RBC cultured from immortalized erythroblasts that express ectopic antigens induce antigen specific immune responses

Abstract Introduction: The exposure to foreign alloantigens upon pregnancy or after receiving red blood cell (RBC) transfusion can trigger humoral immunity. In contrast, recent studies also showcase the possibility of using RBCs as tolerogenic antigen carriers by exploiting the natural route of RBC clearance through the eryptotic pathway where RBC alloantigens can be presented in an immunosuppressive microenvironment. The current evidence points to the role of RBCs as regulators of the immune system that could be utilized as a cellular therapeutic capable of providing antigen specific immune tolerance. Understanding the factors that control an immune response versus a tolerogenic response are thus important. We aim to discover fundamental aspects that dictate the immunological responses in mouse models, after a mismatched transfusion of in vitro cultured mouse RBC (cRBC) that express the hen egg lysozyme (HEL)-Ovalbumin-Duffy (HOD) antigen. Methods: C57BL/6 murine bone marrow was isolated, and erythroid cells were expanded to purity. Erythroblast cell lines were generated through immortalized using CRISPR Cas9-mediated Trp53 gene editing targeting exon 4 to generate a knockout. The immortalized erythroblasts (iEBL) were lentivirally transduced to express the hen egg lysozyme (HEL)-Ovalbumin-Duffy (HOD) antigen and clonal cell lines were obtained through FACS that express high, medium or low HOD. Differentiation of iEBL to cRBC was induced using StemPro34 medium supplemented with 1mg/mL holo-transferrin and 5U/mL Erythropoietin in presence of 5% knock-out serum replacement. BALB/c mice elucidate a stronger humoral immune response towards foreign antigens than C57BL/6 mice. To realize our objective, 5 mice from each inbred strain were transfused with cRBC expressing high HOD through tail vein intravenous injection. The level of IgM and IgG was determined by incubating cRBC expressing high HOD with the collected sera from the mouse followed by a staining with a conjugated anti mouse secondary antibody. Results: The gene editing efficiency of sgRNA-Cas9 ribonuclear protein complexes (RNPs) against Trp53 was assessed. The chosen RNP allowed a total editing efficiency of 52.3% on basis of a Tide analysis of Trp53 exon 4 sequence from the gene edited 3T3 mouse fibroblast cell line. Afterwards, erythroblasts cultured from C57Bl/6 murine bone marrow were electroporated with this specific RNP and cultured for 6 weeks to obtain a continuously proliferating gene edited immortalized erythroid bulk culture. Following the immortalization, iEBLs were transduced to express the HOD antigen. FACS sorting and clonal outgrowth were used to establish erythroblast lines that have varying densities of HOD surface antigen. The HOD expressing murine erythroblasts were differentiated in vitro to HOD-cRBC and achieved 80-90% enucleation rate. To assess the alloimmunization differences between C57BL/6 and BALB/c mice, both strains were transfused with high HOD expressing cRBC. C57BL/6 mice showed an incremental increase in IgM and IgG antibody titer towards HOD cRBC which peaked at day 6, and 21, respectively. The BALB/c IgG response peaked at day 12 followed by a decrease at day 21. 1*10^8 cRBC were sufficient to mount an alloimmune reaction towards HOD in comparison to literature whereby 1*10^9 are recommended. Conclusion: The knockout out of Trp53 allows the generation of iEBL. These iEBL can be further altered using lentiviral transduction to express any antigen of interest. This particular immortalization scheme, expansion and differentiation to cRBCs thus circumvents the use of transgenic animals as a source for genetically modified RBCs and constitutes an important resource We showcased that a singular injection of 1*10^8 HOD expressing cRBC in C57BL/6 mice was safe and sufficient to induce a robust humoral immune response against HOD as seen in the doubling of the HOD-specific IgG titers at day 21 post transfusion. BALB/c mice displayed an equally robust anti-HOD IgG response at day 12 compared to C57BL/6 mice which was the highest at day 21. We conclude that BALB/c mice do indeed elicit a different adaptive immune response towards foreign alloantigens than C57BL/6 mice.