Granzyme B Expression in T-Regulatory Cells Is a Strong Predictor of Acute Graft-Versus-Host Disease after Day +30 in Patients with "Classic" Immunosuppression after Allo-HSCT
Authors: Drokov Mikhail Yu. , Parovichnikova Elena N. , Davydova Julia , Kuzmina Larisa A. , Galtseva Irina V , Kapranov Nikolay M , Vasilieva Vera , Dubnyak Darya , Koroleva Olga , Mikhalcova Ekaterina , Popova Natalia , Savchenko Valery G 
Published: October 2019

Abstract
Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (T-regs) influence on T-effectors cells. Despite the well-known fact that T-regs participate in pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence, data about functional status of T-regs and aGVHD is still limited.
Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30 after allo-HSCT. We use PBMC from 29 patients with hematological malignancies obtained by density gradient media. This method was used due to lymphopenia in this group of patients. Group with no aGVHD consist of 22 patients (AML=12, ALL n=5, LPD=1, CML=2, AA=1, MDS=1) after allo-HSCT n=17 from MUD, n=5 from mismatch unrelated donor. MAC conditioning regimen was used in 17 cases, 5 patients receive RIC. The group of aGVHD after day +30 include 7 patients. (AML=4, ALL n=3) after allo-HSCT from MUD (n=5), mismatch unrelated donor (n=1) and n=1 from sibling HLA-identical donor. Two patients receive MAC and 5 patients receive RIC conditioning regimen. All patients in both groups receive standard immunosuppression (MMF+CSA+ATG). All patients developed II-IV grade aGVHD (II (n=1); III (n=4); IV (n=2)) with a median time onset on day +50 (34-150).
The anti-CD4-APC-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population. A Bland-Altman plot (difference plot) was used in analyzing the agreement between the two different methods of T-regs assay (CD4+CD25high and CD4+CD25highCD127low) - differences were not found (p=0,942). Due to this fact in our experiments CD4+CD25high cells were identified as T-reg cells (Gregg et al., 2005). 30000 of CD4+ cells were analyzed on a BD FACSCanto II to achieve sufficient statistical power (Becton Dickinson, USA).
Results. As we can see on chart 1 level of Granzyme B was higher in patients who never developed aGVHD. In accordance with chart 2 percentage of Granzyme B positive T-regs in group of patients who never developed aGVHD was 7,26±1,89% in comparison with 2,04±0,93% in group who developed aGVHD after day +30 (p=0,02*). We should note that according to our previous experiments bacterial or viral infection (e.g CMV) and HLA-disparity does not affect the level of granzyme B expression in T-regs. Using ROC- curve analysis (see Chart 3) we obtained area under curve (AUC) 0,74.
Conclusion. Our data shows that Granzyme B in T-reg cells after allo-HSCT in patients with standard IST may predict aGVHD onset after day +30 with satisfactory test results (AUC=0,74, «cut-off» - 4,15%; sensitivity - 85,71%; specificity - 45,45%).). According to data of our transplant center (for 2006-2016), "granzyme B-based strategy" can help us to predict up to 47,3% of all aGVHD.
Disclosures: No relevant conflicts of interest to declare.
Chart 1: Example of Granzyme B expression (right; shown on horizontal axis) in T-regs (left; upper right quadrant) on day +30 after allo-HSCT in group with standard immunosuppression
Chart 2: Percentage of Granzyme B positive T-reg cells in patient without aGVHD and patients who develop aGVHD after day +30.
Chart 3: ROC curve analysis of Granzyme B in T-regs as predictor of aGVHD.
Figure Figure. Figure Figure. Figure Figure.

Disclosures
No relevant conflicts of interest to declare.