Abstract B39: Neutrophils G-CSF stimulated promotes specific protection against graft vs. host disease and keeps the graft vs. leukemia effect
Authors: Perobelli Suelen Martins , Mercadante Ana Carolina Terra , Gonçalves-Silva Triciana , Galvani Rômulo , Pereira-Neves Antônio , Benchimol Marlene , Nobrega Alberto , Bonomo Adriana 
Published: October 2015

Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat a series of hematological diseases. While the presence of T cells mediates the graft-versus-leukemia (GVL) effect and improves engraftment, it is also responsible for the graft-versus-host disease (GVHD), which is the main barrier of HSCT. Patients transplanted with cells from G-CSF treated donors show an unexpected low rate of acute GVHD given the high numbers of T cells present. We have previously shown that blood from G-CSF treated donors have high numbers of inhibitory granulocytes. Experimentally these granulocytes are able to prevent aGVHD in a semi-allogeneic mouse model. Here, our goal is to characterize the granulocytes and its mechanism of action. When we compare this granulocytes from the spleens of 5 day G-CSF treated donors and its untreated control, we observed on flow cytometry that the percentage of this granulocytes/neutrophils Ly6G+ increases from about 2% to about 20%. These cells have some differences among themselves after treatment as increased burst oxidative (measured by DHR) and phagocytosis of E.coli, as well as differences in the pattern of expression of some important surface molecules, such as MHC-II. Levels of myeloperoxidase are lower after treatment with G-CSF, and we confirmed by transmission electron microscopy that these neutrophils have a degranulated profile, with 83% fewer cytoplasmic granules than controls. Indeed, the spleens from 5 day G-CSF treated donors together with control bone marrow cells (G-B6) injected into irradiated F1(B6XBALB/c) mice shows survival rate of 100%, against 10% in non-G-B6 transplants. This protection depends on Gr1+ cells and presents milder pathological and clinical manifestations in gastrointestinal mucosa, skin and liver. However, when these G-B6 neutrophils are from IL-10 KO mice, protection is abolished both in survival curve and in clinical score. Surprisingly, 25 days after transplantation the percentage of the subtype of T regulatory Foxp3+ cells in protected chimeras is increased in all lymphoid organs analyzed and this can be associated with the long term specific tolerance observed. Finally, the anti-tumoral effect in G-B6 chimeras is as potent as in the B6 control chimera and this effect was maintained for more than 20 weeks after HSCT. So we can conclude that treatment with G-CSF generates degranulated neutrophils that reduce GVHD in an IL-10 dependent manner while keeping GVL effect opening a promising road in the prevention of human aGVHD. Financial Support: FAF, INCA, Swiss Bridge, INCT Cancer, FAPERJ, CNPQ, CAPES, Fiocruz.
Citation Format: Suelen Martins Perobelli, Ana Carolina Terra Mercadante, Triciana Gonçalves-Silva, Rômulo Galvani, Antônio Pereira-Neves, Marlene Benchimol, Alberto Nobrega, Adriana Bonomo. Neutrophils G-CSF stimulated promotes specific protection against graft vs. host disease and keeps the graft vs. leukemia effect. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B39.