Int J Stem Cells.  2018 Jun;11(1):78-86. 10.15283/ijsc17047.

PD-L1 Expression on Circulating CD34+ Hematopoietic Stem Cells Closely Correlated with T-cell Apoptosis in Chronic Hepatitis C Infected Patients

• Abdellatif H
• Shiha G

• KMID: 2413503
• DOI: http://doi.org/10.15283/ijsc17047

Abstract

BACKGROUND AND OBJECTIVES
Lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered stem cell research. Programmed death-1 (PD-L1) has been reported on parenchymal cells in patients with chronically inflamed livers and found to play an essential role in T cell homeostasis regulation. However, the bidirectional interaction between HSCs and lymphocytes remains elusive. Here, we aimed to get more insight into circulating CD34+ HSCs PD-L1 expression and T cell apoptosis in chronic HCV infected patients.
METHODS
CD34+ HSCs were isolated and purified by immunomagnetic separation. PD-L1 expression was analyzed by quantitative PCR and flow cytometry. Furthermore, co-culture experiments between CD34+ HSCs and T-lymphocytes were established. T-cell lymphocyte apoptosis in peripheral blood and in cultures was detected.
RESULTS
CD34+ HSCs constitutively express low levels of PD-L1. Its expression is up-regulated in chronic HCV infected patients. Moreover, PD-L1 expression on circulating CD34+ HSCs enhanced T cell apoptosis in peripheral blood and co-culture.
CONCLUSION
Our results suggest novel bidirectional interplay between HSCs and lymphocytes mediated by PD-L1 expression on CD34+ HSCs. PD-L1 expression correlated with T-cell lymphocyte apoptosis. This may contribute to immunomodulatory properties of HSCs which improves its use for allogeneic transplantation.

Keyword

CD34+ HSCs; PD-L1; B7-H1; T-lymphocytes; Apoptosis; HCV

MeSH Terms

Apoptosis*
Coculture Techniques
Flow Cytometry
Hematopoietic Stem Cells*
Hepatitis C, Chronic*
Hepatitis, Chronic*
Homeostasis
Humans
Immune System
Immunomagnetic Separation
Liver
Lymphocytes
Polymerase Chain Reaction
Stem Cell Research
T-Lymphocytes*
Transplantation, Homologous

Figure

• 

  Fig. 1 PD-L1 expression on CD34+ HSCs in peripheral blood of chronic HCV-infected patients by flow cytometry. The peripheral blood CD34+ cells of patients and healthy controls were isolated by using MiniMacs high-gradient magnetic separation column and subsequently analyzed by flow cytometry using anti-CD45 and anti-CD34 antibodies. (A) A representative plot of enriched leukocytes shown by forward versus side scatter. The P1 gate represents the isolated PBMCs region. (B) The events in gate P1 are then displayed on a CD45 vs. SSC dot plot and a second gate (LR2) is produced to include the cluster of CD45+ events. (C) This plot is obtained by plotting the events that are fulfilling the criteria of gates P1 and LR2 (sequential gating). Cells forming a cluster with characteristic positivity to CD34 antibody are displayed in this plot creating the region LR3. (D) This plot is created by displaying cells that are positive for both CD45 and CD34 antibodies (LR3). Cells in lower right quadrant LR-4 are those with positive expression of PD-L1.

• 

  Fig. 2 Elevation of CD34+ HSCs in the peripheral blood of CHC patients. A Percentage of CD34+ cells in PBMCs fraction. Data represent the percentages of circulating CD34+ cells in the PBMCs fractions of 30 CHC patients and 20 healthy controls. Values are expressed as mean±SD. *indicates significant deviation from the control as indicated by p value<0.05.

• 

  Fig. 3 PD-L1 expression on CD34+ HSCs in peripheral blood of CHC patients (▲) and healthy controls (●). Data were presented as percentage of positive cells (A) and mean fluorescence intensity (B). Graphs showed vertical scatter plots and mean columns. *indicates significant deviation from the control as indicated by p value<0.05. (C) Detection of PD-L1 transcript in purified CD34+ HSCs of CHC patients by RT-PCR. cDNA was synthesized from total RNA of purified CD34+ cells and β-actin was used as an internal control to demonstrate equal loading and integrity of RNA. M, marker, lane 1 healthy control (cont.) and lane 2 CHC patients.

• 

  Fig. 4 Correlation analysis of expression of PD-L1 on CD34+HSCs and T-cell apoptosis (CD8+ Annexin V+ cells) in chronic HCV infected patients. (A) Percentage (%) of PD-L1 expression (B) Mean fluorescence intensity (MFI). r=0.8979 and 0.8448 respectively. p<0.0001.

• 

  Fig. 5 Expression of PD-L1 on CD34+ HSCs mediated CD-8 T-cell apoptosis in peripheral blood of chronic HCV-infected patients. Apoptosis in T-cells was examined by flow cytometric analysis using AnnexinV/PI staining. Numbers of apoptotic cells (positive for Annexin V stain) are indicated in plot D. One of three independent experiments is depicted.

Reference

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