ISSN 1866-8836
Клеточная терапия и трансплантация

Impact of Post-Transplant Cyclophosphamide after Hematopoietic stem cell transplantation on chimerism in T-regulatory cells

Darya S. Dubnyak1, Natalya V. Risinskaya1, Mikhail Y. Drokov1, Natalia S. Kostritsa2, Julia O. Davydova1, Larisa A. Kuzmina1, Vera A. Vasilyeva1, Natalia N. Popova1, Ekaterina D. Mikhaltsova1, Olga M. Koroleva1, Nikolay М. Kapranov1, Zoya V. Konova1, Irina V. Galtseva1, Andrey B. Sudarikov1, Elena N. Parovichnikova1, Valery G. Savchenko1
1National Research Center for Hematology, Moscow, Russian Federation
2School of Medicine, Lomonosov Moscow State University
doi 10.18620/1866-8836-2015-4-1-2-47-57

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Cellular Therapy and Transplantation (CTT)
Volume 6, Number 3



Immunological incompatibility between donor and recipient is the main cause of alloimmune complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A key role of T-regulatory cells (Treg) is supporting immunological tolerance after transplantation. This cell population prevents the development of acute graft-versus host disease (aGVHD). Activation of T cells occurs on day +3,+4 after allo-HSCT that is associated with T cell-mediated recognition of the peptides presented by donor (direct allorecognition) or recipient (indirect allorecognition) antigen-presenting cells. Over last years, post-transplant regimen with high-dose cyclophosphamide (PTCy) at 50 mg/kg (day +3 and +4) becomes a widespread immunosuppressive treatment which leads to GVHD prevention, due to deletion of alloreactive T-cells. However, its influence upon other subpopulations has not been studied yet. Our aim was to evaluate an impact of a high-dose PTCy after hematopoietic stem cell transplantation upon chimerism in T-regulatory cell population.

Patients and methods

The analysis included 27 patients after allo-HSCT (ALL, n=6; AML, n=20; CMML, n=1). Detailed characteristics of the group are presented in Table 1. Peripheral blood samples were collected by the day +30 after allo-HSCT. Peripheral blood mononuclear cells (PBMCS) were isolated using a ficoll density gradient (1.077 g/L). CD4+CD25+ cells (T-reg cells) were isolated by means of immunomagnetic separation (CD4+CD25+ Regulatory T Cell Isolation Kit, Milteney Biotec, Germany). A control test for “purity” of the enriched cell population was performed in some patients using flow cytometry (Figure 1). DNA extraction from cell suspension was carried out using DNA/RNA isolation kits “Ampli Prime RIBO-prep”. Gene chimerism in DNA samples was assessed by the STR-PCR analysis (polymerase chain reaction with a panel of primers for loci of short tandem repeats). The percentage of donor chimerism was calculated using standard formulas. Statistical analysis was performed using SPSS ver 23. (IBM, Chicago,Ill., USA). A Mann-Whitney U test was used for nonparametric data analysis. Fisher’s exact test was used for 2×2 tables. A p-value less than 0.05 was considered as significant.


Data on genetic chimerism in T-reg cell populations are presented in Figure 2. The proportion of host hemopoiesis is significantly lower in T-reg cells in case of using PTCy on day +3, +4, in contrast to classical immunosuppressive regimen (p=0,03*). A median proportion of these cells with host genotype (with interquartile interval) was 0% (0-2.5%) with PTCy, and 10% (5-27%) after standard immunosuppressive therapy.



Using of PTCy is considered to decrease GVHD rate even in case of sufficient HLA mismatch between patient and donor. However, the mechanism of this phenomenon is still unknown. PTCy leads to deletion of alloreactive T cells as shown by several authors. In present study, we have revealed that PTCy caused deletion of T-regulatory cells, as seen from the course of post-transplant recovery of T-reg, due to predominance of donor cell genotype in the PTCy group.


Allogeneic hematopoietic stem cell transplantation, post-transplant high dose cyclophosphamide, chimerism, T-regulatory (Treg) cells, graft-versus host disease.

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