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Blood, Vol. 113, Issue 22, 5624-5627, May 28, 2009
Regulatory T-cell status in red cell alloimmunized responder and nonresponder mice
Blood Bao et al.
113: 5624
Supplemental materials for: Bao et al
Files in this Data Supplement:
- Figure S1. Groups of mice (n=3, 2–3 mice per group, 3 separate experiments) were injected with CpG ODN and after six weeks (JPG, 34 KB)
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Their splenic CD4+CD25– (effector) and CD4+CD25+ (suppressor) cells were sorted and stimulated with their accessory cells plus anti-CD3 antibodies alone or cocultured at varying suppressor/effector ratios (1:0, 1:1, 1:2, 1:4, 1:8, and 1: 16). Proliferation was measured by incorporation of 3Hthymidine. The mean percent proliferative responses of stimulated CD4+CD25– cells in the presence of autologous CD4+CD25+ cells (dashed line, open squares) was calculated as (cpm incorporated in the coculture)/cpm of effector cells alone) × 100%. For comparison, percent proliferation of CD4+CD25− from responders (filled circles) and non-responders (open circles) in presence of autologous CD4+CD25+ cells from Fig. 2C is also shown. The suppressive activity of mice injected with CpG ODN alone was statistically different from both responder and non-responders at suppressor/effector ratios of 1:4 (compared to responder, p=0.003; compared to non-responder, p=0.003) and at 1:16 (compared to responder, p=0.03; compared to non-responder, p=0.006).
- Figure S2. Mice were adoptively transferred with 106 sorted splenic CD4+CD25− from responder and non-responder mice (JPG, 43.1 KB)
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After 24 hours, the mice were given intravenous transfusions of buffy-coated/granulocyte depleted huGPA red cells (equivalent to 1–2 packed units) with CpG-ODN adjuvant followed by weekly transfusions of red cells alone for another 3 weeks. After that, levels of alloantibodies (IgG) in the plasma was measured using diluted plasma (1 in 4) followed by analysis using flow cytometry and is expressed in fluorescent units on the y axis. Levels of alloanti-huGPA in untransfused (“control”) and non-adoptively–transferred transfused mice (“RBC transfused”) from Fig. 1A is also shown. Levels of alloantibodies in adoptively transferred mice from either responder or non-responder effector T cells were not statistically different from those of mice transfused with huGPA red cells alone (p>0.05).
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