G-CSF Stem Cell Mobilization Is Protective Against Acute Graft-Versus-Host Disease (aGvHD) by Increasing Myeloid-Derived Suppressor Cell (MDSC) Frequencies

In mice, graft-versus-host disease (GvHD) can be abrogated by ex vivo expanded, bone marrow derived myeloid-derived suppressor cells (MDSCs) generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al). Whether MDSCs play a role in human allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still unclear. We hypothesized that G-CSF stem cell mobilization may be protective from acute GvHD (aGvHD) by increasing MDSC frequencies.

G-CSF-mobilized peripheral blood (PB) samples were collected from 40 healthy unrelated donors (median age 34, range 20–43, male/female 31/9) who received G-CSF (Filgrastim) at 10 μg/kg/day for 5 days. Donor selection had been performed according to standard criteria, including molecular typing for HLA-A,-B,-C, DRB1, and DQB1. Donors were 10/10 HLA-matched (MUD) in 20 cases, 9/10 in 13 cases and 8/10 in the remaining 7 cases (MMUD). Patients (median age 46, range 18–67) received reduced intensity conditioning based or low-dose total body irradiation (TBI 2Gy) (8), Fludarabine-Rabbit Antithymocyte Globulin (ATG) (9) or Thiotepa-ATG (23). Diagnosis were lymphomas (29), myelomas (8), acute myeloid leukemia (3). GvHD prophylaxis was cyclosporine plus either methotrexate (36) or mycophenolate mofetil (4). As controls, PB samples were collected from 10 healthy adults. Informed consent was obtained from all subjects. Cells were characterized using flow cytometry with Abs against CD3;CD14;CD16;CD19;CD20;CD56;CD11b;HLADR;CD33. The frequencies of MDSCs and T regulatory cells (Tregs, CD4+CD25+CD127-FoxP3+) in the grafts were correlated with the clinical characteristics and outcome of the 40 patients. To verify that G-CSF treatment increases the number of MDSCs that are transferred with the graft and prevent GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was also used. After lethal irradiation, recipient BALB/c mice received spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=5) or BM cells only (negative control, n=3). To generate MDSC enriched allografts, donor mice were treated with G-CSF (5 μg/d for 5 days) and thereafter BM cells and spleen cells were transferred in recipient mice (MDSC cohort, n=5).

Expansion of MDSCs (Lin^-/LoHLADR⁻CD33⁺CD11b⁺) in the PB of G-CSF-treated unrelated donors was found with respect to steady state control individuals (p< 0.03, Mann Whitney-U). Acute GvHD occurred in 16 of 40 patients (40%). There was no significant correlation between the incidence of aGvHD and the degree of HLA incompatibility or the presence of donor-recipient sex mismatches. Neither the conditioning regimens nor the GvHD prophylaxis had effect on risk of aGvHD. There was no correlation between the number of Tregs infused and the occurrence of GvHD.Conversely, aGvHD patients received grafts containing significantly lower number of MDSCs when compared to non- aGvHD patients (p < 0.006, Mann Whitney-U). The ability of MDSC levels in the graft to predict the occurrence of aGvHD was determined by the receiver operating characteristic (ROC) curve: sensitivity was 100%, specificity 60%, AUC=0.768. The immunosuppressive activity of the MDSCs on activated T lymphocytes was confirmed in vitro. To confirm these results, we set up a MHC mismatched HSCT mouse model. G-CSF treatment induced a significant increase in MDSCs (up to four fold, p<0.05) whereas the frequencies of CD34+ and CD3+ cells were not affected. The MDSC-enriched BM cells transferred into lethally irradiated recipient mice together with spleen cells, resulted in a less severe aGvHD as confirmed by monitoring the clinical signs of GvHD (weight loss, generalized erythema of the skin, poor fur quality and overall survival) compared to the GvHD cohort not receiving G-CSF.

G-CSF mobilization significantly increases circulating MDSC both in Matched Unrelated Donors and in mice. In humans, a significant correlation between the frequencies of MDSC present in the graft and the incidence of aGvHD was found. This result was further confirmed using a MHC mismatched HSCT mouse model. Mice treated with G-CSF received grafts containing higher numbers of MDSCs and developed less severe aGvHD than the untreated mice. Taken together, these findings strongly suggest that G-CSF stem cell mobilization is protective against aGvHD through MDSCs augmentation and might be relevant to modify GvHD prophylaxis.

No relevant conflicts of interest to declare.