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Antiviral Cytotixic T Lymphocytes Can be Rapidly Generated Against an Extended Spectrum of Viruses
AB88 Abstracts
288
Maternal DNA Methylation of TH17 Cytokine Genes in Second Half of Pregnancy Changes with Parity
SATURDAY
Orpita Nilormee1, Gabrielle A. Lockett, PhD2, Sabrina Iqbal3, John W. Holloway, PhD2, Syed H. Arshad, DM, FRCP4,5, Hongmei Zhang, PhD1, Wilfried Karmaus, MD, Dr.med, MPH6; 1University of Memphis, Memphis, TN, 2University of Southampton, Southampton, United Kingdom, 3University Of Memphis, Memphis, TN, 4The David Hide Asthma and Allergy Research Centre, United Kingdom, 5University of Southampton, United Kingdom, 6Division of Epidemiology, Biostatistics, and Environmental Health, School of Public Health, University of Memphis, Memphis, TN. RATIONALE: Pregnancy is an immunological condition with a series of changes. To explore whether DNA-Methylation (DNA-M) is related to these changes in consecutive gestations, we analyzed the DNA-M of TH1, TH2, TH17 and Treg pathway genes in two consecutive pregnancies of multiparous women. METHODS: Blood samples were collected in first (weeks 8-21) and second (weeks 22-38) halves of pregnancy from the Isle of Wight birth cohort to measure DNA-M using Illumina Infinium Human Methylation450 beadchip. DNA-M was obtained in first (13 mothers) and second (8 mothers) halves for two consecutive pregnancies. First, mixed linear models were used to compare DNA-M across two consecutive pregnancies, separately for first and second halves, to detect cytosine_0.05) in TH1 phosphate-guanine (CpG) sites with significant changes (p< (155 CpGs, 19 genes), TH2 (77 CpGs, 12 genes), TH17 (106 CpGs, 15 genes) and Treg (10 CpGs, 2 genes). Then we compared the proportion of significant CpGs to determine whether T cell pathway CpGs were more often different than 20 random samples each containing 348 CpGs. RESULTS: Only Th17 CpGs in the second half of pregnancy were differentially methylated in two consecutive pregnancies. Among the 28 significantly differentially methylated Th17 CpGs, 24 belong to IL17-family and four to IL21-family. Compared to changes in random samples (14.1-17.8%), the methylation of more CpGs in TH17 (26.4%) were significantly more often changed in the second half of pregnancy. CONCLUSIONS: Significant more methylation changes in TH17 genes in the second half of gestation comparing consecutive pregnancies may suggest changes in methylation with parity.
289
Epicutaneous Immunotherapy Using PlasmaDerived Factor VIII Reduces the Inhibitory Immune Response to Therapeutic Factor VIII in Experimental Hemophilia a
Sebastien Lacroix-Desmazes1, Adeline Porcherie2, Sandrine Delignat1, Mathieu Ing1, Pierre-Henri Benhamou, MD2, Lucie Mondoulet, PhD2; 1 Centre de Recherche des Cordeliers - Equipe 16 INSERM UMRS 1138, Paris, France, 2DBV Technologies, Bagneux, France. RATIONALE: Hemophilia A is a rare X-linked bleeding disorder that results from a lack of functional pro-coagulant factor VIII (FVIII). Treatment of bleeding by administration of exogenous FVIII is complicated by the onset of inhibitory anti-FVIII antibodies in up to 30% of the patients. Here, we hypothesized that the epicutaneous delivery of FVIII imposes immune tolerance to FVIII. METHODS: FVIII-deficient C57BL/6 mice were sensitized by intravenous administration of recombinant human FVIII (2 mg/mouse, rFVIII) once a week for 3 weeks. Mice were then treated with epicutaneous patches containing PBS (sham) or 25 mg of plasma-derived FVIII (pdFVIII) once a week for 48 hours over 8 weeks. Two weeks after the last treatment, mice were injected intravenously once a week for 4 weeks with rFVIII (0.5 mg/ mouse) to mimic replacement therapy. Plasma levels of FVIII inhibitory antibodies were measured in a coagulation assay. FVIII-specific cellular responses were evaluated in spleen following in vitro stimulation. RESULTS: Mice exposed to pdFVIII-containing patches developed significantly lower levels of FVIII inhibitors than sham mice (10346356
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
vs 271561025 Bethesda Units/mL, p50.041). Likewise, following stimulation of splenocytes, numbers of IL-10+CD4+ T cells were greater in mice exposed to pdFVIII-patches than in sham animals (13.265.4 vs 7.161.6 cells/106 splenocytes, p50.031). CONCLUSIONS: Epicutaneous exposure to pdFVIII of hemophilic mice sensitized to FVIII, reduces the immune response to therapeutic FVIII during replacement therapy. Further experiments are required to optimize the dose and duration of epicutaneous FVIII exposure, and to investigate the potential regulatory role of the induced splenic IL-10+CD4+T cells.
290
Antiviral Cytotixic T Lymphocytes Can be Rapidly Generated Against an Extended Spectrum of Viruses
Michael Keller, MD1, Patrick Hanley, PhD2, Haili Lang1, Catherine Bollard, MD2; 1Children’s National Medical Center, Washington, DC, 2 Children’s National Medical Center. RATIONALE: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocytes (VST) has been successful in restoring antiviral immunity in patients with primary immunodeficiency. We hypothesized that ex vivo expanded VST generated using a rapid GMP compliant protocol will be (i) polyclonal and polyfunctional and (ii) elicit broad activity against multiple viral antigens in a single culture. METHODS: VST were cultured by stimulating peripheral blood mononuclear cells from 5 healthy donors with overlapping peptide pools encompassing 17 epitopes from 8 viruses (CMV, EBV, adenovirus, BK virus, human herpesvirus 6B, RSV, influenza (H3N2), and varicella), followed by 10-12 days of culture with cytokines. VST were tested via flow cytometry, IFN-g ELISpot against viral epitopes, and for non-alloreactivity via 51Cr cytotoxicity assays using HLA-mismatched PHA blasts. RESULTS: VST predominantly expressed CD3+ (mean 92%, range 88-95%) and CD4+ (mean 66%) or CD8+ (mean 29%), with few B-cells or natural killer cells (<1%). Central memory T-cells (CD3+/CD45RA-/CCR7+) were present in all VST lines (mean 14.6%, range 9-19%). Viral specificity was seen against a median of 7 viruses (range 3-8), with T-cells responding to a median of 12 epitopes tested on Inf-g elispot (range 5-17). VST demonstrated no killing (<5% specific lysis) of mismatched PHA blasts in cytotoxicity assays. CONCLUSIONS: VST can be rapidly expanded with specificity against 8 viruses, with CD4, CD8, and central memory populations suggesting polyclonality. VST are non- alloreactive in vitro, and may be beneficial for prevention and treatment of a multitude of viral infections in patients with primary immunodeficiency.
288
Maternal DNA Methylation of TH17 Cytokine Genes in Second Half of Pregnancy Changes with Parity
SATURDAY
Orpita Nilormee1, Gabrielle A. Lockett, PhD2, Sabrina Iqbal3, John W. Holloway, PhD2, Syed H. Arshad, DM, FRCP4,5, Hongmei Zhang, PhD1, Wilfried Karmaus, MD, Dr.med, MPH6; 1University of Memphis, Memphis, TN, 2University of Southampton, Southampton, United Kingdom, 3University Of Memphis, Memphis, TN, 4The David Hide Asthma and Allergy Research Centre, United Kingdom, 5University of Southampton, United Kingdom, 6Division of Epidemiology, Biostatistics, and Environmental Health, School of Public Health, University of Memphis, Memphis, TN. RATIONALE: Pregnancy is an immunological condition with a series of changes. To explore whether DNA-Methylation (DNA-M) is related to these changes in consecutive gestations, we analyzed the DNA-M of TH1, TH2, TH17 and Treg pathway genes in two consecutive pregnancies of multiparous women. METHODS: Blood samples were collected in first (weeks 8-21) and second (weeks 22-38) halves of pregnancy from the Isle of Wight birth cohort to measure DNA-M using Illumina Infinium Human Methylation450 beadchip. DNA-M was obtained in first (13 mothers) and second (8 mothers) halves for two consecutive pregnancies. First, mixed linear models were used to compare DNA-M across two consecutive pregnancies, separately for first and second halves, to detect cytosine_0.05) in TH1 phosphate-guanine (CpG) sites with significant changes (p< (155 CpGs, 19 genes), TH2 (77 CpGs, 12 genes), TH17 (106 CpGs, 15 genes) and Treg (10 CpGs, 2 genes). Then we compared the proportion of significant CpGs to determine whether T cell pathway CpGs were more often different than 20 random samples each containing 348 CpGs. RESULTS: Only Th17 CpGs in the second half of pregnancy were differentially methylated in two consecutive pregnancies. Among the 28 significantly differentially methylated Th17 CpGs, 24 belong to IL17-family and four to IL21-family. Compared to changes in random samples (14.1-17.8%), the methylation of more CpGs in TH17 (26.4%) were significantly more often changed in the second half of pregnancy. CONCLUSIONS: Significant more methylation changes in TH17 genes in the second half of gestation comparing consecutive pregnancies may suggest changes in methylation with parity.
289
Epicutaneous Immunotherapy Using PlasmaDerived Factor VIII Reduces the Inhibitory Immune Response to Therapeutic Factor VIII in Experimental Hemophilia a
Sebastien Lacroix-Desmazes1, Adeline Porcherie2, Sandrine Delignat1, Mathieu Ing1, Pierre-Henri Benhamou, MD2, Lucie Mondoulet, PhD2; 1 Centre de Recherche des Cordeliers - Equipe 16 INSERM UMRS 1138, Paris, France, 2DBV Technologies, Bagneux, France. RATIONALE: Hemophilia A is a rare X-linked bleeding disorder that results from a lack of functional pro-coagulant factor VIII (FVIII). Treatment of bleeding by administration of exogenous FVIII is complicated by the onset of inhibitory anti-FVIII antibodies in up to 30% of the patients. Here, we hypothesized that the epicutaneous delivery of FVIII imposes immune tolerance to FVIII. METHODS: FVIII-deficient C57BL/6 mice were sensitized by intravenous administration of recombinant human FVIII (2 mg/mouse, rFVIII) once a week for 3 weeks. Mice were then treated with epicutaneous patches containing PBS (sham) or 25 mg of plasma-derived FVIII (pdFVIII) once a week for 48 hours over 8 weeks. Two weeks after the last treatment, mice were injected intravenously once a week for 4 weeks with rFVIII (0.5 mg/ mouse) to mimic replacement therapy. Plasma levels of FVIII inhibitory antibodies were measured in a coagulation assay. FVIII-specific cellular responses were evaluated in spleen following in vitro stimulation. RESULTS: Mice exposed to pdFVIII-containing patches developed significantly lower levels of FVIII inhibitors than sham mice (10346356
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
vs 271561025 Bethesda Units/mL, p50.041). Likewise, following stimulation of splenocytes, numbers of IL-10+CD4+ T cells were greater in mice exposed to pdFVIII-patches than in sham animals (13.265.4 vs 7.161.6 cells/106 splenocytes, p50.031). CONCLUSIONS: Epicutaneous exposure to pdFVIII of hemophilic mice sensitized to FVIII, reduces the immune response to therapeutic FVIII during replacement therapy. Further experiments are required to optimize the dose and duration of epicutaneous FVIII exposure, and to investigate the potential regulatory role of the induced splenic IL-10+CD4+T cells.
290
Antiviral Cytotixic T Lymphocytes Can be Rapidly Generated Against an Extended Spectrum of Viruses
Michael Keller, MD1, Patrick Hanley, PhD2, Haili Lang1, Catherine Bollard, MD2; 1Children’s National Medical Center, Washington, DC, 2 Children’s National Medical Center. RATIONALE: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocytes (VST) has been successful in restoring antiviral immunity in patients with primary immunodeficiency. We hypothesized that ex vivo expanded VST generated using a rapid GMP compliant protocol will be (i) polyclonal and polyfunctional and (ii) elicit broad activity against multiple viral antigens in a single culture. METHODS: VST were cultured by stimulating peripheral blood mononuclear cells from 5 healthy donors with overlapping peptide pools encompassing 17 epitopes from 8 viruses (CMV, EBV, adenovirus, BK virus, human herpesvirus 6B, RSV, influenza (H3N2), and varicella), followed by 10-12 days of culture with cytokines. VST were tested via flow cytometry, IFN-g ELISpot against viral epitopes, and for non-alloreactivity via 51Cr cytotoxicity assays using HLA-mismatched PHA blasts. RESULTS: VST predominantly expressed CD3+ (mean 92%, range 88-95%) and CD4+ (mean 66%) or CD8+ (mean 29%), with few B-cells or natural killer cells (<1%). Central memory T-cells (CD3+/CD45RA-/CCR7+) were present in all VST lines (mean 14.6%, range 9-19%). Viral specificity was seen against a median of 7 viruses (range 3-8), with T-cells responding to a median of 12 epitopes tested on Inf-g elispot (range 5-17). VST demonstrated no killing (<5% specific lysis) of mismatched PHA blasts in cytotoxicity assays. CONCLUSIONS: VST can be rapidly expanded with specificity against 8 viruses, with CD4, CD8, and central memory populations suggesting polyclonality. VST are non- alloreactive in vitro, and may be beneficial for prevention and treatment of a multitude of viral infections in patients with primary immunodeficiency.