Evaluación del factor estimulante de colonias de granulocitos y macrófagos bovino recombinante (rbGM-CSF) producido en dos sistemas eucariotas

Abstract

Dendritic cells (DC) are professional antigen presenting cells (APC) capable of capturing, processing and presenting antigens to CD4+ T lymphocytes by expressing major histocompatibility complex class II (MHC-II) molecules forming stable complexes with degraded antigenic peptides in the endosomal-lysosomal compartment. DC can be obtained in two stages, the first is the immature stage, whose capacity is to capture and process antigens, and the second is the mature stage characteristic of DC with the capacity to generate a specialized immune response through antigenic presentation. Dendritic cells are generated in vitro from myeloid cells (MoDC) by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4), two cytokines specialized in cell activation, regulation, proliferation and differentiation. In the present study, the bovine cytokine GM-CSF was expressed in Komagataella phaffii NRRLY-11430 and mammalian cell line COS-7, with a yield of 374.8 μg and 643.9 μg, respectively, from 450 mL of culture supernatant for K. phaffii and 100 mL of culture supernatant for COS-7 cells which was obtained stable line. Subsequently, the ability of rbGM-CSF to differentiate monocytes to dendritic cells (MoDC) was evaluated by obtaining a morphology of cytoplasmic prolongations characteristic of DC and a phenotype determined by an expression of MHC-II+, CD80+, CD86+ and CD14- cell surface markers when DC are in mature stage, established by microscopy and flow cytometry, respectively. Additionally, the ability of differentiated bovine MoDC to present antigens was analyzed by forming peptide/MHC II complexes with OVA[323-335] and VP1 in two groups of cells, MoDC without preloading and MoDC preloaded with CLIP; The results showed greater binding to the VP1 peptide at 25 min of incubation when the cells were preloaded with CLIP, being this ten times more binding to the VP1 peptide than the OVA peptide, allowing them to exhibit higher rates of active MHC-II molecules on their surface.