Engineering antigens for in situ erythrocyte binding induces T-cell deletion

Abstract

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Fig. 1.

ERY1-OVA binds the equatorial periphery of mouse erythrocytes with high affinity. (A) Schematic of conjugation of ERY1 peptide to OVA, resulting in binding to erythrocyte surface glycophorin-A. (B) High-resolution confocal microscopy images of mouse erythrocytes labeled ex vivo with (green) anti-mouse glycophorin-A (GYPA) and (red) either OVA (Upper) or ERY1-OVA (Lower). (Scale bar = 5 μm.) (C) Binding of each OVA conjugate and intermediate, characterized by flow cytometry. ERY1, erythrocyte-binding peptide WMVLPWLPGTLD; MIS, MIS peptide PLLTVGMDLWPW; SMCC, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, used to conjugate ERY1 to OVA. (D) Equilibrium binding of ERY1-OVA to erythrocytes demonstrates the low dissociation constant of ERY1-OVA (R² = 0.97, one-site binding), determined by flow cytometry.

Fig. 2.

ERY1-conjugated antigen biospecifically binds circulating healthy and eryptotic erythrocytes on i.v. administration, inducing uptake by specific APC subsets. (A) OVA (gray-filled histogram) and ERY1-OVA (black-filled histogram) binding to erythrocyte (CD45⁻) and nonbinding to leukocyte (CD45⁺) populations in vivo compared with noninjected mice (empty histogram), determined by flow cytometry. (B) ERY1-OVA binding and OVA nonbinding to circulating eryptotic (annexin-V⁺) and healthy (annexin-V⁻) erythrocytes, determined by flow cytometry. (C) Cell surface t_1/2 of bound ERY1-OVA to circulating erythrocytes, determined by geometric mean fluorescence intensity of flow cytometry measurements (n = 2; R² = 0.98, one-phase exponential decay), and time-dependent ERY1-OVA cell surface concentration, determined by ELISA, at an administered dose of 150 μg (n = 2).

Fig. 3.

Erythrocyte-bound allophycocyanin uptake by splenic DC subsets and nonprofessional APCs in the liver. (A) Increased cellular uptake of ERY1-allophycocyanin by MHC II⁺ CD11b⁻ CD11c⁺ and MHC II⁺ CD8α⁺ CD11c⁺ CD205⁺ splenic DCs at 12 and 36 h postinjection, compared with MIS-allophycocyanin. (B) Increased cellular uptake of ERY1-allophycocyanin in the liver by hepatocytes (CD45⁻ MHCII^low CD1d⁻) and hepatic stellate cells (CD45⁻ MHC II⁺ CD1d⁺) but not by liver DCs (CD45⁺ CD11c⁺) or Kupffer cells (CD45⁺ MHC II⁺ F4/80⁺), compared with MIS-allophycocyanin, 36 h following i.v. administration (n = 2). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Data represent mean ± SE. (C) Spleen microscopy images of mice 24 h following administration of 10 μg OVA (Left) or ERY1-OVA (Right), stained for OVA (green), F4/80 (red), and DAPI nuclear staining (blue). (Scale bar = 50 μm.) (D) Liver microscopy images of mice 24 h following administration of 10 μg OVA (Left) or ERY1-OVA (Right), stained for MHC I H-2Kb-SIINFEKL (green), CD45 (red), and DAPI for nuclear staining (blue). (Scale bar = 50 μm.)

Fig. 4.

Erythrocyte-binding antigen formulations enhance cross-priming and apoptotic fate deletional proliferation of antigen-specific OTI CD8⁺ T cells in vivo. (A) Proliferation of CFSE-labeled splenic OTI CD8⁺ T cells (CD3ɛ⁺ CD8α⁺ CD45.2⁺) 5 d following i.v. administration of 10 μg of ERY1-GST (Left), 10 μg of OVA (Center), or 10 μg of ERY1-OVA (Right). (B) Dose-dependent quantified proliferative populations of OTI CD8⁺ T-cell proliferation from A, as well as an identical 1-μg dosing study; data represent mean ± SD (n = 5). **P < 0.01; ^##P < 0.01. (C) Proliferation of CFSE-labeled OTI CD8⁺ T cells 5 d following i.v. injection of 7 μg of TER119-SIINFEKL (Right), an equimolar dose of SIINFEKL (Center), or saline (Left). (D) Quantified proliferative populations of OTI CD8⁺ T cells from C. (E) Heat map representing OTI CD8⁺ T-cell proliferation generations exhibiting larger PD-1⁺ populations on ERY1-OVA or TER119-SIINFEKL administration compared with OVA or SIINFEKL, respectively (Left); the cumulative PD-1⁺ population (as a percentage of all OTIs) for each group is denoted (Right). Data represent mean ± SD (n = 5). ***P < 0.0001; ^###P < 0.0001. (F) Heat map representing OTI CD8⁺ T-cell proliferation generations exhibiting larger apoptotic (annexin-V⁺) populations on ERY1-OVA or TER119-SIINFEKL administration compared with OVA or SIINFEKL, respectively (Left); the cumulative annexin-V⁺ population (as a percentage of all OTIs) for each group is denoted (Right). Data represent mean ± SD (n = 5). ***P < 0.0001; ^###P < 0.0001. All data were determined by multiparameter flow cytometry.

Fig. 5.

Erythrocyte binding induces resistance to antigen challenge. (A) OTI CD8⁺ T-cell adoptive transfer tolerance model displays experimental protocol for experimental as well as challenge and naive control groups (n = 5). i.d., intradermal. (B) Flow cytometric detection of OTI CD8⁺ T-cell populations (CD3ɛ⁺ CD8α⁺ CD45.2⁺). (C) OTI CD8⁺ T-cell population quantification in the draining lymph nodes (inguinal and popliteal) 4 d following antigen challenge in CD45.1⁺ mice. **P < 0.01. (D) IFN-γ–expressing OTI CD8⁺ T cells in the draining lymph nodes 4 d following antigen challenge and restimulation with SIINFEKL peptide. **P < 0.01. (E) IFN-γ concentrations in lymph node cell culture media 4 d following restimulation with SIINFEKL peptide, determined by ELISA. **P < 0.01. (F) OVA-specific serum IgG titers at day 19. *P < 0.05. Data represent mean ± SE. (G) Combination OTI- and OVA-expressing EL4 thymoma (E.G7-OVA) tumor tolerance model displays experimental protocol for experimental as well as control groups (n = 4 and n = 3, respectively). (H) Quantification of nonproliferating (generation 0) OTI CD8⁺ T cells circulating in blood 5 d following adoptive transfer; data represent median ± minimum to maximum. ***P = 0.0002. (I) Growth profile of E.G7-OVA tumors that were s.c. injected 9 d following OTI adoptive transfer; data represent mean ± SE. *P < 0.05.

Fig. 6.

Erythrocyte-binding autoantigen protects mice from T cell-induced autoimmune type 1 diabetes. (A) Increased proliferation and deletion of adoptively transferred diabetogenic BDC2.5 CD4⁺ T cells in the spleen and pancreatic lymph nodes 4 d following administration of TER119-p31 compared with p31, as determined by dilution of CFSE fluorescence via flow cytometry (n = 4). *P < 0.01. (B) Microscopy images of pancreatic islets stained for insulin (red), CD3ɛ (green), and DAPI nuclear staining (blue), demonstrating marked T-cell infiltration and islet destruction of saline- and p31-treated mice but not of TER119-p31-treated mice. (Scale bar = 100 μm.) (C) Glycemia monitoring as measured by daily blood glucose measurements following adoptive transfer of diabetogenic BDC2.5 CD4⁺ T cells and a tolerogenic treatment regimen of either saline, p31, or TER119-p31 (n = 8, n = 9, and n = 9, respectively). ***P < 0.0001. (D) Diabetes incidence rate quantified by measurements in C; arrows indicate antigen administration time points. ***P < 0.0001.

Fig. P1.

Protein antigens engineered to bind to circulating erythrocytes after injection drive deletion of antigen-specific T cells. (A) Antigens are engineered for erythrocyte binding by chemically conjugating a glycophorin A-specific peptide (ERY1) or by fusing the antigen to an erythrocyte-specific antibody fragment (TER119). Following i.v. injection in mice, the modified antigens bind to erythrocytes, as depicted in the micrographs of a mouse erythrocyte stained for the erythrocyte-specific glycophorin-A membrane protein (green) and for the antigen (red). Only the ERY1-conjugated antigen binds erythrocytes, colocalizing with glycophorin-A on the periphery of the cell. (Scale bar = 5 μm.) Erythrocyte-bound antigens are then processed by immune cells in a manner that drives deletion of antigen-specific T cells, whereas nonbinding antigens may induce the expansion of armed and/or quiescent antigen-specific T cells, depending on the context of delivery. (B) Administration of an islet autoantigen fused to the TER119 antibody fragment protects mice from hyperglycemia (high blood glucose) and the onset of diabetes by deleting the islet-reactive T cells within the first week of treatment, whereas administration of soluble nonbinding antigen offers no protection from disease induction. Micrographs of islets from mice administered erythrocyte-binding islet autoantigen demonstrate protection from T-cell infiltration and attack (insulitis), and show robust insulin expression, similar to islets from healthy mice. Islets of mice treated with the corresponding nonbinding antigen, however, are targeted for insulitis, thereby losing insulin expression and leading to diabetes onset. (Scale bar = 100 μm.)