Plasmacytoid dendritic cells are proportionally expanded at diagnosis of type 1 diabetes and enhance islet autoantigen presentation to T-cells through immune complex capture

Abstract

Objective: Immune-mediated destruction of beta-cells resulting in type 1 diabetes involves activation of proinflammatory, islet autoreactive T-cells, a process under the control of dendritic cells of the innate immune system. We tested the hypothesis that type 1 diabetes development is associated with disturbance of blood dendritic cell subsets that could enhance islet-specific autoimmunity.

Research design and methods: We examined blood dendritic cells (plasmacytoid and myeloid) in 40 patients with recent-onset diabetes (median duration 28 days) and matched control subjects. We also examined the relative ability of different dendritic cell subsets to process and present soluble or immune complexed islet cell autoantigen (the islet tyrosine phosphatase IA-2) to responder CD4 T-cells.

Results: The balance of blood dendritic cells was profoundly disturbed at diabetes diagnosis, with a significantly elevated proportion of plasmacytoid and reduction of myeloid cells compared with control subjects. Dendritic cell subset distribution was normal in long-standing disease and in patients with type 2 diabetes. Both dendritic cell subsets processed and presented soluble IA-2 to CD4 T-cells after short-term culture, but only plasmacytoid dendritic cells enhanced (by as much as 100%) autoantigen presentation in the presence of IA-2(+) autoantibody patient serum.

Conclusions: The plasmacytoid subset of dendritic cells is overrepresented in the blood close to diabetes onset and shows a distinctive ability to capture islet autoantigenic immune complexes and enhance autoantigen-driven CD4 T-cell activation. This suggests a synergistic proinflammatory role for plasmacytoid dendritic cells and islet cell autoantibodies in type 1 diabetes.

FIG. 1.

Example of flow cytometry analysis and gating strategy for identification and quantification of blood dendritic cell subsets. The mononuclear cell population in whole blood is identified by a combination of forward and side scatter properties (A) followed by exclusion gating of live CD14⁺ and CD19⁺ mononuclear cells and dead cells (B), before the identification and enumeration of plasmacytoid dendritic cell (pDC), myeloid dendritic cell 1 (mDC1), and myeloid dendritic cell 2 (mDC2) subsets, as shown in C–E with the relevant blood dendritic cell (BDC) marker. F: Total dendritic cell populations as a percentage of mononuclear cells in 40 patients with new-onset type 1 diabetes and matched control subjects. (Please see http://dx.doi.org/10.2337/db08-0964 for a high-quality digital representation of this figure.)

FIG. 2.

Dendritic cell subsets in new-onset type 1 diabetic patients and control subjects showing plasmacytoid dendritic cells (A), type 1 myeloid dendritic cells (B), and type 2 myeloid dendritic cells (C). Results are expressed as percentage of total dendritic cell population. Mean values for each group are indicated by a horizontal line, and P values are given where significant.

FIG. 3.

Dendritic cell subsets in long-standing type 1 diabetic patients (LS T1D) and age-matched control subjects showing total dendritic cell population as a percentage of mononuclear cells (A) and the plasmacytoid dendritic cell (B), type 1 myeloid dendritic cell (C), and type 2 myeloid dendritic cell (D) subsets as a percentage of total dendritic cells. Mean values for each group are indicated by a horizontal line and were similar between study groups. Similarly, E–G show plasmacytoid dendritic cell (E), type 1 myeloid dendritic cell (F), and type 2 myeloid dendritic cell (G) subsets as a percentage of total dendritic cells in patients with type 2 diabetes and an appropriately age-matched control group. Mean levels are similar in the two groups (NS).

FIG. 4.

Plasmacytoid (A) and myeloid (B) dendritic cell subsets as a percentage of total dendritic cells in five patients with new-onset type 1 diabetes measured at diagnosis and 2 years later. There is a trend in three of the patients for plasmacytoid dendritic cell levels to decline and in three of the patients for myeloid dendritic cell levels to increase/remain unchanged. C: Dendritic cell subset levels (expressed as a percentage of dendritic cells) in a nondiabetic control individual studied on seven separate occasions over the same 2-year period.

FIG. 5.

IFN-α secretion by isolated plasmacytoid dendritic cells in type 1 diabetic patients and control subjects in response to stimulation with increasing concentrations of the unmethylated CpG oligonucleotide 2,216. Each point represents the mean value of IFN-α secretion from nine different patients (▴) and nine control subjects (□). Vertical bars represent SEs.

FIG. 6.

Effect of islet cell autoantibody–positive serum on presentation by plasmacytoid dendritic cells (A), myeloid dendritic cells (B), and CD14⁺ monocytes (C) of the islet cell autoantigen IA-2ic-HA_307–319 to CD4 T-cells (JNZ-1 clone) specific for the influenza HA_307–319 epitope. CD4 T-cell clone responses are indicated by levels of IFN-γ produced in the culture supernatants. A: In the presence of soluble 10 μg/ml IA-2ic-HA_307–319 without serum supplementation (□), IA-2ic is processed and presented with comparable efficiency by plasmacytoid dendritic cells and myeloid dendritic cells, with CD14⁺ monocytes markedly less proficient. Addition of IA-2⁺ autoantibody (AAb) serum (3,500 World Health Organization [WHO] units) at 1, 5, and 10% (▪) considerably and significantly enhances the presentation of IA-2ic by plasmacytoid dendritic cells but has minimal effects on myeloid dendritic cells and CD14⁺ monocytes (*P < 0.05 vs. presence of antigen alone; □). GAD65⁺ autoantibody serum (1,700 WHO units; ; 1–10%) has no effect on presentation. Likewise, pooled AB serum at these concentrations has no effect (data not shown). In the presence of 5% serum but no IA-2ic-HA_307–319, there is no T-cell activation. Data represent one of four experiments using four different cell donors. D: Pooled data from a total of four donors (including the one used in A) for plasmacytoid dendritic cell enhancement of IA-2ic-HA_307–319 (▪) presentation to JNZ-1 clone cells (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. presence of antigen alone; □). The additional sera contained 3,875.1, 3,263.1, and 3,007.8 WHO units of IA-2 autoantibodies. Bars represent means ± SE from quadruplicate wells of individual experiments. E: IA-2⁺ serum–enhanced IA-2ic-HA_307–319 presentation is dependent on the serum IgG fraction. Serum has been adsorbed using Protein A matrix. Serum IgG level before adsorption, 6.54 g/l; after adsorption, 0.22 g/l. Serum was used at 5% concentration in these experiments. Serum IA-2 autoantibody level before adsorption, 2,152 WHO units; after adsorption, IA-2 autoantibodies were not detectable. *Significant reduction in IFN-γ production by clone JNZ-1 in the presence of adsorbed compared with nonadsorbed serum (P < 0.05).