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Expansion Of Circulating T Follicular Helper Cells In CVID Patients With Autoimmune Cytopenias
AB162 Abstracts
562
SUNDAY
Expansion Of Circulating T Follicular Helper Cells In CVID Patients With Autoimmune Cytopenias Dr. Neil D. Romberg, MD1, Dr. Ida Hsu, MD1, Dr. Christina C. Price, MD1, Dr. Charlotte Cunningham-Rundles, MD, PhD, FAAAAI2, Dr. Eric Meffre, PhD1; 1Yale University School of Medicine, New Haven, CT, 2Mt. Sinai Medical Center, New York, NY. RATIONALE: Peripherally circulating T cells, resembling germinal center T follicular helper cells, have been proposed as a biomarker in rheumatologic diseases. We have examined the peripheral blood of CVID patients with and without autoimmune cytopenias for the presence of these cells. METHODS: We enumerated circulating CD4+CXCR5+PD-1hi T cells in the blood of CVID patients and healthy donors by flow cytometry. When present, we compared this cell population to classical T follicular helper cells from the tonsils of healthy donors by measuring the expression of characteristic co-activation markers and the transcription factor BCL6. For each patient we correlated the frequency of CD4+CXCR5+PD-1hiT cells with other laboratory and clinical features relevant to CVID. RESULTS: Many CVID patients displayed abundant circulating populations of CD4+CXCR5+PD-1hi T cells that expressed co-activation molecules (ICOS, CD40L) and the transcription factor BCL6. The frequency of circulating CD4+CXCR5+PD-1hi T cells was linearly related to the the frequency of CD21-/lo B cells (R250.62, p<0.0001) and inversely related to the frequency of FOXP3+ T regulatory cells (R250.59, p<0.0001) in peripheral blood. An elevated frequency of circulating T follicular helper-like cells in CVID patients accurately predicted the presence of comorbid autoimmune cytopenias. CONCLUSIONS: Circulating CD4+CXCR5+PD-1hi T cells in CVID patients resemble conventional T follicular helper-like cells and may be a useful biomarker of CVID related autoimmunity.
563
Treatment Of Murine Chronic Granulomatous Disease (CGD) With The PPARg Agonist Pioglitazone Enhances Phagocyte Mitochondrial Reactive Oxygen Species (ROS) Production and Antimicrobial Responses Dr. Donna Bratton, MD, Dr. S. Courtney Frasch, PhD, Dr. Kenneth Malcolm, PhD, Dr. Ruby Fernandez-Boyanapalli, PhD; National Jewish Health, Denver, CO. RATIONALE: PPARg agonists enhance mitochondrial biogenesis and ROS production via ‘‘starvation signaling.’’ Mitochondrial ROS contribute to bactericidal activity of phagocytes. It was hypothesized that PPARg agonist treatment of CGD mice would enhance phagocyte mitochondrial ROS production and microbicidal activity. METHODS: Following pioglitazone treatment (5 days), blood and exudate neutrophils and monocytes/macrophages from wild type (WT) and gp91phox2/2 (CGD) mice were characterized for ROS production and killing of Staphylococcus aureus(SA). RESULTS: Blood neutrophils and monocytes from WT and CGD mice treated with pioglitazone (or vehicle) were tested for ROS production by dihydrorhodamine fluorescence following ex vivo PMA stimulation. Pioglitazone (but not vehicle) treatment of CGD mice resulted in a subpopulation of neutrophils (30%) and monocytes (38%) that produced ROS to levels comparable with WT phagocytes. Similar results were demonstrated for exudate phagocytes lavaged from zymosan-inflamed peritonea. Pretreatment of phagocytes from both genotypes and treatment groups with the inhibitor diphenyleneiodonium ablated stimulated ROS production indicating an alternative flavochrome(s). Further characterization of ROS from the phagocytes of pioglitazone-treated CGD mice showed that superoxide was produced (cytochrome c reduction inhibitable with superoxide dismutase), and the source was mitochondria. Phagocytes from pioglitazone-treated CGD mice showed significantly enhanced killing of SA in vitro: 44% of WT killing (vs. 11% for vehicle-treated). In vivo killing was also enhanced: 24h SA recovery following peritoneal injection of
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
2x10e7 CFU was 4.2x10e5 for WT and 2.6x10e6 in pioglitazone-treated CGD mice (compared to 4.2x10e7 with vehicle treatment). CONCLUSIONS: PPARg agonism significantly enhances mitochondrial ROS production and antimicrobial responses in CGD phagocytes warranting further investigation.
564
Patient Specific Targeted Gene Therapy In The Treatment Of X-Linked Hyper-IgM Syndrome Caroline Y. Kuo, MD1, Alok Joglekar, PhD2, Donald B. Kohn, MD3; 1 Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, 2Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, 3Department of Pediatrics and Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles, Los Angeles, CA. RATIONALE: X-linked hyper-IgM Syndrome (XHIM) is a primary immunodeficiency with absent IgG, IgA, IgE and normal/elevated IgM due to defective CD40 ligand (CD40L). Although stem cell transplantation is curative, it is frequently complicated by significant risks. Previous gene therapy trials with a constitutively expressed CD40L transgene in mice resulted in lymphoproliferation. An alternative approach is targeted gene repair with TAL effector nucleases (TALENs), which target specific DNA sequences and create double-strand breaks (DSBs), combined with homologous donor sequences serving as repair templates to allow homology directed repair of DSBs and physiologic expression of CD40L. METHODS: TALEN pairs are created following a previously published protocol. After electroporation of K562 cells with TALEN plasmids, allelic disruption is quantified by a surveyor endonuclease assay (Cel-1). Donor molecules containing the correct DNA sequence are introduced with TALEN plasmids into hematopoietic cells from CD40L deficient patients to assess for restored CD40L expression. RESULTS: T cells were isolated from an XHIM patient, transformed with HTLV-1, and has been in continuous culture for nine months. This patient was found to have a spice site mutation in the first base pair of intron 3 and three TALEN pairs targeting this location have been assembled. Preliminary studies using TALENs targeting a different locus of the CD40L gene have demonstrated allelic disruption in up to 31% and targeted gene insertion of GFP in up to 10% of K562 cells. CONCLUSIONS: This approach allows site-specific correction and physiologic expression of the endogenous CD40L gene to safely provide permanent immune reconstitution.
562
SUNDAY
Expansion Of Circulating T Follicular Helper Cells In CVID Patients With Autoimmune Cytopenias Dr. Neil D. Romberg, MD1, Dr. Ida Hsu, MD1, Dr. Christina C. Price, MD1, Dr. Charlotte Cunningham-Rundles, MD, PhD, FAAAAI2, Dr. Eric Meffre, PhD1; 1Yale University School of Medicine, New Haven, CT, 2Mt. Sinai Medical Center, New York, NY. RATIONALE: Peripherally circulating T cells, resembling germinal center T follicular helper cells, have been proposed as a biomarker in rheumatologic diseases. We have examined the peripheral blood of CVID patients with and without autoimmune cytopenias for the presence of these cells. METHODS: We enumerated circulating CD4+CXCR5+PD-1hi T cells in the blood of CVID patients and healthy donors by flow cytometry. When present, we compared this cell population to classical T follicular helper cells from the tonsils of healthy donors by measuring the expression of characteristic co-activation markers and the transcription factor BCL6. For each patient we correlated the frequency of CD4+CXCR5+PD-1hiT cells with other laboratory and clinical features relevant to CVID. RESULTS: Many CVID patients displayed abundant circulating populations of CD4+CXCR5+PD-1hi T cells that expressed co-activation molecules (ICOS, CD40L) and the transcription factor BCL6. The frequency of circulating CD4+CXCR5+PD-1hi T cells was linearly related to the the frequency of CD21-/lo B cells (R250.62, p<0.0001) and inversely related to the frequency of FOXP3+ T regulatory cells (R250.59, p<0.0001) in peripheral blood. An elevated frequency of circulating T follicular helper-like cells in CVID patients accurately predicted the presence of comorbid autoimmune cytopenias. CONCLUSIONS: Circulating CD4+CXCR5+PD-1hi T cells in CVID patients resemble conventional T follicular helper-like cells and may be a useful biomarker of CVID related autoimmunity.
563
Treatment Of Murine Chronic Granulomatous Disease (CGD) With The PPARg Agonist Pioglitazone Enhances Phagocyte Mitochondrial Reactive Oxygen Species (ROS) Production and Antimicrobial Responses Dr. Donna Bratton, MD, Dr. S. Courtney Frasch, PhD, Dr. Kenneth Malcolm, PhD, Dr. Ruby Fernandez-Boyanapalli, PhD; National Jewish Health, Denver, CO. RATIONALE: PPARg agonists enhance mitochondrial biogenesis and ROS production via ‘‘starvation signaling.’’ Mitochondrial ROS contribute to bactericidal activity of phagocytes. It was hypothesized that PPARg agonist treatment of CGD mice would enhance phagocyte mitochondrial ROS production and microbicidal activity. METHODS: Following pioglitazone treatment (5 days), blood and exudate neutrophils and monocytes/macrophages from wild type (WT) and gp91phox2/2 (CGD) mice were characterized for ROS production and killing of Staphylococcus aureus(SA). RESULTS: Blood neutrophils and monocytes from WT and CGD mice treated with pioglitazone (or vehicle) were tested for ROS production by dihydrorhodamine fluorescence following ex vivo PMA stimulation. Pioglitazone (but not vehicle) treatment of CGD mice resulted in a subpopulation of neutrophils (30%) and monocytes (38%) that produced ROS to levels comparable with WT phagocytes. Similar results were demonstrated for exudate phagocytes lavaged from zymosan-inflamed peritonea. Pretreatment of phagocytes from both genotypes and treatment groups with the inhibitor diphenyleneiodonium ablated stimulated ROS production indicating an alternative flavochrome(s). Further characterization of ROS from the phagocytes of pioglitazone-treated CGD mice showed that superoxide was produced (cytochrome c reduction inhibitable with superoxide dismutase), and the source was mitochondria. Phagocytes from pioglitazone-treated CGD mice showed significantly enhanced killing of SA in vitro: 44% of WT killing (vs. 11% for vehicle-treated). In vivo killing was also enhanced: 24h SA recovery following peritoneal injection of
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
2x10e7 CFU was 4.2x10e5 for WT and 2.6x10e6 in pioglitazone-treated CGD mice (compared to 4.2x10e7 with vehicle treatment). CONCLUSIONS: PPARg agonism significantly enhances mitochondrial ROS production and antimicrobial responses in CGD phagocytes warranting further investigation.
564
Patient Specific Targeted Gene Therapy In The Treatment Of X-Linked Hyper-IgM Syndrome Caroline Y. Kuo, MD1, Alok Joglekar, PhD2, Donald B. Kohn, MD3; 1 Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, 2Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, 3Department of Pediatrics and Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles, Los Angeles, CA. RATIONALE: X-linked hyper-IgM Syndrome (XHIM) is a primary immunodeficiency with absent IgG, IgA, IgE and normal/elevated IgM due to defective CD40 ligand (CD40L). Although stem cell transplantation is curative, it is frequently complicated by significant risks. Previous gene therapy trials with a constitutively expressed CD40L transgene in mice resulted in lymphoproliferation. An alternative approach is targeted gene repair with TAL effector nucleases (TALENs), which target specific DNA sequences and create double-strand breaks (DSBs), combined with homologous donor sequences serving as repair templates to allow homology directed repair of DSBs and physiologic expression of CD40L. METHODS: TALEN pairs are created following a previously published protocol. After electroporation of K562 cells with TALEN plasmids, allelic disruption is quantified by a surveyor endonuclease assay (Cel-1). Donor molecules containing the correct DNA sequence are introduced with TALEN plasmids into hematopoietic cells from CD40L deficient patients to assess for restored CD40L expression. RESULTS: T cells were isolated from an XHIM patient, transformed with HTLV-1, and has been in continuous culture for nine months. This patient was found to have a spice site mutation in the first base pair of intron 3 and three TALEN pairs targeting this location have been assembled. Preliminary studies using TALENs targeting a different locus of the CD40L gene have demonstrated allelic disruption in up to 31% and targeted gene insertion of GFP in up to 10% of K562 cells. CONCLUSIONS: This approach allows site-specific correction and physiologic expression of the endogenous CD40L gene to safely provide permanent immune reconstitution.