Post-Transplant Cyclophosphomide Spares Granzyme B Expression in T Regulatory Cells (Treg), but Not in CD8+ T and NK Cells after Allogeneic HSCT
Authors: Drokov Mikhail , Parovichnikova Elena N. , Kuzmina Larisa A. , Vasilieva Vera , Mikhaltsova Ekaterina , Koroleva Olga , Dubnyak Daria , Galtseva Irina , Savchenko Valeri 
Published: October 2019

Abstract
Introduction
Allogenic bone marrow transplantation (allo-HSCT) with high-dose, post-transplantation cyclophosphamide (CY) nowadays has become an alternative for standard immunosuppression. Many groups have shown that high dose CY after allo-HSCT selectively deplete alloreactive T effector cells and downregulate Granzyme B expression in CD8+ T and NK cells after allogeneic HSCT. Despite that fact there is no data about other T cell populations, such as Tregs and their immunosuppression abilities after post-transplant CY. We studied Granzyme B expression in Treg on day +14 after allo-HSCT with post-transplant CY and standard immunosuppression.
Patients and methods.
Peripheral blood samples were collected in EDTA-tubes at day +14 after allo-HSCT in patients with hematological malignancies with post-BMT-CY alone (n=8) and patients with standard immunosuppression (post-HSCT CSA, MMF or MTX at standard dose) (n=18). The anti-CD4-PE-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine Treg cells population as CD4+ CD25high CD127low. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms and etc. CD4- lymphocytes (certainly containing CD8+ and NK-cells population that obligatorily express granzyme B) were used as internal positive control to define population of CD4+ CD25high CD127low GranzymeB+ cells and gMFI (geometric mean fluorescence intensity). 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA).
Results. Mann-Whitney U test was used to test for differences between Granzyme B+ Treg cells after post-transplant-CY alone and in a group of standard immunosuppression. The percent of CD4+ CD25high CD127low GranzymeB+ cells among CD4+ cells at day +14 after post-transplant CY alone was statistically higher (30.1±21,9; p=0.018*) than in patients with standard immunosuppression (3,52±1,56) (see Chart 1). There is no differences in Granzyme B expression (gMFI) in CD4+ CD25high CD127low cells after post-transplant-CY (2131,8±412,7) alone and in a group of standard immunosuppression (1718,17±316,8; p=0.541). It's worth to note that probably this mechanism in combination with depletion of alloreactive T effector cells and downregulation of Granzyme B expression in CD8+ T and NK cells help to prevent aGVHD in this group of patients.
Conclusion We suggest that post-transplant CY spares Granzyme B expression in Tregs in a patients with post-transplant-CY and help to prevent aGVHD development in this group of patients. Despite the fact that the analyzed group is small, obtained data is important and needs further investigation.
Figure 1. Granzyme B expression in Tregs after post-transplant-CY alone and in a group of standard immunosuppression. Figure 1. Granzyme B expression in Tregs after post-transplant-CY alone and in a group of standard immunosuppression.

Disclosures
No relevant conflicts of interest to declare.