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ABSTRACT
Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.
List of abbreviations
| iNKT cells | = | Invariant natural killer T cells |
| NK cells | = | natural killer cells |
| NP | = | Nanoparticle |
| IFN | = | Interferon |
| PD-1 | = | Programmed cell death protein 1 |
| MHC | = | Major histocompatibility complex |
| IL | = | Interleukin |
| α-GalCer | = | α-galactosylceramide |
| PLGA | = | poly(lactic-co-glycolic acid) |
| OVA | = | Ovalbumin protein |
| IMM60 | = | threitolceramide 6 |
| TCR | = | T cell receptor |
| CAD | = | Charged Aerosol Detector |
| Ig | = | Immunoglobulin |
| Iv | = | intravenous injection |
| Sc | = | subcutaneous injection |
| Inod | = | intranodal injection |
| Spl | = | Spleen |
| LN | = | Lymph node |
| Axil | = | Axillary |
| Ing | = | Inguinal |
| ALT | = | Alanine aminotransferase |
Acknowledgments
We are grateful to Abderraouf Selmi, Mustafa Diken and Ugur Sahin (TRON, Mainz) for their sharing of experimental knowledge related to intranodal injections. We thank the bio technicians of the PRIME animal unit, Bianca Lemmers, Karin Haas-Cremers, Kitty Lemmens-Hermans, and Mike Peters for their efforts in mouse interventions and care.
We would like to show our great respect to our co-author and colleague, Prof. Vincenzo Cerundolo. After submission of this manuscript Enzo passed away in January of 2020. We will miss him on a daily basis, an excellent scientist, a true gentleman and dedicated director of the MRC Human Immunology Unit, with passion for research pertaining to immuno-oncology and vaccination, who made key discoveries being taken forward to the clinic. We greatly benefitted from his warm, open and encouraging approach. He will be greatly missed and remembered as a dear friend.
Author contributions
Y.D., M.V. and C.G.F conceived the research question, Y.D. and M.V. designed, planned, carried out all experiments and discussed the results, Y.D. processed the experiment data, designed the figures and drafted the manuscript, U.G. put valuable contribution to the experiment design, discussions interpretation of the results and revising the final manuscript, O.T. produced and characterized fluorescent nanoparticles, E.v.D. produced and analysed all non-fluorescent nanoparticles, contributed to tissue processing and ELISA’s, K.R. provided assistance for in vivo experiments, E.J. and M.H. develop the method for the IMM60 quantification and analyzed the nanoparticles for IMM60 contents, V.C. encouraged to investigate checkpoint combination therapy, DC maturation and supervised the findings, M.V., U.G., O.T., V.C., C.G.F supplemented, commented and edited the manuscript, C.G.F supervised the project and revised the final manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Disclosure of potential conflicts of interest
V.C. is serving as a consultant for iOx Therapeutics, which has an interest in the development of iNKT-cell-targeted therapeutics. All other authors declare no financial or commercial conflict of interest.
Ethics Approval
All animal experiments were performed according to guidelines for animal care of the Nijmegen Animal Experiments Committee in accordance with the ethical standards described in the declaration of Helsinki (Project No: DEC2015-019).
Supplementary material
Supplemental data for this article can be accessed on the publisher’s website.