Peripheral blood Th9 cells reconstitution and its relationship with acute graft-versus-host disease after matched-sibling peripheral blood hematopoietic stem cell transplantation

Abstract

T helper type 9 (Th9) cells have recently been identified as a new effector T cell subset. This study is to analyze the reconstitution of Th9 cell after matched sibling peripheral blood hematopoietic stem cell transplantation (MS-PBSCT) and the relationship between Th9 cell and acute graft-versus-host disease (aGVHD). Flow cytometry and ELISA were used to analyze the percentages of Th9 cell, levels of IL-9, TGF-β, IFN-γ, and IL-4. The results showed that for patients without aGVHD, Th9 cells recovery started from day 60 after transplantation and reached normal level on day 90. Serum TGF-β, IL-4, and IFN-γ reached normal levels on day 60, 60, and 90 post transplantation respectively. The serum IL-9 recovery is slower than that of IFN-γ and IL-4. For patients suffering from aGVHD, they had declined Th9 cell numbers, lower IL-9 and TGF-β levels, but higher serum IFN-γ level when compared with those without aGVHD after transplantation. Serum IFN-γ/IL-9 ratios increased linearly with grades of aGVHD. In conclusion, Th9 cells recovery is delayed after MS-PBSCT in patients with aGVHD but early in patients without aGVHD, indicating quick immune reconstitution of Th9 cells and IL-9 after MS-PBSCT may promote the immune tolerance.

Keywords: Allogeneic peripheral blood hematopoietic stem cell transplantation (allo-PBSCT); IL-9; Th9 cells; acute graft-versus-host disease (aGVHD).

Figure 1

Changes of Th9 cell in patients at various time points after MS-PBSCT. Subtypes of CD4⁺ IL-9⁺ Th9 cells in peripheral blood were detected by flow cytometry. Cells were first gated on CD3⁺ T cells and then on CD4⁺ IL-9⁺ T cells. A. The representative flow cytometric dot-plots of CD4⁺ IL-9⁺ Th9 cells in the blood of the control, 30-day, 60-day, 90-day and 150-day after transplantation. Numbers within each quadrant indicated percentages of cells within each dot-plot. B. Comparisons of the percentages of CD4⁺ IL-9⁺ Th9 cells in the peripheral blood. C. Comparisons of the percentages of Th9 cell in the peripheral blood of the HC, and patients after transplantation. The horizontal bars indicated means. Comparison was performed using one-way analysis of variance. HC: healthy controls. MS-PBSCT: matched-sibling peripheral blood hematopoietic stem cells transplantation.

Figure 2

Serum cytokine levels in patients without aGVHD after transplantation. Serum levels of IL-9, IFN-γ, TGF-β, and IL-4 in the patients from the MS-PBSCT group (on days 30, 60, 90, and 150 after transplantation), as well as in the healthy control (HC) subjects, were detected with ELISA. Compared with the HC group, *P < 0.05; compared with day 30 after transplantation, ΔP < 0.05; compared with the day 60 after transplantation, ※P < 0.05.

Figure 3

Dynamic change of IL-9, IFN-γ, TGF-β, and IL-4 in patients without aGVHD. IL-9 and TGF-β levels were significantly increased and then gradually recovered after transplantation.

Figure 4

Correlation analysis of cytokines in patients without aGVHD after transplantation. Data was analyzed with Spearman correlation analysis. IL-9 was positively correlated with the concentration of TGF-β (P=0.000, r=0.381), but not IFN-γ (P=0.561) and IL-4 (P=0.298).

Figure 5

Th9 cell percentage and associated cytokine levels in patients with or without aGVHD after transplantation. (A) Th9 cell percentage in the patients with or without aGVHD. Serum levels of IL-9 (B), TGF-β (C), IFN-γ (D), and IL-4 (E) in the patients with or without aGVHD after transplantation were detected with ELISA. In the aGVHD(+) group, IL-9 and TGF-β concentration was lower in the aGVHD(+) group than in aGVHD(-) group, but IFN-γ concentration was higher in the aGVHD(+) group.

Figure 6

Correlation between the IFN-γ/IL-9 ratio and aGVHD grade. Correlation between the IFN-γ/IL-9 ratio and aGVHD grade was analyzed by the Spearman correlation analysis. Patients without aGVHD (grade 0), patients with mild aGVHD (grade 1 to 2), patients with sever aGVHD (grade 3 to 4). IFN-γ/IL-9 ratio was positively correlated with the severity of aGVHD (P=0.000, r=0.723).