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

Abstract

ABSTRACTchondria but is released following MOMP. BH3 profiling thus provides an assessment of mitochondrial susceptibility to MOMP that integrates the functional activity of all of the BCL2 family proteins that regulate the intrinsic apoptosis pathway in individual cells. This method also allows us to simultaneously compare BH3 peptideinduced mitochondrial membrane depolarization ('priming') in different T-cell subsets: Treg, conventional CD4 T cells (Tcon), and CD8 T cells. Using this approach, our analysis of apoptotic pathways demonstrated that Treg are more susceptible to apoptosis through both intrinsic and extrinsic pathways than other T-cell subsets. This difference is evident in healthy donors indicating that increased susceptibility to apoptosis is reflective of normal Treg homeostasis. In addition, mitochondrial apoptotic priming of Treg and other T-cell subsets is significantly increased after HSCT and particularly in patients with active chronic GvHD (cGvHD). Methods Patients and sample collectionBlood samples were obtained from 57 patients who had undergone allogeneic HSCT at the Dana-Farber Cancer Institute and Brigham and Women's Hospital (Boston, MA, USA). Written informed consent was obtained from each patient before sample collection in accordance with the Declaration of Helsinki. This protocol has been reviewed and approved by the Institutional Review Board of the Dana-Farber Harvard Cancer Center. The severity of cGvHD was classified according to NIH criteria. We also studied 25 healthy individuals. BH3 profiling assayThe BH3 profiling assay has been previously described. 21,25,26 PBMC were incubated with anti-CD4, anti-CD25, anti-CD127, and anti-CD8 at 4°C for 20 min. After incubation, 2.4 x 10 6 cells were washed and suspended in T-EB Buffer. 26 Individual BH3 peptides were added to 50 μl of cell suspension for 30 min at room temperature. After incubation, 12.5 μl of 22.5 µM TMRE (Invitrogen) was added for 30 min. 25 Cells were analyzed on the BD LSRFortessa using FACS Diva software (BD Biosciences). CD4 T-cell subsets were defined by surface antigen expression: Tcon as CD4 + CD25 neg-low CD127 med Other methodsAdditional flow cytometry methods are detailed in the Online Supplementary Methods. Statistical analysisDescriptive statistics were used for patients' and transplantrelated characteristics. Fisher's exact test or a c 2 test was used for group comparisons for categorical variables in Table 1. The Wilcoxon rank sum test was used for pair-wise group comparisons for continuous variables. The Wilcoxon signed test was used for the difference in BH3 profiling, expression levels of BCL2 family proteins and proliferation and functional assays between two Tcell subsets within the same blood samples. For the Wilcoxon signed test, percent change was calculated between two T-cell subsets and an absolute change of 10% or higher was regarded as significantly different between two T-cell subsets. Multivariate linear regression analysis was also performed to compare T-cell subsets after adjusting for...

Circulating T follicular helper cells with increased function during chronic graft-versus-host disease

“…Cell death mechanisms through either extrinsic (CD95/Fas) or intrinsic (BCL-2) apoptosis pathways have thus been shown to play a role in the differential regulation of different T-cell subsets. 42,43 BCL-2 is a critical antiapoptotic molecule that protects mitochondrial integrity during activation of the intrinsic apoptosis pathway. In murine models, BCL-2 helps to protect activated TFH from negative regulation.…”

Unbalanced recovery of regulatory and effector T cells after allogeneic stem cell transplantation contributes to chronic GVHD

“…32 Susceptibility to apoptosis was assessed by intensity of surface membrane expression of CD95 (Fas) and intracellular expression of BCL2. 9,33 Prior to analysis, peripheral blood mononuclear cells were purified by density gradient centrifugation. peripheral blood mononuclear cells were incubated with fluorochrome-conjugated Mabs directed against cell surface antigens for 20 minutes at room temperature.…”

“…[1][2][3][4] Although most studies have focused on reconstitution of effector T cells, several studies have also examined recovery of CD4 regulatory T cells (CD4Tregs). [5][6][7][8][9] These studies suggest that CD4Treg deficiency can enhance alloreactivity and promote graftversus-host disease (GVHD). [10][11][12][13][14] Conversely, prompt recovery of CD4Tregs can prevent GVHD while also supporting recovery of a broad T-cell repertoire.…”

Mechanistic approaches for the prevention and treatment of chronic GVHD

“…Although there is a compensatory increased homeostatic proliferation of circulating memory Tregs, this is ultimately insufficient to prevent Treg deficiency, due in part to proliferation-induced telomere shortening, 66 enhanced death signaling, and apoptosis of rapidly dividing Treg. 57,67 Treatment strategies attempting to enhance Treg number and function are therefore attractive for chronic GVHD therapy, offering the possibility of therapeutic immune modulation without generalized immunosuppression.…”