Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ......
Transcript of Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ......
Figure S1
Figure S1. Characterization of MSCs.
The morphology of human MSCs (passage 5) as revealed by phase contrast microscopy
(A). Oil O staining shows the adipogenic differentiation of human MSCs (B). Alizarin
red S staining shows the osteogenic differentiation of human MSCs (C). Flow
cytometric analysis of cell surface markers of human MSCs (D). Scale bar=200 μm.
Figure S2
Figure S2. Population doubling time (PDT) of MSCs
12 MSCs samples from healthy donors were cultured and counted at Passage 3 and
Passage 6, respectively. PDT were calculated according to the following formula:
(t−t0)∙log2/(logN−logN0), where t−t0 is culture time (h), N the number of harvested
cells and N0 is the number of cells in the initial. Mean PDT ± SEM of MSCs at passage
3 and passage 6 (n=12)
Figure S3
Figure S3. The frequency and number of T cells in cGVHD patients after MSC
treatment.
The frequency and number of CD3+ T cells (A, E), CD4+ T cells (B, F), CD8+ T cells
(C, G), and Tregs (D, H) in the cGVHD patients were detected before and after MSCs
treatment (n=20) and compared with those in the non-GVHD patients (n=11). The
symbols represent individual samples, the horizontal bars represent the mean, and the
error bars show the SEM. Significant differences are indicated as follows: *P<0.05 and
**P<0.01.
Figure S4
Figure S4. The increased number of CD5+ B cells in the cGVHD patients after
MSCs treatment.
The numbers of CD19+ B cells (A), CD5+ B cells (B), and CD5- B cells (C) in the
cGVHD patients were detected before and after MSCs treatment (n=20) and compared
with that of the non-GVHD patients (n=11). The symbols represent individual samples,
the horizontal bars represent the mean, and the error bars show the SEM. Significant
differences are indicated as follows: *P<0.05 and **P<0.01.
Figure S5
Figure S5. CD5+IL10+ B cell frequency in NR cGVHD patients before and after
MSCs treatment.
The flow cytometry plots represent the frequencies of CD5+IL-10+ B cells in the NR
cGVHD patients (A) before and after MSCs treatment. The symbols represent the
frequency of IL-10-producing CD5+ B cells in individual NR cGVHD patients (B). The
correlation analysis between the frequency of CD5+IL-10+ B cells in the NR cGVHD
patients and the average NIH score is shown (C).
Figure S6
Figure S6. Correlation between the frequency of CD5+IL-10+ B cells in cGVHD patients
and the organ NIH scores.
There is a significant negative correlation between the frequency of CD5+IL-10+ B cells in
the CR/PR cGVHD patients and each organ NIH score, including that for the skin, mouth,
liver, eye, gastrointestinal tract, lung and joint (A). The same correlation analysis in the NR
cGVHD patients is shown (B).
Figure S7
Figure S7. IL-10 level in the supernatant of CD5+ B cells from CR/PR cGVHD patients
before and after MSCs treatment.
Purified CD5+ B cells from CR/PR cGVHD patients (n=5) before and after MSCs treatment
were cultured in vitro, and the concentration of IL-10 was detected by ELISA. The symbols
represent individual samples. Significant differences are indicated as follows: *P<0.05.
Figure S8
Figure S8. The phenotypic characteristics of CD5+ B cells in cGVHD patients and
non-GVHD patients.
The histograms represent the cell surface phenotypes of peripheral CD5+ B cells in
cGVHD (n=20) and non-GVHD patients (n=11) (A). The horizontal bars represent the
mean, and the error bars show the SEM (B). Significant differences are indicated as
follows: **P<0.01.
Figure S9
Figure S9. The expression of CD86 on CD5+B cells from CR/PR cGVHD patients was
increased after MSCs treatment.
The mean fluorescence intensity (MFI) of CD86 expressed on CD5+B cells from CR/PR
cGVHD patients before and after MSCs treatment was detected by flow cytometry and
analyzed by Flowjo. Symbols represent individual samples. Significant differences are
indicated as follows: *P<0.05.
Figure S10
Figure S10. Activated CD5+ B cells from healthy donors inhibit CD3+ T cell
cytokine production.
Activated CD5+ CD19+ B cells inhibited TNF-α production by CD3+ T cells at B: T
ratios of 1:1 and 1:2.
Figure S11
Figure S11. CD5-CD19+ B cells have no regulatory effect on T cells.
Neither resting nor activated CD5- B cells from either healthy donors (A) or cGVHD
patients (B) regulated the TNF-α production by CD3+ T cells. Neither resting nor
activated CD5+ B cells from either healthy donors (C) or cGVHD patients (D)
influenced the proliferation of CD3+ T cells. The results are representative of three
independent experiments.
Figure S12
Figure S12. Lower IL-10 production by CD5+ B cells from cGVHD patients after
stimulation.
Purified CD5+ B cells from both healthy donors (n=3) and cGVHD patients (n=3) were
treated with or without LPS in vitro, and IL-10 production was detected by ELISA (A)
and ELISPOT (B). The bars represent the mean, and the error bars show the SEM.
Significant differences are indicated as follows: *P<0.05 and **P<0.01.
Figure S13
Figure S13. MSCs increased the frequencies of IL-10-producing B cells in CD5+ B
cells.
Isolated CD19+ B cells from either cGVHD patients or healthy donors were
cultured alone or co-cultured with MSCs for 3 d or 5 d. PMA, ionomycin, and
BFA were added before the end of the culture period, and the expression of CD5
and IL-10 was detected by flow cytometry. The frequency of IL-10-producing B
cells in CD5+ B cells from cGVHD patients in the presence of MSCs were higher
than that in the absence of MSCs at day 5 (A). The frequencies of
IL-10-producing B cells in CD5+ B cells from healthy donors co-cultured with
MSCs were significantly higher than those without MSCs at day 3 and day 5
(B). The results are representative of five independent experiments. Significant
differences are indicated as follows: *P<0.05 and **P<0.01.
Figure S14
Figure S14. Activated CD5+ B cells induce functional IDO in MSCs through IFN-γ.
The supernatants of MSCs alone, MSCs treated with IFN-γ, MSCs co-cultured with
activated CD5+ B or CD5- B cells were collected. The IDO activity in these supernatant
was evaluated through the kynurenine to tryptophan ratio by HPLC (A). Using western
blot, The IDO expression levels in MSCs alone, MSCs treated with IFN-γ or LPS,
MSCs co-culture with CD5+ B or CD5- B cells were detected (B). After LPS
stimulation for 72h, the frequency of IFN-γ producing B cells in CD5+ B or CD5- B
cells are shown in a dot plot (C). The bars represent the mean, and the error bars show
the SEM. Significant differences are indicated as follows: *P<0.05.
Figure S15
Figure S15. MSCs increase the survival of CD5+ B cells partially through IDO.
Purified CD5+ B cells and CD5- B cells from healthy donors were co-cultured with
MSCs in the presence of the IDO inhibitors D-1MT or L-1MT, as well as with IDO
knock-down MSCs, and B cell survival was evaluated by annexin V and PI staining (A).
The bars represent the mean, and the error bars show the SEM (B, C). Significant
differences are indicated as follows: *P<0.05 and **P<0.01.
Figure S16
Figure S16. MSCs promote the proliferation of CD5+ B cells partially through
IDO.
Purified CD5+ B cells and CD5- B cells from healthy donors were co-cultured with
MSCs in the presence of the IDO inhibitors D-1MT or L-1MT as well as with IDO
knock-down MSCs, and B cell proliferation was evaluated by EdU staining (A). The
bars represent the mean, and the error bars show the SEM (B, C). Significant
differences are indicated as follows: *P<0.05 and **P<0.01.