Dab2, a negative regulator of DC immunogenicity, is an attractive molecular target for DC-based immunotherapy

Abstract

Dab2 is an adapter protein involved in receptor-mediated signaling, endocytosis, cell adhesion, hematopoietic cell differentiation, and angiogenesis. It plays a pivotal role in controlling cellular homeostasis. In the immune system, the Dab2 is a Foxp3 target gene and is required for regulatory T (Treg) cell function. Dab2 expression and its biological function in dendritic cells (DCs) have not been described. In this study, we found that Dab2 was significantly induced during the development of mouse bone marrow (BM)-derived DCs (BMDCs) and human monocyte-derived DCs (MoDCs). Even in a steady state, Dab2 was expressed in mouse splenic DCs (spDCs). STAT5 activation, Foxp3 expression, and hnRNPE1 activation mediated by PI3K/Akt signaling were required for Dab2 expression during GM-CSF-derived BMDC development regardless of TGF-β signaling. Dab2-silencing was accompanied by enhanced IL-12 and IL-6 expression, and an improved capacity of DC for antigen uptake, migration and T cell stimulation, which generated strong CTL in vaccinated mice. Vaccination with Dab2-silenced DCs inhibited tumor growth more effectively than did vaccination with wild type DCs. Dab2-overexpression abrogated the efficacy of the DC vaccine in DC-based tumor immunotherapy. These data strongly suggest that Dab2 might be an intrinsic negative regulator of the immunogenicity of DCs, thus might be an attractive molecular target to improve DC vaccine efficacy.

Keywords: BAT, blocking the TGF-β-activated translation element; BM, bone marrow; CFSE, 5, 6-carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocyte; DCs, dendritic cells; Dab2; Dab2, disabled-2 adaptor protein; Dab2KD, Dab2-knockdown; Foxp3, forkhead box P3; GM-CSF, granulocyte-macrophage colony stimulating factor; OT-1 and OT-2 mice, OVA257–264 and OVA323–339-peptide-specific T cell receptor transgenic mice; OVA, ovalbumin; PI3K, phosphoinositide-3 kinase; STAT5, transducer and activator of transcription 5; TGF-β, transforming growth factor-β; Treg, regulatory T; WT, wild type; dendritic cells; hMoDC, human monocyte-derived dendritic cell; hnRNP E1, heterogeneous nuclear ribonucleoprotein E1; imDC, immature DC; immunogenicity; mDC, mature DC; molecular target.

Figure 1.

Dab2 expression in BMDC, splenic DC and human monocyte-derived DC (A and B) Dab2 expression was assessed during BM (C57 BL/6) cell-derived DC (BMDC) development. Cells developing into BMDCs were harvested on days 0, 2, 4, and 6, and Dab2 mRNA and protein levels were assessed by quantitative real-time (qRT)-PCR with Fast SYBR® Green Master Mix kit (Life Technologies) and by Western blot with rabbit anti-mouse Dab2 polyclonal antibody (Protein Tech), respectively. qRT-PCR data are shown as mean ± SD of nine samples pooled from three independent experiments. (C) Cells were harvested on days 0, 2, 4, and 6 during DC development, and assessed by flow cytometry after staining with FITC-labeled CD11c and PE-labeled intracellular Dab2 antibodies. (D) Splenic DCs were isolated from mouse (C57BL/6) spleen using CD11c⁺ isolation kit (Miltenyi Biotech) and treated with or without LPS (100 ng/mL) for 24 h. Intracellular Dab2 expression in splenic DCs was assessed by FACS (left) and Western blot assay (right). (E) Dab2 mRNA and protein expression were assessed by real-time (RT)-PCR and by Western blot with rabbit anti-mouse Dab2 polyclonal antibody (Protein Tech), respectively, during human monocyte-derived DC (hMoDC) development (left) as described in Materials and Methods . Intracellular Dab2 expression in MoDCs was also assessed by FACS (right).

Figure 2 (See previous page).

Dab2 expression in DCs requires STAT5 signaling, Foxp3 expression, and hnRNPE1 activation but has no association with TGF-β signaling. (A) Mouse (C57BL/6) BMDCs were treated with 100 μM STAT5 inhibitor for 4 h before harvest. Harvested cells were subjected to Western blot analysis of STAT5 and Dab2 expression with STAT5, phospho-STAT5, and Dab2 antibodies. (B) DC precursor cells on day 4 during BMDC development were transfected with control and Foxp3 siRNAs and then harvested after 48 h. Dab2 expression was assessed by Western blot with Dab2 and Foxp3 antibodies. (C) mRNA (left) and protein (right) expressions of hnRNPE1 and Dab2, together with hnRNPE1 phosphorylation, were assessed during BMDC ←development by RT-PCR and Western blot analysis. (D) BMDC precursor cells on day 4 were transfected with si-con and si-hnRNPE1 and harvested after 48 h. Dab2 and hnRNPE1 expression was assessed by Western blot with Dab2 and hnRNPE1 antibodies. (E) BMDCs were treated with 50 μM PI3K inhibitor (Calbiochem) for 20 min before harvest and subjected to Western blot analysis of Akt and Dab2. hnRNPE1 was immunoprecipitated from BMDC lysates with anti-hnRNPE1 antibody, followed by immunoblot analysis with phospho-hnRNPE1 (phospho-serine) and phospho-Akt antibodies. (F) BMDC precursor cells on day 4 were transfected with Dab2-specific siRNAs (si-Dab2) or control siRNA (si-con). After 48 h, the level of TGF-β in culture supernatant was examined using TGF-β ELISA kit (BioLegend). (G) BMDCs were treated or untreated with 5 ng/mL TGF-β for 24 h in the presence or absence of SB431524 (TGFβRI). Dab2 and Smad2 expressions, and phospho-Smad2 were assessed by Western blot.

Figure 3.

Effects of Dab2-silencing on BMDC surface phenotypes. (A) BMDC precursor cells on day 4 were transfected with si-Dab2 or si-con and harvested on day 6 as WT or Dab2^KD BMDCs. Dab2 silencing was measured by qRT-PCR (left), Western blot (middle) and flow cytometry (right). qRT-PCR data are shown as mean ± SD of n = 9 samples pooled from three independent experiments. (B) Representative flow cytometry data showing surface phenotypes of Dab2^KD (si-Dab2) and WT (si-con) immature (imDC) and mature (mDC) BMDCs. (C) Geometric mean fluoescence intensities (gMFI) of each BMDC surface molecule shown in flow cytometry (B) are presented as mean ± SD of nine samples pooled from three individual experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control WT DCs, Student's t-test.

Figure 4 (See previous page).

Effects of Dab2-silencing on antigen uptake, migration, and cytokine secretion of BMDCs. (A) WT or Dab2^KD imDCs and LPS-stimulated (100 ng/mL for 24 h) mDCs (2 × 10⁵ cells) were equilibrated with 1 mg/mL FITC-conjugated dextran for 1 h at 37°C or 4°C, respectively. Cells were washed and analyzed by flow cytometry. Representative flow cytometry of FITC⁺ cell populations is shown (left) with the mean ± SD of 9 samples pooled from three independent experiments (right). **p < 0.01, *p < 0.05 compared with control imDCs and mDCs. Student's t-test. (B) WT or Dab2^KD imDCs and LPS-stimulated mDCs were stained with PE-conjugated anti-CCR7 mAb followed by FACS analysis. Representative flow cytometry analysis of the CCR7⁺ cell populations (%) in imDCs and mDCs (left) and accumulated statistical data (right) are shown with the mean ± SD of 9 samples pooled from three independent experiments. **p < 0.01, Student's t-test. (C) The migration of WT and Dab2^KD imDCs and mDCs was assessed by an in vitro chemotaxis assay as described in the materials and methods. The data are shown as mean ± SD of nine samples pooled from three independent experiments. *p < 0.05, Student's t-test. (D) In vivo migration assay with Dab2^KD BMDC was performed as described in materials and methods. CFSE-labeled Dab2^KD or WT mature DCs (1 × 10⁶ cells) were inoculated s.c in the right flank region of C57BL/6 mice. After 24 h, CFSE-labeled DCs in the inguinal lymph nodes were examined by flow cytometry. Mitomycine C (MMC)-treated (50 μg/mL for 30 min) CFSE-labeled BMDCs were used as a negative control. Representative FACS data (left) and statistical data from three independent experiments are shown as mean ± SD (right). ***p < 0.001. (E) Cytokine production was assessed by ELISA of WT and Dab2^KD BMDC culture supernatants after stimulating with LPS (100 ng/mL) for 24 h. The data are shown as mean ± SD of nine samples pooled from three independent experiments. *p < 0.05 compared with control mDCs, Student's t-test.

Figure 5 (See previous page).

The effects of Dab2-silencing on the ability of BMDCs to stimulate T cell proliferation. (A) WT and Dab2^KD mDCs (from C57BL/6 mice) that were pulsed with OVA_257–264 or OVA_323–329 peptides were co-cultured with CFSE-labeled OT-1 or OT-2 T cells, respectively, for 4 d at three different ratios. CFSE-positive proliferated (CFSE-diluted) T cells were gated, calculated, and represented by fold increases as described in materials and methods. Data are shown as mean ± SD of six samples pooled from three independent experiments. *p < 0.05 and **p < 0.01, Student t-test. (B) OVA peptide (OVA_257–264 or OVA_323–329)-pulsed WT and Dab2^KD mDCs were co-cultured with CFSE-labeled OT-1 or OT-2 T cells, respectively, for 4 d at different T:DC cell ratios. CFSE-labeled proliferating T cells were gated and represented by histogram. (C) Th1 (IFNγ), Th17 (IL-17), and Th2 (IL-4) cytokines in the supernatant of DC/T cell co-cultures at a 1:10 ratio were assessed by ELISA at day 3. The data are shown as mean ± SD of six samples pooled from three independent experiments. *p < 0.05 compared with WT (si-con) DC, student t-test. (D) Treg cell populations were assessed by intracellular Foxp3 staining and CD25 surface staining from co-cultures of mDCs (C57BL/6) pulsed with OVA_323–339 peptide and OT-2 T cells at a ratio 1:10 for 5 d. CD25⁺ Foxp3⁺ Treg cells were assessed by flow cytometry and are shown as mean ± SD of six samples pooled from three independent experiments. (E) OVA_323–329 peptide-pulsed WT and Dab2^KD mDCs were co-cultured with OT-2 T cells for 3 d, and Th17 cells among the OT-2 cells were assessed by FACS after intracellular staining with anti-IL-17 antibody (left). Statistical data (right) are shown as mean ± SD of six samples pooled from three independent experiments. *p < 0.05, Student t-test.

Figure 6 (See previous page).

Dab2^KD DC vaccine was more effective than WT DC vaccine in the induction of antitumor immunity in tumor immunotherapy. (A) CD8⁺ T cells proliferation in vivo by WT or Dab2^KD BMDCs was assessed by tetramer assay as described in materials and methods. Naive C57BL/6 mice were transferred i.v with OT-1 T cells together with OVA-pulsed Dab2^KD or WT mDCs, and then boosted with OVA_257–264 peptide on day 7. Three day later, splenocytes from the mice were stained with PE-labeled H-2K^b/OVA_257–264 tetramer and FITC-anti-CD8 antibodies, and subsequently analyzed by flow cytometry. Shown is representative FACS data (upper) and the number of tetramer(Tet)⁺CD8⁺ T cells is represented as mean± SD of six samples from two mice (lower). *p < 0.05, Student t-test. (B) The splenocytes prepared for tetramer assay in (A) were activated with phorbol myristate acetate (PMA)/ionomycin (40 ng/mL each, Sigma–Aldrich) for 4 h. Cells were then examined by flow cytometry after surface and intracellular staining with FITC-anti-CD8 antibody and PerCP/Cy-anti-IFNγ antibody, respectively. Shown is representative FACS data (upper), and the number of IFNγ+CD8+ T cells is represented as mean ± SD of six samples from two mice (lower). ***p < 0.001, Student t-test. (C) OT-1 mice were immunized twice at a 1-week interval with 1 × 10⁶ OVA peptide (OVA_257–264)-pulsed Dab2^KD or WT mBMDCs (C57BL/6). Cells from the spleen and DLN of vaccinated mice were harvested and cultured for 5–7 d in the presence of OVA_257–264 peptide. The level of IFNγ in the culture supernatants on day 2 of culture was assessed by ELISA, and the data are shown as mean ± SD of six simples from two independent experiments. *p < 0.05, Student t-test. (D) CTL activity in the DLNs of vaccinated OT-1 mice was assessed by flow cytometry using CFSE-labeled E.G7 and EL4 as target cells. Quantitative CTL activities are shown as mean ± SD (n = 3). *p < 0.05, Student t-test. (E) C57BL/6 mice were inoculated s.c. with E.G7 and EL4 tumor cells (5 × 10⁵) in the right flank and immunized twice on day 3 and day 10 with 1 × 10⁶ Dab2^KD (si-Dab2) or WT (si-con) mDCs (from C57BL/6 BM cells) that were pulsed with OVA peptide. Tumor growth was monitored and represented as mean ± SD of four mice from each of two experiments (bottom). *p < 0.05, Student t-test.

Figure 7.

Dab2-overexpression impaired the efficacy of the DC vaccine for tumor immunotherapy. (A) Dab2 expression plasmid (pEF-Myc/Dab2) and control vector (pEF-Myc/con) were transfected into DC precursor cells on day 4 during BMDC development. The transfected cells were harvested after 48 h, and matured with LPS (100 ng/mL) for 24 h. Dab2 expression in the transformed mDCs was assessed by Western blot (upper) and FACS analysis (lower) using anti-Myc and anti-Dab2 antibodies. (B) The surface phenotypes of Dab2 transfected mDCs were assessed by flow cytometry, and the gMFI value of each DC surface marker from three independent experiments is shown as mean ± SD of nine samples. *p < 0.05, **p < 0.01 compared with control vector-transfected DCs, Student's t-test. (C) IL-12 levels were assessed by ELISA of the culture supernatant of Dab2-transfected DCs and are shown as mean ± SD of nine samples. *p < 0.05 compared with control vector transfected DCs, Student's t-test. (D) C57BL/6 mice were inoculated s.c. with E.G7 tumor cells (5 × 10⁵) in the right flank, and then immunized on day 3 and day 10 with 1 × 10⁶ Dab2-expressing mDCs (OVA-DC-Dab2) or control vector-transfected mDCs (OVA-DC-con), which were derived from C57BL/6 BM cells and pulsed with OVA peptides (OVA_257–264 and OVA_323–339). Representative images of E.G7 tumors are shown on day 20 after DC vaccination (left). Tumor growth was monitored every 2–3 d and presented as mean ± SD of four mice from each of two experiments. *p < 0.05, Student's t-test.