Using carbon magnetic nanoparticles to target, track, and manipulate dendritic cells - PubMed (original) (raw)

Using carbon magnetic nanoparticles to target, track, and manipulate dendritic cells

Heidi A Schreiber et al. J Immunol Methods. 2010.

Abstract

Dendritic cells (DCs) are crucial in the initiation of immune responses and are primary targets in vaccination. Here, we describe fluorescent, carbon magnetic nanoparticles (CMNPs) within the 20-80 nm size range that are non-toxic and preferentially endocytosed by DCs. These attributes allow for DC tracing in vitro, ex vivo and in vivo, by both fluorescence and MRI. We show that CMNPs conjugated with an array of proteins are able to induce strong immune responses in mice. The addition of TLR ligand, CpG, to the CMNPs along with protein results in both T cell activation, but also a selective IFNgamma response. The magnetism afforded by the CMNPs facilitates a simple DC enrichment ex vivo by magnetic means from both secondary lymphoid organs, and sites of chronic inflammation. The magnetic and fluorescent properties of the CMNPs allow for visualization, recovery, and potentially the facilitation of directed DC migration. These particles may support more efficient immunization protocols or new diagnostic assays to characterize functionalities of DCs from patients.

PubMed Disclaimer

Figures

Figure 1. Characterization of CMNPs

A, TEM image of CMNPs shows irregular, crystalline-shaped particles with a 20–80 nm size distribution. B, Ferromagnetic resonance spectroscopy confirming magnetism of carbon (0.1% iron) particles. C, Direct conjugation of proteins to CMNPs. Flow cytometric analysis of CMNP conjugated with HEL protein and anti-DEC205 monoclonal mouse IgG specific for DCs (right) or unconjugated (left). D, Flow cytometric analysis of Avidin-FITC conjugated CMNPs (green) compared to non-labeled particles (black).

Figure 2. Dendritic cell uptake of CMNPs

Top, In vitro BMDC culture pulsed with CMNPs. Light microscopy (top left- DC with dendrite protrusions and nearby, round macrophage/monocyte-like cell) and SEM (top right) of BMDC culture pulsed with CMNPs over night. Middle, EM images also reveal CMNPs in endocytic vesicles in DCs (arrows point to CMNPs). Bottom, fluorescent microscopy of BMDC culture pulsed with FITC-CMNPs for 30 min at 37°C (bottom left) and CD11c-EYFP BMDC culture pulsed with PE-CMNPs (arrows, bottom right).

Figure 3. Enhanced targeting of CMNPs to DCs by DC-specific antibodies

A, BMDC culture pulsed overnight with HEL-conjugated CMNPs or HEL/DEC205-conjugated CMNPs. Light microscopy (400× magnification (upper) and 1000× magnification (lower). B, Quantification of the percentage of DCs with CMNPs from three independent experiments.

Figure 4. CMNP traffic in vivo

A, 4.7T MRI scan of mouse 15 and 75 minutes post i.v. injection of CMNPs. Red arrows point to particle accumulation in spleen, kidney and inguinal lymph node. B, Hematoxylin and Eosin staining of formalin fixed tissue samples two days and one week post i.v. injection of CMNPs. Arrows point to extracellular CMNP aggregates.

Figure 5. DC targeting and selective enrichment by CMNPs

A, Selective enrichment of DEC205+MHCII+ DCs from BMDC culture pulsed with CMNPs and exposed to Dynal magnet (bottom FACS panel) compared to non-magnet exposed BMDC-pulsed culture (top FACS panel). B, After 5 days post i.v. injection of FITC-CMNPs, splenocytes were isolated and stained with antibodies recognizing DCs. C, Fluorescent confocal image of liver section taken from a 3 week BCG-infected mouse injected 5 days prior to harvest with FITC-CMNPs. White arrows point to FITC-CMNP extra cellular aggregates and red arrows point to dsRED BCG bacilli. D, Liver granuloma cells passed through Miltenyi magnetic separation column (inset figure). Flow cytometric analysis using anti-CD11c reveals enrichment of DCs using FITC-CMNPs.

Figure 6. Redirecting in vivo DC traffic using a magnetic field

A, Sleeping mice were injected i.v. with PE-CMNPs. A neodymium ringed-magnet was positioned to encompass their inguinal lymph nodes for 30 minutes. FACs plots of pooled inguinal and cervical lymph nodes (left and right plots, respectively) show staining with anti-CD11c and expression of PE from CMNPs. B, Light and fluorescent microscopy images of CD11c-EYFP BMDC containing NanoLink particles (red arrows point to particles). N52 grade neodymium magnets were positioned juxtaposed to left inguinal lymph node of sleeping mice. Magnetically enriched bead-containing BMDCs were s.c. injected at the base of the tail and left for three hours. C, Both inguinal lymph nodes, cervical nodes and spleen were removed, and analyzed on flow cytometry for the presence of CD11c-EYFP cells (histogram plots generated from live cell gate). D, Fluorescent and light microscopy reveals the presence of bead-containing CD11c-EYFP cells in magnet-exposed inguinal lymph node (black arrow points to particles).

Figure 7. In vitro and in vivo CD4+ T cell activation by antigen-conjugated CMNPs

A, 18 hr in vitro activation of anti-HEL 3A9 TCR transgenic T cells. FACS analysis staining with 1G12, anti-3A9 clonotypic antibody, and CD4. B, Percentage of CD69 expression on 3A9 T cells after 18 hr in vitro activation. C, IFNγ 5 hour recall with anti-CD3 following three day in vitro activation of 3A9 splenocytes. Cells were treated with either HEL protein alone, CMNPs conjugated with HEL or CPG alone, or HEL and CPG. D, Upper plots, In vivo expansion of adoptively transferred CFSE-labeled 3A9 T cells into wild type mice 6 days after transfer and 7 days post immunization of HEL-bound CMNPs (left) and HEL-CpG-bound CMNPs (right). Lower plots, intracellular IFNγ cytokine staining following 5 hour recall with anti-CD3 ex vivo. Plots shown are from adoptively transferred Tg T cell gate of CD4+1G12+. Representative plots from two independent experiments with 3–5 mice per group.

References

1. Akagi T, Wang X, Uto T, Baba M, Akashi M. Protein direct delivery to dendritic cells using nanoparticles based on amphiphilic poly(amino acid) derivatives. Biomaterials. 2007;28:3427–3436. -PubMed
1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. -PubMed
1. Baumjohann D, Hess A, Budinsky L, Brune K, Schuler G, Lutz MB. In vivo magnetic resonance imaging of dendritic cell migration into the draining lymph nodes of mice. Eur J Immunol. 2006;36:2544–2555. -PubMed
1. Bharali DJ, Pradhan V, Elkin G, Qi W, Hutson A, Mousa SA, Thanavala Y. Novel nanoparticles for the delivery of recombinant hepatitis B vaccine. Nanomedicine. 2008;4:311–317. -PubMed
1. Borges O, Cordeiro-da-Silva A, Tavares J, Santarem N, de Sousa A, Borchard G, Junginger HE. Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. Eur J Pharm Biopharm. 2008;69:405–416. -PubMed

Using carbon magnetic nanoparticles to target, track, and manipulate dendritic cells - PubMed (original) (raw)