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Blood, Vol. 113, Issue 19, 4575-4585, May 7, 2009
The molecular signature of CD8+ T cells undergoing deletional tolerance
Blood Parish et al.
113: 4575
Supplemental materials for: Parish et al
T-cell enrichment, CFSE labeling and ppERK1/2 staining
CD8+ T cells were enriched from spleen and lymph nodes by incubating single cell suspensions with monoclonal Abs against Mac-1 (M1/70), macrophages (F4/80), red blood cells (Ter119), Gr1 (RB6-8C5), MHC class II (M5/114), and CD4 (GK1.5) on ice for 30 min. The Ab-labeled cells were then depleted using goat anti-rat IgG antibody-coupled magnetic beads (QIAGEN). For microarray transfers, CD44hi cells were depleted at this point with CD44-PE antibody and anti-PE antibody coated MACS beads using an AutoMACS machine (Miltenyi Biotech, Germany) according to manufacturer’s instructions. Purified CD8+ T cells were CFSE labeled in PBS (containing 0.1% bovine serum albumin (Sigma-Aldrich, MO)) with 5 µM CFSE (Invitrogen, CA) at 37°C for 10 min. For intracellular ppERK1/2 staining, lymph node (RIP-OVAhi) or spleen (OCS/LPS) cell suspensions were stimulated with either 1 µg/mL OVA257-264 peptide (AusPep, Australia) or 100 ng/mL PMA (Sigma-Aldrich) for 5 min. Cells were then fixed and permeabilised using the BD PhosFlow Lyse/Fix buffer and BD PhosFlow Perm Buffer III (BD Biosciences, CA) according to manufacturer’s instructions, and analyzed by flow cytometry. Real-time PCR validation
For real-time PCR validation, cDNA was prepared from cRNA using the SuperScript™ III Reverse Transcriptase Kit (Invitrogen). TaqMan® real-time PCR was performed in duplicate according to manufacturer’s guidelines using the appropriate Taqman probes (Applied Biosystems). RNA levels were normalised by quantitative PCR for the house-keeping gene, Protein Kinase C-Iota. Threshold cycle (Ct) values from the PCR amplification plots were converted to arbitrary copy number using the formula 105/2(Ct-17) (where a Ct value of 17 was set to 105 copies) and mRNA changes were expressed as fold change relative to naïve cells.
Files in this Data Supplement:
- Table S1. Genes up-regulated in tolerance (unfiltered) (PDF, 114 KB) -
Only genes that were significantly up-regulated in Tol.Naïve, but not Imm.Naïve or Rag.Naïve, are shown in this table. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. “NA” in the Gene Symbol column indicates probesets for which there is no gene annotation.
- Table S2. Genes down-regulated in tolerance (unfiltered) (PDF, 107 KB) -
Only genes that were significantly down-regulated in Tol.Naïve, but not Imm.Naïve or Rag.Naïve, are shown in this table. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. “NA” in the Gene Symbol column indicates probesets for which there is no gene annotation.
- Table S3. Changes in apoptosis related genes (PDF, 111 KB) -
p-values were calculated independently for this gene group as described in the methods. Bold numbers represent a statistically significant change in gene expression. “No shifts” means that no statistically significant shifts were observed within any of the comparisons listed for the probeset(s) in question. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. * means that the shift was significantly greater in Tol.Naïve relative to either Imm.Naïve or Rag.Naïve (ie. exhibited statistically significant up-regulation in both the Tol.Imm and Tol.Rag comparisons). Bold gene symbols highlight those genes up-regulated to a greater degree in Tol.Naïve (in one or more probesets) than in the other two comparisons.
- Table S4. Changes in genes up-regulated by Nur77 over-expression (PDF, 53.4 KB) -
p-values were calculated independently for this gene group as described in the
methods. Bold numbers represent a statistically significant change in gene expression. “No shifts” means that no statistically significant shifts were observed within any of the comparisons listed for the probeset(s) in question. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. * means that the shift was significantly greater in Tol.Naïve relative to either Imm.Naïve or Rag.Naïve (ie. exhibited statistically significant up-regulation in both the Tol.Imm and Tol.Rag comparisons). Bold gene symbols highlight those genes up-regulated to a greater degree in Tol.Naïve (in one or more probesets) than in the other two comparisons, ie. genes that are up-regulated in both deletion and the Nur77 over-expression gene group.
- Table S5. Changes in genes up-regulated in exhausted CD8+ T cells (PDF, 133 KB) -
p-values were calculated independently for this gene group as described in the
methods. Bold numbers represent a statistically significant change in gene expression. “No shifts” means that no statistically significant shifts were observed within any of the comparisons listed for the probeset(s) in question. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. * means that the shift was significantly greater in Tol.Naïve relative to either Imm.Naïve or Rag.Naïve (ie. exhibited statistically significant up-regulation in both the Tol.Imm and Tol.Rag comparisons). Bold gene symbols highlight those genes up-regulated to a greater degree in Tol.Naïve (in one or more probesets) than in the other two comparisons, ie. genes that are up-regulated in both deletion and exhaustion.
- Table S6. Changes in genes down-regulated in exhausted CD8+ T cells (PDF, 99.5 KB) -
p-values were calculated independently for this gene group as described in the methods. Bold numbers represent a statistically significant change in gene expression. “No shifts” means that no statistically significant shifts were observed within any of the comparisons listed for the probeset(s) in question. The numbers represent Log (base 2) fold change. The comparison abbreviations Tol.Naïve, Imm.Naïve and Rag.Naïve have the same meaning as in Fig. 2. * means that the shift was significantly greater in Tol.Naïve relative to either Imm.Naïve or Rag.Naïve (ie. exhibited statistically significant down-regulation in both the Tol.Imm and Tol.Rag comparisons). Bold gene symbols highlight those genes down-regulated to a greater degree in Tol.Naïve (in one or more probesets) than in the other two comparisons, ie. genes that are down-regulated in both deletion and exhaustion.
- Figure S1. Real-time PCR confirmation of genes differentially expressed during deletion (JPG, 73.8 KB)
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cRNA used for array hybridisation was used for real-time PCR validation of some deletion-specific gene expression changes seen within the both the filtered (A to C) and unfiltered (D) datasets. Genes examined are Egr2 (A), Nr4a1 (or Nur77; B), Nr4a3 (or Nor1; C) and Cblb (D). The bar graphs show gene expression changes relative to Naïve and represent mean data ± SEM from the duplicate array samples (with the exception of the Rag sample, where there was only a single replicate). Naïve = Naïve cells, Tol = Deleted cells, Imm = Primed cells and Rag = Cells undergoing lymphopenia-induced proliferation.
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