Creation and Characterization of Erythrocyte-Binding OVA.

As one approach to obtain strong and specific biophysical targeting of an antigen to erythrocyte cell surfaces in situ, we used a synthetic 12-aa peptide (ERY1) that we discovered by phage display to bind to mouse glycophorin-A specifically (16), which is present uniquely on erythrocytes. We used the model antigen OVA, so as to enable initial analysis in a transgenic mouse strain (OTI), the CD8⁺ T-cell population of which expresses the T-cell receptor specific for the MHC I immunodominant OVA epitope, SIINFEKL. We chemically conjugated approximately three copies of the ERY1 peptide to OVA to create an OVA variant (ERY1-OVA) that binds mouse erythrocytes with high affinity and specificity (Fig. 1A). High-resolution confocal microscopy showed localization to the cell membrane equatorial periphery (Fig. 1B), and flow cytometric analysis demonstrated binding to be sequence-specific (Fig. 1C). ERY1-OVA bound to erythrocytes with high affinity, exhibiting a dissociation constant (K_d) of 6.2 ± 1.3 nM, as determined by equilibrium binding measurements (Fig. 1D). Based on this approximate value, an entire 10 μg dose of ERY1-OVA would be bound to the surface of erythrocytes following i.v. injection in a mouse.

We validated ERY1-OVA binding in vivo to circulating erythrocytes following i.v. administration in mice. Whole-blood samples taken 30 min following injection of 150 μg of either OVA or ERY1-OVA confirmed the specific erythrocyte binding capability of ERY1-OVA even amid the complex heterogeneous milieu of blood and the vasculature (Fig. 2A). Consistent with glycophorin-A association, ERY1-OVA bound to erythrocytes (CD45⁻) but not to leukocytes (CD45⁺). ERY1-OVA binding was unbiased as to the eryptotic state of the erythrocytes, binding strongly to both annexin-V⁺ and annexin-V⁻ CD45⁻ populations (Fig. 2B). Pharmacokinetic studies of the OVA conjugate demonstrated that ERY1-OVA erythrocyte binding was long-lived in vivo, exhibiting a cell surface t_1/2 of 17.2 h (Fig. 2C). ERY1-OVA remained bound to erythrocytes for as long as 72 h following administration; during this time frame, ∼13% of erythrocytes are cleared in the mouse (17). Quantification of erythrocyte-bound ERY1-OVA in vivo showed a relatively high loading of 1.174 ± 0.005 ng of OVA per 10⁶ erythrocytes. Erythrocyte binding by ERY1-OVA elicited no detectable differences in hematocrit, corpuscular volume, or hemoglobin content, compared with administration of OVA (Fig. S1), demonstrating that glycophorin-A–mediated erythrocyte binding with antigen did not alter their hematological parameters.

Erythrocyte Bound Antigen Uptake and Biodistribution.

To reveal the cellular targets of antigen as it is collected after binding to erythrocytes after administration, we i.v. injected mice with the highly fluorescent allophycocyanin protein, conjugated to either ERY1 (ERY1-allophycocyanin) or a mismatch (MIS) scrambled peptide (MIS-allophycocyanin) (binding shown in Fig. S2A). Flow cytometric analysis of splenic dendritic cell (DC) populations 12 and 36 h following administration showed 9.4-fold enhanced uptake of ERY1-allophycocyanin by MHC II⁺ CD11b⁻ CD11c⁺ DCs compared with MIS-allophycocyanin yet similar uptake of ERY1-allophycocyanin and MIS-allophycocyanin by MHC II⁺ CD11b⁺ CD11c⁺ DCs (Fig. 3A). Additionally, MHC II⁺ CD8α⁺ CD11c⁺ CD205⁺ splenic DCs were found to uptake ERY1-allophycocyanin to a threefold greater extent than MIS-allophycocyanin, although the absolute magnitude was markedly lower than for other DC populations in the spleen. Such targeting of antigen toward nonactivated and CD8α⁺ CD205⁺ splenic DCs could strengthen the tolerogenic potential of erythrocyte binding, because these populations have been extensively implicated in apoptotic cell-driven tolerogenesis (7, 18). Such flow cytometry-based results were validated using confocal microscopy. Spleen sections taken from mice 24 h following i.v. injection of OVA or ERY1-OVA showed OVA staining in areas distinct from F4/80⁺ macrophage regions, with ERY1-OVA–injected mice displaying more overall OVA staining than OVA-injected mice (Fig. 3C). In the liver, ERY1-allophycocyanin also greatly enhanced uptake by hepatocytes (CD45⁻ MHC II^low CD1d⁻, by 78.4-fold) and hepatic stellate cells (CD45⁻ MHC II⁺ CD1d⁺, by 60.6-fold) compared with MIS-allophycocyanin (Fig. 3C); both populations have been reported as APCs that trigger CD8⁺ T-cell deletional tolerance (19–21). Interestingly, preferential uptake was not seen in liver DCs (CD45⁺ CD11c⁺) or Kupffer cells (CD45⁺ MHC II⁺ F4/80⁺), which serve as members of the reticuloendothelial system that aids in clearance of erythrocytes and complement-coated particles. Analysis of liver sections confirmed the flow cytometry measurements. Although no intact-OVA staining was seen in the liver 24 h following injection (perhaps due to antigen processing), only ERY1-OVA–treated mouse livers stained strongly with the 25.d1.16 mAb, which specifically recognizes the immunodominant OVA peptide SIINFEKL displayed on H-2kb MHC I (Fig. 3D). In addition to supporting observations of hepatic cell uptake of ERY1-allophycocyanin, the presence of MHC I–SIINFEKL complexes on hepatic cell surfaces suggests their role in cross-presentation of exogenous antigens.

T-Cell Phenotype in Response to ERY1- and Single-Chain Fv Antigen Constructs.

We next determined whether erythrocyte-bound antigen could be efficiently cross-presented and could correspondingly cross-prime reactive CD8⁺ T cells. We adoptively transferred carboxyfluorescein succinimidyl ester (CFSE)-labeled OTI CD8⁺ T cells (CD45.2⁺) into CD45.1⁺ mice and measured proliferation of the OTI CD8⁺ T cells 5 d following i.v. administration of 10 μg of OVA; 10 μg of ERY1-OVA; or 10 μg of an irrelevant erythrocyte-binding antigen, ERY1-GST (binding shown in Fig. S2B). OTI CD8⁺ T-cell proliferation, determined by dilution of the fluorophore CFSE as measured by flow cytometry (Fig. 4A), was markedly enhanced over threefold in mice administered ERY1-OVA compared with OVA (Fig. 4B), demonstrating that erythrocyte binding increased antigen-specific CD8⁺ T-cell cross-priming compared with the soluble antigen. Similar results were also obtained by administration of a 10-fold lower antigen dose of 1 μg, demonstrating the wide dynamic range of efficacy of OTI CD8⁺ T-cell proliferation induced by ERY1-conjugated antigen.

To address the technical hurdle of conjugating ERY1 to small peptide antigens insufficiently large for conjugation of multiple copies of the ERY1 peptide, we engineered a second molecular implementation of our concept, namely, an erythrocyte-specific single-chain Fv (scFv) antibody fragment to display peptide antigens. The murine glycophorin A-binding TER119 mAb was sequenced and converted into the scFv format with a C-terminal SIINFEKL peptide fusion. We characterized the resulting erythrocyte-binding peptide fusion, TER119-SIINFEKL (binding shown in Fig. S2C), in an analogous in vivo OTI proliferation study as with ERY1-OVA, with even more striking cross-priming results (Fig. 4C). TER119-SIINFEKL induced proliferation of the entire (95%) OTI CD8⁺ T-cell population 5 d following adoptive transfer, indicating strong proteosomal processing and cross-presentation of the peptide antigen when fused to the erythrocyte-binding TER119 scFv (Fig. 4D). Although administration of soluble SIINFEKL at an equimolar dose induced similar proliferation rates, it is important to note that in this format, SIINFEKL can directly bind to MHC I and present to OTI CD8⁺ T cells without first being proteolytically processed by APCs. Our results on cross-presentation and cross-priming are consistent with those of other studies concerning tolerogenic antigen presentation on MHC I by APCs engulfing antigen from apoptotic cell debris (4, 22).

To distinguish T cells being expanded into a functional effector phenotype from those being expanded and deleted, we analyzed the proliferating OTI CD8⁺ T cells for programmed death-1 (PD-1), a marker of T-cell exhaustion, and annexin-V binding, a hallmark of apoptosis, and thus deletion. ERY1-OVA induced much higher total numbers of PD-1⁺ (23.2 ± 4.9%) and annexin-V⁺ (20.7 ± 7.4%) proliferating OTI CD8⁺ T cells 4 d after treatment than did OVA (7.6 ± 1.1% and 4.8 ± 1.3%, respectively) (Fig. 4 E and F, upper two rows), suggesting an exhausted and apoptotic fate that would eventually lead to clonal deletion. Even more striking results were obtained with the TER119-SIINFEKL treatment (Fig. 4 E and F, lower two rows), with very high PD-1 (43.1 ± 10%) and annexin-V⁺ (35.7 ± 9.1%) levels; although the free SIINFEKL peptide was able to induce CD8⁺ T-cell proliferation (above), it did not induce high levels of these markers (8.3 ± 3.6% and 7.5 ± 2.9%, respectively). This phenotype of proliferating CD8⁺ T cell is consistent with other reported OTI adoptive transfer models in which regulated antigen-specific T-cell receptor engagement by APCs fails to induce inflammatory responses (23).

OTI Challenge Model.

Using an established OTI challenge model (12) (Fig. 5A), we demonstrated the ability of ERY1-OVA to prevent postdeletion immune responses to vaccine-mediated antigen challenge, even with a very strong bacterially derived adjuvant. To delete, we i.v. administered 10 μg of either OVA or ERY1-OVA 1 and 6 d following adoptive transfer of OTI CD8⁺ (CD45.2⁺) T cells to CD45.1⁺ mice. After an additional 9 d to allow potential deletion of the transferred T cells, we then challenged the recipient mice with OVA adjuvanted with LPS by intradermal injection. Characterization of draining lymph node and spleen cells, as well as their inflammatory responses, 4 d after challenge allowed us to determine if deletion actually took place.

The i.v. administration of ERY1-OVA resulted in profound reductions in OTI CD8⁺ T-cell populations in the draining lymph nodes (Fig. 5C, gating shown in Fig. 5B) and spleens (Fig. S3, gating shown in Fig. S3A) compared with administration of unmodified OVA, demonstrating deletion. Draining lymph nodes from ERY1-OVA–treated mice contained over 11-fold fewer OTI CD8⁺ T cells compared with OVA-treated mice and 39-fold fewer than challenge control mice that did not receive i.v. injections of antigen; responses in spleen cells were similar (Fig. S3B). This effective clonal deletion exhibited in mice administered with ERY1-OVA supported our earlier observations of enhanced OTI CD8⁺ T-cell cross-priming (Fig. 4); furthermore, it shows that splenic and/or liver-localized cross-priming occurred in the absence of APC presentation of costimulatory molecules, leading to T-cell deletion.

To evaluate the immune response following antigen challenge further, the inflammatory nature of resident lymph node and spleen cells was characterized by expression of IFN-γ by OTI CD8⁺ T cells. Following challenge with OVA and LPS, the lymph nodes of mice previously treated with ERY1-OVA harbored 53-fold fewer IFN-γ–expressing OTI CD8⁺ T cells compared with those of challenge control mice (previously receiving no antigen) and over 19-fold fewer IFN-γ–expressing cells compared with those of mice previously treated with an equivalent dose of OVA (Fig. 5D), demonstrating the importance of erythrocyte binding in deletional protection to challenge; responses in spleen cells were similar (Fig. S3D, gating shown in Fig. S3C). In addition, of the small OTI CD8⁺ T-cell population present in the lymph nodes and spleens of mice previously treated with ERY1-OVA, a lower percentage expressed IFN-γ, suggesting clonal inactivation (Figs. S3E and S4). Furthermore, the magnitude of total IFN-γ levels produced by cells isolated from the draining lymph nodes on SIINFEKL restimulation was substantially reduced in mice previously treated with ERY1-OVA (Fig. 5E), with erythrocyte binding reducing IFN-γ levels 16-fold compared with OVA administration and over 115-fold compared with challenge controls. Interestingly, the suppressive phenomenon was also correlated with down-regulated IL-10 expression by lymph node cells (Fig. S5). Typically considered a regulatory CD4⁺ T cell-expressed cytokine in the context of APC–T-cell communication to dampen T helper 1 cell responses (24, 25), IL-10 expression was dispensable for desensitization to challenge. Similar IL-10 down-regulation has been implicated in CD8⁺ T cell-mediated tolerogenesis (8, 26, 27). Similar OTI responses were seen when using TER119-SIINFEKL as the deletional antigen (Fig. S6).

Erythrocyte binding also substantially attenuated humoral immune responses against antigen, because mice treated with ERY1-OVA exhibited 100-fold lower antigen-specific serum IgG titers compared with mice treated with soluble OVA (Fig. 5F). A similar reduction in OVA-specific IgG titer reduction as a result of erythrocyte binding was seen in nonadoptively transferred C57BL/6 (CD45.2⁺) mice. Following two i.v. injections of 1 μg of OVA or ERY1-OVA 7 d apart, ERY1-OVA–treated mice exhibited 39.8-fold lower OVA-specific serum IgG levels 19 d after the first antigen injection (Fig. S7). This apparent reduction in B-cell activation, following erythrocyte binding by the antigen, corroborates current hypotheses concerning noninflammatory antigen presentation during tolerance induction (3, 4, 28).

E.G7-OVA Thymoma Tolerance Model.

To validate the induction of antigen-specific immune tolerance in response to the engineered erythrocyte-targeting antigen further, we combined the OTI challenge model with an OVA-expressing tumor graft model (Fig. 5G). Similar to the previous experimental design, mice were tolerized by means of two i.v. injections of 10 μg of ERY1-OVA or 10 μg of OVA following adoptive transfer of OTI CD8⁺ T cells. Marked T-cell deletion was detected 5 d following the first antigen administration, because ERY1-OVA–injected mice harbored 2.9-fold fewer nonproliferating (generation 0) OTI CD8⁺ T cells in the blood (Fig. 5H). To determine the functional responsiveness of proliferating OTI CD8⁺ T cells in the absence of a strong exogenously administered adjuvant, OVA-expressing EL-4 thymoma cells (E.G7-OVA) were intradermally injected into the back skin of mice 9 d following adoptive transfer. To assess the protective efficacy of erythrocyte-bound antigen, tumor-bearing mice were challenged with LPS-adjuvanted OVA 6 d following tumor grafting, analogous in dose and schedule to the challenge model. Robust tumor growth was continuously observed in ERY1-OVA–treated mice compared with OVA-treated or nontreated control mice through to 8 d following tumor grafting (Fig. 5I), confirming that ERY1-OVA–driven OTI CD8⁺ T-cell proliferation induced functional immune nonresponsiveness to OVA. That tumor size was arrested to a steady state 8 d following grafting may be indicative of residual OTI CD8⁺ T cells that had yet to undergo ERY1-OVA–driven deletion.

Taken together, these results demonstrate that erythrocyte binding by ERY1-OVA induces antigen-specific T-cell deletion to a strongly adjuvanted challenge, as well as implanted cellular grafts expressing a xenoantigen. Moreover, this response was achieved by functional inactivation and deletion of reactive CD8⁺ T cells through interaction with antigen present on circulating erythrocytes.

BDC2.5 Diabetes Induction Model.

To explore erythrocyte-binding antigen-based deletion of pathogenic T cells in an autoimmune model, we used the accelerated onset model of autoimmune type 1 diabetes (29, 30). We demonstrated that a peptide islet β-cell autoantigen fused to the erythrocyte-binding antibody fragment TER119 protected mice from diabetes onset by deleting the aggressive adoptively transferred activated diabetogenic T cells (Fig. 6). We adoptively transferred transgenic diabetogenic CD4⁺ T cells from the nonobese diabetic (NOD) BDC2.5 mouse model previously activated ex vivo with the p31 mimetope peptide (YVRPLWVRME) to induce rapid diabetes onset; the BDC2.5 clonal epitope has recently been identified as the islet antigen chromogranin-A processed and displayed on I-A^g7 MHC II (31, 32).

To determine if TER119-p31 induced analogous antigen-specific CD4⁺ T-cell proliferation and deletion as TER119-SIINFEKL did in the OTI CD8⁺ T-cell context, activated BDC2.5 T cells were labeled with CFSE and adoptively transferred into NOD hosts. Mice were i.v. administered 10 μg of TER119-p31 or an equimolar dose of p31 8 h following adoptive transfer and were euthanized 4 d later. Splenocytes and pancreatic lymph node cells were harvested, and BDC2.5 CD4⁺ T-cell proliferation was assessed by dilution of the CFSE fluorophore via flow cytometry. Consistent with results obtained with ERY1-OVA and TER119-SIINFEKL, erythrocyte-binding p31 induced potent BDC2.5 T-cell proliferation and deletion, because TER119-p31–treated mice harbored 20-fold and 28-fold fewer nonproliferated BDC2.5 CD4⁺ T cells compared with mice treated with p31 in the spleen and pancreatic lymph nodes, respectively (Fig. 6A).

To assess the effects of TER119-p31–driven proliferation in a pathological autoimmune setting, we performed an analogous yet longer term adoptive transfer study to allow the diabetogenic BDC2.5 CD4⁺ T cells to induce autoimmune diabetes. Following adoptive transfer of activated BDC2.4 CD4⁺ T cells, NOD mice were i.v. administered 10 μg of TER119-p31 or an equimolar dose of p31 every 3 d, for a total of three doses. Mice treated with p31 peptide or saline exhibited profound hyperglycemia as early as 3 d following adoptive transfer (Fig. 6C), with 100% of mice developing diabetes by days 5 and 6, respectively (Fig. 6D). Conversely, mice treated with TER119-p31 remained normoglycemic throughout the experimental time course (Fig. 6C), with 0% incidence of diabetes 62 d following adoptive transfer (Fig. 6D). Histological confirmation of pathological findings showed low insulin expression and extensive T-cell infiltration (CD3ɛ⁺) into pancreatic islets of mice treated with saline or p31 peptide, compared with robust insulin expression and no evident T-cell presence in mice administered TER119-p31 (Fig. 6B). These data demonstrate the efficacy of erythrocyte-binding antigens in driving clonal deletional protection even in the context of preactivated, pathogenic CD4⁺ T cells in an autoimmune setting.

Animals.

The Swiss Veterinary Authority and the EPFL Centre d'Application du Vivant previously approved all animal procedures. Female C57BL/6 mice (Charles River Laboratories) aged 8–12 wk were used for in vivo binding studies and as E.G7-OVA tumor hosts. C57BL/6-Tg(TcraTcrb) 1100Mjb (OTI) mice (Jackson Laboratories) were bred in specific pathogen-free (SPF) conditions at the Ecole Polytechnique Fédérale Lausanne Animal Facility, and females were used for splenocyte isolation at 6–12 wk of age. Female B6.SJL-Ptprc^aPepc^b/Boy (CD45.1) mice (Charles River Laboratories) aged 8–12 wk were used as recipient hosts for OTI CD8⁺ T-cell adoptive transfer and tolerance induction studies. NOD.Cg-Tg(TcraBDC2.5, TcrbBDC2.5)1Doi/DoiJ mice (BDC2.5 mice), which express the T-cell receptor transgene for specific recognition of the chromogranin-A islet antigen, were bred in SPF conditions at the Ecole Polytechnique Fédérale Lausanne Animal Facility, and females were used for cell isolations. NOD/ShiLtJ mice (Charles River Laboratories) were used as BDC2.5 adoptive transfer hosts.

ERY1 Peptide Design.

The ERY1 peptide was H₂N-WMVLPWLPGTLDGGSGCRG-CONH₂, and the MIS peptide was H₂N-PLLTVGMDLWPWGGSGCRG-CONH₂. The underlined sequence is the ERY1 12-mer sequence that we previously discovered by phage display as a mouse glycophorin-A binder (16). The GGSG region served as a linker to the cysteine residue used for conjugation; the flanking arginine residue served to lower the pK_a, and thus to increase the reactivity of the cysteine residue (46). SIINFEKL and p31 (YVRPLWVRME) peptides were purchased from Genscript. Detailed methods are provided in SI Materials and Methods.

ERY1-Antigen Design.

The ERY1 or MIS peptide was conjugated via its free cysteine thiol to lysine side chains on OVA using the sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate linker (Thermo Scientific). As a control, the ERY1 peptide was conjugated to GST derived from Escherichia coli. For biodistribution studies, the ERY1 or MIS peptide was conjugated to allophycocyanin (Innova Biosciences). Methods are shown in SI Materials and Methods.

TER119-Antigen Development.

The TER119 scFv antibody fragment was created as described elsewhere (45). In brief, forward and reverse primer mixtures were used to amplify the variable domains of the heavy and light chains of cDNA of the TER119 hybridoma clone (provided by Shozo Izui, University of Geneva, Geneva, Switzerland). The products were digested and ligated into the pMAZ360 subcloning vector, and the resultant sequence was queried in the ImMunoGeneTics database (www.imgt.org) to determine the relevant VH and VL domains. Using assembly PCR, the VH and VL genes were then fused with a (G₄S)₃ linker and cloned into the SfiI and XbaI sites on pSecTagA (Invitrogen). From this parent vector, SIINFEKL and/or p31 was then additionally cloned into the 3′ end of the TER119 scFv gene using assembly PCR. The TER119-antigen constructs were expressed in suspension culture of HEK293E cells under serum-free conditions with 3.75 mM valproic acid (Sigma–Aldrich) (47) for 5 d and purified from supernatant using immobilized metal ion affinity chromatography on an Akta FPLC system (GE Healthcare). Purified proteins were analyzed for purity using SDS/PAGE, for endotoxin level using THP-1 × Blue cells (InvivoGen), and for concentration using bicinchoninic acid assays (Thermo Scientific). The final product was sterile-filtered and stored at −80 °C in working aliquots.

OTI T-Cell Adoptive Transfer.

CD8⁺ T cells from OTI (CD45.2⁺) mouse spleens were isolated using a CD8 magnetic bead negative selection kit (Miltenyi Biotec) as per the manufacturer’s instructions. Freshly isolated CD8⁺ OTI cells were resuspended in PBS and labeled with 1 μM CFSE (Invitrogen) for 6 min at room temperature, and the reaction was quenched for 1 min with an equal volume of Iscove’s modified Dulbecco’s medium (IMDM) with 10% (vol/vol) FBS (Gibco). Cells were washed, counted, and resuspended in pure IMDM before injection. A total of 3 × 10⁶ CFSE-labeled CD8⁺ OTI cells were injected i.v. into the tail vein of recipient CD45.1⁺ mice. For short-term proliferation studies, 10 μg of ERY1-OVA or OVA or an equimolar dose of SIINFEKL or TER119-SIINFEKL in a 100-μL volume was injected 24 h following adoptive transfer. Splenocytes were harvested 5 d following antigen administration and stained for analysis by flow cytometry.

OTI Challenge Model.

A total of 3 × 10⁵ CFSE-labeled OTI CD8⁺ T cells were injected into CD45.1⁺ recipient mice as described above. At 1 and 6 d following adoptive transfer, mice were i.v. administered 10 μg of ERY1-OVA or OVA or an equimolar dose of SIINFEKL or TER119-SIINFEKL in 100 μL of saline into the tail vein. At 15 d following adoptive transfer, mice were challenged with 5 μg of OVA and 25 ng of ultrapure E. coli LPS (InvivoGen) in 25 μL of saline injected intradermally into each rear leg pad (Hock method: total dose of 10 μg of OVA and 50 ng of LPS). Mice were killed 4 d following challenge, and spleen and draining lymph node cells were isolated for restimulation. For flow cytometry analysis of intracellular cytokines, cells were restimulated in the presence of 1 mg/mL OVA or 1 μg/mL SIINFEKL peptide (Genscript) for 3 h. Brefeldin-A (5 µg/mL; Sigma) was added, and restimulation was resumed for an additional 3 h before staining and flow cytometry analysis. For ELISA measurements of secreted factors, cells were restimulated in the presence of 100 μg/mL OVA or 1 μg/mL SIINFEKL peptide for 4 d. Cells were spun, and the media were collected for ELISA analysis using IFN-γ and IL-10 Ready-Set-Go kits (eBioscience) as per the manufacturer’s instructions. OVA-specific serum IgG was detected by incubating mouse serum at varying dilutions on OVA-coated plates, followed by a final incubation with goat anti-mouse IgG-HRP (Southern Biotech).

OTI E.G7-OVA Tolerance Model.

A total of 1 × 10⁶ CFSE-labeled OTI CD8⁺ T cells were injected into 8- to 12-wk-old female C57BL/6 mice as described above. At 1 and 6 d following adoptive transfer, mice were i.v. administered 10 μg of ERY1-OVA or 10 μg of OVA in 100 μL of saline into the tail vein. Blood was drawn 5 d following adoptive transfer for characterization of OTI CD8⁺ T-cell proliferation by flow cytometry. OVA-expressing EL-4 thymoma cells (E.G7-OVA, CRL-2113; American Type Culture Collection) were cultured as per American Type Culture Collection guidelines. In brief, cells were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) FBS, 10 mM Hepes, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, 1% puromycin/streptomycin (Invitrogen Gibco), and 0.4 mg/mL G418 (PAA Laboratories). Just before injection, cells were expanded in media without G418 and resuspended on harvest in HBSS (Gibco). At 9 d following adoptive transfer, mice were anesthetized with isoflurane, the back area was shaved, and 1 × 10⁶ E.G7-OVA cells were injected intradermally between the shoulder blades. At 4 d following E.G7-OVA graft, tumor dimensions were measured every 24 h with a digital caliper and tumor volume was calculated as an ellipsoid ( where V is volume, l is length, w is width, and h is the height of the tumor). At 15 d following adoptive transfer, mice were challenged with 5 μg of OVA and 25 ng of ultrapure E. coli LPS in 25 μL of saline injected intradermally into each rear leg pad (total dose of 10 μg of OVA and 50 ng of LPS).

BDC2.5 Diabetes Induction Model.

Freshly isolated splenocytes from female BDC2.5 transgenic mice were stimulated for 4 d in DMEM supplemented with 10% (vol/vol) FBS, 0.05 mM β-mercaptoethanol, 1% puromycin/streptomycin, and 0.5 μM p31 peptide. Following stimulation, cells were washed with basal DMEM and analyzed for purity by flow cytometry, and 5 × 10⁶ T cells were i.v. injected into normoglycemic NOD/ShiLtJ mice. At 8 h following adoptive transfer, mice were i.v. administered saline, 10 μg TER119-p31, or an equimolar dose of p31 peptide every 3 d for three doses. Diabetes onset was monitored by measuring nonfasting blood glucose levels using an Accu-Check Aviva glucometer (Roche). Mice were considered diabetic at blood glucose readings ≥250 mg/dL. After three hyperglycemic readings, mice were euthanized and organs were harvested for histology.

Data Analysis.

All flow cytometry data were analyzed using FlowJo (v8.8.6 and v9.2; TreeStar). Graphing and statistical analyses of worked-up data were performed using Prism (v5; GraphPad). Mann–Whitney U tests were used for interpreting flow cytometry population data, two-tailed Student t tests were used for interpreting ELISA data, and ANOVA was used for interpreting OTI generation data (***P < 0.001; **P < 0.01; *P < 0.05). Log rank (Mantel–Cox) tests were used to analyze Kaplan–Meier plots of diabetes onset data.

Additional detailed materials and methods are available in SI Materials and Methods.