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. 2015 Oct;64(10):3521-31.
doi: 10.2337/db14-1880. Epub 2015 Jun 12.

DCIR2+ cDC2 DCs and Zbtb32 Restore CD4+ T-Cell Tolerance and Inhibit Diabetes

Jeffrey D Price  1 , Chie Hotta-Iwamura  1 , Yongge Zhao  1 , Nicole M Beauchamp  1 , Kristin V Tarbell  2
Affiliations

DCIR2+ cDC2 DCs and Zbtb32 Restore CD4+ T-Cell Tolerance and Inhibit Diabetes

Jeffrey D Price et al. Diabetes. 2015 Oct.

Abstract

During autoimmunity, the normal ability of dendritic cells (DCs) to induce T-cell tolerance is disrupted; therefore, autoimmune disease therapies based on cell types and molecular pathways that elicit tolerance in the steady state may not be effective. To determine which DC subsets induce tolerance in the context of chronic autoimmunity, we used chimeric antibodies specific for DC inhibitory receptor 2 (DCIR2) or DEC-205 to target self-antigen to CD11b(+) (cDC2) DCs and CD8(+) (cDC1) DCs, respectively, in autoimmune-prone nonobese diabetic (NOD) mice. Antigen presentation by DCIR2(+) DCs but not DEC-205(+) DCs elicited tolerogenic CD4(+) T-cell responses in NOD mice. β-Cell antigen delivered to DCIR2(+) DCs delayed diabetes induction and induced increased T-cell apoptosis without interferon-γ (IFN-γ) or sustained expansion of autoreactive CD4(+) T cells. These divergent responses were preceded by differential gene expression in T cells early after in vivo stimulation. Zbtb32 was higher in T cells stimulated with DCIR2(+) DCs, and overexpression of Zbtb32 in T cells inhibited diabetes development, T-cell expansion, and IFN-γ production. Therefore, we have identified DCIR2(+) DCs as capable of inducing antigen-specific tolerance in the face of ongoing autoimmunity and have also identified Zbtb32 as a suppressive transcription factor that controls T cell-mediated autoimmunity.

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Figures

Figure 1
Figure 1
Stimulation of BDC2.5 T cells by DCIR2+ DCs delays diabetes development in NOD.scid mice. A: Percent of NOD.scid mice that are diabetes free after injection with CD4+CD25− BDC2.5 T cells and the indicated treatments. Statistical analysis was performed with log-rank test. P < 0.001 for αDEC-BDC vs. αDCIR2-BDC; P = 0.452 for PBS vs. αDEC-BDC. Summation of four experiments. n = 28 mice treated with PBS, n = 20 mice treated with αDEC-BDC, and n = 19 mice treated with αDCIR2-BDC. BDC2.5 T cells (5 × 104) were transferred to NOD.scid mice and treated with the indicated conditions. Graphs indicate the total BDC2.5 T-cell number (B) and the ratio of Foxp3+ BDC2.5 T cells (C) in NOD.scid mice on day 5. Average of three independent experiments ± SEM. Statistical analysis was performed with one-way ANOVA with Bonferroni posttest. *P < 0.05. LN, lymph nodes; NS, not significant.
Figure 2
Figure 2
DEC-205+ DCs, but not DCIR2+ DCs, elicit sustained expansion of BDC2.5 T cells in NOD mice. A: Activation markers (CD69 and CD25) on BDC2.5 T cells in spleen and lymph nodes (LNs) after stimulation of BDC2.5 T cells in NOD mice with the indicated dose of αDEC-BDC or αDCIR2-BDC (or PBS control, 0 ng). Average of two independent experiments ± SEM. B: Histograms of carboxyfluorescein succinimidyl ester (CFSE) dilution for BDC2.5 T cells stimulated with the indicated antibodies at day 3 or day 10. Numbers indicate the percentage of divided T cells. C: Fold expansion (over PBS controls, dotted line at fold expansion 1) of BDC2.5 T cells after the indicated treatments. Statistical analysis was performed with one-way ANOVA with Bonferroni posttests. *P < 0.05 for αDEC-BDC vs. αDCIR2-BDC.
Figure 3
Figure 3
DEC-205+ DCs, but not DCIR2+ DCs, stimulate sustained IL-2 and IFN-γ production by T cells. Percent of BDC2.5 T cells expressing IL-2 or IFN-γ after the indicated treatments for 3 or 10 days in NOD mice after the indicated treatments. Average of four independent experiments ± SEM. Statistical analysis was performed with one-way ANOVA with Bonferroni posttests. *P < 0.05; **P < 0.01; ***P < 0.001. LN, lymph nodes.
Figure 4
Figure 4
NOD CD8+ DCs express higher levels of CD40 than CD11b+ DCs. Geometric mean fluorescence intensity (MFI) of CD40 on spleen CD8+ or CD11b+ DCs in C57Bl/6 (B6) or NOD mice. Each dot represents a single mouse, representative of three independent experiments. Statistical analysis was performed with two-way ANOVA with Bonferroni posttests. **P < 0.0001.
Figure 5
Figure 5
DEC-205+ and DCIR2+ DCs induce similar Treg proliferation, with little Treg induction and no increase in the Treg-to-Teff ratio. A: BDC2.5 Foxp3+ T cells and BDC2.5 Foxp3− T cells, sorted from BDC2.5.Foxp3-GFP mice, were stimulated in vivo with αDEC-BDC or αDCIR2-BDC for 3 days in NOD mice. B: The number of GFP+ cells was assessed among transferred GFP+ cells after treatment with the indicated antibodies. Average of three independent experiments ± SEM. C: The number of GFP+ cells converted from transferred GFP− cells (left) and the number of GFP− cells (right) expanded by the indicated treatments were assessed. Average of three independent experiments ± SEM. D: NOD mice were injected with BDC2.5 T cells, and 5 days after treatment with the indicated antibodies, the total percentage of BDC2.5 T cells expressing Foxp3 was measured. Average of two independent experiments ± SEM. No significant differences were observed by statistical analysis with one-way ANOVA (B and C) or two-way ANOVA (D). LN, lymph nodes; ND, not detected; NS, not significant; pLN, pancreatic lymph nodes.
Figure 6
Figure 6
αDCIR2-BDC increases BDC2.5 T-cell apoptosis and decreases responsiveness to antigen rechallenge. A: BDC2.5 cells from spleen were assessed for caspase activation and mitochondrial membrane potential after 4 days with the indicated treatments in NOD mice. B: The average percentage of BDC2.5 cells within the apoptotic gate in spleen and lymph nodes (LNs). Average of three experiments ± SEM. *P < 0.05, **P < 0.005. C: NOD mice injected with BDC2.5 T cells were treated with either PBS or αDCIR2-BDC intraperitoneally. Ten days after the initial treatment, mice were challenged with αDCIR2-BDC + LPS + poly(I:C) + αCD40 or given PBS as a control. Five days later (day 15 after initial treatment), numbers of transferred cells in spleen and LNs were assessed. Each dot represents cells from a single mouse, representative of three individual experiments. Statistical analysis was performed with one-way ANOVA with Bonferroni posttests. *P < 0.05. NS, not significant.
Figure 7
Figure 7
In vivo stimulation with αDCIR2-BDC induces distinct gene expression changes in BDC2.5 T cells. BDC2.5 T cells were transferred to NOD mice, followed by treatment with αDEC-BDC or αDCIR2-BDC. Fourteen hours after treatment, BDC2.5 T cells were sorted from spleen and lymph nodes and RNA was obtained and then analyzed by microarray. Genes differentially regulated by DCIR2+ and DEC-205+ DCs are analyzed by a volcano plot of genes (dark lines indicate a P value of 0.05 [horizontal] and a twofold change [vertical]) (A) and a pathway analysis of immune system process in gene ontology ID 2376 (P < 0.05, false discovery rate < 0.75) (B). From the data in B, leukocyte activation-related genes are shown in C (P < 0.05, false discovery rate < 0.75).
Figure 8
Figure 8
Overexpression of Zbtb32 in BDC2.5 T cells inhibits T-cell expansion, IFN-γ production, and diabetes development. A: BDC2.5 T cells were transferred to NOD mice, followed by the indicated treatments. Fourteen hours after treatment, BDC2.5 T cells were sorted from spleen and RNA was obtained and then analyzed by NanoString nCounter analysis. **P < 0.001; *P < 0.05. B: BDC2.5 T cells were transfected with the indicated plasmids and allowed to rest for 14 h before being lysed. Western blotting was performed using anti-Flag antibodies (top), with anti-actin antibodies (bottom) as a loading control. Transfected BDC2.5 T cells were stimulated with the indicated concentration of plate-bound anti-CD3 antibody and anti-CD28 antibody in vitro for 3 days to see the proliferation (**P < 0.001, *P < 0.01) (C) and IFN-γ secretion after the phorbol myristic acid and ionomycin stimulation (*P < 0.05) (D). E–G: BDC2.5 T cells were transfected with the indicated plasmids and transferred to NOD.scid mice. E: Percent of NOD.scid mice that were diabetes free at the indicated time points after BDC2.5 T-cell injection is shown. P < 0.0001 (n = 15 mice). Six days after T-cell injection, the pancreata were harvested and scored for the degree of insulitis, with at least 20 islets per pancreas analyzed by two independent examiners (F), and the numbers of transferred cells in spleen, lymph nodes (LNs), and pLN (pooled from three mice) were assessed (G). **P < 0.01; ***P = 0.0005 (n = 3 mice). Statistical analysis was performed with two-way ANOVA with Bonferroni posttests (A and C), Student t test (D and G), and log-rank test (E). pLN, pancreatic lymph nodes.
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