Targeting antigens through blood dendritic cell antigen 2 on plasmacytoid dendritic cells promotes immunologic tolerance

Abstract

The C-type lectin receptor blood dendritic cell Ag 2 (BDCA2) is expressed exclusively on human plasmacytoid DCs (pDCs) and plays a role in Ag capture, internalization, and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4(+) T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4(+) T cells and preservation of Foxp3(+) regulatory T cells (Tregs). Reduction in Treg numbers following Ag delivery to BDCA2 restored both CD4(+) T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.

Figure 1. Characterization of anti-human BDCA2 mAbs and human BDCA2 transgenic mice

A, upper panel, NIH-3T3 cells transfected with BDCA2 or an empty vector control were stained with 10 μg/ml anti-BDCA2 mAb, UW80.1 or mouse IgG1 isotype control (G28-1). Lower panel, NIH-3T3-BDCA2 transfectants were incubated with either UW80.1 anti-BDCA2, mIgG1 isotype control or left untreated. Following washes to remove unbound mAbs, cells were stained with 5 μl PE-conjugated anti-BDCA2, (AC144). B, Analysis of pDC-specific expression of BDCA2. Splenocytes from control B6 or B6.BDCA2 transgenic mice were stained with fluorochrome-conjugated mAbs against CD11c, PDCA-1, Siglec-H, B220, and BDCA2 (UW80.1) or mIgG1 isotype control. Upper panel shows BDCA2 expression on PDCA-1⁺ pDCs from B6 and B6.BDCA2 Tg mice. Lower panel displays Siglec-H and B220 expression on BDCA2-and CD11c-gated cells from B6.BDCA2 Tg mice. Numbers in plots denote frequency among total scatter-gated splenocytes. Data shown are representative of >10 animals analyzed. C, Splenocytes from B6.BDCA2 Tg mice were stained with mAbs against CD19, CD4, CD8, NK1.1, CD11c, Siglec-H, PDCA-1, and BDCA2 or mIgG1 isotype control. Data shown are representative of 3 animals analyzed. D, Magnetically enriched pDCs from B6.BDCA2 Tg mice were stimulated in vitro with 10 μg/ml CpG-A with or without 2 or 10 μg/ml BDCA2 mAb (AC144) or mIgG1 isotype control (G28-1). 18 h later supernatants were collected and assayed for IFN-α and IL-12p40 by ELISA. Experiment shown is representative of 4 independent experiments.

Figure 2. BDCA2 can serve as a target for Ag delivery in B6.BDCA2 mice

A, Groups of B6.BDCA2 mice were injected i.v. with 2 μg AlexaFluor647-conjugated anti-BDCA2 (UW80.1) or mIgG1 isotype control and sacrificed one hour following injection. Splenocytes from injected mice were stained with mAbs specific for PDCA-1, Siglec-H, and CD11c, or IgD and CD19. Flow plots (left) depict gating for Siglec-H⁺PDCA-1⁺ pDCs (upper), CD11c⁺ myeloid DCs (middle), and CD19⁺IgD⁺ B cells (bottom). Numbers within flow plots denote frequency among total splenocytes. Histograms (right) show AlexaFluor647 fluorescence among the gated populations from anti-BDCA2-647-injected or mIgG1-647-injected mice. Numbers within histograms denote frequency of AlexaFluor647-positive cells among the gated population from mice that received anti-BDCA2-647. A representative experiment of five is shown using 2-3 mice/group. B, Groups of B6.BDCA2 mice were immunized i.v. with 10 μg OVA-conjugated anti-BDCA2, mIgG1 isotype control or anti-DCIR2 with or without 50 μg CpG-A. Graph depicts mean ± SEM of anti-OVA IgG in the serum 10 d post-immunization. A representative experiment of three is shown using 3-4 mice/group.

Figure 3. Targeting Ag to BDCA2⁺ pDCs leads to a reduction of Ag-specific T cells

A, Schematic for immunization and subsequent analysis. B, B6.BDCA2 recipients of CFSE-labeled OT-II cells were injected i.v. with PBS or 1 μg OVA-isotype, OVA-BDCA2 or OVA-DEC205 and sacrificed on d 4 following injection. Flow plots (top) depict gating of CD4⁺Ly5.1⁺ cells. Histograms (bottom) depict CFSE fluorescence among gated CD4⁺Ly5.1⁺ population in each group. C-D, Cumulative data from B showing the percentage of divided cells (C) and cell proliferation index (D) defined as total CFSE fluorescence (MFI) of PBS group divided by total CFSE fluorescence (MFI) for each group. Data in B-D are from 2 independent experiments using 2-4 mice per group. E, Cohorts of B6.BDCA2 recipients were immunized as in (A) and the frequency of CD4⁺Ly5.1⁺ and CD4⁺Ly5.1⁻ cells were determined at d 4. Numbers within flow plots denote frequency among total splenocytes. F, Scatter plots depict the total number of CD4⁺Ly5.1⁺ splenocytes from mice immunized as in (A) 4, 7 and 14 d post-immunization. Data in E-F are from three independent experiments using 3 mice/group. G, The number of endogenous CD4⁺ T cells per spleen 7 d p.i. from mice immunized as in (A) are plotted. H, The percentage of apoptotic cells within gated CD4⁺Ly5.1⁺ splenocytes at d 4 and d 7 was determined by AnnexinV staining in B6.BDCA2 recipients immunized as in (A). Data in C, F and G are pooled data from three independent experiments where each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. Cell numbers in F and G were calculated based on the total cell number per spleen. * p<0.05, ** p<0.01, *** p<0.001, as determined by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey post-test.

Figure 4. Targeting Ag to BDCA2⁺ pDCs leads to increased frequencies of Ag-specific T_reg cells

A-C, Cohorts of B6.BDCA2 recipients were immunized as in (Fig. 3A) and sacrificed 7 d post-immunization. A, The frequency of Foxp3⁺ cells within CD4⁺Ly5.1⁺ and CD4⁺Ly5.1⁻ splenocytes is shown. B and C, Combined data from three independent experiments showing the frequency and number of Foxp3⁺CD4⁺Ly5.1⁺ (B) and Foxp3⁺CD4⁺Ly5.1⁻ splenocytes (C). D, Histograms depict CFSE dilution among gated Foxp3⁺CD4⁺Ly5.1⁺ splenocytes 4 d following immunization as indicated. E, Representative histograms show the expression of Helios and CD25 among gated Foxp3⁺CD4⁺Ly5.1⁺ cells 7 d p.i.. F, Representative histograms show CD44 expression among gated Foxp3⁺CD4⁺Ly5.1⁺ cells 7 d p.i. For B, C and F, each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. Cell numbers in B and C were calculated based on the total cell number per spleen. * p<0.05, ** p<0.01, *** p<0.001, as determined by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey post-test.

Figure 5. Ag delivery to BDCA2⁺ pDCs alters secondary T-cell responses and leads to increased frequencies of Foxp3⁺ T cells

A, Schematic for immunization and subsequent analysis for panels B-E. B, Flow plots depict gating strategy for analysis of CD44 and CD62L expression among transferred CD4⁺Ly5.1⁺ Foxp3⁻ and Foxp3⁺ splenocytes. Numbers in quadrants represent percentages within the gated population. C, Data showing the frequencies and numbers of CD4⁺Ly5.1⁺ cells. D, Scatter plots depict the frequency and number of Foxp3⁺ CD4⁺ Ly5.1⁺ cells. E, Scatter plots show the ratio of Foxp3⁺ CD4⁺ Ly5.1⁺ to Foxp3⁻ CD4⁺ Ly5.1⁺ splenocytes as defined in B. Data shown in C, D and E are pooled data from three independent experiments. Each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. Cell numbers in C and D were calculated based on the total cell number per spleen. F. Cohorts of B6.BDCA2 recipients of OT-II T cells were injected with PBS, 1 μg OVA-isotype 1 (mouse IgG1), OVA-BDCA2, OVA-isotype 2 (rat IgG2a), OVA-SiglecH or OVA-DEC205; mice were otherwise treated as in A and analyzed as in B. Scatter plot depicts the frequency of Foxp3⁺CD4⁺Ly5.1⁺ splenocytes within each group. Data are pooled from two independent experiments. Each dot represents an individual animal with the mean indicated for each group (horizontal bars). * p<0.05, ** p<0.01, *** p<0.001, as determined by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey post-test.

Figure 6. OVA-BDCA2 immunization suppresses Ag-specific Ab responses

A, Schematic for immunization and subsequent analysis. B, Cohorts of B6.BDCA2 recipients of OT-II T cells were primed with 1 μg OVA-BDCA2 or OVA-isotype, or given PBS or 50μg OVA plus alum one day following adoptive transfer. 14 d later mice were boosted with 50 μg OVA plus alum and then bled weekly for 4 wks to collect serum. Line graph depicts quantities of OVA-specific serum IgG as determined by ELISA. Data shown are a representative experiment of 3 independent experiments using 3-4 mice/group. C, B6.BDCA2 recipients of OT-II T cells were primed with 1 μg OVA-BDCA2 or OVA-isotype and challenged 14 d later with either 50 μg OVA or CGG plus alum and bled weekly for 4 wks to collect serum. Line graph shows quantities of OVA-specific serum IgG as determined by ELISA. A representative experiment of 3 independent experiments using 3-4 mice/group is shown.

Figure 7. Depletion of T_regs by anti-CD25 Ab restores Ag-specific Ab responses following Ag re-challenge

A, Schematic for immunization, injections and subsequent analysis. B, Flow plots depict frequencies of CD4⁺Ly5.1⁺ and CD4⁺Ly5.1⁻ cells (among total splenocytes) and Foxp3⁺ and Foxp3⁻ cells (among gated CD4⁺Ly5.1⁺ cells) in mice given anti-CD25 or isotype control mAbs 8 d previously. C, Scatter plot showing the frequency of Foxp3⁺ cells among gated CD4⁺Ly5.1⁺ splenocytes for the indicated groups. D, Scatter plot showing the frequency of CD4⁺Ly5.1⁺ cells among total splenocytes for the indicated groups. E, Flow plots depicting CD44 and CD62L expression among gated Foxp3⁻ CD4⁺ Ly5.1⁺ splenocytes. F, Scatter plot depicts the combined frequency of activated CD44^hiCD62L^hi/lo cells gated in (E). Data in B-F are from three independent experiments using 2-3 mice/group. For C, D and F, each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. G, Line graph depicts quantities of OVA-specific serum IgG as determined by ELISA for the indicated groups. Data shown are the combined results from 3 independent experiments using 3-5 mice/group. * p<0.05, ** p<0.01, *** p<0.001, as determined by one-way ANOVA with Tukey post-test (B-F) or one-way ANOVA with repeated measures (G).

Figure 8. TLR7 agonist treatment at the time of Ag delivery prevents T_eff cell deletion and restores Ag-specific Ab responses following Ag re-challenge

Cohorts of B6.BDCA2 mice were immunized as in Fig. 3A with or without 50 μg R848. A, Scatter plots showing the frequency (left) and number (right) of total transferred Ly5.1⁺ CD4⁺ splenocytes for the indicated groups 7 d p.i. B, Scatter plots showing the frequency (left) and number (right) of Foxp3⁺ cells among CD4⁺ Ly5.1⁺ splenocytes quantified in A. C, Scatter plot depicts the ratio of the frequencies of Foxp3⁺ CD4⁺ Ly5.1⁺ to Foxp3⁻ CD4⁺ Ly5.1⁺ for the indicated groups at d 7 p.i. Data shown in A-C are from 2 independent experiments using 3-4 mice/group. Each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. Cell numbers were calculated based on the total cell number per spleen. E. Line graph shows the quantity of OVA-specific serum IgG for the indicated groups as determined by ELISA. One representative experiment of three independent experiments using 3-5 mice/group is shown. Each dot represents an individual animal with the mean indicated for each group (horizontal bars) ± SEM. * p<0.05, ** p<0.01, *** p<0.001, as determined by one-way ANOVA with Tukey post-test.