Assistant Professor University of Illinois at Urbana Champaign, United States
Introduction:: Erythrocytes or red blood cells (RBCs) are an attractive engineering target for numerous applications such as drug delivery, imaging vaccination, etc. In addition to their long lifespan, the clearance process of aged or damaged RBCs is also an interesting property. As aged or damaged RBCs are removed by phagocytotic cells (e.g. macrophages) in a non-inflammatory manner, autoantigen-conjugated RBCs or RBC-like materials have been adoptively transferred to induce antigen-specific tolerance (Kontos S, Kourtis IC, Dane KY, Hubbell JA. Proc Natl Acad Sci U S A. 110(1):E60-8 (2013).). However, the underlying mechanism behind this immunotolerance phenomenon remains to be explained. Moreover, a safe and effective approach of in vivo surface engineering of RBCs are yet to be developed.
Here we propose an approach for in vivo metabolic glycan labeling of RBCs which introduces unique chemical tags such as azido groups onto the membranes of RBCs. This labeling process allows subsequent in vivo conjugation of desirable cargos (e.g. dibenzocyclooctyne (DBCO)-bearing autoantigens in our case) onto RBCs via click chemistry (Wang, H., Mooney, D.J. Nat. Chem. 12, 1102–1114 (2020).). We hypothesize that the conjugated RBCs will induce amplified antigen specific immune tolerance through interacting with macrophages in a non-inflammatory manner. A scheme of our hypothesis is shown in figure 1.
Materials and Methods:: Chemicals: DBCO modification of SIINFEKL (ovalbumin peptide) was achieved by mixing the peptide solution and DBCO-Sulfo-NHS ester solution (in DMSO) at 1:2 molar ratio. Reaction was carried out through incubation under 4c overnight. The remaining DMSO and unreacted NHS ester were removed through dialysis.
Cells: Bone marrow derived macrophages (BMDMs) were isolated and differentiated from C57BL/6 mice. In vivo metabolic labeling of RBCs was achieved by intravenous injection of azido sugar (Tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz)) into C57BL/6 mice (2mg sugar per dose, 6 doses per mouse). RBCs were obtained by tail vein blood collection.
In vitro study: The labeling effect of RBCs was examined by FACS assay after in vitro conjugating DBCO-Cy5 with the RBCs collected from the sugar injected mice and control mice. Phagocytosis of RBCs by BMDMs were demonstrated by adding DBCO-Cy5 conjugated RBCs to the macrophage culture and subsequent FACS assay. Antigen presentation by macrophages was verified by in vitro coculturing of DBCO-SIINFEKL conjugated RBCs with BMDMs at a ratio of RBC: BMDM = 10:1 overnight and subsequent FACS assay. The antigen-specific immune tolerance was examined in vitro by first coculturing DBCO-SIINFEKL conjugated RBCs with BMDMs at a ratio of RBC:BMDM=100:1 overnight, then removing the unphagocytosed RBCs before adding freshly isolated OT1 cells. The OT1 cells were labeled with CFSE for proliferation analysis. During all experiments, RBCs were thoroughly washed after coculturing with DBCO-cargo.
Results, Conclusions, and Discussions:: First, we confirmed that the azido-sugar (Ac4ManAz) successfully labeled RBCs with azido groups in vitro, as detected by DBCO-Cy5 under flow cytometry analysis. (figure 2) The higher intensity of Cy5 from the RBCs from sugar injected mice indicates a higher percentage of Cy5 conjugated to RBCs through click chemistry between the DBCO and azido groups, verifying the successful in vivo azido labeling of RBCs.
Next, we confirmed that BMDMs can phagocytose the metabolically labeled RBCs. After coculturing DBCO-Cy5 conjugated RBCs with BMDMs for 6 hours, we measure the mean fluorescent intensity of Cy5 among BMDM population. As shown in figure 3, BMDMs show much higher Cy5 signal after cocultured with DBCO-Cy5 conjugated RBCs compared with coculturing with only RBCs, indicating that RBCs can be efficiently phagocytosed by BMDMs. And it is reasonable that the highest Cy5 intensity is from the azido labeled RBC group.
Then we change to DBCO-SIINFEKL in order to further investigate the antigen presentation as well as antigen specific immune effect. The presentation of SIINFEKL by BMDMs after their phagocytosis of SIINFEKL conjugated RBCs can be verified by direct detecting of SIINFEKL with H-2Kb monoclonal antibody. As shown in figure 4, the presence of SIINFEKL on the surface of BMDMs verifies the antigen presentation capability of BMDMs. Moreover, our metabolic glycan labeling leads to a more efficient antigen presentation effect, indicated by the highest level of SIINFEKL.
After demonstrating the phagocytosis and antigen presentation process when RBCs interact with BMDMs, we further investigate the immune suppression effect by coculturing BMDMs (previously cocultured with SIINFEKL conjugated RBCs) with OT1 T cells. Through analyzing the division and proliferation index (figure 5), we concluded that antigen specific immune tolerance was generated, indicated by the lower division and proliferation index of the groups where the antigen SIINFEKL is present. The proliferation status of OT1 can also be confirmed by measuring the MFI of CFSE. The metabolic labeling of RBCs induced stronger immune tolerance, characterized by the division index.
Next, we plan to move forward with in vivo study to demonstrate the immune tolerance effect using autoimmune disease models.
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