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Blood, Vol. 105, Issue 5, 2206-2213, March 1, 2005
Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells
Blood Kim et al.
105: 2206
Supplemental materials for: Kim et al, Vol 105, Issue 5, 2206-2213
Files in this Data Supplement:
- Figure S1. Effect of 3H3 treatment on host splenocyte populations in cGVHD. (PDF, 38 KB) -
cGVHD was induced and mice were treated with antibody as described in Methods. Spleens were collected on days 5, 14 and 21 after disease induction, and analyzed for (A) host CD4+ and (B) CD8+ T cells, and (C) granulocytes. Cell numbers are shown as (mean ± SD) × 10-6. n = 4-6 mice per group. *p < 0.05, between the two groups at the indicated times.
- Figure S2. 3H3 treatment induces apoptosis of CD4+ T cells and B cells in cGVHD. (PDF, 50 KB) -
cGVHD was induced and mice were treated with antibody as described in Methods. Splenocytes were harvested on day 14 after disease induction, and analyzed for apoptosis of T and B cells. Shown are (A) representative FACS plots of CD3+CD4+ T cells and Annexin-V+CD4+ T cells, (B) CD3+CD8+ T cells and Annexin-V+CD8+ T cells, and (C) CD3-B220+ B cells and Annexin-V+B220+ B cells. Data shown are representative of three experiments with n = 3-5 per group.
- Figure S3. Donor CD8+ T cells may function as suppressor T cells in cGVHD. (PDF, 53 KB) -
Donor CD8+ T cells were depleted (>90%) before transfer as described in Methods. (A) Anti-DNA IgG1 levels were measured every 2 weeks after disease induction. OD values are shown as means ± SD of n = 4 per group at 10-fold dilution of samples. (B) Survival curves. n = 10 per group.
- Figure S4. CD11b+Gr-1+ granulocytes are not required for 3H3-mediated inhibition of autoantibody production. (PDF, 33 KB) -
(A and B) cGVHD was induced and mice were treated with antibody as described in Methods. Splenocytes were collected on day 7 after disease induction and stained with anti-CD11b and anti-Gr-1 with or without anti-FasL. Shown are (A) representative FACS plots of CD11b+Gr-1+ granulocytes and (B) FasL expression on gated CD11b+Gr-1+ granulocytes. (C) Granulocytes were depleted in vivo by treating with 200 µg anti-Gr-1 mAb on days –2, 0, and 2 (>99% depletion). Two weeks after disease induction, anti-DNA IgG1 levels were measured. OD values are shown as means ± SD of n = 5 per group at 10-fold dilution of samples. *p <0.05, between the control Ig-treated group without anti-Gr-1 mAb and the indicated group.
- Figure S5. 4-1BB costimulation activates IFN-γ-producing CD11c+CD8+ T cells in cGVHD. (PDF, 108 KB) -
(A) Splenocytes were harvested from 7-day cGVHD mice and triple-stained with anti-CD8 and anti-CD11c plus mAb to various surface molecules. Note that 4-1BB stimulation induced expansion of CD11c+CD8+ T cells with an activation phenotype. (B) Splenocytes were harvested from 7-day cGVHD mice, cultured in the presence of anti-CD3 mAb for 24 hours, and stained for CD8 combined with intracellular staining for IFN-
 .
- Figure S6. Blockade of 4-1BB/4-1BB ligand interactions does not affect autoantibody production in cGVHD. (PDF, 29 KB) -
cGVHD was induced by transferring 8 × 107 DBA/2 spleen/lymph node cells into BDF1 mice. 200 ㎍ of anti-4-1BB ligand mAb or control Ig was injected on days 0, 2, and 4. Serum samples were collected every 2 weeks, and assayed in duplicate by ELISA for IgG1 anti-DNA autoantibody. OD values are shown as means ± SD (n = 5 per group) at 10-fold dilution of samples.
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