Innate Immune Function Research Articles

Effect of supplementation of vitamin D3 or vitamin D2 on serum concentrations of free and total 25-hydroxyvitamin D and the expression of genes involved in immune function in peripheral blood mononuclear cells of weaned pigs

ABSTRACT The present study aimed to compare the effects of vitamin D2 and vitamin D3 supplementation on concentrations of total and free 25(OH)D in plasma and the expression of genes involved in the innate immune system in peripheral blood mononuclear cells (PBMC) in weaned pigs. Five groups of pigs (with an initial body weight of around 9 kg) received basal diets supplemented with either 500 (control group), 1000 or 2000 IU vitamin D3/kg diet or 1000 or 2000 IU vitamin D2/kg diet for a period of 4 weeks. Vitamin D supplementation did not influence feed intake, body weight gain, feed conversion ratio, apparent total tract digestibility of calcium and phosphorus, and serum concentrations of calcium, inorganic phosphate and parathyroid hormone. Supplementation of vitamin D3 led to a dose-dependent increase of the concentrations of total and free 25(OH)D in serum. In contrast, pigs supplemented with 1000 or 2000 IU vitamin D2/kg diet did not have higher concentrations of total and free 25(OH)D in serum than the control group. The ratio of free/total 25(OH)D in serum was not influenced by vitamin D3 supplementation, whereas the group supplemented with 2000 IU vitamin D2/kg diet had a higher free/total 25(OH)D ratio than the groups supplemented with 1000 or 2000 IU vitamin D3/kg diet. Genes involved in vitamin D signalling (CYP27B1, VDR), as well as pro-inflammatory and immune regulatory genes (TLR4, TNF, IL1B and TGFB1) and genes encoding porcine protegrins (NPG1, NPG4), proteins belonging to the group of antimicrobial peptides, in PBMC were not different among groups supplemented with vitamin D3 or vitamin D2 and the control group. Therefore, the study indicates that supplementation of vitamin D2 causes much lower levels of total 25(OH)D than supplementation of vitamin D3 and that supplementation of vitamins D2 or D3 at moderate levels does not have an impact on the innate immune function in healthy pigs.

Uncovering the role of the local environment in shaping antiviral immunity of the female reproductive tract

Abstract The female reproductive tract (FRT) is a unique barrier site. It tolerates foreign entities like sperm and the fetus for reproduction but must also be immunologically active enough to control pathogens, like viruses. Millions of women use antibiotics or anti-retrovirals, and these treatments alter the environment of the FRT by changing the microbiota or inhibiting endogenous retroviruses, but the impact of this on FRT immunity and function are understudied. Human cohort data has revealed that the vaginal microbiota profoundly influences risk of virus infection, but the mechanisms for this are not well understood. Endogenous retroviruses (ERV) are necessary for reproduction, as an ERV envelope protein is essential for placental formation, but how these elements shape immunity of the FRT is barely characterized. Preliminarily, I have found that both the microbiota and ERVs shape innate and adaptive immune cell populations and functions in the FRT. Additionally, I found that microbial derived ligands such as LPS are sufficient to induce ERV expression in the uterine epithelium. Therefore, the main hypothesis tested by my project is that the vaginal microbiota induces ERV expressionin the epithelium of the reproductive tract which in turn activates host nucleic acid sensing pathways and interferon production in the epithelium and that this response promotes local antiviral protection. Y.B. was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID; ZIAAI001115-09, ZIAAI001132-07, ZIAAI001133-08). E.S. was supported by the Training Office and OITE of the National Institute of Allergy and Infectious Diseases (Intramural AIDS Research Fellowship).

SARS-CoV-2 Envelope Protein Tolerizes Macrophage Response to Secondary Inflammatory Stimuli

Abstract Exposure of innate immune cells to bacteria or viruses may result in epigenetic rewiring that either enhances or weakens innate immune responses to a secondary challenge. One primary mechanism of innate immune cell function is through toll-like receptors (TLRs) that sense a variety of bacterial and viral molecules and cause immune activation. SARS-CoV-2 Envelope protein (E protein) has been shown to mediate innate immune activation, through engagement of TLR2. In this study, we determined the secretome of chemokines, cytokines and growth factors from monocytes in response to E protein stimulation and compared this response with other viral antigens and TLR agonists. Next, we stimulated monocytes with E protein followed by secondary stimulation with lipopolysaccharide (LPS) on monocyte-derived macrophages (MDM) 1 week later. Reduced expression of CCL3, CCL4, CCL5, CXCL10, TNF-A, and IL-12 upon secondary LPS or autologous E protein stimulation were statistically reflecting a more tolerant MDM state that is imprinted after E protein exposure. Finally, we demonstrated that immunizing neonatal mice with SARS-CoV-2 E protein induced proinflammatory cytokine secretion and lung tissue inflammation pathology and demonstrated a long-term impact on immune cell function and secondary response to LPS. Thus, E protein induces macrophage proinflammatory networks that lead to tissue inflammation, but also induce a more tolerant state after return to a resting state that could impact the ability to respond to secondary infection. Further understanding of the molecular pathways that drive this immune activation and tolerance could identify treatment targets to restore immunity after SARS-CoV-2 infection.

Passive antibody transfer from pregnant women to their fetus are maximized after SARS-CoV-2 vaccination irrespective of prior infection

Abstract Pregnancy is associated with a higher risk of adverse symptoms and outcomes for most infectious diseases, including SARS-CoV-2. These adverse risks are also observed in neonates. Antibodies provide protection against SARS-CoV-2 infection, and have been reported after maternal vaccination, maternal infection, and through passive transfer from mother to fetus in utero. However, it is unclear whether the magnitude or quality of maternally-derived fetal antibodies differs in the context of infection or vaccination. Here, we evaluated 93 paired maternal and neonatal umbilical cord blood plasma samples collected between October 2020 and February 2022 from a birth cohort of pregnant women from the greater New Orleans area. Histories of SARS-CoV-2 infection or vaccination were obtained. Humoral immunity was profiled for the levels of spike-specific antibodies and induction of antiviral humoral immune functions, including neutralization and Fc-mediated innate immune effector functions. We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies from naïve subjects. Importantly, vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. These results show that the most effective passive transfer of functional antibodies against SARS-CoV-2 in utero is achieved through vaccination, highlighting the importance of vaccination in pregnant women. Supported by grants from NIH (NCI U54CA260581-02, NHLBI Grant HL-061007) and USDA (APHIS NBAF NSTP)

Self antigen-driven, undifferentiated memory-phenotype CD4 T lymphocytes can induce mild and systemic inflammation by differentiating into effector and regulatory T cells

Abstract In adaptive immune responses, naïve CD4 +T lymphocytes that have T cell receptors (TCRs) reactive to challenged foreign antigens are activated to differentiate into antigen-specific effector and memory cells. We have recently reported that in steady state, peripheral naïve precursors can also recognize self antigens to spontaneously acquire a memory phenotype (MP) and that such naturally arising “MP cells” can exert innate immune function in host defense. Given the self-reactivity of MP cells, it is likely that the same T lymphocyte population also has the capacity to induce autoimmune and/or inflammatory disease. Here we show that in lymphopenic environment, self antigen-driven MP cells can evoke mild and persistent inflammation in various organs including colon, lungs, kidneys, and liver. The MP cell-driven colitis is dependent on commensal microbiota as well as cytokines IFN-γ and IL-17A while inflammation in the other organs is independent of these factors. On the other hand, we demonstrate using combined single cell RNA sequencing, TCR sequencing, and flow cytometric approaches that MP as compared to naïve CD4 +T lymphocytes more efficiently differentiate into regulatory T (Treg) cells in a TGF-β-dependent fashion, thereby partially ameliorating inflammation. We further provide evidence arguing that these Th1, Th17, and Treg cells are generated from preexisting, undifferentiated T-bet −rather than T-bet +MP subset. Altogether, our results identify self-driven T-bet −MP cells as a multi-potent CD4 +T lymphocyte population that can induce systemic and mild inflammation by differentiating into Th1 and Th17 effector as well as Treg cells. This work was supported by the Japan Society for the Promotion of Science, Astellas Foundation for Research on Metabolic Disorders, Bristol-Myers Squibb, Cell Science Research Foundation, Chemo-Sero-Therapeutic Research Institute, Daiichi Sankyo Foundation of Life Science, GlaxoSmithKline, Gonryo Foundation for the Promotion of Medical Science, G-7 Scholarship Foundation, Harmonic Ito Foundation, Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care, Inamori Foundation, Intelligent Cosmos Foundation, Japan Allergy Foundation, Japan Intractable Diseases Research Foundation, Kanae Foundation for the Promotion of Medical Science, Kobayashi Foundation, Life Science Foundation of Japan, Mitsubishi Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, MSD Life Science Foundation, Nakajima Foundation, Nakayama Foundation for Human Science, Ohyama Health Foundation, Okinaka Memorial Institute for Medical Research, Pharmacodynamics Research Foundation, Senri Life Science Foundation, Senshin Medical Research Foundation, Sumitomo Foundation, Takeda Science Foundation, Ube Industries Foundation, Uehara Memorial Foundation, and Waksman Foundation of Japan. Akihisa Kawajiri was supported by the PhD Scholarship (Kibou Project) from Japanese Society for Immunology.

Effects of Hermetia illucens larvae meal on the Pacific white shrimp (Litopenaeus vannamei) revealed by innate immunity and 16S rRNA gene sequencing analysis

The larvae of the black soldier fly, Hermetia illucens, are now attracting attention and becoming promising sources for aquafeed ingredient due to the nutritious substance. However, the introduction of a novel ingredient into the recipe may have unpredictable effects on the innate immune function and gut bacteria composition of crustaceans. Therefore, the present study aimed to evaluate how dietary black soldier fly larvae meal (BSFLM) affected the antioxidant ability, innate immunity and gut microbiome of shrimp (Litopenaeus vannamei) fed with a practical diet, including the gene expression of Toll and immunodeficiency (IMD) pathways. Six experimental diets were formulated by replacing gradient levels of fish meal (0 %, 10 %, 20 %, 30 %, 40 % and 50 %) based on a commercial shrimp diet. Four replicates of shrimp were fed different diets three times daily for 60 days. Growth performance linearly decreased with increasing BSFLM inclusion. Results of antioxidative enzyme activities and gene expression suggested that low dietary BSFLM levels activated the antioxidant capacity of shrimp, while dietary BSFLM levels up to 100 g/kg may induce oxidative stress and inhibit glutathione peroxidase activity. Although traf6, toll1, dorsal and relish were significantly upregulated in different BSFLM groups, the expression of tak1 was significantly downregulated in groups containing BSFLM, implying the immune susceptibility may be weakened. Gut flora analysis indicated dietary BSFLM altered both beneficial and opportunistic pathogenic bacterial abundance, with low levels of dietary BSFLM increased the abundance of bacteria that may contribute to carbohydrate utilization, while high levels of dietary BSFLM may cause intestinal disease and low intestinal immune response. To conclude, 60–80 g/kg of dietary BSFLM showed no adverse effects on the growth, antioxidant capacity and gut flora of shrimp, which was the adequate level in shrimp diet. While 100 g/kg dietary BSFLM may induce oxidative stress and potentially weaken the innate immunity of shrimp.

Senescent CD8+ T cells acquire NK cell-like innate functions to promote antitumor immunity.

It has been suggested that aging of the immune system (immunosenescence) results in a decline in the acquired immune response, which is associated with an increase in age-related tumorigenesis. T-cell senescence plays a critical role in immunosenescence and is involved in the age-related decline of the immune function, which increases susceptibility to certain cancers. However, it has been shown that CD8+ T cells with the senescent T-cell phenotype acquire an natural killer (NK) cell-like function and are involved in tumor elimination. Therefore, the role of senescent CD8+ T cells in tumor immunity remains to be elucidated. In this study, we investigated the role of senescent CD8+ T cells in tumor immunity. In a murine model of transferred with B16 melanoma, lung metastasis was significantly suppressed in aged mice (age ≥30 weeks) in comparison to young mice (age 6-10 weeks). We evaluated the cytotoxic activity of CD8+ T cells in vitro and found that CD8+ T cells from aged mice activated in vitro exhibited increased cytotoxic activity in comparison to those from young mice. We used Menin-deficient effector T cells as a model for senescent CD8+ T cells and found that cytotoxic activity and the expression of NK receptors were upregulated in Menin-deficient senescent CD8+ T cells. Furthermore, Menin-deficient CD8+ T cells can eliminate tumor cells in an antigen-independent manner. These results suggest that senescent effector CD8+ T cells may contribute to tumor immunity in the elderly by acquiring NK-like innate immune functions, such as antigen-independent cytotoxic activity.

Leukolectin-proteins are synthesized and secreted by lectocytes, a distinct category of fish embryonic mucus cells

Fish perivitelline fluid (PVF) is a vital extra-embryonic compartment. At hatching, PVF-contents dissolve into the hatching fluid (HF). Analysis of Atlantic salmon HF reveals nearly a hundred distinct proteins, most of which were identified by advanced mass-spectrometry. However, one entity with an apparent molecular weight 26 kDa, necessitated identification from its tryptic peptides. Subsequent cloning and sequencing revealed novel leukolectin-proteins. From bioinformatic analysis, leukolectins (LL) belong in the tectonin protein-family, with recognized functions in innate immunity. This study aims to identify LL-expressing cells in diverse fish species, and to characterize the LL-gene in order to predict bio-functions of leukolectins. LL-proteins were detected in HF from several fish species and one invertebrate, using polyclonal LL-specific IgGs. Embryonic LL-immunoreactive cells were numerous in Atlantic salmon, rainbow trout, fewer in Atlantic cod, and rare in Atlantic halibut and Oikopleura dioica. LL-immunoreactive cells were termed lectocytes, which corresponded to peridermal mucuscells stained by PAS, but unstained by eosin. Hence, lectocytes and hatching-gland cells were clearly distinguished. Northern blots revealed two salmon LL-transcripts at mid-embryogenesis. Such transcripts were detected in epithelial cells of the periderm, gills and oral cavity. LL-transcripts predominated in the periderm, while choriolysin-transcripts were dominant in the gills. No co-expression of choriolysins and LL-transcripts was detected. BAC-library screening yielded salmon LL's genestructure with 4 introns, 5 exons, TATA-box, multiple upstream putative transcription-factor bindingsites and polyadenylation site. LL-gene location on chromosome ssa17 was identified in Ssal_v3.1, the 2021version of the salmon genome. In conclusion, larvae from several fish species are outfitted with mucus enriched by LL-proteins. Mucus cells are present in embryos of all fishes, but embryonic lectocyte-numbers are far higher in species with near total larval survival. When (maternal) chorionic first-line immuno-defence is lost at hatching, leukolectin-enriched mucus may provide vital protection for larvae.

Ozone-oxidized black carbon particles change macrophage fate: Crosstalk between necroptosis and macrophage extracellular traps

The impacts of environmental PM 2.5 on public health have become a major concern all over the world. Many studies have shown that PM 2.5 still poses a threat to public health even at very low levels. Physical or chemical reactions occur between primary particles and other components in the environment, which changes the properties of primary particles. Such newly formed particles with changed properties are called secondary particles. Ozone-oxidized black carbon (oBC) is a key part of PM 2.5 and a representative secondary particle. Macrophages extracellular traps (METs) is a means for macrophages to capture and destroy invading pathogens, thereby exercising innate immunity. Necroptosis is a kind of programmed cell death, which is accompanied by the destruction of membrane integrity, thus inducing inflammatory reaction. However, there is no research on the crosstalk mechanism between necroptosis and MET after oBC exposure. In our study, AO/EB staining, SYTOX Green staining, fluorescent probe, qPCR, Western blot, and immunofluorescence were applied. This experiment found that under normal physiological conditions, when macrophages receive external stimuli (such as pathogens; in our experiment: phorbol 12-myristate 13-acetate (PMA)), they will form METs, capture and kill pathogens, thus exerting innate immune function. However, exposure to oBC can cause necroptosis in macrophages, accompanied by increased levels of reactive oxygen species (ROS) and cytosolic calcium ions, as well as the expression disorder of inflammatory factors and chemokines, and prevent the formation of METs, lose the function of capturing and killing pathogens, and weaken the innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibited the formation of METs. This study was the first to explore the crosstalk mechanism between necroptosis and METs after oBC exposure.

Assessment of glucocorticoids, sex steroids, and innate immunity in wild red-eared slider turtles (Trachemys scripta elegans)

When access to resources is limited, organisms must shift energy investment among physiological processes to survive, reproduce, and respond to unpredictable events. The shifting of these limited resources among processes may result in physiological tradeoffs, often mediated by glucocorticoids. We assessed relationships among the physiological processes of immunity, reproduction, and the stress response in wild adult red-eared slider turtles (Trachemys scripta elegans). Red-eared sliders exhibit a multi-clutching reproductive strategy that requires high energetic investment in reproduction at the beginning of the nesting season in females. Males mate in spring and undergo spermatogenesis and mating in late summer/early fall. We expected to observe tradeoffs when investment toward reproductive processes was particularly demanding. To test this, we subjected 123 individuals to a standardized acute stressor and collected blood to measure innate immunocompetence and circulating steroid hormone concentrations. Tradeoffs between female reproduction and immunocompetence occurred early in the nesting season. This high reproductive investment was evident by heightened circulating progesterone and reduced baseline innate immunity. Corticosterone (CORT) was also high during this period, indicating a role in facilitating allocation of energy. Tradeoffs were not as evident in males, though males upregulated innate immune function, baseline CORT, and testosterone prior to fall spermatogenesis and mating. Throughout the entire sampling period, both males and females increased CORT and immunocompetence following the acute standardized stressor. Taken together, we concluded that reproduction requires shifts in energy allocation in during the highest reproductive period for females but all individuals in this population remain able to respond to the standardized stressor even during increased reproductive investment. These findings reinforce the continuing evidence that physiological relationships are context-dependent and resource demands are dynamic across the reproductive season.

1,4-Naphthoquinone-Coated Black Carbon, a Kind of Atmospheric Fine Particulate Matter, Affects Macrophage Fate: New Insights into Crosstalk between Necroptosis and Macrophage Extracellular Traps

1,4-Naphthoquinone-coated BC (1,4 NQ-BC) is an important component of PM2.5 and a representative secondary particle. However, there is no research on the crosstalk mechanism between necroptosis and macrophage extracellular traps (METs) after 1,4 NQ-BC exposure. In this study, we treated RAW264.7 cells with 50, 100, and 200 mg/L 1,4 NQ-BC for 24 h, with 10 μM necrostatin-1 for 24 h, and with 2.5 μM phorbol 12-myristate 13-acetate (PMA) for 3 h. Our experiment revealed that under normal physiological conditions, when macrophages receive external stimuli (such as pathogens; in this experiment, PMA), they will form METs and capture and kill pathogens, thus exerting innate immune function. However, exposure to 1,4 NQ-BC can cause necroptosis in macrophages, accompanied by increased levels of reactive oxygen species (ROS) and cytosolic calcium ions, as well as the expression disorder of inflammatory factors and chemokines, prevent the formation of METs, lead to loss of the function of capturing and killing pathogens, and weaken the innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibited the formation of METs. Our study was the first to explore the crosstalk mechanism between necroptosis and METs. This experiment will enrich the mechanism of macrophage injury caused by 1,4 NQ-BC exposure.

Playing Russian Roulette with Alzheimer's Disease Patients: Do the Cognitive Benefits of Lecanemab Outweigh the Risk of Edema, Stroke and Encephalitis?

The questionable approval of aducanumab and the recent approval of lecanemab (Leqembi; Eisai and Biogen) by the FDA has raised the issue of safety (stroke, meningitis, and encephalitis) over efficacy (slowing of cognitive decline). This communication recounts the important physiological functions of amyloid-β as a barrier protein with unique sealant and anti-pathogenic activities important for maintaining vascular integrity coupled with innate immune functions that prevent encephalitis and meningitis. The approval of a drug that obviates both of these purposive functions increases the risk of hemorrhage, edema and downstream pathogenic outcomes and should be clearly outlined to patients.

Abstract 2925: In vitro characterization of next generation therapeutic antibodies

Abstract Therapeutic antibodies are an established drug class. Checkpoint inhibitors, such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies for cancer indications, and anti-TNF-α monoclonal antibody against autoimmune disorders represent some of most successful therapeutics in the past decade. Over 100 therapeutic targets suitable for antibody therapy have been identified for various disease indications, with several currently in late-stage clinical trials. In addition, there has been an explosion in the various types of therapeutic antibody modalities due to the increasing number of antibody engineering platforms. The new emerging bi-specific T-cell engaging antibodies (BiTEs) along with antibody-drug conjugates (ADCs) have demonstrated superior therapeutic qualities over the traditional monoclonal antibodies. Here, we describe a pipeline to extensively characterize the various antibody therapeutics in vitro utilizing multiple innovative technologies. Assays to determine target expression and binding potency provide a thorough understanding of the target engagement. Furthermore, we present data from an innovative technology platform, the z-Movi, which measures cell avidity to predict invitro effector functions of T-cell engaging therapies, enabling more robust read-outs and could drive the lead selection of said therapies. Several mechanisms of action of different antibodies were investigated and relevant case studies describing the innate immune effector functions and the signaling pathway modulation are presented. Using a unique label-free approach, the xCELLigence, we report on the kinetics of target cell killing mediated by lymphocytes upon recognition of antigen-antibody complexes. Finally, safety assessment studies focusing on on-target/off-tumor effects of antibodies on primary cells were conducted. The cytokine profiling and cell viability assays to assess potential risk due to exaggerated levels of cytokine, reiterate the specificity and safety of the next generation of antibody therapeutics and support Investigational New Drug (IND) applications. In conclusion, we provide a comprehensive report on the in vitro characterization of the advanced antibody therapeutics, showcasing a pipeline for development and functional characterization of various antibody modalities, including bispecific T-cell engagers. This workflow can be used to progress a wide range of engineered antibodies from pre-clinical to the clinical phase of the drug discovery process. NJ and GM contributed equally to the work. Citation Format: Benita Quist, Hamed Janbazacyabar, David Cobeta Lopez, Namrata Jayanth, Gemma Moiset. In vitro characterization of next generation therapeutic antibodies [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 2925.

Abstract 4423: XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses

Abstract XMT-2056 is a systemically administered Immunosynthen STING agonist antibody-drug conjugate (ADC) that targets a novel HER2 epitope and induces complete tumor regressions with a single dose in multiple tumor models. We have previously shown that XMT-2056 delivers its STING agonist payload into tumor cells and FcγRI (CD64)-expressing myeloid cells, activating STING signaling in both cell types, leading to type I interferon (IFN) and anti-tumor innate immune responses. Here, we demonstrate that XMT-2056 exhibits ADCC (antibody-dependent cell-mediated cytotoxicity) function, which synergizes with STING pathway activation and induces potent cancer cell-killing activity in co-cultures of HER2-expressing cancer cells and FcγRIII+ (CD16+) immune cells. We show that both XMT-2056 and HT-19 (the unconjugated parental antibody) retain significant cancer cell-killing activity in an Fc-dependent manner in PBMC co-cultures depleted of FcγRI-expressing myeloid cells. This activity is abrogated by co-depletion of FcγRIII+ immune cells, illustrating the ADCC function of XMT-2056. In this setting, XMT-2056 cancer cell-killing activity was significantly increased compared to HT-19, suggesting that the STING payload contributes to the differential activity observed with XMT-2056 treatment. Indeed, co-treatment of cancer cell and immune cell co-cultures with HT-19 and free STING agonist payload enhanced the anti-tumor responses, although to a lesser extent than XMT-2056, suggesting a synergy between the ADCC function and STING pathway activation. Consistently, XMT-2056 treatment of HER2-expressing cancer cells co-cultured with unstimulated CD56+/FcγRIII+ NK cells induced granzyme b and IFN-γ cytokine production, expression of NK cell activation markers, and cancer cell-killing activity. The ADCC activity of HT-19 was comparable to that of trastuzumab in NK cell co-cultures. Finally, we found that depletion of FcγRI+ cells inhibited the cancer cell-killing activity of XMT-2056 in cancer cell and PBMC co-cultures more substantially compared to depletion of FcγRIII+ cells or CD56+ NK cells, indicating a greater contribution of myeloid cells to the XMT-2056 mechanism of action in this setting. Notably, XMT-2056 was capable of engaging both FcγRI+ myeloid cells and FcγRIII+ NK cells, activating both STING-mediated innate immune responses and ADCC function in triple cultures with HER2-expressing cancer cells. Collectively, our data reveal a synergy between ADCC function and STING pathway induction both mediated by XMT-2056, which enhances the cancer cell-killing activity of FcγRIII+ cells. This additional mechanism of action of XMT-2056 can potentially impact the overall anti-tumor immune responses in tumors infiltrated by FcγRIII+ cells. Citation Format: Jahna Soomer-James, Marc Damelin, Naniye Malli. XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses. [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 4423.

191 Treatment with GM-CSF Reverses TBI-Induced Immune Suppression in a Combined Injury Model

INTRODUCTION: Traumatic brain injury (TBI) is a potentially devastating injury for which there are no pharmacotherapies. Frequently, TBI is accompanied by injuries outside the CNS, and we have previously shown that TBI with concurrent extra-cranial injury reliably leads to post-injury immunosuppression, which puts patients at risk for infection and ultimately a poor outcome. To reverse post-injury immune suppression, we have proposed the use of the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF). METHODS: To induce post-injury immunosuppression, we used a previously described model. 28 day old rats were injured via controlled cortical impact and then had 25% blood volume removed. Following injury, animals were treated with vehicle, 5 μg/kg, or 50 μg/kg GM-CSF daily for 7 days. Blood was obtained pre-injury, and on 1, 3, and 7 days post-injury. Brain and spleen were harvested at 7 days post-injury. Adaptive and innate immune function (measured by ex vivo cytokine production in response to an immunostimulant) were assessed in blood and spleen. Flow cytometry was performed on blood and spleen samples. Cytokine levels at the lesion and penumbra were measured by ELISA. RESULTS: As shown previously, injury induced reduction of both innate and adaptive immune function (approximately 50% decrease), which was reversed with GM-CSF. Treatment with GM-CSF significantly expanded the percentage of monocytes. In spleen, injury was associated with an increase in percentage of CD4 cells expressing programmed death-1 (PD-1), and this was also reversed with GM-CSF. ELISA for cytokines in brain samples from lesion and penumbra from GM-CSF treated animals were not significantly different from vehicle. CONCLUSIONS: These data add to a body of literature suggesting that severe trauma leads to immune suppression. Our findings suggest that treatment with GM-CSF may normalize post-injury immune response.

A new frontier for fat: dietary palmitic acid induces innate immune memory.

Dietary saturated fats have recently been appreciated for their ability to modify innate immune cell function, including monocytes, macrophages, and neutrophils. Many dietary saturated fatty acids (SFAs) embark on a unique pathway through the lymphatics following digestion, and this makes them intriguing candidates for inflammatory regulation during homeostasis and disease. Specifically, palmitic acid (PA) and diets enriched in PA have recently been implicated in driving innate immune memory in mice. PA has been shown to induce long-lasting hyper-inflammatory capacity against secondary microbial stimuli in vitro and in vivo, and PA-enriched diets alter the developmental trajectory of stem cell progenitors in the bone marrow. Perhaps the most relevant finding is the ability of exogenous PA to enhance clearance of fungal and bacterial burdens in mice; however, the same PA treatment enhances endotoxemia severity and mortality. Westernized countries are becoming increasingly dependent on SFA-enriched diets, and a deeper understanding of SFA regulation of innate immune memory is imperative in this pandemic era.

Acute/chronic exposure to bisphenol A induced immunotoxicity in zebrafish and its potential association with pancreatic cancer risk

Previous studies have confirmed that bisphenol A (BPA) induced immune toxicity and affected diseases, however, the underlying mechanism remains unknown. In the present study, zebrafish was employed as the model to assess the immunotoxicity and the potential disease risk of BPA exposure. Upon BPA exposure, a series of abnormalities were found, which included the increased oxidative stress, damaged innate and adaptive immune functions and the elevated insulin and blood glucose levels. According to the target prediction and RNA sequencing data of BPA, the differential expression genes were found enriched in immune- and pancreatic cancer-related pathway and process, and the potential role of stat3 in the regulation of these processes was revealed. The key immune- and pancreatic cancer-related genes were selected for further confirmation by RT-qPCR. Based on the changes in the expression levels of these genes, our hypothesis that BPA induced the occurrence of pancreatic cancer by modulating immune responses was further evidenced. Deeper mechanism was further disclosed by molecular dock simulation and survival analysis of key genes, proving that BPA stably bound to STAT3 and IL10 and STAT3 may serve as the target of BPA-inducing pancreatic cancer. These results are of great significance in deepening the molecular mechanism of immunotoxicity induced by BPA and our understanding of the risk assessment of contaminants.

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

Regulation of the Innate Immune System as a Therapeutic Approach to Supporting Respiratory Function in ALS

Amyotrophic lateral sclerosis (ALS) is a clinical diagnosis used to define a neurodegenerative process that involves progressive loss of voluntary muscle function and leads to death within 2-5 years after diagnosis, in most cases because of respiratory function failure. Respiratory vital capacity (VC) measurements are reproducible and strong predictors of survival. To understand the role of the innate immune response in progressive VC loss we evaluated ALS clinical trial and biomarker results from a 6-month phase 2 study of NP001, a regulator of innate immune function. All ALS baseline values were similar between treated and controls except for those > 65 years old who were excluded from analysis. Treated patients with plasma CRP ≥ 1.13 mg/L (high CRP) showed a 64% slower rate of VC decline compared with placebo and those with plasma CRP < 1.13 mg/L (low CRP) who showed no response. High CRP patients showed no age associated loss of VC whereas low CRP patients showed an age dependent loss of VC function. Plasma levels of serum amyloid A (SAA) were similarly elevated in high CRP patients consistent with ongoing innate immune activation. Plasma TGFB1 in high CRP treated patients was 95% higher than placebo at 6-months, confirming the activation and release of this anti-inflammatory factor by the innate immune alpha 2 macroglobulin (A2M) system. This report is the first to link a biomarker confirmed regulation of the innate immune system with a therapeutic approach for controlling VC loss in ALS patients.

APOE and immunity: Research highlights.

At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.