated LDL supplementation in our model. Supplementation of LDL significantly improves gDT mediated killing, with induction of apoptosis seen in 52% of the PDAC PDO area (p=0.003). This effect is mimicked by the small molecule SR9243 (an LXR inverse agonist), with 56% of PDAC PDO demonstrating apoptotic induction at 48 hours. Conclusions A PDAC PDO model system can be used to investigate gDT mediated PDAC killing when applied in low-volume medium throughput screening applications. Modulation of LXR activity and microenvironment LDL results in substantially increased immune-mediated killing of PDAC. This screening platform identifies potentially clinically actionable modulators of immune response to PDAC, and suggests potential future immunologically relevant targets that may improve the efficacy of immunotherapy. Citation Format: Johnathan D Ebben, David Turicek, Md Shahadat Hossan, Austin Stram, Ethan Lin, Nicholas Hess, Zachary Mayhew, Melissa A Kinney, Devin Burpee, Anikait Patel, Christian M Capitini, Jeremy D Kratz. Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A071.

Immune Cells, Cytokines, and Gut Microbiota Landscape Along Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) Evolution

Introduction: Multiple myeloma (MM) is an incurable hematologic cancer characterized by abnormal plasma cell growth in the bone marrow (BM). It is preceded by two premalignant conditions: monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Understanding the transition from MGUS to MM is crucial for better risk stratification, early interventions, and targeted treatments. This study aims to gain insights into immunological changes and fecal microbiota variations along the MGUS to MM transition. Methods: To comprehensively evaluate the immune composition (myeloid/lymphoid T/B/NK subpopulations and immune checkpoint distribution) in both bone marrow (BM) and peripheral blood (PB), we designed six 10-color custom and two 8-color flow cytometry panels. We analyzed samples from 13 MGUS, 12 SMM, and 63 newly diagnosed MM patients. Additionally, we evaluated cytokine/chemokine abundance in BM and PB using a 48-plex Luminex plate on 72 samples from the same MGUS, SMM, and MM patients, along with 4 healthy donors (HD). Furthermore, we performed fecal microbiome profiling on samples from 3 MGUS, 6 SMM, and 9 MM patients to investigate potential relationships between bacterial composition and MM evolution. Statistical analysis was performed in R by using specific packages (such as FlowCT). Results: Through unsupervised analysis of T cells by flow cytometry, we identified a significant increase in circulating TEMRA CD8 T cells, mostly for CD57+ cells (mean, 7.5% for MGUS vs 20.1% for SMM vs 21.6% for MM; p<0.05) and a significant reduction in naïve CD8 T TIGIT+ cells (mean, 12.4% for MGUS vs 5.98% for SMM vs 4.16% for MM; p<0.001) and naïve CD8 T TIGIT+ TIM3+ (mean, 2.18% for MGUS vs 1.24% for SMM vs 1.17% for MM; p<0.001). Regarding CD4 T subsets, we observed a reduction in BM effector memory phenotype along disease evolution (mean, 7.07% for MGUS vs 4.54% for SMM vs 3.72% for MM; p<0.05). Interestingly, we confirmed the increase of BM CD4 T cells production of IL17 (mean: 6.7% for MGUS vs 22% for MM; p<0.05) in MM patients compared to those with MGUS. In the context of myeloid cell subsets, circulating non-classical monocytes expressing HLA-DR and CD11c (mean, 1.45% for MGUS vs 0.95% for SMM vs 0.69% for MM; p<0.05) as well as granulocytes (mean, 74.18% MGUS vs 73.86% SMM vs 65.59% MM; p<0.05) were found to be reduced along MM progression. No major differences were observed for other evaluated lineages. Regarding cytokines and chemokines, a concurrent decrease in GM-CSF, IFN-a2, IFN-g, IL-1b, IL-2, IL-2Ra, IL-3, IL-10, IL-13, and MCP-1/CCL2 levels within the BM plasma throughout MM progression was observed, thus supporting a global reduction in myeloid function as well as in T cell effector activity. Moreover, PDGF-BB, IFN-a2, IL-16, and IL-8 decrease was observed in PB plasma along MM progression. Lastly, while no differences were observed in term of alpha and beta diversity, we observed an overall increase in total abundance of fecal bacteria along MM evolution (maybe related to loss of myeloid/lymphoid control according to what previously described), with several bacterial genus significantly over-represented in MM patients, including Bariatricus, CAG-83, Alistipes, Bifidobacterium, Streptococcus, Ruminococcus D, Slackia A, and Coprococcus A, while others (such as Parabacteroides B) was found to be more represented in MGUS and SMM patients. The analysis of phyla abundance indicated Firmicutes D and Lactobacillales as more abundant in MM patients, while Actinobacteriota dominated in SMM patients, and Proteobacteria and Bacteroidota were more prominent in MGUS patients. Conclusions: Overall, this study demonstrated a progressive impairment of both myeloid and lymphoid compartment in term of cellular composition, cytokine production and release and control over gut microbiome. Recovering from T cell exhaustion and IL17 production remains a major challenge for these patients.

Cold to Hot: Tumor Immunotherapy by Promoting Vascular Normalization Based on PDGFB Nanocomposites.

Immunotherapy is a promising cancer therapeutic strategy. However, the "cold" tumor immune microenvironment (TIME), characterized by insufficient immune cell infiltration and immunosuppressive status, limits the efficacy of immunotherapy. Tumor vascular abnormalities due to defective pericyte coverage aregradually recognized as a profound determinant in "cold" TIME establishment by hindering immune cell trafficking. Recently, several vascular normalization strategies by improving pericyte coverage have been reported, whereas have unsatisfactory efficacy and high rates of resistance. Herein, a combinatorial strategy to induce tumor vasculature-targeted pericyte recruitment and zinc ion-mediated immune activation with aplatelet-derived growth factor B (PDGFB)-loaded, cyclo (Arg-Gly-Asp-D-Phe-Lys)-modified zeolitic imidazolate framework 8 (PDGFB@ZIF8-RGD) nanoplatform is proposed. PDGFB@ZIF8-RGD effectively induced tumor vascular normalization, which facilitated trafficking and infiltration of immune effector cells, including natural killer (NK) cells, M1-like macrophages and CD8+ T cells, into tumor microenvironment. Simultaneously, vascular normalization promoted the accumulation of zinc ions inside tumors to trigger effector cell immune activation and effector molecule production. The synergy between these two effects endowed PDGFB@ZIF8-RGD with superior capabilities in reprogramming the "cold" TIME to a "hot" TIME, thereby initiating robust antitumor immunity and suppressing tumor growth. This combinatorial strategy for improving immune effector cell infiltration and activation is a promising paradigm for solid tumor immunotherapy.

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.