Figure 1S.
Comparison of clinical GVHD in two chemotherapy-based, miHA-
mismatched allo-HSCT models (LP/J→C57BL/6 and 129S2/SvPasCrl→C57BL/6).
Survival, weight loss and GVHD Score after BMT in syngeneic and allogeneic
transplanted mice. Allogeneic transplanted animals of both miHA-mismatched allo-HSCT
models showed similarities in GVHD progression. Survival data was analysed using
Mantel-Cox log-rank test. Animals were regularly scored for five clinical parameters
(weight loss, posture, activity, fur and skin) on a scale from 0 to 2. Clinical GVHD score
was generated by summation of these five parameters. N=10 per group. (P<0.05 *,
P<0.01 **, P<0.001 ***).
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Figure 2S.
Allogeneic transplanted mice showed histopathologic signs of chronic
GVHD in liver and skin. 64 days after BMT, syngeneic and allogeneic transplanted
animals were sacrificed for histologic examination. Representative H&E-stained sections
of liver and abdominal skin from each group are shown. Liver of allogeneic transplanted
mice exhibited periportal mononuclear infiltration and fibrosis with beginning of septum
formation (black arrow). Whereas skin of syngeneic transplanted mice showed normal
skin structure with adnexae and low cell density, skin of allogeneic transplanted mice
showed fibrosis with high cell and fibrous density and atrophy of adnexal structures.
V=Vein. Original magnification was 40x for liver samples and 200x for skin samples.
2
3
Figure 3S.
Systemic Inflammation during GHVD. (a) FACS analysis of CD3, CD11b
in peripheral blood of syngeneic and allogeneic transplanted animals on different days
after BMT. Absolute numbers are shown. Immune cell populations were gated on donor
cells. Data from 1 representative of 3 individual experiments, N=3-10 per group. (b)
CD4+CD62L-, CD8+CD62L-, CD4+CD69+ T cells were increased in allogeneic
transplanted mice at d+15 after BMT. FACS analysis was performed in blood, spleen
and lymph nodes. Percentage of gated cells from CD3+ cells are shown. Data pooled
from two individual experiments. N=5 per group. (c) FACS analysis of different immune
cell subsets in bone marrow at d+15 after BMT. Cell concentrations in the bone marrow
cell preparation are shown. Immune cell populations were gated on donor cells. Data
from 1 representative of 2 individual experiments, N=5 per group. (d) FACS analysis of
different immune cell subsets in spleen at d+15 after BMT. Cell concentrations in the
splenic cell preparation are shown. Immune cell populations were gated on donor cells.
Data from 1 representative of 2 individual experiments, N=5 per group. (e) Serum levels
of IL-2, IL-4, IL-17A and IL-10 after BMT. Serum was collected from naïve C57BL/6,
chemotherapy-treated and allogeneic and syngeneic transplanted animals 4, 8, 11 and
15 days after BMT. Three individual experiments were performed, N=5-8 per group.
(n.s.= not significant, P<0.05*, P<0.01 **, P<0.001***).
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Table 1S.
GVHD Scoring. Mice were individually scored twice a week for five clinical
parameters (posture, activity, fur, skin and weight loss) on a scale from 0-2. Clinical
GVHD score was assessed by summation of these parameters. Animals were sacrificed
when reaching a single score of 2 or exceeding clinical GVHD score of 6.
Table 2S.
Recovery of thymopoiesis at late phase after BMT. FACS analysis of
thymus cell preparations of transplanted mice at d+15 and d+52-64 after BMT revealed
recovered CD3+ T cell number in transplanted mice at late phase. Allogeneic
transplanted mice showed recovery of CD4/CD8 thymocytes ratio. Cell count of CD3+ T
cells in 10 mg thymus and percentage of CD4/CD8 gated cells from CD3+ cells are
shown. Data pooled from two individual experiments. N=5 per group. FACS data from
d+52 and d+64 showed similar values and were summarized in one group.
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