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Functional and Phenotypic Characterization of CD8+CD28- T Cells in Atopic and Non-Atopic Individuals
S246 Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Inverse Correlation between Circulating CD4+CD25high Cells and CD4+IL4+ Cells In Vivo K. Orihara1,2, T. Tobe2, A. Akasawa3, H. Saito1, K. Matsumoto1; 1Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, JAPAN, 2Graduate School of Pharmaceutical Sciences, Showa University, Tokyo, JAPAN, 3Department of Interdisciplinary Medicine, National Center for Child Health and Development, Tokyo, JAPAN. RATIONALE: While CD25 molecule had been well known as a surface marker of activated lymphocytes, it has been recently shown that CD25 is also a practical marker of regulatory T cells. Circulating CD4+CD25high cells in human as well as CD4+CD25+ cells in mice are reported to be capable of down-regulating Th1 responses against self-antigens both in vitro and in vivo. However, the effects of these cells against Th2 res ponses are less well understood especially in human. In order to clarify the role of circulating CD4+CD25high cells in Th2 immune responses in human, we investigated the correlation between cell count of CD4+CD25high cells in PBMCs and clinical markers of Th2 responses, in vivo. METHODS: PBMCs were obtained from 168 healthy volunteers after obtaining written informed consent. The number of CD4+CD25high cell was measured by flow cytometry after staining with anti-CD4 and antiCD25 mAbs. Th1 and Th2 immune responses were determined by intracellular staining of IFN-gamma/IL-4 in CD4+ cells after stimulation with PMA and ionomycin. Various laboratory data was also obtained. RESULTS: There was a significantly inverse correlation between CD4+CD25high cell count and CD4+IL4+ cell count. On the other hand, the CD4+CD25high cell count was not correlated with CD4+IFN+ cell count, serum IgE level or eosinophil count. CONCLUSIONS: Our findings imply that circulating CD4+CD25high cells regulate Th2 immune responses in vivo, and thus, exogenous induction of CD4+CD25high cells may suppress allergic inflammations.
951
Gr-1+ Cells Induce the Expression of Matrix Metalloproteinases (MMPs) that Are Crucial for Antigen-Stimulated Recruitment of Th Cells into Murine Airways Y. W. Jung1, C. L. Zindl1, C. T. Weaver2, D. D. Chaplin1; 1Microbiology, University of Alabama at Birmingham, Birmingham, AL, 2Pathology, University of Alabama at Birmingham, Birmingham, AL. RATIONALE: In order to define innate mechanisms by which Th1 and Th2 cells are recruited into the airways following antigenic challenge, we examined the role of Gr-1+ cells and their mediators in the recruitment of these effector T cells. METHODS: After depletion of Gr-1+ cells using an anti-Gr-1 mAb in vivo, we measured the recruitment of adoptively transferred Th cells into the airways of antigen-challenged mice. To identify mediators of the action of Gr-1+ cells, we characterized the expression and activity of MMPs and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) using RNase protection assay, immunostaining, and in situ and PAGE-zymography. In addition, we tested the roles of MMP-8 and MMP-9 in the development of airway inflammation using specific inhibitors. RESULTS: Systemic depletion of Gr-1+ cells significantly reduced the numbers of both Th1 and Th2 cells recruited into the airways after intranasal antigen challenge. Challenged lungs over-expressed both MMP-9 and TIMP-1 mRNAs with the over-expression of MMP-9 being Gr-1+ cell-dependent. Additionally, the enzymatic activities of MMP-9 and collagenase were reduced in lungs after Gr-1+ cells were depleted. Treatment of mice with inhibitors of MMP-8 and MMP-9 reduced antigen-induced recruitment of Th1 and Th2 cells, resulting in significantly reduced eosinophilic airway inflammation. CONCLUSIONS: These data support a model in which airway antigen challenge activates Gr-1+ cells and consequently MMPs to support the recruitment of Th1 and Th2 cells in the airway inflammatory response. Funding: NIH
953
Functional and Phenotypic Characterization of CD8+CD28T Cells in Atopic and Non-Atopic Individuals O. M. M. Lourenco, A. R. S. Rafael, A. M. L. Fonseca, L. M. P. Taborda-Barata; Department of Medical Sciences, University of Beira Interior, Health Sciences Research Centre, Covilha, PORTUGAL. RATIONALE: The aim of the present study was to analyse possible differences in phenotype and functional properties of human CD8+CD28- T cells upon allergen-specific PBMC proliferation between atopic and non-atopic individuals. METHODS: Peripheral blood was obtained from 44 atopic subjects and 40 non-atopic controls. Initial phenotypic studies were performed using flow cytometry in whole blood, gradient-separated lymphocytes and immunomagnetic-isolated T cells. Phenotypic markers studied included NK cell markers, chemokine receptors, activation markers as well as and TCR chains. Purified CD8+CD28+ or CD8+CD28 T cells were mixed with PBMC at different ratios (1:1, 1:2 and 1:4) and stimulated with the allergen Dermatophagoides pteronyssinus (Der p) or PHA in 6-day co-cultures. Proliferation was analysed using thymidine incorporation. RESULTS: Of all markers studied, only expression of TCR on CD8+CD28- T cells was different between atopic and non-atopic volunteers. In terms of allergen-induced proliferation, CD8+CD28- T cells significantly increased proliferation of PBMC (p < 0.05; Wilcoxon-signed rank test) but only in non-atopic individuals. CONCLUSIONS: These preliminary data suggest that peripheral blood CD8+CD28- T cells from non-atopic individuals may contribute to proliferation of PBMC in response to allergens that act as recall antigens. However, this effect should be further correlated with effects upon cytokine profiles. Funding: Fundacao Para a Ciencia e a Tecnologia
954
952
MONDAY
CD4+CD25++ T Cells in Peripheral Blood Correlate with Total and Pollen-Specific IgE in Young Children K. A. Burmeister1, C. Seroogy2, C. Tisler1, M. Evans3, L. Franco1, L. Pleiss1, R. Gangnon3, R. F. Lemanske, Jr.2, J. E. Gern2; 1Dept of Pediatrics, University of Wisconsin-Madison, Madison, WI, 2Dept of Medicine, University of Wisconsin-Madison, Madison, WI, 3Dept of Biostatistics and Medical Informatics and Population Health Sciences, University of Wisconsin-Madison, Madison, WI. RATIONALE: There is evidence that CD4+CD25++ T-regulatory cells help to establish tolerance to allergens in childhood. METHODS: To test this theory, a cohort of 6-year-old children (n=46) at risk for developing allergy and asthma was analyzed for the presence of T-regulatory cells by flow cytometry and the results were compared to clinical and biological markers of atopy. The percentage of T-regulatory cells (CD3+CD4+CD25++) in PBMC was estimated by flow cytometry. Total and allergen-specific IgE in plasma were determined by fluoroenzyme immunoassays (Unicap 100, Pharmacia and Upjohn Diagnostics). RESULTS: There was a highly significant correlation between percent CD4+CD25++ T cells and total IgE levels (r=0.57, p<0.0001). In addition, mean CD4+CD25++ T cell numbers were higher in children with IgE specific for ragweed (p=0.046) and birch (p=0.039), with similar trends (p<0.1) for children with IgE to egg and cat. There was no relationship between percent CD4+CD25++ T cells and atopic dermatitis (p = 0.82), wheezing (p = 0.16), or sensitization to alternaria (p=0.26) or house dust mite (p=0.25 dp and p=0.65 df). CONCLUSIONS: The strong positive correlation between CD4+CD25++ cells and total and pollen-specific IgE in young children does not support a straightforward relationship between T-regulatory cell numbers and allergen tolerance. Additional studies are in progress to determine the relationships between IgE and T regulatory cell functional activity, and the expression of FOXP3 and other associated genes. Funding: NIH
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Inverse Correlation between Circulating CD4+CD25high Cells and CD4+IL4+ Cells In Vivo K. Orihara1,2, T. Tobe2, A. Akasawa3, H. Saito1, K. Matsumoto1; 1Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, JAPAN, 2Graduate School of Pharmaceutical Sciences, Showa University, Tokyo, JAPAN, 3Department of Interdisciplinary Medicine, National Center for Child Health and Development, Tokyo, JAPAN. RATIONALE: While CD25 molecule had been well known as a surface marker of activated lymphocytes, it has been recently shown that CD25 is also a practical marker of regulatory T cells. Circulating CD4+CD25high cells in human as well as CD4+CD25+ cells in mice are reported to be capable of down-regulating Th1 responses against self-antigens both in vitro and in vivo. However, the effects of these cells against Th2 res ponses are less well understood especially in human. In order to clarify the role of circulating CD4+CD25high cells in Th2 immune responses in human, we investigated the correlation between cell count of CD4+CD25high cells in PBMCs and clinical markers of Th2 responses, in vivo. METHODS: PBMCs were obtained from 168 healthy volunteers after obtaining written informed consent. The number of CD4+CD25high cell was measured by flow cytometry after staining with anti-CD4 and antiCD25 mAbs. Th1 and Th2 immune responses were determined by intracellular staining of IFN-gamma/IL-4 in CD4+ cells after stimulation with PMA and ionomycin. Various laboratory data was also obtained. RESULTS: There was a significantly inverse correlation between CD4+CD25high cell count and CD4+IL4+ cell count. On the other hand, the CD4+CD25high cell count was not correlated with CD4+IFN+ cell count, serum IgE level or eosinophil count. CONCLUSIONS: Our findings imply that circulating CD4+CD25high cells regulate Th2 immune responses in vivo, and thus, exogenous induction of CD4+CD25high cells may suppress allergic inflammations.
951
Gr-1+ Cells Induce the Expression of Matrix Metalloproteinases (MMPs) that Are Crucial for Antigen-Stimulated Recruitment of Th Cells into Murine Airways Y. W. Jung1, C. L. Zindl1, C. T. Weaver2, D. D. Chaplin1; 1Microbiology, University of Alabama at Birmingham, Birmingham, AL, 2Pathology, University of Alabama at Birmingham, Birmingham, AL. RATIONALE: In order to define innate mechanisms by which Th1 and Th2 cells are recruited into the airways following antigenic challenge, we examined the role of Gr-1+ cells and their mediators in the recruitment of these effector T cells. METHODS: After depletion of Gr-1+ cells using an anti-Gr-1 mAb in vivo, we measured the recruitment of adoptively transferred Th cells into the airways of antigen-challenged mice. To identify mediators of the action of Gr-1+ cells, we characterized the expression and activity of MMPs and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) using RNase protection assay, immunostaining, and in situ and PAGE-zymography. In addition, we tested the roles of MMP-8 and MMP-9 in the development of airway inflammation using specific inhibitors. RESULTS: Systemic depletion of Gr-1+ cells significantly reduced the numbers of both Th1 and Th2 cells recruited into the airways after intranasal antigen challenge. Challenged lungs over-expressed both MMP-9 and TIMP-1 mRNAs with the over-expression of MMP-9 being Gr-1+ cell-dependent. Additionally, the enzymatic activities of MMP-9 and collagenase were reduced in lungs after Gr-1+ cells were depleted. Treatment of mice with inhibitors of MMP-8 and MMP-9 reduced antigen-induced recruitment of Th1 and Th2 cells, resulting in significantly reduced eosinophilic airway inflammation. CONCLUSIONS: These data support a model in which airway antigen challenge activates Gr-1+ cells and consequently MMPs to support the recruitment of Th1 and Th2 cells in the airway inflammatory response. Funding: NIH
953
Functional and Phenotypic Characterization of CD8+CD28T Cells in Atopic and Non-Atopic Individuals O. M. M. Lourenco, A. R. S. Rafael, A. M. L. Fonseca, L. M. P. Taborda-Barata; Department of Medical Sciences, University of Beira Interior, Health Sciences Research Centre, Covilha, PORTUGAL. RATIONALE: The aim of the present study was to analyse possible differences in phenotype and functional properties of human CD8+CD28- T cells upon allergen-specific PBMC proliferation between atopic and non-atopic individuals. METHODS: Peripheral blood was obtained from 44 atopic subjects and 40 non-atopic controls. Initial phenotypic studies were performed using flow cytometry in whole blood, gradient-separated lymphocytes and immunomagnetic-isolated T cells. Phenotypic markers studied included NK cell markers, chemokine receptors, activation markers as well as and TCR chains. Purified CD8+CD28+ or CD8+CD28 T cells were mixed with PBMC at different ratios (1:1, 1:2 and 1:4) and stimulated with the allergen Dermatophagoides pteronyssinus (Der p) or PHA in 6-day co-cultures. Proliferation was analysed using thymidine incorporation. RESULTS: Of all markers studied, only expression of TCR on CD8+CD28- T cells was different between atopic and non-atopic volunteers. In terms of allergen-induced proliferation, CD8+CD28- T cells significantly increased proliferation of PBMC (p < 0.05; Wilcoxon-signed rank test) but only in non-atopic individuals. CONCLUSIONS: These preliminary data suggest that peripheral blood CD8+CD28- T cells from non-atopic individuals may contribute to proliferation of PBMC in response to allergens that act as recall antigens. However, this effect should be further correlated with effects upon cytokine profiles. Funding: Fundacao Para a Ciencia e a Tecnologia
954
952
MONDAY
CD4+CD25++ T Cells in Peripheral Blood Correlate with Total and Pollen-Specific IgE in Young Children K. A. Burmeister1, C. Seroogy2, C. Tisler1, M. Evans3, L. Franco1, L. Pleiss1, R. Gangnon3, R. F. Lemanske, Jr.2, J. E. Gern2; 1Dept of Pediatrics, University of Wisconsin-Madison, Madison, WI, 2Dept of Medicine, University of Wisconsin-Madison, Madison, WI, 3Dept of Biostatistics and Medical Informatics and Population Health Sciences, University of Wisconsin-Madison, Madison, WI. RATIONALE: There is evidence that CD4+CD25++ T-regulatory cells help to establish tolerance to allergens in childhood. METHODS: To test this theory, a cohort of 6-year-old children (n=46) at risk for developing allergy and asthma was analyzed for the presence of T-regulatory cells by flow cytometry and the results were compared to clinical and biological markers of atopy. The percentage of T-regulatory cells (CD3+CD4+CD25++) in PBMC was estimated by flow cytometry. Total and allergen-specific IgE in plasma were determined by fluoroenzyme immunoassays (Unicap 100, Pharmacia and Upjohn Diagnostics). RESULTS: There was a highly significant correlation between percent CD4+CD25++ T cells and total IgE levels (r=0.57, p<0.0001). In addition, mean CD4+CD25++ T cell numbers were higher in children with IgE specific for ragweed (p=0.046) and birch (p=0.039), with similar trends (p<0.1) for children with IgE to egg and cat. There was no relationship between percent CD4+CD25++ T cells and atopic dermatitis (p = 0.82), wheezing (p = 0.16), or sensitization to alternaria (p=0.26) or house dust mite (p=0.25 dp and p=0.65 df). CONCLUSIONS: The strong positive correlation between CD4+CD25++ cells and total and pollen-specific IgE in young children does not support a straightforward relationship between T-regulatory cell numbers and allergen tolerance. Additional studies are in progress to determine the relationships between IgE and T regulatory cell functional activity, and the expression of FOXP3 and other associated genes. Funding: NIH