In vivo Tracking of Dendritic Cell using MRI Reporter Gene, Ferritin

Abstract

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R2*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R2*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R2* in both in vivo and ex vivo T2*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.

Fig 1. Analysis of dendritic cell (DC) transduced with myc-tagged human ferritin heavy chain (FTH) and green fluorescence prtein (GFP) using lentivirus.

(A) Representative GFP fluorescence image (green) and FTH immunofluorescence image (red) detected with anti-myc antibody in DCs and FTH-DCs. (B) Representative Western blots for FTH from whole cell lysate of DCs and FTH-DCs detected by using anti-myc antibody.

Fig 2. Analyses of proliferation and migration activities and co-stimulatory molecules expressions in dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC).

(A) A standard 3-,5-diphenyltetrazolium bromide (MTT) assay for proliferation activity of DCs and FTH-DCs cultured for 24 h, 48 h and 72 h. (B and C) Trans-well assay for migration abilities of DCs and FTH-DCs incubated with TNF-α (20 ng/mL) and IFN-γ (20 ng/mL) in the presence or absence of CCL19 and CCL21 for 24 h. Representative fluorescent image of nuclear stained with diamidino-2-phenylindole (DAPI) in DCs and FTH-DCs that migrated to the lower chamber. (D) RT-PCR analysis of C-C chemokine receptor type-7 (CCR-7) in DCs and FTH-DCs. Both DCs and FTH-DCs, which were incubated in the medium supplemented with TNF-α (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h, highly expressed the CCR-7 as compared to untreated cells. (E and F) Representative flow cytometric analysis of co-stimulatory molecules such as CD40, CD80 and CD86 in DCs and FTH-DCs treated with or without LPS (100 ng/mL) for 24 h. Flow cytometric results obtained from 3 independent experiments. All data are presented as the mean ± standard deviations of at least three independent experiments. *, p ≤0.05.

Fig 3. Cellular iron staining, iron amount measurement and in vitro MRI analysis of dendritic cell (DC) and human ferritin heavy chain-transduced DC (FTH-DC).

(A) Representative prussian blue staining of cellular iron in DCs and FTH-DCs incubated with or without 250 μM ferric ammonium citrate (FAC) for 24 h. (B) Average cellular iron amount measured from DCs and FTH-DCs incubated with 25 μM and 250 μM FAC. (C) Representative T₂*-weighted images and color-coded of the R₂* values in the DCs and FTH-DCs phantoms. (D) Average R₂* values measured from DCs and FTH-DCs phantoms. All data are presented as the mean ± standard deviations of at least three independent experiments. *, p ≤0.05.

Fig 4. in vivo and ex vivo MRI of popliteal lymph nodes (LNs) of mouse injected with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC).

(A) Representative in vivo T₂*-weighted images of popliteal LNs (circle) of mouse before and at 48 h after injection of 1x10⁷ DCs and FTH-DCs. (B) Average R₂* values measured from in vivo popliteal LNs with DCs and FTH-DCs. (C) Representative ex vivo T₂*-weighted images of popliteal LNs isolated from mouse at 48 h after injection of DCs and FTH-DCs. (D) Average R₂* values measured from ex vivo popliteal LNs with DCs and FTH-DCs. All data are presented as the mean ± standard deviations of at least three independent experiments. *, p≤0.05

Fig 5. Histological analysis of cryosectioned popliteal lymph nodes (LNs) with dendritic cell (DC) and ferritin heavy chain-transduced DC (FTH-DC).

(A) Representative GFP fluorescence images of popliteal LNs isolated from mouse at 48 h after injection of DCs and FTH-DCs. (B) Representative hematoxylin and eosin staining of cryosectioned popliteal LNs with DCs and FTH-DCs. (C) Immunofluorescence images of GFP (green) and FTH (red) detected with anti-GFP and anti-myc antibodies in LNs with DCs and FTH-DCs. (D) Merge images (yellow) of co-immunofluorescence staining of activation marker, CD25 (red) and GFP (green) detected with anti-CD25 and anti-GFP antibodies in FTH-DCs of cryosectioned LNs. Nuclear was stained with diamidino-2-phenylindole (DAPI, blue).