Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ......

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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.

Transcript of Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ......

Page 1: Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ... including that for the skin, mouth, liver, eye, gastrointestinal tract, lung and joint

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.

Page 2: Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ... including that for the skin, mouth, liver, eye, gastrointestinal tract, lung and joint

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)

Page 3: Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ... including that for the skin, mouth, liver, eye, gastrointestinal tract, lung and joint

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.

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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.

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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).

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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).

Page 7: Figure S1 - Nature · Oil O staining shows the adipogenic differentiation of human MSCs (B). ... including that for the skin, mouth, liver, eye, gastrointestinal tract, lung and joint

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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.