Yonsei Med J.  2017 Sep;58(5):1031-1039. 10.3349/ymj.2017.58.5.1031.

Immune Tolerance of Human Dental Pulp-Derived Mesenchymal Stem Cells Mediated by CD4⁺CD25⁺FoxP3⁺ Regulatory T-Cells and Induced by TGF-β1 and IL-10

• Hong JW
• Lim JH
• Chung CJ
• Kang TJ
• Kim TY
• Kim YS
• Roh TS
• Lew DH

• KMID: 2418942
• DOI: http://doi.org/10.3349/ymj.2017.58.5.1031

Abstract

PURPOSE
Most studies on immune tolerance of mesenchymal stem cells (MSCs) have been performed using MSCs derived from bone marrow, cord blood, or adipose tissue. MSCs also exist in the craniofacial area, specifically in teeth. The purpose of this study was to evaluate the mechanisms of immune tolerance of dental pulp-derived MSC (DP-MSC) in vitro and in vivo.
MATERIALS AND METHODS
We isolated DP-MSCs from human dental pulp and co-cultured them with CD4⁺ T-cells. To evaluate the role of cytokines, we blocked TGF-β and IL-10, separately and together, in co-cultured DP-MSCs and CD4⁺ T-cells. We analyzed CD25 and FoxP3 to identify regulatory T-cells (Tregs) by fluorescence-activated cell sorting (FACS) and real-time PCR. We performed alloskin grafts with and without DP-MSC injection in mice. We performed mixed lymphocyte reactions (MLRs) to check immune tolerance.
RESULTS
Co-culture of CD4⁺ T-cells with DP-MSCs increased the number of CD4⁺CD25⁺FoxP3⁺ Tregs (p<0.01). TGF-β or/and IL-10 blocking suppressed Treg induction in co-cultured cells (p<0.05). TGF-β1 mRNA levels were higher in co-cultured DP-MSCs and in co-cultured CD4⁺ T-cells than in the respective monocultured cells. However, IL-10 mRNA levels were not different. There was no difference in alloskin graft survival rate and area between the DP-MSC injection group and the non-injection group. Nonetheless, MLR was reduced in the DP-MSC injected group (p<0.05).
CONCLUSION
DP-MSCs can modulate immune tolerance by increasing CD4⁺CD25⁺FoxP3⁺ Tregs. TGF-β1 and IL-10 are factors in the immune-tolerance mechanism. Pure DP-MSC therapy may not be an effective treatment for rejection, although it may module immune tolerance in vivo.

Keyword

Dental pulp; mesenchymal stem cell; immune tolerance

MeSH Terms

Allografts/immunology
Animals
Biomarkers/metabolism
CD4 Antigens/*metabolism
Cell Proliferation/drug effects
Coculture Techniques
Dental Pulp/*cytology
Forkhead Transcription Factors/metabolism
Graft Survival/immunology
Humans
*Immune Tolerance/drug effects
Interleukin-10/*pharmacology
Interleukin-2 Receptor alpha Subunit/*metabolism
Lymphocyte Culture Test, Mixed
Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/cytology/*immunology
Mice, Inbred BALB C
Real-Time Polymerase Chain Reaction
Skin Transplantation
T-Lymphocytes, Regulatory/drug effects/*immunology
Transforming Growth Factor beta1/*pharmacology
Biomarkers
CD4 Antigens
Forkhead Transcription Factors
Interleukin-2 Receptor alpha Subunit
Transforming Growth Factor beta1
Interleukin-10

Figure

• 

  Fig. 1 DP-MSC surface marker analysis using flow cytometry. (A) CD13 positive, (B) CD44 positive, (C) CD73 positive, (D) CD90 positive, and (E) CD105 positive. (F) CD31 negative, (G) CD34 negative, and (H) CD45 negative. These signals are shown as blue lines, while isotype matched control antibodies are shown as red lines. DP-MSC, dental pulp-derived mesenchymal stem cell.

• 

  Fig. 2 Changes in CD4+CD25+FoxP3+ Tregs upon DP-MSC co-culture with and without cytokine blocking. Statistical differences were assessed by paired t tests (n=9). CD4+CD25+FoxP3+ Tregs increased in the co-cultured group (p=0.003) and decreased in the double-block group (p<0.01). The single-block groups also showed statistical differences (p<0.05). *p<0.05, †p<0.01. DP-MSC, dental pulp-derived mesenchymal stem cell; Tregs, regulatory T-cells.

• 

  Fig. 3 Semi-quantitative PCR of CD25 (A) and FoxP3 (B). DP-MSC, dental pulp-derived mesenchymal stem cell.

• 

  Fig. 4 Real-time PCR of monocultured CD4+ T-cells and CD4+ T-cells co-cultured with DP-MSCs. (A and B) CD25 and FoxP3 expression was higher in the co-cultured CD4+ T-cells (p=0.001, Mann-Whitney U test). (C and D) TGF-β1 expression was higher in DP-MSCs co-cultured with CD4+ T-cells than in monocultured DP-MSCs (p=0.002, Mann-Whitney U test). There was no statistical difference in IL-10 expression. (E and F) TGF-β1 expression was higher in CD4+ T-cells co-cultured with DP-MSCs than in monocultured CD4+ T-cells (p=0.007, Mann-Whitney U test). Although IL-10 expression was higher in the co-cultured cells, the difference was not statistically significant. *p<0.01. DP-MSC, dental pulp-derived mesenchymal stem cell.

• 

  Fig. 5 Skin survival rate and area. Although the DP-MSC injection group showed elevated data, there were no statistical differences. DP-MSC, dental pulp-derived mesenchymal stem cell.

• 

  Fig. 6 MLR analysis. The PBS-injection group showed a greater increase than the DP-MSC-injection group. (A) Responder cell incubation in PBS injection group. (B) MLR with stimulator cells in the PBS injection group. (C) Responder cell incubation in DP-MSC injection group. (D) MLR with stimulator cells in DP-MSC group. (E) The CFSE-labeled cells were analyzed. The number of labeled cells of the DP-MSC-injection group was smaller than that of the PBS-injection group. There were statistical differences (p=0.04, Mann-Whitney U test). *p<0.05. DP-MSC, dental pulp-derived mesenchymal stem cell; MLR, mixed lymphocyte reaction.

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