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Su.86. Automated High-dimensional Flow Cytometric Data Analysis
S152 d-7) the administration of SU5416 (d0). To determine whether there is a threshold time period during which the splenocytes must be applied in order to prevent SPH, i.e. the protection of SPH by splenocyte in the athymic rats are timedependent, we are injecting euthymic rats (rnu/+) originated splenocytes into athymic rats (rnu/rnu) at -7, - 3, 0, 3, 7, 10, 14 days relative to SU5416 administration to evaluate whether cell-based protection strategies have a therapeutic window. Echocardiographic, physiologic, immunohistochemical and western blot results will analyze phenotypic changes occurring around these time periods to best assess changes in conferred protection. doi:10.1016/j.clim.2008.03.435
Su.85. Preliminary Analytical Performance of Natural T Regulatory Cell Assays using CD127 and hFoxP3 Christopher Boyce, Ravi Hingorani, Jason Vidal, Li Li. BD-Biosciences, San Diego, CA Expression of human CD127 and CD25 on CD4 natural Tregs (nTreg) correlates well with expression of FoxP3 in the same cells. Using CD127dim surface, rather than FoxP3 intracellular expression to identify nTregs provides advantages for ex vivo and in vitro assays. It is known that Tregs can be more efficiently sorted using CD127, rather than by CD25 alone. However, less well defined is the performance of CD127 direct immune monitoring assay in peripheral blood mononuclear cells (PBMC) or Whole Blood (WB). We studied analytical performance and methodology using CD127 (hIL7R-M21.3) and/or Foxp3 (259D) employing rare-event flow cytometry model protocols. An absolute counting approach was also evaluated. Evalutions of PBMC and WB from normal donors are ongoing. We demonstrate the intraassay precision, the biological range and variability, and feasibility of a CD4/CD25/CD127 cocktail and/or FoxP3 for nTreg enumeration. We identified nTreg populations via typical 259D staining or by the phenotype CD4+CD25+/++ CD127dim for comparison. Results indicate the CD127dim population correlates well to FoxP3+ expression (N N 4). The CD4+ CD25+/++CD127dim population shows a biological % positive mean = 6.8, with range of 5.5-8.9 and SD = 1.3. While CD4+CD25+/++CD127dim nTregs also showed intra-assay precision of a mean %CV = 6.1, median %CV = 4.3, with a %CV range 0.7-10.2 (N N 8). CD127 is therefore a useful surface marker for evaluation of nTreg cells. CD127 is for research use only and is not intended for diagnostic purposes. doi:10.1016/j.clim.2008.03.436
Su.86. Automated High-dimensional Flow Cytometric Data Analysis Xinli Hu,1 Saumyadipta Pyne,2 Elizabeth Rossin,1 Pablo Tamayo,2 David Hafler,3 Jill Mesirov,2 Philip De Jager. 3 1 Broad Institute of Harvard and MIT, Cambridge, MA; 2The Broad Institute of Harvard and MIT, Cambridge, MA; 3 Brigham and Women's Hospital, Boston, MA
Abstracts FACS has matured as a sophisticated cytometric tool allowing simultaneous detection of a large number of markers. However, current methods of FACS data analysis rely on manual or semi-automatic gating based on 2D visualization and pre-determined regions of interest, which quenches the potential of the high-dimensional nature of FACS data. FLow analysis with Automated Multivariate Estimation (FLAME) is a new tool which automatically clusters and analyzes high-throughput cytometric data with minimal operator interference. FLAME models the natural mixture of heterogeneous populations coexisting within biological samples as a mixture of mathematical distributions. The parameters (e.g. mean, variance, frequency, etc.) of each component describe the features of each population. We extract features that best characterize and distinguish samples belonging to different classes (e.g. healthy vs. diseased), thus enabling classification of new samples. Recognizing that cytometric data often do not follow Gaussian distributions, we incorporate Student's-t, skewnormal, and skew-t distributions for optimal fitting. In three different applications of FLAME, we demonstrate that (1) modeling enhances phenotyping of HLA DQ expression in 3 genotypic classes of 270 lymphoblastic cell lines, (2) its automated clustering algorithm discovers a 3.7% subset of CD4+CD25highHLADR+FoxP3+ regulatory T cells in PBMCs, and (3) its meta-clustering of cell populations across different classes of human subjects reveals differences in phosphorylation events in naïve and memory T cells. Marked by complete automation, FLAME is a statistically rigorous but flexible tool with a wide array of potential applications stemming from the identification of known cell populations and the discovery of new cell populations. doi:10.1016/j.clim.2008.03.437
Su.87. Suppression of T Cell Cytokine Expression in Short-term Assays as an Indicator of Regulatory T Cell Functional Potential Joyce Ruitenberg, Smita Ghanekar. BD Biosciences, San Jose, CA Human regulatory T cells (Treg) play a critical role in the induction and maintenance of peripheral immune tolerance. Phenotypically, Treg are characterized as T cells that are CD4+, CD25++, CD127- and FoxP3+. We have demonstrated that highly purified and in vitro expanded Treg are capable of suppressing T-cell specific cytokine responses in 7 hr intracellular cytokine assays. For practical reasons, the short term assay would seem to be a more valuable tool for assessing the potency of Treg compared to the routinely used 5-day proliferation assays. In this study, the ability of Treg to inhibit expression of inflammatory cytokines (IFNγ, TNFα, and IL2) and CD69 by autologous T cells, was evaluated to optimize Treg (effector) and PBMC (responder) co-culture times, Treg to PBMC ratio, and the relationship between Treg potency (cytokine suppression) and the amount of FoxP3 expressed by the Treg. The Treg were purified from healthy donor PBMC by sorting the CD4+ CD25++ CD127- cells with a BD FACSAria flow cytometer. The functional ability of the highly purified Treg was evaluated either after activation for
Su.85. Preliminary Analytical Performance of Natural T Regulatory Cell Assays using CD127 and hFoxP3 Christopher Boyce, Ravi Hingorani, Jason Vidal, Li Li. BD-Biosciences, San Diego, CA Expression of human CD127 and CD25 on CD4 natural Tregs (nTreg) correlates well with expression of FoxP3 in the same cells. Using CD127dim surface, rather than FoxP3 intracellular expression to identify nTregs provides advantages for ex vivo and in vitro assays. It is known that Tregs can be more efficiently sorted using CD127, rather than by CD25 alone. However, less well defined is the performance of CD127 direct immune monitoring assay in peripheral blood mononuclear cells (PBMC) or Whole Blood (WB). We studied analytical performance and methodology using CD127 (hIL7R-M21.3) and/or Foxp3 (259D) employing rare-event flow cytometry model protocols. An absolute counting approach was also evaluated. Evalutions of PBMC and WB from normal donors are ongoing. We demonstrate the intraassay precision, the biological range and variability, and feasibility of a CD4/CD25/CD127 cocktail and/or FoxP3 for nTreg enumeration. We identified nTreg populations via typical 259D staining or by the phenotype CD4+CD25+/++ CD127dim for comparison. Results indicate the CD127dim population correlates well to FoxP3+ expression (N N 4). The CD4+ CD25+/++CD127dim population shows a biological % positive mean = 6.8, with range of 5.5-8.9 and SD = 1.3. While CD4+CD25+/++CD127dim nTregs also showed intra-assay precision of a mean %CV = 6.1, median %CV = 4.3, with a %CV range 0.7-10.2 (N N 8). CD127 is therefore a useful surface marker for evaluation of nTreg cells. CD127 is for research use only and is not intended for diagnostic purposes. doi:10.1016/j.clim.2008.03.436
Su.86. Automated High-dimensional Flow Cytometric Data Analysis Xinli Hu,1 Saumyadipta Pyne,2 Elizabeth Rossin,1 Pablo Tamayo,2 David Hafler,3 Jill Mesirov,2 Philip De Jager. 3 1 Broad Institute of Harvard and MIT, Cambridge, MA; 2The Broad Institute of Harvard and MIT, Cambridge, MA; 3 Brigham and Women's Hospital, Boston, MA
Abstracts FACS has matured as a sophisticated cytometric tool allowing simultaneous detection of a large number of markers. However, current methods of FACS data analysis rely on manual or semi-automatic gating based on 2D visualization and pre-determined regions of interest, which quenches the potential of the high-dimensional nature of FACS data. FLow analysis with Automated Multivariate Estimation (FLAME) is a new tool which automatically clusters and analyzes high-throughput cytometric data with minimal operator interference. FLAME models the natural mixture of heterogeneous populations coexisting within biological samples as a mixture of mathematical distributions. The parameters (e.g. mean, variance, frequency, etc.) of each component describe the features of each population. We extract features that best characterize and distinguish samples belonging to different classes (e.g. healthy vs. diseased), thus enabling classification of new samples. Recognizing that cytometric data often do not follow Gaussian distributions, we incorporate Student's-t, skewnormal, and skew-t distributions for optimal fitting. In three different applications of FLAME, we demonstrate that (1) modeling enhances phenotyping of HLA DQ expression in 3 genotypic classes of 270 lymphoblastic cell lines, (2) its automated clustering algorithm discovers a 3.7% subset of CD4+CD25highHLADR+FoxP3+ regulatory T cells in PBMCs, and (3) its meta-clustering of cell populations across different classes of human subjects reveals differences in phosphorylation events in naïve and memory T cells. Marked by complete automation, FLAME is a statistically rigorous but flexible tool with a wide array of potential applications stemming from the identification of known cell populations and the discovery of new cell populations. doi:10.1016/j.clim.2008.03.437
Su.87. Suppression of T Cell Cytokine Expression in Short-term Assays as an Indicator of Regulatory T Cell Functional Potential Joyce Ruitenberg, Smita Ghanekar. BD Biosciences, San Jose, CA Human regulatory T cells (Treg) play a critical role in the induction and maintenance of peripheral immune tolerance. Phenotypically, Treg are characterized as T cells that are CD4+, CD25++, CD127- and FoxP3+. We have demonstrated that highly purified and in vitro expanded Treg are capable of suppressing T-cell specific cytokine responses in 7 hr intracellular cytokine assays. For practical reasons, the short term assay would seem to be a more valuable tool for assessing the potency of Treg compared to the routinely used 5-day proliferation assays. In this study, the ability of Treg to inhibit expression of inflammatory cytokines (IFNγ, TNFα, and IL2) and CD69 by autologous T cells, was evaluated to optimize Treg (effector) and PBMC (responder) co-culture times, Treg to PBMC ratio, and the relationship between Treg potency (cytokine suppression) and the amount of FoxP3 expressed by the Treg. The Treg were purified from healthy donor PBMC by sorting the CD4+ CD25++ CD127- cells with a BD FACSAria flow cytometer. The functional ability of the highly purified Treg was evaluated either after activation for