Selective delivery of beta cell antigen to dendritic cells in vivo leads to deletion and tolerance of autoreactive CD8+ T cells in NOD mice

Abstract

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet beta cells. Cytotoxic CD8(+) T cells, reactive to beta cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8(+) T cells specific for beta cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8(+) T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with beta cell antigens leads to deletion of autoreactive CD8(+) T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.

Fig. 1.

Presentation of peptides derived from anti-DEC-205/MimA2 is restricted to CD11c⁺ cells in vitro. Splenic DCs (CD11c⁺ cells) (Left) or CD11c⁻ cells (Right) were isolated from NOD mice and incubated overnight with the indicated antibodies. CD8⁺ T cells were purified from the spleens of NOD.AI4αβTg mice, and 5 × 10⁴ AI4 T cells were incubated at the indicated ratios with APCs in 96-well plates. After 3 d of coculture, proliferation was monitored by [³H]thymidine incorporation. Data shown are representative of at least two similar experiments. Significant differences between corresponding anti-DEC-205 and control Ig values are indicated: *, P < 0.05; **, P < 0.01.

Fig. 2.

In vivo targeting of peptide-linked anti-DEC-205 results in similar proliferation of CD8⁺ T cells specific for a self or foreign peptide. NOD.NON.Thy1^a mice were injected i.v. with (A) 2 × 10⁶ CFSE-labeled AI4 T cells purified from NOD.AI4αβTg mice or (B) 2 × 10⁶ CFSE-labeled CD8⁺ T cells purified from NOD.LCMV TCR Tg mice. Twenty-four hours later, recipient mice were treated i.p. with (A) 10 μg of anti-DEC-205/MimA2 or control Ig/MimA2 or PBS, or (B) 10 μg of anti-DEC-205/GP_33–41 or PBS. Three days after hybrid antibody injection, lymphoid organs were harvested, and the proliferation of Thy1.2⁺ CD8⁺ cells was evaluated by flow cytometry for CFSE dilution. Data shown are representative of at least two experiments.

Fig. 3.

In vivo DC depletion blocks CD8⁺ T cell proliferation in response to anti-DEC-205/MimA2. NOD.CD11c-DTR.Thy1^a mice were injected i.v. with 2 × 10⁶ CFSE-labeled AI4 T cells and 16 h later with 4 ng/g DT i.p. or PBS. Eight hours after the DT or PBS injection, recipient mice were given 10 μg of anti-DEC-205/MimA2 i.p. (A) Three days after antibody treatment, spleens were dissociated with collagenase D and the CD11c⁺ CD8⁺ and CD11c⁺ CD8⁻ DC subsets were identified after enrichment into a low-density cell fraction and analysis by flow cytometry, with samples gated on B220⁻ CD3⁻ cells. (B) Lymphoid organs were harvested to evaluate the proliferation of Thy1.2⁺ CD8⁺ AI4 T cells by flow cytometry for CFSE dilution. Data shown are representative of two similar experiments.

Fig. 4.

Anti-DEC-205/MimA2 targeting of DCs in vivo leads to deletion of AI4 T cells. (A) NOD.NON.Thy1^a mice were injected i.v. with 2 × 10⁶ AI4 T cells from NOD.AI4αβTg mice and, after 24 h, i.p. with 10 μg of anti-DEC-205/MimA2 or control Ig/MimA2. Twelve days later, lymphoid organs of recipient mice were harvested, and Thy1.2⁺ CD8⁺ cells were enumerated by flow cytometry. (B) Same as in A, but the transferred T cells were from NOD.Rag1^null. AI4αβTg mice. (C) The pancreata of the same mice used in B were perfused and islets were picked and cultured for 2 d. Islet infiltrates were harvested and evaluated for Thy1.2⁺ CD8⁺ cells by flow cytometry. In A-C, numbers denote the percentage of CD8⁺ cells present in the indicated gates. Data shown are representative of at least two experiments.

Fig. 5.

In vivo targeting of DCs with anti-DEC-205/MimA2 induces tolerance to MimA2. (A) NOD.NON.Thy1^a mice were injected i.v. with 2 × 10⁶ AI4 T cells from NOD.AI4αβTg mice and i.p. with 10 μg of anti-DEC-205/MimA2 or control Ig/MimA2. Twelve days after hybrid antibody administration, mice were boosted with 50 μg of MimA2 peptide in CFA. Three days later, peripheral lymph nodes were harvested and stimulated with free MimA2 peptide and AI4 T cells evaluated for production of intracellular IFN-γ. (B) Same as in A, but the transferred T cells were from NOD.Rag1^null.AI4αβTg mice and the recipient mice were given anti-DEC-205/MimA2 i.p. with or without anti-CD40 (25 μg) and poly I:C (50 μg).