TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells afterStaphylococcus aureusEnterotoxin A Inhalation

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared

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IMMU-45. CUSTOMIZED LIPID NANOPARTICLES DELIVER NUCLEIC ACIDS TO IMMUNE CELLS IN BRAIN TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.475 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii114-ii114

Author(s):

Adam Grippin ◽

Brandon Wummer ◽

Hector Mendez-Gomez ◽

Tyler Wildes ◽

Kyle Dyson ◽

...

Keyword(s):

Brain Tumors ◽

Nucleic Acids ◽

Immune Cells ◽

White Blood Cells ◽

Lipid Nanoparticles ◽

Intracranial Tumor ◽

Innate Immune ◽

Checkpoint Blockade ◽

Normal Brain ◽

Innate Immune Cells

Abstract BACKGROUND Brain tumors are notoriously difficult to treat in part due to their isolation behind the blood brain barrier and their power to suppress antitumor immune responses. We have previously reported cationic liposome formulations capable of delivering immune modulatory nucleic acids to immune cells in various peripheral organs, but there is currently no reliable method to deliver nucleic acids into brain tumors without direct injection into the tumor site. OBJECTIVE Here, we report the development of a customized lipid nanoparticle to deliver immune modulatory nucleic acids to immune cells in brain tumors. APPROACH Cationic liposomes composed of varying lipid combinations were evaluated for their ability to deliver functional mRNA and siRNA to innate immune cells in vitro and in intracranial tumor models. Nucleic acids were labelled with Cy3 to monitor particle distribution in vivo. RESULTS Lipids composed of DOTAP and cholesterol selectively delivered mRNA and siRNA to intracranial GL261 and KR158b tumors. Interestingly, these particles selectively delivered these nucleic acids to CD45+ white blood cells with minimal delivery to CD45- tumor cells or normal brain tissue. Encapsulation of siRNA blocking programmed death ligand 1 (PDL1) significantly reduced PDL1 expression on innate immune cells in brain tumors, with the greatest effects on tumor-associated macrophages. Co-administration of systemic checkpoint blockade with intravenous administration of these lipid nanoparticles bearing PDL1 siRNA enabled systemic and intratumoral checkpoint blockade, leading to 37% long term survivorship in an otherwise fatal intracranial tumor model. CONCLUSIONS Our customized lipid nanoparticles enable potent intratumoral immune modulation via delivery of nucleic acids to immune cells in brain tumors.

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