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Contributions of Two Distinct T Cell Subsets (CD4+, CD8+CD60+) to Induction of Specific Memory IgE Responses
Abstracts AB187
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Eosinophil Production of PGD2 in AspirinExacerbated Respiratory Disease
John W. Steinke, PhD, FAAAAI1, Julie Negri, BS2, Mary Grace Baker, MD3, Spencer Payne, MD4, Larry Borish, MD, FAAAAI5, Xin Feng6; 1Asthma and Allergic Disease Center, Carter Center for Immunology Research, University of Virginia, Charlottesville, VA, 2University of Virginia, Charlottesville, VA, 3Yale-New Haven Hospital, New Haven, CT, 4University of Virginia Department of Otolaryngology, Division of Rhinology and Endoscopic Sinus Surgery, Charlottesville, VA, 5Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 6 QiLu Hospital of Shandong University, Shandong, China. RATIONALE: Aspirin-exacerbated respiratory disease (AERD) differs from aspirin tolerant disease in part due to high tissue eosinophilic infiltration and the over-expression of arachidonic acid metabolic pathway components driving secretion of cysteinyl leukotrienes (CysLT) and prostaglandin D2 (PGD2). We had previously shown the capacity of interferon (IFN)-g to drive CysLT expression pathways. We investigated eosinophils as a source for PGD2 production in AERD and the ability of (IFN)-g to drive expression. METHODS: Eosinophils were enriched from tissue and peripheral blood obtained from control, aspirin-tolerant, and AERD subjects. mRNA was extracted and evaluated for expression of hematopoietic PGD synthase (hPGDS). Expression of hPGDS protein was confirmed with western blot and immunohistochemical staining. Cells were stimulated with aspirin and secretion of PGD2 quantified. Finally, CD34+ progenitor cells were isolated and matured into eosinophils in the presence or absence of IFN-g and hPGDS mRNA and PGD2 release measured. RESULTS: Gene expression analysis revealed that eosinophils from AERD tissue and blood display increased levels of hPGDS compared with asthmatic and control samples. Western blot confirmed the increase in hPGDS gene expression translated to increased protein expression. Using immunofluorescence, mast cells and eosinophils from AERD and asthmatic subjects demonstrated hPGDS with higher levels in AERD eosinophils. Incubation of eosinophils from blood and tissue with aspirin stimulated PGD2 release. IFN-g-matured eosinophil progenitors showed enhanced hPGDS expression and increased levels of PGD2 release at baseline and following stimulation with aspirin. CONCLUSIONS: In addition to mast cells, eosinophils represent an important source of PGD2 in AERD at baseline and following exposure to aspirin.
611
Contributions of Two Distinct T Cell Subsets (CD4+, CD8+CD60+) to Induction of Specific Memory IgE Responses
Charles J. Kim, BS1, Bryan McCarthy, BS2, Jonathan I. Silverberg, MD, PhD, MPH3, Seto M. Chice, MS2, Yitzchok M. Norowitz, BS4, Maja Nowakowski, PhD5, Stephan Kohlhoff, MD2,6, Rauno Joks, MD2,7, Tamar A. Smith-Norowitz, PhD2,8, Helen G. Durkin, PhD2,9; 1Center for Allergy and Asthma Research at SUNY Downstate Medical Center, 2Center for Allergy and Asthma Research at SUNY Downstate Medical Center, Brooklyn, NY, 3Department of Dermatology, Northwestern University School of Medicine, Chicago, IL, 4Center for Allergy and Asthma Research at SUNYDownstate Medical Center, 5Department of Pathology, 6Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY, 7SUNY Downstate Medical Center, Brooklyn, NY, 8Department of Pediatrics, SUNY Downstate Medical Center, Brooklyn, NY, 9Department of Pathology at SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: T cells and their cytokines (IL-4) are required for human IgE responses and phosphorylated p38 MAP kinase (p38MAPK) correlates with IL-4. We reported that two distinct T cell subsets (CD4+, CD8+CD60+) are required for induction of human specific memory IgE responses (SmithNorowitz, et al. J. Immunology). We now report the interrelationships between CD4+ and CD8+CD60+ T cells in induction of these responses. METHODS: Blood/serum was obtained from ragweed sensitized humans (n56) and CD4+ and CD8+CD60+ T cell expression of p38MAPK (MESF),
CD4+IL-4+ and CD8+CD60+IL-4+ T cell numbers and IgE levels (IU/ml) were determined (flow cytometery, fluoroenzymeimmunoassay). PBMC (1x106) were cultured for 0-12 days 6 varying concentrations of ragweed antigen and IgE levels in supernatants determined (ng/ml)(ELISA). RESULTS: CD4+ and CD8+CD60+ T cells were ;40% and ;5% of blood lymphocytes, respectively. CD4+ and CD8+ T cell expression of p38MAPK correlated with serum IgE (p50.001, p50.005, respectively). When CD4+ or CD8+CD60+ T cells were depleted before, or anti-IL-4 was included in culture, no specific memory IgE responses were induced; reconstitution of memory IgE responses required bothsubsets. Of the CD4+ T cells only 5% were CD4+IL-4+, whereas of the CD8+CD60+ T cells, >99% were CD8+CD60+IL-4+ (all were p38MAPK+); the individual T cell subsets (;20,000, 50,000 cells, respectively) did not produce sufficient IL-4 for induction of memory IgE responses by PBMC (1 million cells), but together they did. CONCLUSIONS: Both CD4+ and CD8+CD60+ T cell subsets are necessary to produce sufficient IL-4 required for induction of specific memory IgE responses of ragweed sensitized humans.
612
Identification of Functional Peanut-Responsive Tregs in Peanut Allergic Human Blood
David Chiang, MS1, Hugh A. Sampson, MD, FAAAAI2, M. Cecilia Berin, PhD2; 1Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 2Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY. RATIONALE: The presence and suppressive capacity of food-allergen specific regulatory T cells in humans remains poorly understood. METHODS: Blood was obtained from peanut allergic (n515) or control (n56) subjects. PBMCs were evaluated by flow cytometry. CD25+ cells were removed by magnetic depletion or Tregs were selectively depleted by FACS, and cells were stimulated with peanut extract or staphylococcal enterotoxin B (SEB). Measured outcomes included activation (CD154, 6-18hrs), proliferation (Ki67, 5 days), and cytokine secretion (5 days). RESULTS: Stimulation of PBMCs from allergic but not healthy subjects with peanut resulted in upregulation of CD154 on Foxp3+CD25+CD127lowcells. CD25 depletion prior to peanut stimulation abolished the appearance of CD154+ Tregs, indicating that these cells were derived from the CD25+ population at baseline. Removal of Tregs by CD25 or FACS resulted in suppressed proliferative responses to peanut, indicating that Tregs contribute to the proliferating pool of cells after peanut stimulation. CD25 depletion led to significant increases in peanutinduced IFNg, TNFa, IL-21, and IL-6, whereas surprisingly there were significant decreases in peanut-induced Th2 cytokines – IL-5, IL-9, and IL-13. Similar patterns were observed with SEB stimulation. Immune profiling of CD25-depleted PBMCs demonstrated depletion of basophils as well as Tregs. Selective Treg-depletion by FACS led to increased Th1 and Th2 cytokine responses to peanut. CONCLUSIONS: Our data demonstrate the presence of functional peanut-responsive Tregs in peripheral blood of peanut allergics, but not healthy controls. Our data also demonstrate that basophils, but not Tregs, contribute substantially to the production of Th2 cytokines central to the pathogenesis of food allergy.
SUNDAY
610
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Eosinophil Production of PGD2 in AspirinExacerbated Respiratory Disease
John W. Steinke, PhD, FAAAAI1, Julie Negri, BS2, Mary Grace Baker, MD3, Spencer Payne, MD4, Larry Borish, MD, FAAAAI5, Xin Feng6; 1Asthma and Allergic Disease Center, Carter Center for Immunology Research, University of Virginia, Charlottesville, VA, 2University of Virginia, Charlottesville, VA, 3Yale-New Haven Hospital, New Haven, CT, 4University of Virginia Department of Otolaryngology, Division of Rhinology and Endoscopic Sinus Surgery, Charlottesville, VA, 5Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 6 QiLu Hospital of Shandong University, Shandong, China. RATIONALE: Aspirin-exacerbated respiratory disease (AERD) differs from aspirin tolerant disease in part due to high tissue eosinophilic infiltration and the over-expression of arachidonic acid metabolic pathway components driving secretion of cysteinyl leukotrienes (CysLT) and prostaglandin D2 (PGD2). We had previously shown the capacity of interferon (IFN)-g to drive CysLT expression pathways. We investigated eosinophils as a source for PGD2 production in AERD and the ability of (IFN)-g to drive expression. METHODS: Eosinophils were enriched from tissue and peripheral blood obtained from control, aspirin-tolerant, and AERD subjects. mRNA was extracted and evaluated for expression of hematopoietic PGD synthase (hPGDS). Expression of hPGDS protein was confirmed with western blot and immunohistochemical staining. Cells were stimulated with aspirin and secretion of PGD2 quantified. Finally, CD34+ progenitor cells were isolated and matured into eosinophils in the presence or absence of IFN-g and hPGDS mRNA and PGD2 release measured. RESULTS: Gene expression analysis revealed that eosinophils from AERD tissue and blood display increased levels of hPGDS compared with asthmatic and control samples. Western blot confirmed the increase in hPGDS gene expression translated to increased protein expression. Using immunofluorescence, mast cells and eosinophils from AERD and asthmatic subjects demonstrated hPGDS with higher levels in AERD eosinophils. Incubation of eosinophils from blood and tissue with aspirin stimulated PGD2 release. IFN-g-matured eosinophil progenitors showed enhanced hPGDS expression and increased levels of PGD2 release at baseline and following stimulation with aspirin. CONCLUSIONS: In addition to mast cells, eosinophils represent an important source of PGD2 in AERD at baseline and following exposure to aspirin.
611
Contributions of Two Distinct T Cell Subsets (CD4+, CD8+CD60+) to Induction of Specific Memory IgE Responses
Charles J. Kim, BS1, Bryan McCarthy, BS2, Jonathan I. Silverberg, MD, PhD, MPH3, Seto M. Chice, MS2, Yitzchok M. Norowitz, BS4, Maja Nowakowski, PhD5, Stephan Kohlhoff, MD2,6, Rauno Joks, MD2,7, Tamar A. Smith-Norowitz, PhD2,8, Helen G. Durkin, PhD2,9; 1Center for Allergy and Asthma Research at SUNY Downstate Medical Center, 2Center for Allergy and Asthma Research at SUNY Downstate Medical Center, Brooklyn, NY, 3Department of Dermatology, Northwestern University School of Medicine, Chicago, IL, 4Center for Allergy and Asthma Research at SUNYDownstate Medical Center, 5Department of Pathology, 6Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY, 7SUNY Downstate Medical Center, Brooklyn, NY, 8Department of Pediatrics, SUNY Downstate Medical Center, Brooklyn, NY, 9Department of Pathology at SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: T cells and their cytokines (IL-4) are required for human IgE responses and phosphorylated p38 MAP kinase (p38MAPK) correlates with IL-4. We reported that two distinct T cell subsets (CD4+, CD8+CD60+) are required for induction of human specific memory IgE responses (SmithNorowitz, et al. J. Immunology). We now report the interrelationships between CD4+ and CD8+CD60+ T cells in induction of these responses. METHODS: Blood/serum was obtained from ragweed sensitized humans (n56) and CD4+ and CD8+CD60+ T cell expression of p38MAPK (MESF),
CD4+IL-4+ and CD8+CD60+IL-4+ T cell numbers and IgE levels (IU/ml) were determined (flow cytometery, fluoroenzymeimmunoassay). PBMC (1x106) were cultured for 0-12 days 6 varying concentrations of ragweed antigen and IgE levels in supernatants determined (ng/ml)(ELISA). RESULTS: CD4+ and CD8+CD60+ T cells were ;40% and ;5% of blood lymphocytes, respectively. CD4+ and CD8+ T cell expression of p38MAPK correlated with serum IgE (p50.001, p50.005, respectively). When CD4+ or CD8+CD60+ T cells were depleted before, or anti-IL-4 was included in culture, no specific memory IgE responses were induced; reconstitution of memory IgE responses required bothsubsets. Of the CD4+ T cells only 5% were CD4+IL-4+, whereas of the CD8+CD60+ T cells, >99% were CD8+CD60+IL-4+ (all were p38MAPK+); the individual T cell subsets (;20,000, 50,000 cells, respectively) did not produce sufficient IL-4 for induction of memory IgE responses by PBMC (1 million cells), but together they did. CONCLUSIONS: Both CD4+ and CD8+CD60+ T cell subsets are necessary to produce sufficient IL-4 required for induction of specific memory IgE responses of ragweed sensitized humans.
612
Identification of Functional Peanut-Responsive Tregs in Peanut Allergic Human Blood
David Chiang, MS1, Hugh A. Sampson, MD, FAAAAI2, M. Cecilia Berin, PhD2; 1Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 2Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY. RATIONALE: The presence and suppressive capacity of food-allergen specific regulatory T cells in humans remains poorly understood. METHODS: Blood was obtained from peanut allergic (n515) or control (n56) subjects. PBMCs were evaluated by flow cytometry. CD25+ cells were removed by magnetic depletion or Tregs were selectively depleted by FACS, and cells were stimulated with peanut extract or staphylococcal enterotoxin B (SEB). Measured outcomes included activation (CD154, 6-18hrs), proliferation (Ki67, 5 days), and cytokine secretion (5 days). RESULTS: Stimulation of PBMCs from allergic but not healthy subjects with peanut resulted in upregulation of CD154 on Foxp3+CD25+CD127lowcells. CD25 depletion prior to peanut stimulation abolished the appearance of CD154+ Tregs, indicating that these cells were derived from the CD25+ population at baseline. Removal of Tregs by CD25 or FACS resulted in suppressed proliferative responses to peanut, indicating that Tregs contribute to the proliferating pool of cells after peanut stimulation. CD25 depletion led to significant increases in peanutinduced IFNg, TNFa, IL-21, and IL-6, whereas surprisingly there were significant decreases in peanut-induced Th2 cytokines – IL-5, IL-9, and IL-13. Similar patterns were observed with SEB stimulation. Immune profiling of CD25-depleted PBMCs demonstrated depletion of basophils as well as Tregs. Selective Treg-depletion by FACS led to increased Th1 and Th2 cytokine responses to peanut. CONCLUSIONS: Our data demonstrate the presence of functional peanut-responsive Tregs in peripheral blood of peanut allergics, but not healthy controls. Our data also demonstrate that basophils, but not Tregs, contribute substantially to the production of Th2 cytokines central to the pathogenesis of food allergy.
SUNDAY
610