DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of diabetogenic CD8⁺ T cells independently of PD-1 and PD-L1

Abstract

CD8⁺ T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) have been implicated in type 1 diabetes in both humans and non-obese diabetic (NOD) mice, in which T cells specific for IGRP₂₀₆₋₂₁₄ are highly prevalent. We sought to manipulate these pathogenic T cells by exploiting the ability of steady-state dendritic cells (DCs) to present antigens in a tolerogenic manner. The endocytic receptor DEC-205 was utilized to deliver an IGRP₂₀₆₋₂₁₄ mimotope to DCs in NOD mice, and the impact of this delivery on a polyclonal population of endogenous islet-reactive cognate T cells was determined. Assessment of islet-infiltrating CD8⁺ T cells showed a decrease in the percentage, and the absolute number, of endogenous IGRP₂₀₆₋₂₁₄-specific T cells when the mimotope was delivered to DCs, compared with delivery of a specificity control. Employing an adoptive transfer system, deletion of CD8⁺ T cells as a result of DEC-205-mediated antigen targeting was found to occur independently of programmed death-1 (PD-1) and its ligand (PD-L1), both often implicated in the regulation of peripheral T-cell tolerance. Given its promise for the manipulation of self-reactive polyclonal T cells demonstrated here, the distinctive characteristics of this antigen delivery system will be important to appreciate as its potential as an intervention for autoimmune diseases continues to be investigated.

Keywords: Autoimmunity; NOD mice; diabetes.

Fig. 1.

Characterization of anti-DEC-205/NRP-V7 which can specifically stimulate reactive CD8⁺ T cells in vitro. The hybrid antibodies anti-DEC-205/ovalbumin and anti-DEC-205/NRP-V7 were produced by transient transfection of 293 T cells with calcium phosphate. (A) SDS-PAGE analysis showing bovine γ-globulin standard (lane 1), anti-DEC-205/NRP-V7 (lane 2) and anti-DEC-205/ovalbumin (lane 3) after affinity purification on a Protein G column. Molecular weight standards are in the left lane. The molecular weights of the light chains and the antigen-linked heavy chains were as expected (light chains, 25 kD; anti-DEC-205/NRP-V7 heavy chain, 51 kD; anti-DEC-205/ovalbumin heavy chain, 93 kD). (B) Binding of anti-DEC-205/NRP-V7 or anti-DEC-205/ovalbumin to CHO cells (left panel) or CHO cells expressing DEC-205 (right panel). Cells were stained with 0.5 μg ml⁻¹ anti-DEC-205/NRP-V7 (thick black line) or anti-DEC-205/ovalbumin (thin gray line) followed by FITC-labeled secondary antibody or secondary antibody alone (goat anti-mouse IgG; filled histogram) and analyzed by flow cytometry. (C) Splenic CD11c⁺ DCs were isolated from NOD mice and incubated overnight in the presence of LPS with 5 μg ml⁻¹ anti-DEC-205/NRP-V7 or anti-DEC-205/ovalbumin or with 10⁻⁶ M NRP-V7 peptide. CD8⁺ T cells from the spleens of 8.3-NOD mice were purified and 2×10⁴ T cells were incubated at the indicated ratios with the DCs. After 3 days of co-culture, proliferation was monitored by BrdU incorporation.

Fig. 2.

DEC-205-mediated antigen delivery in NOD mice leads to deletion of endogenous cognate CD8⁺ T cells. Female NOD mice were treated with either anti-DEC-205/NRP-V7 or anti-DEC-205/ovalbumin at 4 weeks and 8 weeks of age. At 12 weeks of age, pancreatic islets were isolated and cultured for 3 days in the presence of IL-2. Islet-infiltrating cells were stained with anti-CD8 antibody and PE-labeled H-2K^d/NRP-V7 tetramers to quantify the IGRP_206–214-specific T cells by flow cytometry (gating on CD8⁺ cells). Representative plots are shown in the top panel. A statistically significant decrease in both the percentage of endogenous IGRP_206–214-specific T cells in the islet infiltrate (P = 0.01; middle panel) and the absolute number of these cells per islet (P = 0.03; bottom panel) was observed in mice treated with anti-DEC-205/NRP-V7 compared with those treated with the specificity control anti-DEC-205/ovalbumin (n = 7 and 8, respectively). *P < 0.05.

Fig. 3.

Anti-PD-1 blocking antibody could not abrogate the deletion of cognate CD8⁺ T cells by DEC-205-mediated antigen targeting. DEC-205⁺, DEC-205⁻ and DCIR2⁺ splenic DC subsets (CD11c⁺) from NOD mice were examined for expression of (A) PD-L1 or (B) PD-L2. The open histograms depict staining with the PD-L1 or PD-L2 antibodies, whereas the filled histograms depict binding of the correspondingisotype controls. (C–G) CD8⁺ T cells were purified from AI4 donors by negative selection using MACS microbeads (Miltenyi Biotec) and injected i.v. into female NOD.NON-Thy1 ^a recipients (6–8 weeks of age). The recipient mice were also injected i.p. with 200 μg of anti-PD-1 blocking antibody (29F.1A12) or its isotype control (rat IgG2a) soon after the cell transfer. The following day, the recipient mice were injected i.p. with 10 μg of anti-DEC-205/MimA2. The blocking antibody or the isotype control treatment was carried on thrice more on days 3, 6 and 9. (C) Spleens and (D) pancreatic lymph nodes were harvested 12 days later and single-cell suspensions were obtained from these organs. (E) Whole pancreas was collagenase digested to enumerate the transferred CD8⁺Thy1.2⁺ T cells in the pancreas. Cells were stained and analyzed for the percentage of transferred CD8⁺Thy1.2⁺ T cells by flow cytometry, gating on live cells. Also, in some experiments, cells were stained with an H-2D^b/MimA2 tetramer to further characterize the transferred cells. No significant difference was found when 29F.1A12-treated mice were compared with those treated with rat IgG2a. (F) Pancreatic islets from both groups of mice were cultured for 2 days in the presence of IL-2, and the percentage or the total number of transferred cells in the islets or per islet was analyzed. No significant differences between the experimental and control groups were observed, thus demonstrating the lack of any effect of the PD-1 blocking antibody on the deletion of the transferred cells by DEC-205-mediated targeting. (G) To ensure that the DEC-205-mediated antigen targeting did not cause TCR down-regulation, the transferred cells were also stained with a TCRβ antibody and shown to have comparable TCR expression between mice treated with anti-DEC-205/MimA2 alone or anti-DEC-205/MimA2 with either 29F.1A12 or rat IgG2a or the specificity control anti-DEC-205/ovalbumin (gating on CD8⁺ cells). Data shown are representative of five independent experiments.

Fig. 4.

PD-L1 is not involved in the deletion of reactive CD8⁺ T cells induced by DEC-205-mediated antigen targeting. CD8⁺ AI4 T cells purified from spleens of NOD-AI4αβTg mice were injected i.v. into female NOD.NON-Thy1 ^a recipients (6–8 weeks of age). The recipient mice were also injected i.p. with 200 μg of anti-PD-L1 blocking antibody or its isotype control soon after the cell transfer. The following day, the recipient mice were injected i.p. with 10 μg of anti-DEC-205/MimA2. The blocking antibody or the isotype control treatment wascarried on thrice more on days 3, 6 and 9. (A) Spleens and (B) pancreatic lymph nodes were harvested 12 days later and analyzed by flow cytometry, gating on live cells. There was no significant difference in the percentage of transferred cells in the spleen or the pancreatic lymph nodes showing that PD-L1 is not required for the deletion by DEC-205-mediated antigen targeting. Data shown are representative of three independent experiments.