Increased mitochondrial apoptotic priming of human regulatory T cells after allogeneic hematopoietic stem cell transplantation

Abstract

CD4 regulatory T cells play a critical role in establishment of immune tolerance and prevention of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. The recovery and maintenance of regulatory T cells is dependent on homeostatic factors including the generation of naïve regulatory T cells from hematopoietic precursor cells, the proliferation and expansion of mature regulatory T cells, and the survival of regulatory T cells in vivo. In this study, quantitation of mitochondrial apoptotic priming was used to compare susceptibility of regulatory T cells, conventional CD4 T cells and CD8 T cells to intrinsic pathway apoptosis in 57 patients after allogeneic hematopoietic stem cell transplantation and 25 healthy donors. In healthy donors, regulatory T cells are more susceptible to mitochondrial priming than conventional T cells. Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T-cell subsets and particularly in patients with chronic graft-versus-host disease. Regulatory T cells express high levels of CD95 and are also more susceptible than conventional T cells to apoptosis through the extrinsic pathway. However, CD95 expression and extrinsic pathway apoptosis is not increased after hematopoietic stem cell transplantation. Decreased expression of BCL2 and increased expression of BIM, a mitochondrial cell death activator protein, in regulatory T cells contributes to increased mitochondrial priming in this T-cell subset but additional factors likely contribute to increased mitochondrial priming following hematopoietic stem cell transplantation.

Figure 1.

Apoptosis pathways in T-cell subsets: healthy donors (n=25). (A) BH3 profiling. Mitochondrial membrane depolarization after challenge with BH3 peptides in each T-cell subset (CD8: green; Tcon: blue; Treg: red). The percentage of depolarization was determined after challenge with individual peptides indicated on the x-axis. (B) Expression of anti-apoptotic proteins and BIM in each T-cell subset. Protein expression was measured by flow cytometry. Relative levels of BCL2, BCLXL, MCL1 and BIM were calculated by dividing the median MFI for each protein by the median MFI of isotype control IgG. (C) Expression of Fas (CD95) and cell proliferation (Ki67) in each T-cell subset were measured by flow cytometry. (D) Apoptosis induction after in vitro stimulation with staurosporine (STS) or anti-CD95 monoclonal antibody. Rapid induction of apoptosis in each T-cell subset was assessed by annexin V/7-AAD co-staining. CD95-induced apoptosis data were log-transformed. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 2.

Apoptosis pathways in T-cell subsets: post-HSCT patients (n=57). (A) BH3 profiling. Mitochondrial membrane depolarization after challenge with BH3 peptides in each T-cell subset (CD8: green; Tcon: blue; Treg: red). The percentage of depolarization was determined after challenge with individual peptides indicated on the x-axis. (A) Expression of anti-apoptotic proteins and BIM in each T-cell subset. Protein expression was measured by flow cytometry. Relative levels of BCL2, BCLXL, MCL1 and BIM were calculated by dividing the median MFI for each protein by the median MFI of isotype control IgG. (B) Expression of CD95 and Ki67 in each T-cell subset was measured by flow cytometry. (C) Apoptosis induction after in vitro stimulation with STS or anti-CD95 monoclonal antibody. Rapid induction of apoptosis in each T-cell subset was assessed by annexin V/7-AAD co-staining. CD95-induced apoptosis data were log-transformed. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 3.

BH3 priming in T-cell subsets after allogeneic HSCT. Percentage of depolarization was determined in each T-cell subset after challenge with individual BH3 peptides. Within each T-cell subset, results are compared for healthy donors (HD), patients without cGvHD (no cGvHD) and patients with cGvHD. *, **, *** denote significant differences between HD and cGvHD and between HD and no cGvHD: *P<0.05; **P<0.01; ***P<0.001. +, ++ denote significant differences between cGvHD and no cGvHD: +P<0.05; ++P<0.01.

Figure 4.

Expression of apoptotic proteins in T-cell subsets in patients with and without cGvHD and healthy donors. (A) Expression of anti-apoptotic (BCL2, BCLXL, MCL1) and pro-apoptotic proteins (BIM) in each T-cell subset. (B) Expression of cell surface CD95 and Ki67 in each T-cell subset. (C) Apoptosis induction after in vitro stimulation with STS or anti-CD95 monoclonal antibody in each T-cell subset. CD95-induced apoptosis data were log-transformed. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 5.

BH3 priming in T-cell subsets in patients with cGvHD. Percentage of depolarization was determined in each T-cell subset after challenge with individual BH3 peptides. Within each T-cell subset, results are compared for patients with mild, moderate or severe cGvHD. *, ** denote significant differences in BH3 priming between mild or moderate cGvHD and severe cGvHD. *P<0.05; **P<0.01. There were no significant differences between mild and moderate cGvHD.

Figure 6.

Expression of apoptotic proteins in T-cell subsets in patients with severe, moderate or mild cGvHD. (A) Expression of anti-apoptotic (BCL2, BCLXL, MCL1) and pro-apoptotic proteins (BIM) in each T-cell subset. (B) Expression of cell surface CD95 and Ki67 in each T-cell subset. (C) Apoptosis induction after in vitro stimulation with STS or anti-CD95 monoclonal antibody in each T-cell subset. CD95-induced apoptosis data were log-transformed. *P<0.05; **P<0.01; ***P<0.001.