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Blood, Vol. 113, Issue 3, 622-625, January 15, 2009
Impaired negative regulation of homeostatically proliferating T cells
Blood Shvets et al.
113: 622
Supplemental materials for: Shvets et al
Mice. Female C57BL/6 mice (5–7 weeks old), or Thy1 or CD45 congenic strains, were either purchased from Charles River (Montreal, Quebec) or from Jackson Laboratories. Use of mice in this study was approved by the animal review committees of our institutes.
Construction of B7-H4/Ig expression vector. B7-H4/Ig cDNA and expression plasmid were constructed by techniques previously described.24
Adoptive transfer. Lymphopenia was induced by sublethal irradiation of mice at 600cGy (Cesium 137 Irradiator, GC 1000 SN 1258) one day before the adoptive transfer of cells. Unless otherwise mentioned, freshly isolated spleen cells (5 × 107 cells) from normal mice were adoptively transferred into syngeneic lymphopenic mice.
Effects of anti–CTLA-4 and B7-H4/Ig on T-cell activation. Unless stated otherwise, freshly isolated spleen cells from normal mice were transfused into lymphopenic mice, and spleen cells were recovered at various time points. T cells were prepared and cultured as previously described,22 and subpopulations sorted at > 97% purity with MagCellect kits (R&D systems). To examine CTLA-4–mediated suppression, T cells were stimulated with plate-bound hamster anti-CD3ε (Pharmingen) and hamster anti-CD28 (Pharmingen), to induce CTLA-4 expression. These cells were collected and restimulated with anti-CD3 in the presence of IgG or anti–CTLA-4 mAb (eBioscience), all immobilized in 96-well plate pre-coated with anti-hamster IgG. Supernatants were collected for ELISA cytokine assays and cell proliferation determined by MTT assay.
To examine the effects of B7-H4/Ig on CD4+ T cells, 96-well round bottom plates were coated with rabbit anti-hamster IgG (Sigma). A mixture of anti-CD3ε and anti-CD28 mAb was the added to the wells along with B7-H4/Ig or control mouse IgG1. B7-H4/Ig also binds to this rabbit anti-hamster IgG, because of cross-reactivity against mouse IgG1-Fc.
Expression of PD-1, CTLA-4, and Foxp3. In some experiments, freshly isolated spleen cells from normal mice were transfused into lymphopenic mice, and spleen cells were recovered at various time points. For induction of PD-1, CTLA-4, and Foxp3 expression, T cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 48 hours, stained for CTLA-4 (cell surface and intracellular), PD-1, and FoxP3 with fluorochrome-conjugated anti–CTLA-4 (clone UC10-489) anti–PD-1 (clone J43) and anti-Foxp3 (clone FJK-16a), and analyzed by flow cytometry. In some other experiments, lymph node T cells were isolated, activated in vitro, labeled with CFSE, and transferred into lymphopenic mice. Two weeks latter T cells were isolated and analyzed for CFSE and PD-1 expression. The stained cells were analyzed by flow cytometry as previously described.1, 25
Statistical analysis. Two-tailed T tests were performed, and p < 0.05 was considered significant. All analyses were performed with GraphPad Prism 3.03 software (San Diego, CA).
Files in this Data Supplement:
- Figure S1. Increase in T-cell populations over time during HP (JPG, 24.2 KB)
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Freshly isolated spleen cells from normal mice were transfused into irradiated mice. At various time points, spleen cells were isolated and analyzed for CD3+, CD8+ and CD4+ subpopulations by flowcytometry. ■, CD3+ cells; ▲,CD4+ cells; ▼, CD8+ cells (results representative of 3 experiments).
- Figure S2. Splenic CD4+CD25− T cells recovered 2 or 3 weeks after cell transfer and stimulated with plate-bound anti-CD3/CD28 (JPG, 26.7 KB)
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After 48 hours, MTT was added (1mg/ml) and incubated for 4 hours. Formazan crystals were solubilized with isopropanol and OD was read at 540 nmλ (OD540). Cell proliferation was expressed as the proliferation index = OD540 of stimulated cells/OD540 of unstimulated cells. HP T-cell proliferation was not significantly different from normal T cells (p > 0.05). The results are the mean ± SEM of 3 mice.
- Figure S3. PD-1 expression is downregulated in the lymphopenic environment in CD4+ and CD8+ T cells (JPG, 63.2 KB)
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(A) T-cell activation upregulates PD-1 in CD4+ and CD8+ T cells. LN T cells from B6.CD45.1 mice were stimulated in vitro for 48 hrs with immobilized anti-CD3/CD28 antibodies, and analyzed by flow cytometry for the expression of PD-1. (B) Activated CD4+ and CD8+ T cells downregulate PD-1 expression during HP. LN T cells from B6.CD45.1 donors were activated in vitro as described in A, and transferred to irradiated (600 rad) B6.CD45.2 recipients (n=3). Two weeks later, PD-1 expression was examined in donor T cells by flow cytometry, after gating in the CD45.1+CD4+ and CD45.1+CD8+ cell populations.
- Figure S4. Homeostatic T-cell proliferation promotes anti-tumor responses (JPG, 30.7 KB)
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As we have previously described (Ref. 1), C57BL/6 (B6) mice (n=10–12/group) were irradiated (or not) with 6 Gy to induce lymphopenia on day −1, challenged with 5 × 105 B78D14 melanoma cells (s.c.) on day 0, and transfused (or not) with 5 × 106 LN cells from unmanipulated B6 mice on day 1. Tumor volume was determined using a caliper and the formula 1/2 × length × (width)2, and expressed as Mean ± SEM. Tumor growth was significantly suppressed (p < 0.05) in lymphopenic versus control mice.
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