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Journal of Neurorestoratology  2018, Vol. 6 Issue (1): 122-133    doi: 10.26599/JNR.2018.9040010
Research Article     
The immunomodulatory function of human amniotic fluid stromal cells on B lymphocytes
Qun Xue1,2,3,4,(✉), Zhou Yin1, Nagam Varshithreddy1, Han-si Liang2,3, Ming-yuan Wang5, Wan-li Dong1, Xueguang Zhang2,3, Yanzheng Gu2,3,(✉), Qi Fang1,2,3,(✉)
1 Department of Neurology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China
2 Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China
3 Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou 215006, China
4 Suzhou Clinical Research Center of Neurological Disease, the Second Affiliatted Hospital of Soochow University, Suzhou 215004, China
5 Soochow Red Cross Blood Center, Suzhou 215006, China
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Abstract  

Current treatments for B cell-mediated disease are mainly based on global B cell depletion, thereby eliminating pathogenic B cells as well as Breg subsets. A more refined modulation of B cell activity could prove beneficial for patient treatment.

Objective:

To investigate the immunomodulatory function of human amniotic fluid stromal cells (hAFSCs) on different subpopulation of B lymphocytes.

Methods:

hAFSCs were isolated and cultured and identified by characteristic phenotypic markers. After coculture of B lymphocytes with hAFSCs, the activation, proliferation, differentiation, as well as apoptosis, cell cycle, and expression of the inhibitory costimulatory molecules B7H1, B7H3, and B7H4 of B lymphocytes were examined in vitro.

Results:

Coculture with hAFSCs significantly decreased the expression of CD80/CD86, Ki-67 and CFSE expression, on activated B lymphocytes. These might be due to the inhibition of B lymphocyte apoptosis and cell cycle arrest. In activated B lymphocytes, coculture with hAFSCs resulted in a reduced proportion of memory B and plasma cells, reduced amounts of immunoglobulins. hAFSCs could balance the B1 to B2 cell subpopulation ratio. hAFSCs could inhibit the expression of the negative co-inhibitory molecule B7H4 and PD-L1 on the activated B lymphocytes.

Conclusion:

hAFSCs could inhibit B cell activation, proliferation, and subpopulation differentiation. These might be due to their affect on B cell apoptosis, cell cycle and the expression of costimulatory molecules of human B lymphocytes. Our experiment provided the evidence for hAFSCs as ideal seed cells with therapeutic potential for treating humoral immunity disorders, which were mainly mediated by B lymphocytes.



Key wordsHuman amniotic fluid stem cells      B lymphocyte      immune regulation      autoimmune diseases     
Received: 25 March 2018      Published: 30 September 2018
Corresponding Authors: Qun Xue,Yanzheng Gu,Qi Fang   
Cite this article:

Qun Xue, Zhou Yin, Nagam Varshithreddy, Han-si Liang, Ming-yuan Wang, Wan-li Dong, Xueguang Zhang, Yanzheng Gu, Qi Fang. The immunomodulatory function of human amniotic fluid stromal cells on B lymphocytes. Journal of Neurorestoratology, 2018, 6: 122-133.

URL:

http://jnr.tsinghuajournals.com/10.26599/JNR.2018.9040010     OR     http://jnr.tsinghuajournals.com/Y2018/V6/I1/122

Fig. 1Culture and identification of hAFSCs. (A) Adherent hAFSCs under an inverted microscope. (a) Primary culture at day 3 (×400); (b) primary culture at day 5 (×400); (c) cellsat the third generation (×200); (d) cellsat the third generation (×400). (B) Flow cytometric analysis of hAFSCsurface markers. hAFSCs (at passage 5) were analyzed after staining with the control isotype IgG (red peaks) or antibodies against the surface proteins CD29, CD44, CD133 CD90, CD73, CD105 , OCT-4, CD166, HLA-DR, MHC-II, CD45, and CD34. (C) Detection of the inhibitory costimulatory molecules B7H1, B7H3, and B7H4 on hAFSCsby flow cytometry. The plot is representative of one out of three experiments.
Fig. 2The effect of hAFSCs on B cell activation and proliferation. (A-1) Cell activation was observed under microscope on the second day after cell culture in the presence or absence of activators (×200): (a) B cell, (b) B cells were stimulated by CpG, (c) B cells cultured in hAFSCs supernatant conditioned median, (d) CpG stimulated B cells cultured in hAFSCs supernatant conditioned median, (e) B cells co-cultured with hAFSCs, (f) CpG stimulated B cells co-cultured with hAFSCs. (A-2) The figure showed the statistical results of the expression of CD80 and CD86 on B cells which were detected by FACS after 72 hs of co-culture with or without hAFSCs. Results are the representative of the average of three independent experiments and bars represent the SD. *Statistically significant (P< 0.05). (B) To examine B-cell proliferation with different ratio of hAFSCs (AFS:B, 1:20; 1:10; 1:5; 1:1), the expression of Ki-67 (B-1, B-2) and CFSE (C-1, C-2) markers was detected by flow cytometry after 72 h. The plot is representative of one out of three experiments. Results are the representative of the average of three independent experiments and bars represent the SD. *Statistically significant (P< 0.05). In B-1 and C-1, (a) unactivated B cells, (b) B cells stimulated by CpG 2006, CD40L, anti-immunoglobulin, IL-4, and IL-2, (abbreviated as CpG later), (c) B cells stimulated by CpG co-cultured with hAFSCs in the ratio of hAFSCS:B=1:20, (d) activated B cells co-culture in the ratio of hAFSCS:B=1:10, (e) activated B cells co-culture in the ratio of hAFSCs:B=1:5, (f) activated B cells co-culture in the ratio of hAFSCS:B=1:1. The image shows the results of a random experiment and the results are shown as the mean±SEM from three independent experiments. (D) To explore the effect of hAFSCs on activated B-cell proliferation, the expression of Ki-67 and CFSE markers was detected by flow cytometry 72 h after B cells stimulated by CpG co-cultured with or without hAFSCs, or in the hAFSCs conditioned media. The plot is representative ofone out of three experiments. Results are the representative of the average of three independent experiments and bars represent the SD. *Statistically significant (P< 0.05) (B) b cells;B+SUP: B cells cultured in hAFSCs supernatant conditioned median; B+AFS: B cells co-cultured with hAFSCs in the ratio of AFS:B=1:5.
Fig. 3The effect of hAFSCs on B cell differentiation and subtype of B lymphocytes. (A) Detection of the surface markers CD19, CD20, and CD27 on B cells was examined by flow cytometry to discriminate the differentiation of cultured B cells, defined as naive B cells (CD19+CD20+CD27-), memory B cells (CD19+CD20+CD27+), and plasma cells (CD19+CD20-CD27+). Quantitative analysis of memory B cells (B), and plasma cells (C). The content of IgM (D), IgA (E), and IgG (F) immunoglobulin secretion in the supernatant was detected by ELISA after 72 h. (G) The percentage of CD20+CD5+B1 cells by flow cytometry. (H) The percentage CD20+IL-10+ Bregs by flow cytometry. The results are shown as the mean±SEM from three independent experiments, *P < 0.05.
Fig. 4The effects of hAFSCs on B lymphocyte apoptosis, cell cycle and the expression of surface molecules and cytokine. (A) Detection of the apoptosis ratio of B lymphocytes labeled with annexin-V/propidium iodide at 72 h; (B) cell cycle markers was detected by flow cytometry after 72 h of B cells co-cultured with or without hASFCs. The plot is representative of one out of three experiments. (C) Quantitative analysis of the B lymphocyte apoptosis ratio, B lymphocyte in the S phase (D), the expression of B7H4 (E) and PD-L1 (F) on the surface of B lymphocytes by flow cytometry. All the results are the representative of the average of three independent experiments and bars represent the SD. *Statistically significant (P< 0.05)
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