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Blood, Vol. 109, Issue 3, 1086-1094, February 1, 2007
Early acquisition of cytolytic function and transcriptional changes in a primary CD8+ T-cell response in vivo
Blood Chiu et al.
109: 1086
Supplemental materials for: Chiu et al, Vol 109, Issue 3, 1086-1094
Files in this Data Supplement:
- Table S1. Differential gene expression levels of genes in NP366-specific CTLs 24 hours after adoptive transfer compared with NP366-specific CTLs from naive F5 Rag–/– mice (PDF 18.6 KB) -
Raw fluorescence data was converted to expression values using MAS, RMA, and dChip, then normalized using GeneSpring, RMA, and dChip. Genes with a raw expression value below 150 were excluded. Genes with differential expression greater than 2-fold using each normalization algorithm were selected, and only those genes fulfilling these criteria in all 3 normalization strategies are displayed. Fold change values are derived from RMA.
- Table S2. Differential gene expression levels of functional groups in NP366-specific CTLs at 96 hours after stimulation compared with unstimulated controls (PDF, 30.2 KB) -
Raw fluorescence data were converted to expression values using MAS, RMA, and dChip, then normalized using GeneSpring, RMA, and dChip. Genes with a raw expression value below 150 were excluded. Genes with differential expression greater than 2-fold using each normalization algorithm were selected, and only those genes fulfilling these criteria in all 3 normalization strategies are displayed. Fold change values are derived from RMA.
- Table S3. Cluster analysis of transcriptional profiles of NP366-specific CTLs up to 96 hours after stimulation compared with unstimulated control (PDF, 48.6 KB) -
Raw fluorescence data were converted to expression values using MAS, RMA, and dChip, then normalized using GeneSpring, RMA, and dChip. Genes with a raw expression value below 150 were excluded. Genes with differential expression greater than 4-fold compared with the unstimulated control were selected. Those genes fulfilling these criteria in all 3 normalization strategies were further analyzed by K-means clustering.
- Table S4. Pathways analysis of differentially expressed genes during the 96-hour time course clustered by K-means clustering (PDF, 18.6 KB) -
Raw fluorescence data were converted to expression values using MAS, RMA, and dChip, then normalized using GeneSpring, RMA, and dChip. Genes with a raw expression value below 150 were excluded. Genes with differential expression greater than 2-fold compared with the unstimulated control were selected. Those genes fulfilling these criteria in all 3 normalization strategies were clustered using K-means. The resulting genes were analyzed using ArrayXPath and assigned to known Biocarta functional pathways. The number of assigned genes identified is expressed as a proportion of the number of unique (and redundant in brackets) genes in the pathway. P is calculated using the Fisher exact test.
- Table S5. Genes with more than 2-fold differential expression compared with unstimulated control in NP366-specific CTLs during a 96-hour time course (PDF, 17.7 KB) -
Raw fluorescence data were converted to expression values using MAS, RMA, and dChip, then normalized using GeneSpring, RMA, and dChip. Genes with a raw expression value below 150 were excluded. Genes with differential expression greater than 2-fold using each normalization algorithm were selected and only those genes fulfilling these criteria in all 3 normalization strategies were further analyzed. The resulting list was probed for genes with functions relating to (A) transcription factors, (B) cell cycle, (C) cytokines and chemokines, (D) cytokine receptors, (E) chemokine receptors, and (F) cytolytic functions.
- Figure S1. Analysis of extralymphoid tissue compartments reveals no reciprocal increase in epitope-specific CTL numbers at 24 hours after stimulation (PDF, 31.8 KB) -
CFSE-labeled splenocytes (107)were transferred from F5 Rag–/– to C57BL/6 mice and the recipients infected 24 hours later with 107 PFU recombinant vaccinia virus expressing NP366-374 by intraperitoneal injection. Epitope-specific cells were tracked using CFSE and tetramer-PE in spleen, blood, abdominal lymph node, liver, and lung. The size of the transferred epitope-specific population is shown as percentage of the total cells. Plots are representative of at least 3 individuals.
- Figure S2. Analysis of the ungated FACS data following stimulation of F5 Rag–/– splenocytes by rVV-NP366 demonstrates no drop in cell numbers at 24 hours after stimulation (PDF, 1.04 MB) -
CFSE-labeled splenocytes (107) were transferred from F5 Rag–/– to C57BL/6 mice and the recipients infected 24 hours later with 107 PFU recombinant vaccinia virus expressing NP366-374 by intraperitoneal injection. Epitope-specific cells were tracked using CFSE and tetramer-PE. The size of the transferred epitope-specific population is shown as percentage of the total cells. All plots show data representative of at least 3 individuals and are ungated.
- Figure S3. Quantitative PCR data correlate closely with microarray expression profiles emphasizing large-scale changes in expression prior to the peak proliferative response at 96 hours after stimulation (PDF, 48.2 KB) -
F5 Rag–/– splenocytes were transferred by intravenous injection into C57BL/6 mice that were infected 24 hours later with recombinant vaccinia virus expressing NP366-374. Spleens were collected from these mice at time points up to 192 hours after infection and epitope-specific cells sorted by flow cytometry. Quantitative PCR of 14 selected genes (dotted lines) was performed using the Roche Lightcycler system. Gene expression ratios were calculated using ALAS1 as an housekeeping gene. Graphs represent data from 3 mice per time point. The corresponding microarray expression profile is shown for comparison (solid lines).
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