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Immune Differences in Asthmatic Children Using Mass Cytometry
Abstracts AB375
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
LATE-BREAKING ABSTRACTS PRESENTED AT SCIENTIFIC SESSIONS AAAAI ANNUAL MEETING MARCH 3-6, 2017 The following abstracts were accepted for presentation after the deadline for the abstract supplement
L1
Airway Exosomes from Asthmatics Differential T Helper Responses
Modulate
Dr Jessy Deshane, PhD, Mr Kenneth Hough, BS, Mr Landon Wilson, BS, Mr Marion Spell, BS, Prof Jennifer L. Trevor, MD, Dr Diptiman Chanda, PhD, Mr Jose Rodriguez Dager, BS, Dr Yong Wang, PhD, Dr Stephen Barnes, PhD, Dr Veena Antony, MD, Dr Chad Steele, PhD, Dr Mark Dransfield, MD, Dr Steven Duncan, MD, Dr Jeevan Prasain, PhD, and Victor Thannickal, MD; University of Alabama at Birmingham, Birmingham, AL. RATIONALE: Recent studies have illustrated a role for exosomes (nanosized membrane vesicles) in the pathogenesis of various diseases. We hypothesize the potential for airway exosomes to mediate CD4+T cell activation and polarization. Furthermore, we characterize unique lipid compositions of airway exosomes from asthmatics and healthy controls. METHODS: Exosomes were isolated from the bronchoalveolar lavage fluid (BALF) of healthy (n59) or asthmatic (n511) subjects. Exosomes were then characterized by transmission electron microscopy, Nanosight nanoparticle tracking, flow cytometry, and the AMNIS ImageStream. Proliferation and polarization of CD4+T lymphocytes were assessed by coculturing exosomes with purified autologous T cells. Lipids were extracted from exosomes using methanol:chloroform (2:1) with 5mM ammonium acetate. Untargeted lipidomics was performed on the exosomes using a SCIEX 5600 TripleTof mass spectrometer in both positive and negative modes, and SWATH analysis was performed using a mass evaluation range of 200-1200 m/z. RESULTS: Airway exosomes isolated from mild asthmatics displayed a heterogeneous population of particles varying in size and granularity. Flow cytometry analyses demonstrated higher expression of HLA-DR, CD54, and CD36 in exosomes from asthmatics. Airway exosomes from asthmatics promoted CD4+T lymphocyte proliferation and enhanced Th2 and Th17 polarization. SWATH lipidomics analysis revealed significantly higher levels of phosphatidylcholines and sphingomyelin in airway exosomes from asthmatics. CONCLUSIONS: Unique lipid species that are known to play a role in inflammation are significantly different in exosomes isolated from asthmatics. Furthermore, airway exosomes from asthmatics can enhance Th2 and Th17 polarization, suggesting a novel role in asthma pathogenesis.
L2
Prostaglindin I2 Orchestrates Regulatory and Effector T Cell-Mediated Autoimmunity
Melissa H. Bloodworth1, Kelli L. Boyd1, Lisa M. Rogers2, David M. Aronoff2, and Stokes Peebles, Jr, MD, FAAAAI3; 1Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN, 2 Division of Infectious Diseases, Vanderbilt University, Nashville, TN, 3 Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University, Nashville, TN. RATIONALE: Tregs maintain tolerance and prevent autoimmune disease by inhibiting T cell activation. Prostacyclin I2 (PGI2) signaling through the I prostanoid (IP) receptor inhibits allergic airway inflammation, but it remains unknown whether PGI2 regulates Tregs and Treg-mediated
inflammation. We investigated the role of PGI2 on autoimmune manifestations and on Treg Foxp3 expression, suppressive function, and polarization. METHODS: Whole animal histopathology was performed on aged WT and IP KO mice. Splenic T cells from WTand IP KO mice were enriched by magnetic selection and subsequently purified by FACS. For Treg suppression assays, Treg and CFSE-stained Teff were stimulated with anti-CD3 and anti-CD28. Induced Tregs (iTregs) were generated from CD4+ T cells by treating with immobilized anti-CD3 in the presence of IL-2 and TGF-b. RESULTS: PGI2 protected against autoimmune manifestations including splenomegaly, perivascular-bronchiolar lymphocytic cuffing, and epicarditis that developed in aged IP KO mice. PGI2 increased Treg Foxp3 expression, suppressive function, and polarization. PGI2 also increased Teff proliferation and susceptibility to Treg-mediated suppression. CONCLUSIONS: PGI2 promotes Treg stability, suppressive function, and polarization. PGI2 simultaneously promotes Teff proliferation and susceptibility to Treg- mediated suppression. There is extensive interest in the application of Tregs in transplantation, autoimmune diseases, and allergy. PGI2 protects against autoimmunity and may represent a novel treatment strategy for Treg-mediated diseases.
L3
Immune Differences in Asthmatic Children Using Mass Cytometry
Ms Tara Paglino1, Mary Prunicki1, Xiaoying Zhou1, and Dr Kari C Nadeau, MD, PhD, FAAAAI2; 1Stanford, Stanford, 2Stanford University, Stanford, CA. RATIONALE: Our objective is to identify immune system differences between asthmatic and non-asthmatic children. Specifically, we are interested in T cell subsets, including na€ıve and memory cells. Increasing our understanding of asthma at the cellular level may facilitate novel treatment strategies. METHODS: Current asthmatic (n520) and non-asthmatic (n520) children were recruited from Fresno, CA. Asthma diagnosis was determined by current NHLBI criteria. PBMCs were stored in liquid nitrogen. Cells were thawed, stimulated for 4 hours using PMA-ION and stained for cell surface markers, intracellular markers, transcription factors and cytokines. Single cell mass cytometry was then used to collect data on 500,000 to 1,000,000 single cells per subject. RESULTS: There was a difference in the number of memory T cell subsets when comparing asthmatics versus non-asthmatics using t-tests, with CD8+ central memory T cells being reduced in asthmatics (p<0. 002), but there were not significant differences between various basic cell types (Monocytes, B cells, T cells, TCRgd). However, when applying Elastic Net Regression Analysis with fivefold cross-validation, there were 4 relevant features that were selected with misclassification error50.22. The frequency of CD8+ terminally differentiated effector memory CCR5+ cells positively predicted asthma. The frequency of CD8+ central memory CD226+ cells, CD8+central memory cells CD49d+cells and CD8+ central memory cells were negative predictors of asthma. CONCLUSIONS: There are differences in T cell subsets of asthmatic versus non-asthmatic children that can be detected using mass cytometry and sophisticated computational modeling, such as elastic net regression.
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
LATE-BREAKING ABSTRACTS PRESENTED AT SCIENTIFIC SESSIONS AAAAI ANNUAL MEETING MARCH 3-6, 2017 The following abstracts were accepted for presentation after the deadline for the abstract supplement
L1
Airway Exosomes from Asthmatics Differential T Helper Responses
Modulate
Dr Jessy Deshane, PhD, Mr Kenneth Hough, BS, Mr Landon Wilson, BS, Mr Marion Spell, BS, Prof Jennifer L. Trevor, MD, Dr Diptiman Chanda, PhD, Mr Jose Rodriguez Dager, BS, Dr Yong Wang, PhD, Dr Stephen Barnes, PhD, Dr Veena Antony, MD, Dr Chad Steele, PhD, Dr Mark Dransfield, MD, Dr Steven Duncan, MD, Dr Jeevan Prasain, PhD, and Victor Thannickal, MD; University of Alabama at Birmingham, Birmingham, AL. RATIONALE: Recent studies have illustrated a role for exosomes (nanosized membrane vesicles) in the pathogenesis of various diseases. We hypothesize the potential for airway exosomes to mediate CD4+T cell activation and polarization. Furthermore, we characterize unique lipid compositions of airway exosomes from asthmatics and healthy controls. METHODS: Exosomes were isolated from the bronchoalveolar lavage fluid (BALF) of healthy (n59) or asthmatic (n511) subjects. Exosomes were then characterized by transmission electron microscopy, Nanosight nanoparticle tracking, flow cytometry, and the AMNIS ImageStream. Proliferation and polarization of CD4+T lymphocytes were assessed by coculturing exosomes with purified autologous T cells. Lipids were extracted from exosomes using methanol:chloroform (2:1) with 5mM ammonium acetate. Untargeted lipidomics was performed on the exosomes using a SCIEX 5600 TripleTof mass spectrometer in both positive and negative modes, and SWATH analysis was performed using a mass evaluation range of 200-1200 m/z. RESULTS: Airway exosomes isolated from mild asthmatics displayed a heterogeneous population of particles varying in size and granularity. Flow cytometry analyses demonstrated higher expression of HLA-DR, CD54, and CD36 in exosomes from asthmatics. Airway exosomes from asthmatics promoted CD4+T lymphocyte proliferation and enhanced Th2 and Th17 polarization. SWATH lipidomics analysis revealed significantly higher levels of phosphatidylcholines and sphingomyelin in airway exosomes from asthmatics. CONCLUSIONS: Unique lipid species that are known to play a role in inflammation are significantly different in exosomes isolated from asthmatics. Furthermore, airway exosomes from asthmatics can enhance Th2 and Th17 polarization, suggesting a novel role in asthma pathogenesis.
L2
Prostaglindin I2 Orchestrates Regulatory and Effector T Cell-Mediated Autoimmunity
Melissa H. Bloodworth1, Kelli L. Boyd1, Lisa M. Rogers2, David M. Aronoff2, and Stokes Peebles, Jr, MD, FAAAAI3; 1Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN, 2 Division of Infectious Diseases, Vanderbilt University, Nashville, TN, 3 Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University, Nashville, TN. RATIONALE: Tregs maintain tolerance and prevent autoimmune disease by inhibiting T cell activation. Prostacyclin I2 (PGI2) signaling through the I prostanoid (IP) receptor inhibits allergic airway inflammation, but it remains unknown whether PGI2 regulates Tregs and Treg-mediated
inflammation. We investigated the role of PGI2 on autoimmune manifestations and on Treg Foxp3 expression, suppressive function, and polarization. METHODS: Whole animal histopathology was performed on aged WT and IP KO mice. Splenic T cells from WTand IP KO mice were enriched by magnetic selection and subsequently purified by FACS. For Treg suppression assays, Treg and CFSE-stained Teff were stimulated with anti-CD3 and anti-CD28. Induced Tregs (iTregs) were generated from CD4+ T cells by treating with immobilized anti-CD3 in the presence of IL-2 and TGF-b. RESULTS: PGI2 protected against autoimmune manifestations including splenomegaly, perivascular-bronchiolar lymphocytic cuffing, and epicarditis that developed in aged IP KO mice. PGI2 increased Treg Foxp3 expression, suppressive function, and polarization. PGI2 also increased Teff proliferation and susceptibility to Treg-mediated suppression. CONCLUSIONS: PGI2 promotes Treg stability, suppressive function, and polarization. PGI2 simultaneously promotes Teff proliferation and susceptibility to Treg- mediated suppression. There is extensive interest in the application of Tregs in transplantation, autoimmune diseases, and allergy. PGI2 protects against autoimmunity and may represent a novel treatment strategy for Treg-mediated diseases.
L3
Immune Differences in Asthmatic Children Using Mass Cytometry
Ms Tara Paglino1, Mary Prunicki1, Xiaoying Zhou1, and Dr Kari C Nadeau, MD, PhD, FAAAAI2; 1Stanford, Stanford, 2Stanford University, Stanford, CA. RATIONALE: Our objective is to identify immune system differences between asthmatic and non-asthmatic children. Specifically, we are interested in T cell subsets, including na€ıve and memory cells. Increasing our understanding of asthma at the cellular level may facilitate novel treatment strategies. METHODS: Current asthmatic (n520) and non-asthmatic (n520) children were recruited from Fresno, CA. Asthma diagnosis was determined by current NHLBI criteria. PBMCs were stored in liquid nitrogen. Cells were thawed, stimulated for 4 hours using PMA-ION and stained for cell surface markers, intracellular markers, transcription factors and cytokines. Single cell mass cytometry was then used to collect data on 500,000 to 1,000,000 single cells per subject. RESULTS: There was a difference in the number of memory T cell subsets when comparing asthmatics versus non-asthmatics using t-tests, with CD8+ central memory T cells being reduced in asthmatics (p<0. 002), but there were not significant differences between various basic cell types (Monocytes, B cells, T cells, TCRgd). However, when applying Elastic Net Regression Analysis with fivefold cross-validation, there were 4 relevant features that were selected with misclassification error50.22. The frequency of CD8+ terminally differentiated effector memory CCR5+ cells positively predicted asthma. The frequency of CD8+ central memory CD226+ cells, CD8+central memory cells CD49d+cells and CD8+ central memory cells were negative predictors of asthma. CONCLUSIONS: There are differences in T cell subsets of asthmatic versus non-asthmatic children that can be detected using mass cytometry and sophisticated computational modeling, such as elastic net regression.