The Effect of Granulocyte-Colony-Stimulating Factor (G-CSF) on T Cell Polarization in Vitro: A Direct Comparison Between Nivestim® and Neupogen®
Authors: Mifsud Nicole , Ismail Amina , de Valle Elisha , Spencer Andrew , Patil Sushrut S. , Grigoriadis George , Gugasyan Raffi 
Published: October 2019

Abstract
Introduction Recombinant granulocyte colony stimulating factor (G-CSF) such as filgrastin (NeupogenÒ) is widely used to mobilize peripheral blood stem cells (PBSC) in healthy donors prior to allogeneic stem cell transplantation (all-SCT). In comparison to the bone marrow harvest, the G-CSF mobilized graft has a higher content of donor-derived T cells, the primary mediators of acute graft versus host disease (aGvHD). However, the incidence of aGvHD is identical suggesting a modulatory effect of Neupogen on T cells. The differentiation of allo-T cells to a T-helper 1 (Th1) phenotype polarizes the cells to produce IFN-g and IL-2, and this in turn promotes the development of GvHD. Conversely, recent evidence suggests that T-helper 2 (Th2) cells that produce IL-4 and IL-10 protects against GvHD. In healthy donors, G-CSF (NeupogenÒ) has been shown to decrease IFN-g and increase IL-4 production. A finding that is also observed under specific stimulatory condition in vitro.
The recent patent expiry on G-CSF (Neupogen®) has prompted the development of alternative forms of G-CSF – called biosimilars. These molecules are structurally equivalent but their biological mode of action is currently under review. However, biosimilar G-CSF has not been accepted for routine stem cell mobilization in healthy donors primarily due to a lack of data about their safety in this cohort. In addition, no data exists of their influence on T cell modulation, which will potentially have a significant effect on aGvHD in the recipient. The primary objective of this study was to investigate the in vitro effects of Nivestim®, a biosimilar G-CSF that has been approved by the Therapeutic Goods Administration in Australia, and compared to the reference product filgrastim (Neupogen®) on human CD4+ T cells obtained from healthy volunteers.
Results PBMC from 10 healthy volunteers, were cultured in the presence of Nivestim® or Neupogen® (both at 100 ng/ml), and stimulated 24 hrs later with either (1) anti-CD3 MoAb; (2) Phytohaemagglutin (PHA) or (3) PMA plus Ionomycin. The effects of Nivestim® and Neupogen® were examined on Th1 and Th2 cytokine profiles (IFN-g and IL-4 respectively), Th-specific transcriptional regulators, and T cell proliferation and survival.
Our findings showed that CD4+ T cells from PBMC cultures produced significant levels of IFN-g following T cell activation. However, in the presence of Nivestim® or Neupogen®, the production of IFN-g was notably reduced and IL-4 levels were increased. These findings were consistent regardless of whether the T cell receptor was stimulated directly, or by-passed through the activation of proximal signaling molecules.
Conclusion Whilst this pattern of Th2 polarization was not observed for all healthy volunteers, the biological effects of Nivestim® was comparable to Neupogen® in all cases. These findings have established that Nivestim® has an immunomodulatory effect on Th1/Th2 polarization in vitro that is equivalent to the reference product Neupogen®. Our findings will assist in facilitating Nivestim’s use for stem cell mobilization in healthy donors.

Disclosures
Mifsud: Hospira: Research Funding. Ismail:Hospira: Research Funding. de Valle:Hospira: Research Funding. Spencer:Hospira: Research Funding. Patil:Hospira: Research Funding. Grigoriadis:Hospira: Research Funding. Gugasyan:Hospira: Research Funding.