Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal–antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal–antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.
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Gene-expression data obtained by RNA-seq are available from the NCBI Gene Expression Omnibus, via the accession number GSE192671. Source data are provided with this paper. All other data needed to evaluate the conclusions of the study are available within the paper and its Supplementary Information.
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We thank B. Jabri and P. A. Savage, both of the University of Chicago, for helpful discussions. We thank the Cytometry and Antibody Core Facility at the University of Chicago. We thank Hooke Laboratories for adoptive transfer EAE studies. We thank the Center for Research Informatics, which is funded by the Biological Sciences Division at the University of Chicago with additional funding from the Institute for Translational Medicine, CTSA grant number UL1 TR000430 from the NIH, and the University of Chicago Comprehensive Cancer Center Support Grant (NIH P30CA014599). Tetramers were provided by the NIH Tetramer Core Facility. We thank B. Delache, S. Langois, O. Lacroix, N. Dhooge, J.M. Robert, T. Prot and C. Dodan for the NHP experiments; L. Bossevot, M. Leonec, A. Chatenet, J. Morin and M. Gomez-Pacheco for the NHP ELISPOT assays and flow cytometry; S. Gomes for assistance with experiments; and L. Shores for assistance with manuscript editing. This work was supported by the University of Chicago’s Chicago Immunoengineering Innovation Center, the Alper Family Foundation and Anokion S.A. The Infectious Disease Models and Innovative Therapies research infrastructure is supported by the ‘Programme Investissements d’Avenir’, managed by the Agence Nationale de la Recherche (ANR) (ANR-11-INBS-0008 and ANR-10-EQPX-02-01).
This work was funded in part by Anokion SA. D.S.W., J.A.H., S.K. and K.M.L. are inventors on patents related to synthetically glycosylated antigens, licensed to Anokion SA. J.A.H. consults for Anokion, is on the Board of Directors of the company and on its Scientific Advisory Board, and holds equity in the company. T.B.T., D.J.B., J.L.H., R.M., and G.P.C. are employees of Anokion. S.K. is on the Scientific Advisory Board of Anokion and holds equity in the company. K.M.L. also holds equity in Anokion. All other authors declare no competing interests.
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OTI and OTII cells were adoptively transferred into mice prior to vaccination with CFA/OVA as in Fig. 1. 4 weeks later mice were treated with either pGal alone, an unconjugated mix of pGal and OVA or saline prior to challenge. (a) Percent recovery of OTI and OTII cells in dLN and spleens as a proportion of the CD8+ and CD4+ T-cell populations, respectively. (b) Expression frequencies of the specified protein markers as indicated on the y-axes on recovered OTI and OII cells from SLO. (c) Three-day restimulations were also carried out for ELISA-based analyses of the indicated cytokines in culture supernatants. Data are shown as means ± SEM. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s test.
a–d) As in Fig. 1, six-hour ex vivo antigen restimulations were performed with recovered lymphocytes and peptides as indicated above each graph, and intracellular flow cytometry was used to detect production of the indicated cytokines in OTI and OTII cells. Data are shown as means ± SEM. Each point represents an individual mouse. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s test.
As in Fig. 2, six-hour ex vivo antigen restimulations were performed with recovered lymphocytes and peptides as indicated above each graph, and intracellular flow cytometry was used to detect production of the indicated cytokines in OTI and OTII cells. Data are shown as means ± SEM. Each point represents an individual mouse. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s test.
(a–d) MFI of the indicated markers on the y-axes in recovered OTI&II cells. (e, f) Expression frequencies of the specified protein markers as indicated on the y-axes on recovered OTIs from SLO. Data are shown as means ± SEM. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s test.
Extended Data Fig. 5 Antigen-mediated suppression of LmOVA/CFA recall response in endogenous CD8+ T cells.
Mice were first inoculated with LmOVA and allowed to clear the infection, then one month later given antigen therapy as described previously, and finally challenged two weeks after the last antigen dose with OVA+CFA s.c. a) Quantification of endogenous OVA-specific CD8+T cells recovered in dLN and spleens as a proportion of total CD8+ T cells as well as total cell number. b) Representative flow cytometry profiles of recovered pentamer-reactive CD8+ T cells in the dLN comparing effects of saline and antigen treatments. c–f) Expression frequencies of the specified protein markers as indicated on the y-axes on recovered pentamer-specific CD8+ T cells from SLO. g–i) Six-hour ex vivo antigen restimulations were performed with recovered lymphocytes and peptides as indicated above each graph, and intracellular flow cytometry was used to detect production of the indicated cytokines in pentamer-reactive CD8+ T cells. *p indicates comparisons between pGal and Saline groups, &p indicates comparisons between OVA and Saline groups, #p indicates comparisons between pGal and OVA groups. Data are shown as means ± SEM. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s post hoc test.
a–c) As in Fig. 4, six-hour ex vivo antigen restimulations were performed with recovered lymphocytes and peptides as indicated above each graph, and intracellular flow cytometry was used to detect production of the indicated cytokines in OTI and OTII cells. Data are shown as means ± SEM. Each point represents an individual mouse. Unless otherwise stated statistical differences in all graphs were determined by one-way ANOVA with Tukey’s test.
(a) Six-hour ex vivo antigen restimulations were performed as indicated and intracellular flow cytometry was used to detect production of the indicated cytokines in CD8+and CD4+T cells. (b) Three-day restimulations were carried out for analysis of IL-17A production in culture supernatants. All statistical comparisons were performed by one-way ANOVA using Tukey correction unless otherwise indicated.
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Tremain, A.C., Wallace, R.P., Lorentz, K.M. et al. Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses. Nat. Biomed. Eng 7, 1142–1155 (2023). https://doi.org/10.1038/s41551-023-01086-2