Résumé
Objective To investigate whether rat adipose-derived stem cells (rASCs) could resist human xenoantibody-dependent complement-mediated lysis and to explore its possible mechanisms.Method SD rat ASCs were isolated,rASCs at passage 2 to 8 were used for the following studies and rat lymphocytes were harvested as control cells.α-Gal expression was detected by flow cytometry.After incubation of rASCs with 20％ normal human serum (NHS) or heat inactivated normal human serum (HINHS),flow cytometry was used to detect cytotoxicity,IgG or IgM binding,and C3c,C4c and C5b-9 deposition.Result We successfully established the method to isolate and culture rASCs.The morphology of rASCs remained unchanged after passages.rASCs were positive for tell surface markers of CD44 and CD90,while negative for CD45 and MHC-Ⅱ.As compared with rLCs,rASCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20％ NHS in vitro (20.42％ ± 2.80％ vs 51.84％ ± 6.70％,P ＜ 0.01).Mechanistically,rASCs expressed lower level of α-Gal (13.97 ± 0.33 vs.24.47 ± 3.03,P＜0.05),which was correlated with decreased binding of human xenoreactive IgG and IgM (IgM:9.4 ± 2.0 vs.107.2± 4.8,P＜0.01; IgG:5.73 ± 1.0 vs.27.49 ± 3.9,P＜0.01) and reduced deposition of complements C3c,C4c and C5b-9 (C3c:294.6 ± 38.02 vs.1924 ± 509.4,P＜0.05; C4c:35.23 ± 3.1vs.177.3 ± 37.17,P＜0.05; C5b-9:5.63 ± 1.74 vs.37.05 ± 7.4,P＜0.01).Conclusion These data demonstrated that the resistance of rASCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen a-Gal and inhibition of MAC (membrane attack complex) formation.
Résumé
Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibodys efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitts lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitts cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.