Maleewan Kitcharoensakkul1, Leonard B Bacharier1, Huiqing Yin-Declue2, Jonathan S Boomer2, Dana Burgdorf2, Brad Wilson3, Kenneth Schechtman3, Mario Castro4. 1. Division of Allergy, Immunology and Pulmonary Medicine, Department of Pediatrics, St. Louis Children's Hospital, Washington University School of Medicine, United States. 2. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, United States. 3. Division of Biostatistics, Washington University School of Medicine, United States. 4. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, United States. Electronic address: castrom@dom.wustl.edu.
Abstract
BACKGROUND: Studies evaluating circulating dendritic cells (DCs) and natural and induced regulatory T cells (nTregs, iTregs) are often obtained at a single time point and difficult to interpret without understanding their intrinsic day-to-day biologic variability. METHODS: We investigated the day-to-day variability in quantifying DCs, nTregs (FoxP3(+)CD25(+)CD4(+)) and cytokine production by iTregs (granzyme B-GZB, Th1/2 cytokines following CD3 plus CD46 in vitro activation) from peripheral blood mononuclear cells (PBMCs) collected on three consecutive days in healthy adults. Intraclass correlation coefficients (ICCs) were used to evaluate intra-individual variability. RESULTS: In 10 healthy adults, the %PBMCs of plasmacytoid (pDC) and myeloid (mDC1 and mDC2) were 0.27 ± 0.12, 0.22 ± 0.10, and 0.02 ± 0.02, with ICC 0.91, 0.90, and 0.17 respectively. Natural Tregs (3.27 ± 1.27% CD4(+) cells) had an ICC of 0.86. Inducible Tregs (GZB-positive, 35.3 ± 17.7% CD4(+) cells) had an ICC of 0.77. The ICCs for IL-10, TNF-α, IFN-γ, IL-4, and IL-5 production by iTregs were 0.49, 0.63, 0.68, 0.74, and 0.82, respectively. There were no significant changes in ICC (<0.1) after adjusting for age, gender and atopy except for IL-4. Substantial variability for iTregs was determined for the control condition (PBS with IL-2). CONCLUSIONS: No meaningful day-to-day biologic variability was observed for the quantification of nTregs, pDC and mDC1 in normal adults; however, there was substantial variability in measuring mDC2 proportions and iTreg production of IL-10. These results suggest obtaining an average of several measurements over time to determine the most representative value of these biologic measures.
BACKGROUND: Studies evaluating circulating dendritic cells (DCs) and natural and induced regulatory T cells (nTregs, iTregs) are often obtained at a single time point and difficult to interpret without understanding their intrinsic day-to-day biologic variability. METHODS: We investigated the day-to-day variability in quantifying DCs, nTregs (FoxP3(+)CD25(+)CD4(+)) and cytokine production by iTregs (granzyme B-GZB, Th1/2 cytokines following CD3 plus CD46 in vitro activation) from peripheral blood mononuclear cells (PBMCs) collected on three consecutive days in healthy adults. Intraclass correlation coefficients (ICCs) were used to evaluate intra-individual variability. RESULTS: In 10 healthy adults, the %PBMCs of plasmacytoid (pDC) and myeloid (mDC1 and mDC2) were 0.27 ± 0.12, 0.22 ± 0.10, and 0.02 ± 0.02, with ICC 0.91, 0.90, and 0.17 respectively. Natural Tregs (3.27 ± 1.27% CD4(+) cells) had an ICC of 0.86. Inducible Tregs (GZB-positive, 35.3 ± 17.7% CD4(+) cells) had an ICC of 0.77. The ICCs for IL-10, TNF-α, IFN-γ, IL-4, and IL-5 production by iTregs were 0.49, 0.63, 0.68, 0.74, and 0.82, respectively. There were no significant changes in ICC (<0.1) after adjusting for age, gender and atopy except for IL-4. Substantial variability for iTregs was determined for the control condition (PBS with IL-2). CONCLUSIONS: No meaningful day-to-day biologic variability was observed for the quantification of nTregs, pDC and mDC1 in normal adults; however, there was substantial variability in measuring mDC2 proportions and iTreg production of IL-10. These results suggest obtaining an average of several measurements over time to determine the most representative value of these biologic measures.
Authors: Eli Silver; Huiqing Yin-DeClue; Ken B Schechtman; Mitchell H Grayson; Leonard B Bacharier; Mario Castro Journal: Pediatr Allergy Immunol Date: 2008-12-22 Impact factor: 6.377
Authors: Edward Morgan; Rudi Varro; Homero Sepulveda; Julia A Ember; John Apgar; Jerry Wilson; Larry Lowe; Roy Chen; Lalita Shivraj; Anissa Agadir; Roberto Campos; David Ernst; Amitabh Gaur Journal: Clin Immunol Date: 2004-03 Impact factor: 3.969