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CD4 T Cells Receive Costimulatory Signals from CpG DNA during Antigen-Specific Stimulation
Abstracts S177
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
FENO 35ppb - Is it Associated with Asthma Control
C. Lopes; Hospital S. João, Porto, PORTUGAL. RATIONALE: A fixed cut value of the fraction of nitric oxide in the exhaled air (FENO) has been recently suggested for the adjustment of inhaled steroid treatment without compromising asthma control. We aimed to determine the association between FENO and asthma control in patients followed at a specialized allergy clinic. METHODS: One hundred and seventy four asthma patients included in a cross-sectional study of dietary intake and asthma severity were analysed. Asthma control was assessed with the 7-item asthma control questionnaire (ACQ)(Juniper, 1999), FeNO 0.05 with Niox (Aerocrine, Sweden) and FEV1 with Piko-1 (Ferraris, UK). Pearson correlation, general linear model and Mann-Whitney U test were used. RESULTS: Eighty-two percent were women, 74% were atopic and 72% with current inhaled steroid. The mean (SD) age was 40(15.2) years, FEV1 87.4(24.7)%, ACQ score was 1.22 (IC 95% 1.07-1.37) and geometric mean (IC95%) FENO was 29.3 (26.1-32.8). We found no significant correlation between FENO and ACQ (r=0.027; p=0.723) even after adjusting for age, sex, atopy, rhinitis, smoking, and inhaled steroid (B =0.15, IC95% -0.84 to -3.89, F=1.63, p=0.204). ACQ sores were not different in patients with FENO values below or above 35ppb. (p=0.828). CONCLUSIONS: In this cross-sectional analysis FENO was not associated with asthma control and the results do not support the use of a fixed cut-value of 35ppb. Differential Expression of NF-B Molecular Species in Th1 and Th2 Cells R. J. Kurnat, U. De Fanis, W. Lee, S. N. Georas, J. Guo, V. Casolaro; Johns Hopkins University, Baltimore, MD. RATIONALE: Increasing evidence points to a critical role of NF- B/Rel transcription factors in T-cell polarization. Recent studies indicate a dichotomy between the function of I B- and Bcl-3-interacting molecular species. To understand whether these species undergo differential regulation by polarizing signals, we conducted a systematic assessment of their relative expression in Th1 and Th2 cells. METHODS: T cells were polarized in vitro from human naïve precursors using established protocols, then restimulated or not with ionomycin (1 g/mL) and PMA (10 ng/mL). RNA levels were measured by real-time RT-PCR against a peptidyl-prolyl-isomerase A (PPIA) loading control. Transient transfections for promoter analysis were carried out by electroporation of Jurkat and primary mononuclear cells. RESULTS: Expression of c-Rel, RelA/p65, RelB, and NF- B1/p50 was not significantly different in Th1 and Th2 cultures. Conversely, stimulated Th2 cells expressed significantly higher levels of NF- B2/p52 than Th1 (33±8% vs. 19±4% of PPIA, respectively; p<0.001). Moreover, stimulation caused markedly downregulated levels of Bcl-3 in Th1 cells (to 32±4% of control; p<0.0001), resulting in significantly lower levels than in Th2 cells (1.0±0.1% vs. 3.3±0.4%; p<0.0001). These findings were confirmed in ex vivo sorted, CRTH2+ Th2 cells. Overexpression of Bcl-3, with or without NF- B1, markedly enhanced activity of IL-4 promoterdriven luciferase constructs in Jurkat and freshly isolated primary T cells. CONCLUSIONS: The relative abundance of certain NF- B species is altered as a result of T-cell polarization. Consistent with findings in knockout mice, Bcl-3, possibly via its interaction with NF- B2, appears to play a central role in Th2 differentiation via direct activation of the IL-4 gene. Funding: NIH/NIAID
690
Immunomodulation of T Cells by Targeting Allergen to Antigen Presenting Cells K. Hulse1, A. J. Reefer1, S. M. Satinover1, M. D. Chapman2, V. Engelhard1, J. A. Woodfolk1; 1University of Virginia, Charlottesville, VA, 2Indoor Biotechnologies, Charlottesville, VA. RATIONALE: Targeting allergen to receptors present on antigen presenting cells (APC) may have therapeutic value by conditioning both CD4+ and CD8+ T cell responses. However, the effect of receptor-targeted allergen on these T cell subsets has not been investigated. METHODS: Allergen-stimulated T cell cultures were established from cat-allergic (IgE+ IgG+) and cat-tolerant (IgE- IgG+) patients (n=10) using freshly isolated peripheral blood mononuclear cells (PBMC), or PBMC depleted of either CD4+ or CD8+ T cells. Cultures were also established using FACS-sorted CD8+ T cells (>99% purity) as responder cells. Cells were stimulated with recombinant Fel d 1 (rFel d 1) or rFel d 1 linked to an anti-CD64 mAb (clone H22), which binds to the high affinity IgG receptor, FcRI (H22-rFel d 1). Cultures were supplemented with IL-7 on day-0 and IL-2 on day-2. Cellular proliferation and cytokine production (day-7) were measured in all cultures by [3H]-thymidine incorporation and multiplex suspension bead array, respectively. RESULTS: Stimulation of PBMC with H22-rFel d 1 enhanced both proliferation and Th1/Th2 cytokine (IL-2, IL-5, IL-10, IL-13, IFN-) responses up to 20 fold compared with rFel d 1, irrespective of allergic status. This effect was also observed in PBMC cultures depleted of either CD4+ or CD8+ T cells. Proliferation of purified CD8+ T cells to H22-rFel d 1 confirmed reactivity of this subset. CONCLUSIONS: Targeting cat allergen to FcRI influences both CD4+ and CD8+ T cell compartments. The receptor-targeted variant of Fel d 1 provides us with a useful tool for evaluating modulation of T cell responses to allergen. Funding: NIH
691
CD4 T Cells Receive Costimulatory Signals from CpG DNA during Antigen-Specific Stimulation D. F. LaRosa, A. E. Gelman, L. A. Turka; University of Pennsylvania, Philadelphia, PA. RATIONALE: Previous work demonstrates activated mouse CD4 T cells selectively express TLR9 protein. Purified CD4 T cells stimulated with anti-CD3 and treated with the TLR9 ligand CpG DNA have enhanced proliferation and survival, which is MyD88-dependent. We asked in an antigen-specific system if CD4 T cells would respond to CpG in an APC-independent manner. METHODS: Purified OT-II CD4 T cells were cultured with BMDCs derived from MyD88-/- mice in a 1: 7 DC-to-T cell ratio. These BMDCs do not upregulate CD86 or CD40 after 24 hours of CpG treatment. Cells were cultured for 72 hour in the presence or absence of OVA peptide (0.1 g/ml), CpG (10 M), or IL-2 (50 U/ml) then collected and analyzed by flow cytometry for proliferation by CFSE dilution and survival by 7-AAD exclusion. Supernatants were assessed for IL-2 by ELISA after 36 hours. RESULTS: CD4 T cells treated with CpG during stimulation with cognate OVA peptide had an increased proportion of cells dividing compared to those untreated or IL-2 treated. CpG-treated cultures had a 2-fold increase in responder frequency and a 3-fold increase in survival, and the supernatants had increased IL-2. CONCLUSIONS: CD4 T cells receive additional costimulatory signals from the TLR9 ligand CpG DNA during APC stimulation with cognate antigen. This effect enhances proliferation and survival, and may be partially mediated by increased IL-2 secretion. TLR ligands may directly modulate T cell function and fate. Funding: NIH
692
SUNDAY
689
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
FENO 35ppb - Is it Associated with Asthma Control
C. Lopes; Hospital S. João, Porto, PORTUGAL. RATIONALE: A fixed cut value of the fraction of nitric oxide in the exhaled air (FENO) has been recently suggested for the adjustment of inhaled steroid treatment without compromising asthma control. We aimed to determine the association between FENO and asthma control in patients followed at a specialized allergy clinic. METHODS: One hundred and seventy four asthma patients included in a cross-sectional study of dietary intake and asthma severity were analysed. Asthma control was assessed with the 7-item asthma control questionnaire (ACQ)(Juniper, 1999), FeNO 0.05 with Niox (Aerocrine, Sweden) and FEV1 with Piko-1 (Ferraris, UK). Pearson correlation, general linear model and Mann-Whitney U test were used. RESULTS: Eighty-two percent were women, 74% were atopic and 72% with current inhaled steroid. The mean (SD) age was 40(15.2) years, FEV1 87.4(24.7)%, ACQ score was 1.22 (IC 95% 1.07-1.37) and geometric mean (IC95%) FENO was 29.3 (26.1-32.8). We found no significant correlation between FENO and ACQ (r=0.027; p=0.723) even after adjusting for age, sex, atopy, rhinitis, smoking, and inhaled steroid (B =0.15, IC95% -0.84 to -3.89, F=1.63, p=0.204). ACQ sores were not different in patients with FENO values below or above 35ppb. (p=0.828). CONCLUSIONS: In this cross-sectional analysis FENO was not associated with asthma control and the results do not support the use of a fixed cut-value of 35ppb. Differential Expression of NF-B Molecular Species in Th1 and Th2 Cells R. J. Kurnat, U. De Fanis, W. Lee, S. N. Georas, J. Guo, V. Casolaro; Johns Hopkins University, Baltimore, MD. RATIONALE: Increasing evidence points to a critical role of NF- B/Rel transcription factors in T-cell polarization. Recent studies indicate a dichotomy between the function of I B- and Bcl-3-interacting molecular species. To understand whether these species undergo differential regulation by polarizing signals, we conducted a systematic assessment of their relative expression in Th1 and Th2 cells. METHODS: T cells were polarized in vitro from human naïve precursors using established protocols, then restimulated or not with ionomycin (1 g/mL) and PMA (10 ng/mL). RNA levels were measured by real-time RT-PCR against a peptidyl-prolyl-isomerase A (PPIA) loading control. Transient transfections for promoter analysis were carried out by electroporation of Jurkat and primary mononuclear cells. RESULTS: Expression of c-Rel, RelA/p65, RelB, and NF- B1/p50 was not significantly different in Th1 and Th2 cultures. Conversely, stimulated Th2 cells expressed significantly higher levels of NF- B2/p52 than Th1 (33±8% vs. 19±4% of PPIA, respectively; p<0.001). Moreover, stimulation caused markedly downregulated levels of Bcl-3 in Th1 cells (to 32±4% of control; p<0.0001), resulting in significantly lower levels than in Th2 cells (1.0±0.1% vs. 3.3±0.4%; p<0.0001). These findings were confirmed in ex vivo sorted, CRTH2+ Th2 cells. Overexpression of Bcl-3, with or without NF- B1, markedly enhanced activity of IL-4 promoterdriven luciferase constructs in Jurkat and freshly isolated primary T cells. CONCLUSIONS: The relative abundance of certain NF- B species is altered as a result of T-cell polarization. Consistent with findings in knockout mice, Bcl-3, possibly via its interaction with NF- B2, appears to play a central role in Th2 differentiation via direct activation of the IL-4 gene. Funding: NIH/NIAID
690
Immunomodulation of T Cells by Targeting Allergen to Antigen Presenting Cells K. Hulse1, A. J. Reefer1, S. M. Satinover1, M. D. Chapman2, V. Engelhard1, J. A. Woodfolk1; 1University of Virginia, Charlottesville, VA, 2Indoor Biotechnologies, Charlottesville, VA. RATIONALE: Targeting allergen to receptors present on antigen presenting cells (APC) may have therapeutic value by conditioning both CD4+ and CD8+ T cell responses. However, the effect of receptor-targeted allergen on these T cell subsets has not been investigated. METHODS: Allergen-stimulated T cell cultures were established from cat-allergic (IgE+ IgG+) and cat-tolerant (IgE- IgG+) patients (n=10) using freshly isolated peripheral blood mononuclear cells (PBMC), or PBMC depleted of either CD4+ or CD8+ T cells. Cultures were also established using FACS-sorted CD8+ T cells (>99% purity) as responder cells. Cells were stimulated with recombinant Fel d 1 (rFel d 1) or rFel d 1 linked to an anti-CD64 mAb (clone H22), which binds to the high affinity IgG receptor, FcRI (H22-rFel d 1). Cultures were supplemented with IL-7 on day-0 and IL-2 on day-2. Cellular proliferation and cytokine production (day-7) were measured in all cultures by [3H]-thymidine incorporation and multiplex suspension bead array, respectively. RESULTS: Stimulation of PBMC with H22-rFel d 1 enhanced both proliferation and Th1/Th2 cytokine (IL-2, IL-5, IL-10, IL-13, IFN-) responses up to 20 fold compared with rFel d 1, irrespective of allergic status. This effect was also observed in PBMC cultures depleted of either CD4+ or CD8+ T cells. Proliferation of purified CD8+ T cells to H22-rFel d 1 confirmed reactivity of this subset. CONCLUSIONS: Targeting cat allergen to FcRI influences both CD4+ and CD8+ T cell compartments. The receptor-targeted variant of Fel d 1 provides us with a useful tool for evaluating modulation of T cell responses to allergen. Funding: NIH
691
CD4 T Cells Receive Costimulatory Signals from CpG DNA during Antigen-Specific Stimulation D. F. LaRosa, A. E. Gelman, L. A. Turka; University of Pennsylvania, Philadelphia, PA. RATIONALE: Previous work demonstrates activated mouse CD4 T cells selectively express TLR9 protein. Purified CD4 T cells stimulated with anti-CD3 and treated with the TLR9 ligand CpG DNA have enhanced proliferation and survival, which is MyD88-dependent. We asked in an antigen-specific system if CD4 T cells would respond to CpG in an APC-independent manner. METHODS: Purified OT-II CD4 T cells were cultured with BMDCs derived from MyD88-/- mice in a 1: 7 DC-to-T cell ratio. These BMDCs do not upregulate CD86 or CD40 after 24 hours of CpG treatment. Cells were cultured for 72 hour in the presence or absence of OVA peptide (0.1 g/ml), CpG (10 M), or IL-2 (50 U/ml) then collected and analyzed by flow cytometry for proliferation by CFSE dilution and survival by 7-AAD exclusion. Supernatants were assessed for IL-2 by ELISA after 36 hours. RESULTS: CD4 T cells treated with CpG during stimulation with cognate OVA peptide had an increased proportion of cells dividing compared to those untreated or IL-2 treated. CpG-treated cultures had a 2-fold increase in responder frequency and a 3-fold increase in survival, and the supernatants had increased IL-2. CONCLUSIONS: CD4 T cells receive additional costimulatory signals from the TLR9 ligand CpG DNA during APC stimulation with cognate antigen. This effect enhances proliferation and survival, and may be partially mediated by increased IL-2 secretion. TLR ligands may directly modulate T cell function and fate. Funding: NIH
692
SUNDAY
689