(H-298) Antigen-loaded extracellular vesicles to regulate adaptive immune responses and drive immunological tolerance

Introduction:: Autoimmune diseases are driven by adaptive immune responses that are inappropriately directed towards an autoantigen. Current treatments primarily rely on broad immunosuppression, which leaves patients susceptible to viral infection and cancer progression without providing a permanent cure. Tolerogenic vaccines capable of tolerizing the immune system towards a particular autoantigen posit a potential treatment without the drawbacks of broad immunosuppression. Unfortunately, the clinical success of such therapies has been limited by their inabilities to: 1) adequately deliver sufficient antigen to the lymphatics, where T cell priming occurs and 2) spatiotemporally co-deliver antigen and tolerizing adjuvant to reprogram antigen presenting cells (APCs) towards a suppressive phenotype. To this end, we have developed an extracellular vesicle (EV)-based platform for the co-delivery of antigens and immunoregulatory cues. EVs are cell membrane enclosed nanoparticles (~50-200 nm) secreted by all cells as a means of intercellular communication via the transfer of various biomolecules. Additionally, many types of EVs are intrinsically immunosuppressive and involved in maintenance of immune tolerance. In this work, we aimed to exploit the size-dependent lymphatic trafficking and intrinsic immunosuppressive effects of exogenously administered EVs to induce tolerogenic T cell priming by APCs, leading to CD4⁺ regulatory T cell (Treg) proliferation and CD8⁺ effector T cell (Teff) deletion/anergy.


Materials and Methods:: EVs were isolated from HEK293SF-3F6, B16.F10, MC38, and BeWo cells using a density gradient ultracentrifugation (UC) technique. Briefly, conditioned media (CM) was serially centrifuged at 300 x g for 3 min, 2000 x g for 20 min, and 10000 x g for 30 min to remove cells, cell debris, and large EVs (lEVs), respectively. CM was then loaded onto a 2 mL ‘cushion’ of 60% iodixanol in an SW32 UC tube and centrifuged at 100,000 x g for 4 h. The bottom 3 mL (40% iodixanol) was transferred to an SW40 UC tube, and 3 mL each of 20%, 10%, and 5% iodixanol were layered on top. Following centrifugation at 150,000 x g for 16 h, purified EVs were collected from the interface of the 20% and 10% iodixanol solutions, pelleted on a tabletop UC by spinning at 100,000 x g for 3 h, and resuspended in a minimal volume of PBS for subsequent experiments. Azide-functionalized EVs (N₃-EVs) were generated by adding 10 μM N-azidoacetylmannosamine-tetraacylated (Ac₄ManNAz) to cell culture media. DBCO-modified moieties were ligated to N₃-EVs via overnight incubation, and EVs were purified with either centrifugal dialysis or FPLC. EVs were characterized using nanoparticle tracking analysis (NTA) and TEM. Biodistribution of Cy5 labeled EVs was evaluated using IVIS and flow cytometry. BASP1-OVA expressing EVs were generated via transfection using Transporter 5 transfection reagent. Immunosuppressive effects of EV source cells was determined using in vitro and in vivo models involving bone marrow dendritic cells (BMDCs), OT-I, and OT-II T cells.


Results, Conclusions, and Discussions:: Results and Discussion:

Density gradient UC yielded a pure, homogenous population of small EVs (sEVs) with a median diameter of 168.7 nm and a characteristic cup-like morphology evident by TEM (Fig. 1A, 1B). To generate azide-functionalized EVs with synthetic azide handles for conjugation of DBCO-functionalized antigens, we used a metabolic glycoengineering strategy in which azide-modified sugar derivatives were doped into producer cell culture media. By ligating DBCO-AF488 to treated cells and measuring fluorescence intensity using flow cytometry, we found that HEK293SF-3F6 cells best incorporated Ac₄ManNAz, and we observed only marginal benefit to increasing the sugar concentration in the culture media above 10 μM (Fig. 1C). We found that this result was consistent at the EV membrane level as well by disrupting EVs in a solution of 2% SDS and correlating fluorescence to a standard curve (Fig. 1C). We also incorporated the model antigen ovalbumin (OVA) into EVs via transfection with a BASP1-OVA fusion protein that packages OVA into the EV lumen (Fig. 1D). In vivo, we found that Cy5-labeled EVs traffic to the draining inguinal lymph node (iLN), spleen, and kidneys (Fig. 1E, 1F). Within the iLN, EVs seemed to accumulate within the major APCs: macrophages and dendritic cells (Fig. 1G). Last, CM from B16.F10 and BeWo cells was able to suppress BMDC inflammation, indicating that these EVs may be inherently immunosuppressive.

Conclusions:

Pure populations of HEK293SF-3F6 EVs can be isolated and engineered to package autoantigen via both endogenous and exogenous approaches. DBCO-modified antigens and/or antigenic peptides can be loaded exogenously following a strain promoted azide-alkyne cycloaddition (SPAAC) reaction to EVs bearing synthetic azide groups after MGE. OVA can be loaded endogenously via expression of a BASP1-OVA fusion. Following SC administration, EVs traffic to the iLN and are taken up by APCs to present antigen to T cells. B16.F10 and BeWo EVs may be inherently immunosuppressive, and merit further investigation as antigen delivery platforms. Using this system, we aim to reprogram autoreactive T cells to exhibit a tolerogenic phenotype and ameliorate autoimmune diseases.


Acknowledgements (Optional): : NSF FMSG 2036809
NIH ITED T32 DK101003
NSF GRFP 2022305770

References (Optional): : 1. Dooley, K., McConnell, R.E., Xu, K., Lewis, N.D., Haupt, S., Youniss, M.R., Martin, S., Sia, C.L., McCoy, C., Moniz, R.J., Burenkova, O., Sanchez-Salazar, J., Jang, S.C., Choi, B., Harrison, R.A., Houde, D., Burzyn, D., Leng, C., Kirwin, K., Ross, N.L., Finn, J.D., Gaidukov, L., Economides, K.D., Estes, S., Thornton, J.E., Kulman, J.D., Sathyanarayanan, S., Williams, D.E., "A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties," Molecular Therapy, 29 (5), 2021. 1729-1743.