Innate Immune Effector Cells Research Articles

Homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung natural killer cells

Natural killer (NK) cells are important innate immune effector cells for controlling tumor growth and metastasis. Differentiated mature NK cells preferentially reside in the peripheral tissues and express higher levels of self-major histocompatibility complex class I (MHC-I)-recognizing inhibitory receptors. MHC-I recognition by NK cells are known to be important for their development and maturation processes, however, the role of homeostatic MHC-I recognition in maintaining effector functions of mature NK cells in the peripheral tissues needs to be elucidated. In this study, we utilized a pan anti-MHC-I blocking monoclonal antibody (anti-MHC-I) to examine the role of homeostatic MHC-I recognition in the response of pulmonary mature NK cells in an experimental lung metastasis model of B16F10 melanoma. Anti-MHC-I treatment showed significant inhibition of the lung metastasis of B16F10 melanoma in NK cell- and IFN-γ-dependent mechanisms. The blockade of homeostatic MHC-I recognition increased mature lung NK cell responsiveness, such as direct cytotoxicity and IFN-γ production, rather than the number of lung NK cells. Mechanistically, the gene expression of activating receptors including DNAX accessory molecule-1 (DNAM-1) was upregulated in NK cells treated with anti-MHC-I, and further the enhanced NK cell cytotoxicity against B16F10 cells was DNAM-1-dependent. Collectively, homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung NK cells by restraining the expression of activating receptors.

Analysis of the Effector Functions of Vδ2 γδ T Cells and NK Cells against Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a rare disease characterized by malignant cells derived from the epithelial cells of the biliary duct system. Despite extensive treatments, the prognosis for CCA remains poor, emphasizing the critical need for the development of novel treatments. Considerable attention has been directed towards innate immune effector cells, which can recognize tumor cells independently of the major histocompatibility complex, laying the foundation for the development of off-the-shelf drugs. In this study, we cultured innate immune cells obtained from the peripheral blood of healthy adults and conducted a comparative analysis of the effector functions against CCA cell lines by Vδ2 γδ T cells and NK cells. This analysis was performed using standard short- and long-term cytotoxicity assays, as well as ELISA for IFN-γ. Vδ2 γδ T cells demonstrated cytotoxicity and IFN-γ production in response to CCA cells in a TCR-dependent manner, particularly in the presence of tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate, a bisphosphonate prodrug. In contrast, direct killing and antibody-dependent cellular cytotoxicity were relatively slow and weak. Conversely, NK cells displayed potent, direct cytotoxicity against CCA cells. In summary, both Vδ2 γδ T cells and NK cells show promise as innate immune effector cells for adoptive transfer therapy in the context of CCA.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Innate Immune Effectors and Leishmania: an Overview of Complexities

Abstract: Leishmaniasis represents a pool of debilitating clinical manifestations affecting several individuals globally. The disease remains a serious global health and affects individuals in tropical and subtropical regions. The disease is endemic in several areas of South America, East Africa, the Indian sub-continent and the Mediterranean basin. The bite of female Phlebotomine sand establishes the infection of the Leishmania parasite within human flies belonging to the family Psychodidae (subfamily: Phlebotominae) of class Diptera. Several species of Leishmania parasite serve as the infectious trigger associated with varying clinical presentation of the disease. The immune response against the different parasitizing species varies, resulting in a complex response by innate immune cells. The present review summarizes some of the key innate immune effector cells involved during the infection with the Leishmania parasite in a quest to provide a deeper understanding of Leishmania- mediated immunobiology. The review also summarizes an up-to-date understanding of several strategies adopted by the parasite to evade immune response mediated by altering the functioning of some key innate immune effector cells. A better understanding of these immuno-biological events within the infected individual would help formulate immune-therapeutical interventions against the disease.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity [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 741.

Age-dependent inflammatory response is altered in an ex vivo model of bacterial pneumonia

BackgroundAging is associated with an increased incidence and mortality of Pseudomonas aeruginosa-induced pneumonias. This might be partly due to age-dependent increases in inflammatory mediators, referred to as inflamm-aging and a decline in immune functions, known as immunosenescence. Still, the impact of dysregulated immune responses on lung infection during aging is poorly understood. Here, we aimed to mimic inflamm-aging using ex vivo precision-cut lung slices (PCLS) and neutrophils – as important effector cells of innate immunity – from young and old mice and investigated the influence of aging on inflammation upon infection with P. aeruginosa bacteria.MethodsMurine PCLS were infected with the P. aeruginosa standard lab strain PAO1 and a clinical P. aeruginosa isolate D61. After infection, whole-transcriptome analysis of the tissue as well as cytokine expression in supernatants and tissue lysates were performed. Responses of isolated neutrophils towards the bacteria were investigated by quantifying neutrophil extracellular trap (NET) formation, cytokine secretion, and analyzing expression of surface activation markers using flow cytometry.ResultsInflamm-aging was observed by transcriptome analysis, showing an enrichment of biological processes related to inflammation, innate immune response, and chemotaxis in uninfected PCLS of old compared with young mice. Upon P. aeruginosa infection, the age-dependent pro-inflammatory response was even further promoted as shown by increased production of cytokines and chemokines such as IL-1β, IL-6, CXCL1, TNF-α, and IL-17A. In neutrophil cultures, aging did not influence NET formation or cytokine secretion during P. aeruginosa infection. However, expression of receptors associated with inflammatory responses such as complement, adhesion, phagocytosis, and degranulation was lower in neutrophils stimulated with bacteria from old mice as compared to young animals.ConclusionsBy using PCLS and neutrophils from young and old mice as immunocompetent ex vivo test systems, we could mimic dysregulated immune responses upon aging on levels of gene expression, cytokine production, and receptor expression. The results furthermore reflect the exacerbation of inflammation upon P. aeruginosa lung infection as a result of inflamm-aging in old age.

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.

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

Neutrophils in Inflammatory Diseases: Unraveling the Impact of Their Derived Molecules and Heterogeneity.

Inflammatory diseases involve numerous disorders and medical conditions defined by an insufficient level of self-tolerance. These diseases evolve over the course of a multi-step process through which environmental variables play a crucial role in the emergence of aberrant innate and adaptive immunological responses. According to experimental data accumulated over the past decade, neutrophils play a significant role as effector cells in innate immunity. However, neutrophils are also involved in the progression of numerous diseases through participation in the onset and maintenance of immune-mediated dysregulation by releasing neutrophil-derived molecules and forming neutrophil extracellular traps, ultimately causing destruction of tissues. Additionally, neutrophils have a wide variety of functional heterogeneity with adverse effects on inflammatory diseases. However, the complicated role of neutrophil biology and its heterogeneity in inflammatory diseases remains unclear. Moreover, neutrophils are considered an intriguing target of interventional therapies due to their multifaceted role in a number of diseases. Several approaches have been developed to therapeutically target neutrophils, involving strategies to improve neutrophil function, with various compounds and inhibitors currently undergoing clinical trials, although challenges and contradictions in the field persist. This review outlines the current literature on roles of neutrophils, neutrophil-derived molecules, and neutrophil heterogeneity in the pathogenesis of autoimmune and inflammatory diseases with potential future therapeutic strategies.

Low Dose Rituximab Efficiently Clears Circulating CLL Cells and Maintains Sensitivity to Antibody Dependent Cellular Phagocytosis (ADCP)

Introduction: We have recently shown that high frequency (2 x week) low dose (50 mg)(HFLD) rituximab (RTX) and acalabrutinib is highly effective initial therapy for patients (n=38) with progressive CLL (ClinicalTrials.gov NCT03788291, PMID:36689726). The first dose of 50 mg of IV rituximab administered at 25 mg/h decreased the median circulating CLL cell count by 84% from pre-treatment baseline at 1h with no further decrease in the count during the remainder of the infusion (PMID:37003030). During the first hour of infusion, median CLL cell membrane CD20 levels decreased to 65% of baseline and were 41% of baseline at the end of the infusion. Median serum rituximab concentration was 2 mcg/ml at 1h and 10 mcg/ml at the end of the infusion. Median complement (CH50) concentration decreased to 91% of baseline at 1 h and to 81% of baseline at the end of the infusion. The cessation of clearance of CLL cells from the circulation within 1h of starting rituximab therapy is thus unlikely to be caused by low rituximab levels, decreased CLL cell CD20 expression or low serum complement levels. We and others have previously shown that ADCP is the primary mechanism of clearance of anti-CD20 mAb opsonized B cells in both humans and mice. We hypothesized that clearance of circulating CLL cells decreases because residual circulating CLL cells are resistant to mAb induced ADCP. To test this hypothesis, we measured in vitro ADCP sensitivity of CLL cells sampled from patients before and after rituximab treatment. Methods: Blood specimens were collected from 13 patients prior to starting therapy (C1D1_pre), 1h after start of IV rituximab infusion (C1D1_1h), at end of rituximab infusion (C1D1_post), and 48 h after initiation of therapy (C1D3_pre). Sensitivity of circulating CLL cells to in vitro ADCP was measured using live cell time-lapse high-content microscopy imaging of phagocytosis of CLL cells by human monocyte derived macrophages (hMDM) as previously described (PMID:32005699). Phagocytosis was quantified using a void index and sensitivity of samples was compared using the area under the curve of the 2h void index plot (AUC). Experiments were done in triplicate with co-cultures of hMDM and CLL cells without mAb as controls. Rituximab (10 mcg/ml) was used to test CD20 mAb mediated ADCP and alemtuzumab (10 mcg/ml) to test non-CD20 mediated ADCP. Results: Figure (median and 95% confidence intervals) shows that neither rituximab nor alemtuzumab induced ex-vivo ADCP as measured by median void index AUC was significantly altered by prior in vivo exposure of CLL cells to rituximab (Wilcoxon Match-Paired Sign test; all p>0.05). Discussion: The rapid decrease in circulating CLL cells induced by IV rituximab occurred only within the first hour after initiation of mAb infusion. Our measurements show that serum rituximab and complement concentrations and CLL cell membrane CD20 levels should be adequate to support additional CLL cell clearance after 1h. We now demonstrate that residual circulating CLL cells remain sensitive to in vitro ADCP by both rituximab and alemtuzumab. This suggests that despite reduced levels of surface antigen, circulating cells remain sensitive to mAb mediated clearance. Alternative explanations for the limited clearance of circulating CLL cells include: 1. Saturation of the limited cytotoxic capacity of the innate immune effector cells (Kupffer cells and splenic macrophages) compatible with our previously reported data on finite macrophage ADCP capacity (PMID:32556153); 2. Mobilization of CLL cells from lymphoid tissue; and 3. Temporary sequestration of rituximab opsonized CLL cells in tissue (e.g. spleen or lungs), which is unlikely given the observed decrease in lymph node size following initiation of therapy. Ongoing research is testing these hypotheses.

Granulocytes subsets and their divergent functions in host resistance to Mycobacterium tuberculosis - a 'tipping-point' model of disease exacerbation.

Granulocytes are innate immune effector cells with essential functions in host resistance to bacterial infections. I will discuss emerging evidence that during Mycobacterium tuberculosis infection, counter-intuitively, eosinophils are host-protective while neutrophils are host detrimental. Additionally, I will propose a 'tipping-point' model in which neutrophils are an integral part of a feedforward loop driving tuberculosis disease exacerbation.

Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4.

Macrophages are key effector cells of innate immunity and play a critical role in the immune balance of disease pathogenesis, especially in the tumor microenvironment. In previous studies, we showed that FimH, an Escherichia coli adhesion portion, promoted dendritic cell activation. However, the effect of FimH in macrophage polarization has yet to be fully examined. In this study, we investigated the potential effect of FimH on macrophages, as well as the polarization from M2 to M1 macrophages, contributing to the overall antitumor effect. Mouse bone marrow derived macrophages and peritoneal macrophages were generated to test the effect of FimH in vitro. The expression of costimulatory molecules and production of cytokines were analyzed. The effect of FimH in the tumor-associated macrophages was examine in the B16F10-tumor bearing C57BL/6. FimH was found to promote M1 macrophage activation. In addition, FimH polarized M2 macrophages, which were induced by interleukin (IL)-4 and IL-13 into M1 macrophages were dependent on toll-like receptor 4 and myeloid differentiation factor 2. Moreover, FimH reprogramed the tumor-associated macrophage (TAM) into M1 macrophages in B16 melanoma tumor-bearing mice and promoted an inflammatory reaction in the tumor microenvironment (TME). Furthermore, FimH promoted M1 macrophage activation, as well as the reversion of M2 macrophages into M1 macrophages in humans. Finally, FimH treatment was found to enhance the anti-cancer immunity of anti-PD-L1 antibody by the induction of M1 polarization from TAM. This study demonstrated the potential effect of FimH on the activation of macrophages, responsible for the repolarization of M2 macrophages into the M1 phenotype via the TLR4 signaling pathway. Moreover, FimH could also reprogram TAM polarization to the M1 status in the TME, as well as enhance the anti-tumor activity of immune checkpoint blockade.

Upregulation of CD226 on subsets of T cells and NK cells is associated with upregulated adhesion molecules and cytotoxic factors in patients with tuberculosis

Human T cells and natural killer (NK) cells are major effector cells of innate immunity exerting potential immune surveillance against tuberculosis infection. CD226 is an activating receptor playing vital roles in the functions of T cells and NK cells during HIV infection and tumorigenesis. However, CD226 is a less-studied activating receptor during Mycobacterium tuberculosis (Mtb) infection. In this study, we used peripheral blood from tuberculosis patients and healthy donors to evaluate CD226 immunoregulation functions from two independent cohorts using Flow cytometry. Here, we found that a subset of T cells and NK cells that constitutively express CD226 exhibit a distinct phenotype in TB patients. In fact, the proportions of CD226+ and CD226- cell subsets differ between healthy people and tuberculosis patients, and the expression of immune checkpoint molecules (TIGIT, NKG2A) and adhesion molecules (CD2, CD11a) in CD226+ and CD226- subsets of T cells and NK cells exhibits special regulatory roles. Furthermore, CD226+ subsets produced more IFN-γ and CD107a than CD226- subsets in tuberculosis patients. Our results imply that CD226 may be a potential predictor of disease progression and clinical efficacy in tuberculosis by mediating the cytotoxic capacity of T cells and NK cells.

The role of the host-Neutrophil biology.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are myeloid cells packed with lysosomal granules (hence also called granulocytes) that contain a formidable antimicrobial arsenal. They are terminally differentiated cells that play a critical role in acute and chronic inflammation, as well as in the resolution of inflammation and wound healing. Neutrophils express a dense array of surface receptors for multiple ligands, ranging from integrins to support their egress from bone marrow into the circulation and from the circulation into tissues, to cytokine/chemokine receptors that drive their navigation to the site of infection or tissue damage and also prime them for a second stimulus, to pattern recognition receptors and immunoglobulin receptors to facilitate the destruction and removal of infective agents or debridement of damaged tissues. When afferent neutrophil signals are proportionate and coordinated they will phagocytose opsonized and unopsonized bacteria, activating the nicotinamide adenine dinucleotide phosphate oxidase (respiratory burst) to generate reactive oxygen species, which augment the proteolytic destruction of microbes secured within the phagosome. A highly orchestrated process of apoptosis follows, forming membrane-bound substructures that are removed by macrophages. Neutrophils are capable of various other forms of programmed cell death, such as NETosis and pyroptotic cell death, as well as nonprogrammed cell death by necrosis. In recent years, research has revealed that neutrophils are capable of far more subtle cell-cell interactions than previously thought possible. This includes synthesis of various inflammatory mediators and also myeloid cell training within bone marrow, where epigenetic and metabolic signals associated with returning neutrophils that undergo reverse egress from tissues into the vasculature and back to bone marrow program a hyperreactive subset of neutrophils during myelopoiesis that are capable of hypersensitive reactions to microbial aggressors. These characteristics are evident in various neutrophil subsets/subpopulations, creating broad heterogeneity in the behavior and biological repertoire of these seemingly schizophrenic immune cells. Moreover, neutrophils are critical effector cells of adaptive and innate immunity, binding to opsonized bacteria and destroying them by extracellular and intracellular methods. The former creates substantial collateral host tissue damage, as they are less specific than T-cytotoxic cell-killing mechanisms, and in conditions such as peri-implantitis, where plasma cells and neutrophils dominate the immune infiltrate, bone and tissue destruction are rapid and appear relentless. Finally, the role of neutrophils as conduits for periodontal-systemic disease connections and for oxidative damage to act as a causal link between the two has only recently been realized. In this chapter, we attempt to expand on these issues, emphasizing the contributions of European scientists throughout a detailed appraisal of the benefits and side effects of neutrophilic inflammation and immune function.

Abstract 2656: Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation

Abstract Modification of effector function has proven to be an effective modality for optimizing activity and tolerability of therapeutic antibodies. Currently available methods to modulate effector function include the introduction of point mutations in the Fc region and glycan engineering of the antibody. Here we present an alternative and complementary method of tuning effector function utilizing a conjugation-based approach. This methodology uses conjugation of polyethylene glycol (PEG) to native cysteines of an antibody to impair FcγR binding of antibodies to innate immune effector cells. Utilizing maleimide or disulfide conjugation techniques, attenuated effector function can be either permanent or restored over time through a de-conjugation process. Impacts of PEGylation on FcγR binding, signaling, and restoration of function were assessed in vitro and in vivo. As a proof-of-concept, the lead technology was applied to an agonist CD40 antibody, which resulted in significant reductions in systemic cytokine production in hCD40 mice and non-human primates, while demonstrating retained efficacy and improved pharmacokinetics. Additionally, we combined the conjugation technology with glycan engineering and FcγR enhancing point mutations to impart unique effector function profiles to clinical antibodies. This simple, modular approach can be rapidly applied to existing antibodies to reduce immune-driven toxicities, such as infusion reactions, and optimize effector function activity. Citation Format: Philip N. Moquist, Chris I. Leiske, Noah A. Bindman, Xinqun Zhang, Nicole Duncan, Weiping Zeng, Serena W. Wo, Abbie Wong, Clark M. Henderson, Karalyne Crowder, Haley D. Neff-LaFord, Django Sussman, Shyra J. Gardai, Matthew R. Levengood. Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2656.

Antibody Fc-chimerism and effector functions: When IgG takes advantage of IgA.

Recent advances in the development of therapeutic antibodies (Abs) have greatly improved the treatment of otherwise drug-resistant cancers and autoimmune diseases. Antibody activities are mediated by both their Fab and the Fc. However, therapeutic Abs base their protective mechanisms on Fc-mediated effector functions resulting in the activation of innate immune cells by FcRs. Therefore, Fc-bioengineering has been widely used to maximise the efficacy and convenience of therapeutic antibodies. Today, IgG remains the only commercially available therapeutic Abs, at the expense of other isotypes. Indeed, production, sampling, analysis and related in vivo studies are easier to perform with IgG than with IgA due to well-developed tools. However, interest in IgA is growing, despite a shorter serum half-life and a more difficult sampling and purification methods than IgG. Indeed, the paradigm that the effector functions of IgG surpass those of IgA has been experimentally challenged. Firstly, IgA has been shown to bind to its Fc receptor (FcR) on effector cells of innate immunity with greater efficiency than IgG, resulting in more robust IgA-mediated effector functions in vitro and better survival of treated animals. In addition, the two isotypes have been shown to act synergistically. From these results, new therapeutic formats of Abs are currently emerging, in particular chimeric Abs containing two tandemly expressed Fc, one from IgG (Fcγ) and one from IgA (Fcα). By binding both FcγR and FcαR on effector cells, these new chimeras showed improved effector functions in vitro that were translated in vivo. Furthermore, these chimeras retain an IgG-like half-life in the blood, which could improve Ab-based therapies, including in AIDS. This review provides the rationale, based on the biology of IgA and IgG, for the development of Fcγ and Fcα chimeras as therapeutic Abs, offering promising opportunities for HIV-1 infected patients. We will first describe the main features of the IgA- and IgG-specific Fc-mediated signalling pathways and their respective functional differences. We will then summarise the very promising results on Fcγ and Fcα containing chimeras in cancer treatment. Finally, we will discuss the impact of Fcα-Fcγ chimerism in prevention/treatment strategies against infectious diseases such as HIV-1.

Interaction between nanomaterials and the innate immune system across evolution.

Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte- and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.

IMMU-38. MYC DRIVEN IMMUNE SUPPRESSION IN GROUP 3 MEDULLOBLASTOMA

Abstract BACKGROUND The expression of MYC in tumor cells regulates the tumor microenvironment through innate and adaptive immune effector cells and immune regulatory cytokines. MYC has also been shown to regulate the expression of the immune checkpoint gene products CD47 and programmed death-ligand 1 (PD-L1). While PD-L1 inhibits adaptive immune response, CD47 on tumor cells binds to SIRPa on macrophages and acts as a “Don’t eat me” signal. We have previously shown that blocking the CD47-SIRPa pathway significantly decreases tumor burden and increases survival in five etiologically distinct adult and pediatric brain tumor xenograft models, including Group 3 or MYC-driven MB, by activating macrophage-mediated phagocytosis. METHODS We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in ATRT, medulloblastoma (n = 14), and glioblastoma (n = 14). This unbiased approach revealed the preferential activity of Class I HDACi in MYC-driven medulloblastoma mediated by induction of apoptosis, reduction in MYC transcription, and release of pro-inflammatory cytokines in MYC-driven medulloblastoma, with little to no activity in non-MYC-driven medulloblastoma, AT/RT, and glioblastoma in vitro. In addition, we tested the combinatorial effect of directly targeting the MYC/MAX interaction using MYCI-975 and the CD47-SIRPa phagocytosis checkpoint pathway using in-vitro phagocytosis assays and in-vivo xenograft models. In two orthotopic mouse models of MYC-driven medulloblastoma, MYCi975 displayed anti-tumoral effects at the primary site and the metastatic compartment. Furthermore, RNA sequencing revealed NFκB pathway induction as a response to MYC treatment, followed by interferon-gamma (IFN-G) release and cell surface expression of engulfment (“eat-me”) signals (such as calreticulin) and enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC-medulloblastoma and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Allergic Rhinitis: Pathophysiology and Treatment Focusing on Mast Cells.

Allergic rhinitis (AR) is a common rhinopathy that affects up to 30% of the adult population. It is defined as an inflammation of the nasal mucosa, develops in allergic individuals, and is detected mostly by a positive skin-prick test. AR is characterized by a triad of nasal congestion, rhinorrhea, and sneezing. Mast cells (MCs) are innate immune system effector cells that play a pivotal role in innate immunity and modulating adaptive immunity, rendering them as key cells of allergic inflammation and thus of allergic diseases. MCs are typically located in body surfaces exposed to the external environment such as the nasal mucosa. Due to their location in the nasal mucosa, they are in the first line of defense against inhaled substances such as allergens. IgE-dependent activation of MCs in the nasal mucosa following exposure to allergens in a sensitized individual is a cardinal mechanism in the pathophysiology of AR. This review is a comprehensive summary of MCs’ involvement in the development of AR symptoms and how classical AR medications, as well as emerging AR therapies, modulate MCs and MC-derived mediators involved in the development of AR.

Human natural killer cells: Form, function, and development

Human natural killer (NK) cells are innate lymphoid cells that mediate important effector functions in the control of viral infection and malignancy. Their ability to distinguish "self" from "nonself" and lyse virally infected and tumorigenic cells through germline-encoded receptors makes them important players in maintaining human health and a powerful tool for immunotherapeutic applications and fighting disease. This review introduces our current understanding of NK cell biology, including key facets of NK cell differentiation and the acquisition and execution of NK cell effector function. Further, it addresses the clinical relevance of NK cells in both primary immunodeficiency and immunotherapy. It is intended to provide an up-to-date and comprehensive overview of this important and interesting innate immune effector cell subset.