Figure 1.
MHC-haploidentical murine GvHD model and suppression of hematopoiesis during GvHD.
(A) A MHC-haploidentical murine GVHD model was established by transplanting BM cells (5×106) plus splenocyts (6×107) from B6.SJL donor mice (CD45.1) into lethally irradiated CB6F1 recipient mice (CD45.1/2) [B6.SJL (CD45.1)→CB6F1 (CD45.1/2)]. Recipent mice received BM cells alone as control groups. (B) Survival of mice receiving HSCT with donor BM plus splenocytes or donor BM only (P<0.05, Log-rank test). (C) Body weight of mice receiving HSCT (N = 20 in each group on day 3; n = 4 in GvHD and n = 20 in BMT respectively on day 21 post-transplantation). (D) Engraftment of donor-derived cells after HSCT in a GvHD mouse. (E–G) Kinetics of WBC, Hgb, and platelet counts after HSCT. (H) MNCs count on day 14 and day 21 after HSCT. (I) Count of Lin−/CD48−/CD150+ cells after HSCT. (J) Percentage of Lin−/CD48−/CD150+ cells in MNCs after HSCT. Data are shown as mean ± SD and from 1 of 3 experiments with similar results. *P<0.05; **P<0.01 (n = 4, t-test).
Figure 2.
Hematopoietic cells derived from GvHD mice are competent for hematopoietic reconstitution (A–E) Continuous transplantation.
Analyses were performed on day 14 after second transplantation. (A) Continuous HSCT: To evaluate the effects of GvHD on hematopoietic cell competency, lethally irradiated C57BL/6 (CD45.2) mice received BM cells(5×106) from either BMT or GvHD recipient mice of [B6.SJL (CD45.1)→CB6F1 (CD45.1/2)] model at 14 days after first transplantation. (B) Representative flow cytometry analysis of.B cells (B220+), granulocytes (Gr-1+), and monocytes (CD11b+) in the recipient BM cells after continuous transplantation. (C) MNC count per tibia. (D) Percentages of B cells, granulocytes and monocytes in MNCs. (E) Counts of B cells, granulocytes and monocytes. (F–L) Competitive transplantation. Analyses were performed on day 30 after second transplantation. (F) Competitive transplantation to further evaluate the competency of hematopoietic cells in GvHD mice: 14 days after the first transplantation, BM cells (2.5×106) from the transplanted mice [B6.SJL (CD45.1)→CB6F1 (CD45.1/2)] were mixed with equal amount (2.5×106) of BM cells from healthy C57BL/6 mice (CD45.2), and transplanted into C57BL/6 recipients (CD45.2) after 8Gy radiotherapy. (G) Total MNC counts and CD45.1 positive cell counts per tibia. (H) Representative flow cytometry profile of HSCs (Lin−CD48−CD150+) analysis. (I) Absolute number of Lin−CD48−CD150+ cells in CD45.1 positive cell. (J) The percentage of Lin−CD48−CD150+ cells in CD45.1 positive cell. (K) and (L) Percentages and absolute number of B cells (B220+), granulocytes (Gr-1+), and monocytes (CD11b+), respectively. All tests were performed on day 30 after transplantation. Data are shown as mean ± SD and from 1 of 3 experiments with similar results. NS: no significant (n = 4, t-test).
Figure 3.
Impairment of BM hematopoietic niche during GvHD.
(A) Re-transplantation: In order to evaluate the effects of GvHD on BM niche, recipient mice in the GvHD and BMT groups received a second transplantation from healthy C57BL/6 BM cells (CD45.2, 5×105) after 200cGy TBI on days 14 after first transplantation. Hematopoiesis was analyzed on day 14 after re-transplantation. (B) Count of MNCs per tibia (left bars) and the percentage of C57BL/6 donor-derived CD45.2+ cells in MNCs (right bars). (C) Absolute number of donor-derived B cells (CD45.2+/B220+), monocytes (CD45.2+/CD11b+) and granulocytes (CD45.2+/Gr-1+). (D) Percentages of donor-derived B cells, monocytes and granulocytes in MNCs. Data are shown as mean ± SD and from 1 of 3 experiments with similar results. *P<0.05; **P<0.01; NS: no significant (n = 4, t-test).
Figure 4.
Destruction of vascular niche in acute GvHD.
In order to verify vascular niche is the target of aGvHD, BM SECs from recipient mice were tested by flow cytometric and histological analysis. (A) Representative flow cytometry profile of SECs (Sca-1-VEGFR2+VEGFR3+). (B) Absolute number of BM SECs per tibia. (C) Percentages of BM SECs in MNCs. (D) HE and Immunohistochemistry analysis of disrupted vascular niche in acute GvHD vs BMT control. Arrows indicate SECS. (E) Representative flow cytometry analysis to assess apoptosis SECs. (F) Apoptosis of SECs, expressed by Annexin V+/PI− and Annexin V+/PI+ to SSClow/VEGFR3+. (G and H) Proliferation assay of SECs by measuring Ki67 by flow cytometry and percentage of proliferating SECs (VEGFR3+/Ki-67+ cells) in MNCs. Data are shown as mean ± SD and from 1 of 3 experiments with similar results. **P<0.01 (n = 4, t-test).
Figure 5.
Dysfunction of vascular niche in acute GvHD.
To evaluate the expression of CXCR4/SDF-1 and SCF/c-Kit in BM cells, flow cytometry and RT-PCR analysis were performed at 14 days after transplantation. (A) Representative flow cytometry profile of CXCR4 expression in Lin−CD48−CD150+ cells. (B–D): Analysis of CXCR4/SDF-1 and SCF/c-Kit pathways. (B) Percentage of CXCR4+ cells in HSCS. (C) Percentage of SDF-1+ cells in VEGFR3+ endothelial cells. (D) RT-PCR for measuring the expression of SCF in sorted BM SECs. Data are shown as mean ± SD. Experiments were performed at least twice. *P<0.05; **P<0.01; NS: no significant (n = 4 for CXCR4/SDF-1 analysis and n = 3 for SCF/C-Kit analysis, t-test).
Table 1.
Concentration of VEGF in BM and serum.
Figure 6.
CD4+ T-cell mediated vascular niche damage in GvHD.
(A) Percentages of CD4+ and CD8+ donor T cells in MNCs in BM at 14 days after transplantation. (B) MHC-II expression in BM SECs. (C) MHC-I expression in BM SECs. (D) Representative flow cytometry profile of Fas expression in BM SECs. (E) Percentages of Fas+ cells in SECs. (F) Expression of Fas and caspase-3 in BM SECs measured by RT-PCR. (G) Representative flow cytometry profile of caspase-3 expression in BM SECs. (H) Percentages of caspase-3 positive cells in SECs. (I) Representative flow cytometry of FasL in donor CD4+ T cells. (J) Percentages of FasL+ CD4+ donor T cells in MNCs. Data are shown as mean ± SD and from 1 of 3 experiments with similar results (Only one experiment for caspase-3 expression). **P<0.01; NS: no significant (n = 4, t-test).