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Tregs in Blood of Patients with Pollen Allergy During Immunotherapy
S16 Abstracts
SATURDAY
59
Thymic Stromal Lymphopoietin Induces Distinct Types of CD25hiCD41 T Cells and This Effect is Influenced by Allergen in Atopic Dermatitis A. J. Reefer1, M. D. Solga1, J. A. Lannigan1, S. F. Ziegler2, J. A. Woodfolk1; 1University of Virginia Health System, Charlottesville, VA, 2Benaroya Research Institute, Seattle, WA. RATIONALE: CD25hiCD41 T lymphocytes with skin-homing potential are increased in the blood of patients with atopic dermatitis (AD). Thymic stromal lymphopoietin (TSLP) is expressed at high levels in AD skin lesions and this cytokine has been shown to expand Th2 cells in vitro. We examined the features of CD25hi T cells induced by TSLP in AD patients and healthy non-atopic subjects without AD. METHODS: CD25hi T cells were analyzed by multi-color flow cytometry using fresh PBMCs or CD41 T cells after co-culture (7 days) with TSLPpulsed CD11c1 dendritic cells. RESULTS: Circulating CD25hi T cells isolated from an AD patient with severe disease were Foxp31 (99%). Differential expression of CD127 indicated these were a mixture of regulatory (CD127lo) and effector (CD127hi) T cells. Priming CD41 T cells from AD and non-AD subjects with TSLP-pulsed dendritic cells induced expansion of CD25hi T cells in vitro which comprised 2 populations (CD127loFoxp31 and CD127lo/ med Foxp3neg). However, an increase in CD25hi T cells expressing the Th2 marker, CRTH2, was only observed for cultures from AD patients. House dust mite extract induced a shift from CD127loFoxp31 to CD127hiFoxp3neg cells within the CD25hi subset. Moreover, CD127hiFoxp3neg T cells were further enhanced after priming with allergen in the presence of TSLP, and this effect was restricted to AD patients. CONCLUSIONS: TSLP induces CD25hi T cells with a regulatory phenotype (CD127loFoxp31) and acts in concert with allergen to enhance induction of CD25hi effector T cells. Our findings suggest that the nature of CD25hi T cells induced by TSLP is influenced by allergen in AD. Funding: NIH/NIAID
60
Interferon Gamma (gIFN) Reverses IL-2 and IL-4 Mediated T Cell Steroid Resistance by Inhibiting p38 MAPK Phosphorylation E. Goleva, L. Li, D. Y. M. Leung; National Jewish Medical and Research Ctr., Denver, CO. RATIONALE: Combination IL-2/IL-4 is known to induce T cell steroid resistance. This can be reversed with gIFN, however, the mechanism by which this occurs is unknown. METHODS: Human PBMC from normal donors were treated with the combination of IL-2/IL-4, IL-2/IL-4/gIFN or media for 48 h. Dexamethasone (DEX)-induced glucocorticoid receptor (GCR)a nuclear translocation in the cells, TransAMä GR transcription factor assay and DEX-induced MKP-1 expression were used to assess cellular responses to steroids. Cell lysates were tested with a human Phospho-MAPK array. RESULTS: Following DEX treatment, both CD41 and CD81 cells showed a significant increase in the amount of nuclear GCR when PBMCs were cultured in media for 48 h. However, when cells were cultured in the presence of IL-2/IL-4 for 48 h, addition of DEX did not induce nuclear GCR translocation. Furthermore, DEX-induced MKP-1 induction, used as a read-out for corticosteroid-induced transactivation, was significantly greater (p < 0.05) in media and IL-2/IL-4/gIFN treated conditions compared to IL-2/IL-4 treated cells. Using a human phospho-MAPK array profiler assay, p38a MAPK (T180/Y182) was the only MAPK activated after 48 h of IL-2/IL-4 treatment. This was further confirmed by phosphorylation of hsp27, the downstream phosphorylation substrate for activated p38 MAPK. Phosphorylation of both molecules was significantly diminished when cells were cultured with IL-2/IL-4/gIFN for 48 h and, importantly, addition of gIFN to the IL-2/IL-4 combination restored GCRa nuclear translocation in response to DEX. CONCLUSIONS: These data indicate that combination IL-2/IL-4 inhibits GCRa nuclear translocation in human T cells, and this effect is reversed by gIFN via inhibition of p38 MAPK activation. Funding: NIH
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
61
Tregs in Blood of Patients with Pollen Allergy During Immunotherapy A. M. Hofman1, J. Karczewski2, T. Hofman1, K. Wiktorowicz2; 1Center of Allergology, Poznan, POLAND, 2Medical University, Department of Biology and Enviromental Protection, Poznan, POLAND. The aim of this study was to establish the changes in lymphocytes CD31 and CD41CD251 (Treg) subsets during two years of hyposensibilitation in patient with pollinosis. METHODS: 24 adult patients with allergy to grass pollen was hyposensibilisated against pollen allergens. All had allergy to grass pollen. In 6 patients second vaccine against other allergens (as house dust mite) was needed. The blood sample was collected 3 times: before the start of immunotherapy (IT), after the maintence dose and after one year of IT. Lymphocytes CD31 and CD41CD251 were examined by flow cytometry. RESULTS: In all patients received the maintence dose the average concentration of CD31 and CD41CD251 increased, and after one year CD31 diminished but Treg yet increased. Patients were devided into two groups- with one and two vaccines. In patients treated with two vaccines slowly increase of CD31, and clear increase of Treg after one year of IT was observed. In patients treated with one vaccine the highest increase of CD31 was observed after the maintence dose, then it diminished, but to higher level comparing to the level before IT. In those patients Tregs had parrallel level after the maintence dose, and slowly increase after one year. CONCLUSIONS: During IT higher increase of Treg was observed in patient with two vaccines than one. In the group of patint with one vaccine lymphocytes CD31 increased after the maintence dose, but diminished after one year of IT.
62
Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase Enhanced Human IgE Production S. Fujieda, H. Yamamoto; University of Fukui, Fukui, JAPAN. RATIONALE: Angiogenesis and/or hypervascularity are critical in many pathogeneses. Neovascularizaton in exaggerated Th2 inflammation and airway remodeling are cornerstones in the pathogenesis of allergic airway disease. In allergic rhinitis CD341 vessel count is associated with total nasal symptom score. As a critical regulator of angiogenesis, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are representative factors. PD-ECGF has also thymidine phosphorylase (TP) activity. TP catalyzes the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose-1-phosphate (2DDR). METHODS: We investigated whether two representative angiogenic factors (VEGF and PD-ECGF) enhanced in vitro IgE production by human PBMC. RESULTS: PD-ECGF enhanced the production of IgE induced by IL-4 plus anti-CD40 mAb, while VEGF did not significantly induced enhancement of IgE production. The addition of 2DDR, which has angiogenic activities, has no effect on IgE production. Stimulation of IL-4 and anti-CD mAb induced messenger RNA (mRNA) expression of PD-ECGF in human PBMC. TP inhibitor (TPI) significantly inhibited IgE production by PBMC without affecting production of IgA. The exposure of IL-4 and anti-CD40 stimulated B cells to TPI blocked phosphorylation of STAT6. CONCLUSIONS: This result suggested that angiogenic activity had no direct enhancement of IgE production. TP might be a critical factor in IgE production in vitro by human PBMC. TPI might be an effective novel method of inducing protection against IgE-related allergic disease. Funding: Japan Ministry of Education, Culture, Sports, Science, and Technology
SATURDAY
59
Thymic Stromal Lymphopoietin Induces Distinct Types of CD25hiCD41 T Cells and This Effect is Influenced by Allergen in Atopic Dermatitis A. J. Reefer1, M. D. Solga1, J. A. Lannigan1, S. F. Ziegler2, J. A. Woodfolk1; 1University of Virginia Health System, Charlottesville, VA, 2Benaroya Research Institute, Seattle, WA. RATIONALE: CD25hiCD41 T lymphocytes with skin-homing potential are increased in the blood of patients with atopic dermatitis (AD). Thymic stromal lymphopoietin (TSLP) is expressed at high levels in AD skin lesions and this cytokine has been shown to expand Th2 cells in vitro. We examined the features of CD25hi T cells induced by TSLP in AD patients and healthy non-atopic subjects without AD. METHODS: CD25hi T cells were analyzed by multi-color flow cytometry using fresh PBMCs or CD41 T cells after co-culture (7 days) with TSLPpulsed CD11c1 dendritic cells. RESULTS: Circulating CD25hi T cells isolated from an AD patient with severe disease were Foxp31 (99%). Differential expression of CD127 indicated these were a mixture of regulatory (CD127lo) and effector (CD127hi) T cells. Priming CD41 T cells from AD and non-AD subjects with TSLP-pulsed dendritic cells induced expansion of CD25hi T cells in vitro which comprised 2 populations (CD127loFoxp31 and CD127lo/ med Foxp3neg). However, an increase in CD25hi T cells expressing the Th2 marker, CRTH2, was only observed for cultures from AD patients. House dust mite extract induced a shift from CD127loFoxp31 to CD127hiFoxp3neg cells within the CD25hi subset. Moreover, CD127hiFoxp3neg T cells were further enhanced after priming with allergen in the presence of TSLP, and this effect was restricted to AD patients. CONCLUSIONS: TSLP induces CD25hi T cells with a regulatory phenotype (CD127loFoxp31) and acts in concert with allergen to enhance induction of CD25hi effector T cells. Our findings suggest that the nature of CD25hi T cells induced by TSLP is influenced by allergen in AD. Funding: NIH/NIAID
60
Interferon Gamma (gIFN) Reverses IL-2 and IL-4 Mediated T Cell Steroid Resistance by Inhibiting p38 MAPK Phosphorylation E. Goleva, L. Li, D. Y. M. Leung; National Jewish Medical and Research Ctr., Denver, CO. RATIONALE: Combination IL-2/IL-4 is known to induce T cell steroid resistance. This can be reversed with gIFN, however, the mechanism by which this occurs is unknown. METHODS: Human PBMC from normal donors were treated with the combination of IL-2/IL-4, IL-2/IL-4/gIFN or media for 48 h. Dexamethasone (DEX)-induced glucocorticoid receptor (GCR)a nuclear translocation in the cells, TransAMä GR transcription factor assay and DEX-induced MKP-1 expression were used to assess cellular responses to steroids. Cell lysates were tested with a human Phospho-MAPK array. RESULTS: Following DEX treatment, both CD41 and CD81 cells showed a significant increase in the amount of nuclear GCR when PBMCs were cultured in media for 48 h. However, when cells were cultured in the presence of IL-2/IL-4 for 48 h, addition of DEX did not induce nuclear GCR translocation. Furthermore, DEX-induced MKP-1 induction, used as a read-out for corticosteroid-induced transactivation, was significantly greater (p < 0.05) in media and IL-2/IL-4/gIFN treated conditions compared to IL-2/IL-4 treated cells. Using a human phospho-MAPK array profiler assay, p38a MAPK (T180/Y182) was the only MAPK activated after 48 h of IL-2/IL-4 treatment. This was further confirmed by phosphorylation of hsp27, the downstream phosphorylation substrate for activated p38 MAPK. Phosphorylation of both molecules was significantly diminished when cells were cultured with IL-2/IL-4/gIFN for 48 h and, importantly, addition of gIFN to the IL-2/IL-4 combination restored GCRa nuclear translocation in response to DEX. CONCLUSIONS: These data indicate that combination IL-2/IL-4 inhibits GCRa nuclear translocation in human T cells, and this effect is reversed by gIFN via inhibition of p38 MAPK activation. Funding: NIH
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
61
Tregs in Blood of Patients with Pollen Allergy During Immunotherapy A. M. Hofman1, J. Karczewski2, T. Hofman1, K. Wiktorowicz2; 1Center of Allergology, Poznan, POLAND, 2Medical University, Department of Biology and Enviromental Protection, Poznan, POLAND. The aim of this study was to establish the changes in lymphocytes CD31 and CD41CD251 (Treg) subsets during two years of hyposensibilitation in patient with pollinosis. METHODS: 24 adult patients with allergy to grass pollen was hyposensibilisated against pollen allergens. All had allergy to grass pollen. In 6 patients second vaccine against other allergens (as house dust mite) was needed. The blood sample was collected 3 times: before the start of immunotherapy (IT), after the maintence dose and after one year of IT. Lymphocytes CD31 and CD41CD251 were examined by flow cytometry. RESULTS: In all patients received the maintence dose the average concentration of CD31 and CD41CD251 increased, and after one year CD31 diminished but Treg yet increased. Patients were devided into two groups- with one and two vaccines. In patients treated with two vaccines slowly increase of CD31, and clear increase of Treg after one year of IT was observed. In patients treated with one vaccine the highest increase of CD31 was observed after the maintence dose, then it diminished, but to higher level comparing to the level before IT. In those patients Tregs had parrallel level after the maintence dose, and slowly increase after one year. CONCLUSIONS: During IT higher increase of Treg was observed in patient with two vaccines than one. In the group of patint with one vaccine lymphocytes CD31 increased after the maintence dose, but diminished after one year of IT.
62
Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase Enhanced Human IgE Production S. Fujieda, H. Yamamoto; University of Fukui, Fukui, JAPAN. RATIONALE: Angiogenesis and/or hypervascularity are critical in many pathogeneses. Neovascularizaton in exaggerated Th2 inflammation and airway remodeling are cornerstones in the pathogenesis of allergic airway disease. In allergic rhinitis CD341 vessel count is associated with total nasal symptom score. As a critical regulator of angiogenesis, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are representative factors. PD-ECGF has also thymidine phosphorylase (TP) activity. TP catalyzes the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose-1-phosphate (2DDR). METHODS: We investigated whether two representative angiogenic factors (VEGF and PD-ECGF) enhanced in vitro IgE production by human PBMC. RESULTS: PD-ECGF enhanced the production of IgE induced by IL-4 plus anti-CD40 mAb, while VEGF did not significantly induced enhancement of IgE production. The addition of 2DDR, which has angiogenic activities, has no effect on IgE production. Stimulation of IL-4 and anti-CD mAb induced messenger RNA (mRNA) expression of PD-ECGF in human PBMC. TP inhibitor (TPI) significantly inhibited IgE production by PBMC without affecting production of IgA. The exposure of IL-4 and anti-CD40 stimulated B cells to TPI blocked phosphorylation of STAT6. CONCLUSIONS: This result suggested that angiogenic activity had no direct enhancement of IgE production. TP might be a critical factor in IgE production in vitro by human PBMC. TPI might be an effective novel method of inducing protection against IgE-related allergic disease. Funding: Japan Ministry of Education, Culture, Sports, Science, and Technology