Low Percent of Granzyme B Positive T-Regulatory Cells (CD4⁺CD25^high) As a Predictor of Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

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 (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting.

Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17).

The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4⁺CD25^high. 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, FoxP3-positivity on activated nonregulatory CD4⁺ cells. CD4^- lymphocytes (CD8⁺ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4⁺CD25^highGranzymeB⁺ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4⁺ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA).

Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4⁺CD25^highGranzymeB⁺ cells among CD4⁺cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4⁺CD25^highGranzymeB⁺ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future.

Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4⁺CD25^highGranzymeB⁺ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation.