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Blood, Vol. 110, Issue 13, 4588-4598, December 15, 2007
Preemptive HMG-CoA reductase inhibition provides graft-versus-host disease protection by Th-2 polarization while sparing graft-versus-leukemia activity
Blood Zeiser et al.
110: 4588
Supplements for Zeiser et al
Files in this Data Supplement:
- Figure S1 (PDF, 119 KB) -
(A) Cytokine levels were measured in the supernatant of co-cultures combining irradiated (30 Gy) APCs (Stimulator, H-2KD) with T cells (Responder, H-2KQ) derived from donors that were fed for 10 days with PBS or atorvastatin 10 mg/kg (AT) as indicated in the respective histogram (NS= not significant, * p<0.05). (B) Mesenteric lymph nodes as an important site for intestinal aGvHD induction were isolated from 3 animals per group and snap frozen in OCT on day 3 after BMT. Staining was performed for CD4 (green) and Thy-1.2 (red, donor). The overlay indicates the amount of donor type CD4 T cell infiltration (CD4+Thy−1.2+). Microscopic evaluation was performed on a Nikon Eclipse TE 300 microscope equipped with 20X/0.45 and 40X/0.60 objective lenses (Nikon, Melville, NY). Magnification is ×100.
- Figure S2 (PDF, 142 KB) -
(A) Adoptively transferred CD8+H-2Kq+ donor T cells were isolated from the indicated organs and analyzed for their intracellular cytokine profile on days 3, 15 and 25. The y-axis depicts the percentage of IL-4, IL-10, TNF or IFN- positive CD8+ T cells within the total CD4+H-2Kq+ donor population. Data presented is derived 3 mice per time point, values are mean ± SD. (B) Expansion of CD8+H-2Kq+ donor T cells on day 3 after BMT by CFSE dilution. Data presented is derived from one of 3 mice per time point, values are mean ± SD.
- Figure S3. Perforin dependent elimination of tumor targets is maintained after atorvastatin treatment (PDF, 209 KB) -
(A) Balb/c mice (H-2d) were given 5 × 106 TCD-BM cells and 5 × 105 CD4+/CD8+ (4:1) T cells (both H-2b) after lethal irradiation with 800 cGy. 1 × 105 A20 leukemia cells (H-2Kd) were injected on day 0 after irradiation. C57BL/6 donor mice that had a defective Fas-ligand gene (Tnfsf6gld/gld) or were deficient in perforin (Prf1−/−) were fed with either PBS or AT and used as donors of BM cells and T cells. Three representative animals of the following groups are shown from the left: recipients of TCD-BM, TCD-BM plus wt T cells/PBS treatment, Tnfsf6gld/gld/PBS treatment, Tnfsf6gld/gld AT treatment, Prf1−/− PBS treatment, Prf1−/− AT treatment. (B) Bone marrow samples were analyzed for the presence of yfp+ A20 cells of Balb/c recipients on days 10 and 17 after transplantation for the indicated groups. (C) Survival of BALB/c mice receiving TCD-BM alone (△, n = 10), TCD-BM plus A20 leukemia cells after pretreatment of donor and recipient with PBS (□, n = 10) or AT (▽, n = 10), Tnfsf6gld/gld PBS (○, n = 10), Tnfsf6gld/gld AT (●, n = 10), Prf1−/− PBS (*, n = 10), Prf1−/− AT (■, n = 10). The transfer of T cells from PBS pretreated Prf1−/− mice resulted in 100% GvHD lethality before day 25 after BMT. Transplantation of Tnfsf6gld/gld T cells from PBS treated donors induced an early death of 40% of animals and delayed death of others. (D) Balb/c mice (H-2) were given 5 × 106 TCD-BM cells and 1.2 × 106 CD4+/CD8+ (4:1) T cells (both H-2q) after lethal irradiation with 800 cGy. BCL1 cells (H-2Kd) were injected on day -5 prior to BMT. Expansion of luc+ BCL1 tumor cells was measured in photons over the total body area (photons/second/mouse) at serial time points. Initial signal loss is due to conditioning with lethal irradiation on day 0. Animals rejecting the BCL1 cells demonstrate a durable signal loss. No significant difference in the anti-tumor effects of PBS versus AT treated T cells is observed (○ versus ▼). (E) Survival of BALB/c mice receiving TCD-BM alone (△, n = 10), BCL1 cells alone (■, n = 10), BCL1 cells plus TCD-BM (□, n = 10) after pretreatment of donor and recipient with PBS (○, n = 10) or AT (▼, n = 10).
- Figure S4. Naturally occurring regulatory T cells and engraftment after statin treatment (PDF, 132 KB) -
(A) Spleens and lymph nodes from donor animals treated with the indicated statins were isolated after 10 days of oral garvage. A representative FACS analysis for splenic CD4+ enriched T cells is depicted. No significant change in the frequency of CD4+FoxP3+ T cells isolated from spleen or LNs in recipients of AT treatment was detected.
Chimerism analysis indicates intact lymphoid and myeloid engraftment in the presence of donor AT treatment
Balb/c recipients transplanted with T cell depleted bone marrow as described for the FVB/N → Balb/c combination were sacrificed at the time points indicated and spleens were harvested. Donors and recipients had been treated with AT 10 mg/kg bodyweight for 10 days. (B) T cell chimerism was calculated by dividing the number of CD4+H-2Kq+ or CD8+H-2Kq+ events by the total number of CD4+ or CD8+ events. Values for individual mice from 1 of 5 similar experiments were averaged and plotted as mean ± s.e.m. The total number of mice analyzed in each group at each time point (days 15 and 30) are 4. There was no statistically significant decrease in T cell chimerism at any time point analyzed in the AT treatment groups. (C) Absolute numbers of donor-derived monocytes/granulocytes (CD11b+ CD11c- H-2Kq+) were calculated by dividing the number of monocyte/granulocyte events by the total viable cell events collected and multiplying by the number of splenocytes recovered for each mouse. Values for individual mice from 1 of 3 similar experiments were averaged and plotted as mean ± s.e.m. The total number of mice analyzed in each group at each time point was 6. There was no statistically significant decrease in monocyte/granulocyte reconstitution at any time point analyzed in recipients of AT treatment.
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