Mesenchymal stromal cells treatment attenuates dry eye in patients with chronic graft-versus-host disease

Abstract

Cell therapy is a promising approach for the treatment of refractory ocular disease. This study investigated the efficacy of mesenchymal stromal cells (MSCs) for the treatment of dry eye associated with chronic graft-versus-host disease (cGVHD) and assessed the immunomodulatory effects of MSCs on regulatory CD8(+)CD28(-) T lymphocytes. A total of 22 patients with refractory dry eye secondary to cGVHD were enrolled. The symptoms of 12 out of 22 patients abated after MSCs transplantation by intravenous injection, improving in the dry eye scores, ocular surface disease index scores and the Schirmer test results. The clinical improvements were accompanied by increasing level of CD8(+)CD28(-) T cells, but not CD4(+)CD25(+) T cells, in the 12 patients who were treated effectively. They had significantly higher levels of Th1 cytokines (interleukin (IL)-2 and interferon-γ) and lower levels of Th2 cytokines (IL-10 and IL-4). In addition, CD8(+) T cells were prone to differentiation into CD8(+)CD28(-) T cells after co-culture with MSCs in vitro. In conclusion, transfusion of MSCs improved the clinical symptoms in patients (54.55%) with refractory dry eye secondary to cGVHD. MSCs appear to exert their effects by triggering the generation of CD8(+)CD28(-) T cells, which may regulate the balance between Th1 and Th2.

Figures

Figure 1

Percentages of CD8+CD28− and CD4+CD25+ T cells detected by flow cytometry. In the effective treatment group, the proportion of CD8+CD28− T cells showed a statistical increase accompanied with improved dry eye symptoms (P = 0.008) whereas CD4+CD25+ T cells had no significant changes before and 3 months after the treatment with MSCs (P = 0.551). In the ineffective treatment group, there were no significant changes in the proportion of CD8+CD28− T cells (P = 0.798) and CD4+CD25+ T cells (P = 0.148). The percentages of CD8+CD28− and CD4+CD25+ T cells are shown in the bar graph.

Figure 2

The proportion of CD8+CD28− T cells in CD8+T cells alone or co-cultured with MSCs. (a) Representative flow cytometric analysis of CD28 and CD8 expression from three experiments. CD8+CD28− cells were determined from purified CD8+ T cells following 3 days and 8 days in culture in the absence (top panel) or presence of MSCs at ratios of 100:1 (middle panel) and 10:1 (low panel). (b) Statistical analysis of the flow cytometry results. After 3 and 8 days of co-culturing CD8+ T cells and MSCs at ratios of 100/1 and 10/1, the percent of CD8+CD28− T cells was significantly increased as compared with the CD8+ T cells alone. After 8 days in co-culture, the percent of CD8+CD28− T cells/CD8+T cells was increased as compared with the percent of cells after 3 days. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3

Enzyme-linked immunosorbent assay measurements of interleukin (IL)-2, interferon (IFN)-γ, IL-10, and IL-4 before and 3 months after the treatment with MSCs. In the effective treatment group, the IL-2 and IFN-γ concentrations increased significantly, whereas IL-10 and IL-4 were statistically reduced. In the ineffective treatment group, there were no significant changes in any of the factors (IL-2, IFN-γ, IL-10, and IL-4).

Figure 4

Correlation between the increment of the proportion of CD8+CD28− T cells and changes in the concentrations of Th1 and Th2 cytokines (interleukin (IL)-2, interferon (IFN)-γ, IL-10, and IL-4) in the effective treatment group. A significant positive correlation was observed in ΔCD8+CD28− T cells and ΔIL-2, and ΔCD8+CD28− T cells and ΔIFN-γ. A marked negative correlation was observed in ΔCD8+CD28− T cells and ΔIL-4. ΔCD8+CD28− T cells and ΔIL-10 had no obvious relevance but some degree of negative correlation. Δ indicates differences in the values post- and pretreatment with MSCs; rp indicates the correlation coefficient.

Figure 5

Characterization of hematopoietic MSCs (hMSCs). Assessment of the differentiation abilities of hMSCs. (a) Phase-contrast microscopy of hMSCs at passage 3. (b) Alizarin red S staining for osteogenic potential. (c) Oil red O staining for adipogenic potential. (d) Toluidine blue staining for chondrogenic potential. Flow cytometric analysis of cell surface antigens of hMSCs. (e) hMSCs were negative for CD34, CD45, and positive for cell surface markers, including CD29, CD44, and CD105. Scale bar = 200 µm. FITC, fluorescein isothiocyanate; PE, phycoerythrin.