Basiliximab impairs regulatory T cell (TREG) function and could affect the short-term graft acceptance in children with heart transplantation

Abstract

CD25, the alpha chain of the IL-2 receptor, is expressed on activated effector T cells that mediate immune graft damage. Induction immunosuppression is commonly used in solid organ transplantation and can include antibodies blocking CD25. However, regulatory T cells (Tregs) also rely on CD25 for their proliferation, survival, and regulatory function. Therefore, CD25-blockade may compromise Treg protective role against rejection. We analysed in vitro the effect of basiliximab (BXM) on the viability, phenotype, proliferation and cytokine production of Treg cells. We also evaluated in vivo the effect of BXM on Treg in thymectomized heart transplant children receiving BXM in comparison to patients not receiving induction therapy. Our results show that BXM reduces Treg counts and function in vitro by affecting their proliferation, Foxp3 expression, and IL-10 secretion capacity. In pediatric heart-transplant patients, we observed decreased Treg counts and a diminished Treg/Teff ratio in BXM-treated patients up to 6-month after treatment, recovering baseline values at the end of the 12-month follow up period. These results reveal that the use of BXM could produce detrimental effects on Tregs, and support the evidence suggesting that BXM induction could impair the protective role of Tregs in the period of highest incidence of acute graft rejection.

Figure 1

In vitro BXM-dependent CD25 blockade in Tregs. (A) CD25 expression within the total CD4+ T-cell population on untreated (-BXM) or BXM treated (+BXM) PBMC from a healthy volunteer after 4 h and 72 h culture. CD25 median fluorescence intensity (MFI) in CD4+ Foxp3+ cells (B) and total CD4+ T cells (C) within untreated (−BXM, solid circle) or BXM treated (+BXM, solid triangles) stimulated PBMC after 4 h culture (n = 6). ***p < 0.001.

Figure 2

BXM decreases Treg frequency and Foxp3 MFI in vitro. Frequency of CD4+ Foxp3+ (gated on CD4+ T cells), total CD4+ and CD8+ T cells (both gated in lymphocytes) after 72 h culture within PBMC without activation (A) or activation (B). CD4+ Foxp3+ (C) and CD4+ T (D) cell number after 72 h in unstimulated PBMC (unstim) or stimulated (Stim) for 72 h. (E) Viability after 72 h culture in stimulated PBMC. (F) Foxp3 MFI; Each line represents values for untreated (–BXM, solid circles) and BXM-treated (+BXM, solid triangles) conditions for each donor (n = 6). ns non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

BXM specifically suppresses Treg proliferation in vitro. (A) Histograms from a representative donor showing proliferation as a reduction in the CFSE intensity in Tregs, and total CD4+ and CD8+ T cells. (B) Values of proliferation based on the loss of CFSE signal (% CFSE lost signal in comparison to CFSE signal on non stimulated cells; Non Stim) in CD4+ Foxp3+, CD4+ and CD8+ T cells within stimulated PBMC. Frequency of Ki-67+ cells on CD4+ Foxp3+ and CD4+ T cells in unstimulated (C) or stimulated (D) PBMC (n = 6). ns non-significant; *p < 0.05; **p < 0.01.

Figure 4

BXM modifies the cytokine secretion pattern of Treg cells in vitro. Percentage of IL-10 (A), IL-17 (B) and IL-4 (C)-secreting cells gated on CD4+ Foxp3+ cells within stimulated PBMC. Each line represents values for untreated (–BXM, solid circles) and BXM-treated (+BXM, solid triangles) conditions for each donor (n = 6). ns non-significant; *p < 0.05.

Figure 5

Proportion of CD25+ and or Foxp3+ cells in CD4+ T cells after transplantation. (A) Representative dot plots of CD25+ FoxP3+ and CD25-Foxp3+ Tregs (in gated CD4+ T cells). (B) Percentages and absolute counts of Tregs from children treated (n = 2; broken line) and non-treated with basiliximab (n = 4; solid line) along 120-days follow-up. (C) Percentage of change from baseline values (before Tx; dotted line) for the frequency (left) and absolute counts (right) of CD4+ Foxp3+ T cells from children treated (n = 2; broken line) and non-treated with basiliximab (n = 4; solid line) along 1-year follow-up. (D) Foxp3 MFI at day 10 post-Tx (left) and 1-month post-Tx (right). Dotted line represents the baseline values before Tx and solid lines the mean values along the follow-up.