Fluorine-18 labeled mouse bone marrow-derived dendritic cells can be detected in vivo by high resolution projection imaging

Abstract

Immunization with ex vivo generated dendritic cells has become a focus for many clinical applications. The optimal site of injection and the migration pattern of these cells remain to be elucidated. We therefore developed a novel method for labeling mouse bone marrow-derived dendritic cells (BMDC) with the positron emitting radioisotope F-18 using N-succinimidyl-4-[F-18]fluorobenzoate, which covalently binds to the lysine residues of cell surface proteins. When we determined the stability of F-18 labeled BMDC, we found that at 4 h only 44±10% of the initial cell-bound activity was retained at 37 °C, whereas considerably more (91±3%) was retained at 4 °C. Labeled cells did not exhibit any significant alteration in cell viability or phenotype as determined by trypan blue exclusion and FACS analysis 24 h after radiolabeling. Furthermore, F-18-labeled BMDC stimulated allogeneic T cells in a mixed leukocyte reaction as potently as did sham-treated BMDC and migrated towards secondary lymphoid tissue chemokine (SLC) in a chemotaxis assay in vitro with the same efficiency as sham-treated BMDC. Migration of F-18-labeled BMDC was studied after footpad injection by (1) ex vivo counting of dissected tissues using a gamma counter and (2) in vivo by imaging mice with PiPET, a 2-mm resolution positron projection imager. After 4 h, the ratio between measured activity in draining vs. contralateral (D/C) lymph nodes (LN) was 166±96 (n=7) in the case of live cell injections, whereas if we injected heat-killed F-18-labeled BMDC or F-18-labeled macrophages the D/C ratios were 17±2 (n=2) and 14±4 (n=2), respectively. Injection of cell-free activity in the form of F-18-labeled 4-fluorobenzoic acid resulted in a D/C ratio of 7±2 (n=3), suggesting that the activity measured in the draining lymph node was associated with migrated F-18-labeled BMDC. When F-18-labeled live cells were injected into the footpad, 0.18±0.04% (n=7) of footpad activity was found in the draining LN within 4 h, whereas none was found in the contralateral LN. Quantitative assessment of cell migration by PET projection imaging of mice confirmed the ex-vivo counting results. These studies indicate that PET imaging offers a new approach for in vivo studies of dendritic cell biodistribution and migration.

Introduction

Active immunotherapy using tumor antigen loaded DC in mice have been extensively studied Celluzzi et al., 1996, Porgador et al., 1996, Zitvogel et al., 1996, Lotze et al., 1997, Lotze et al., 2000, Tuting et al., 1997, Fields et al., 1998a, Fields et al., 1998b, Nestle et al., 1998, Salgaller et al., 1998. Other studies demonstrated protection against viruses (De Bruijn et al., 1998) and bacteria (Mbow et al., 1997). Furthermore, human trials utilizing DC loaded with antigen are underway in several institutions. The induction of immunity depends on the interaction between DC and T cells. However, the best route of DC administration for ensuring migration to the T cell areas of lymph nodes, thereby ensuring optimal interactions, is uncertain, in part, because the sites where human DCs localize after injection are not fully known. Although bone marrow-derived DCs (BMDC) or their precursors circulate in the peripheral blood and subsequently reside in peripheral tissues, acquire antigen and migrate to regional lymph nodes in physiological circumstances [Steinman, 1991 #63; Banchereau, 1998 #62; Flores-Romo, 2001 #1], the migration pattern is still under intensive investigation. Migration of dendritic cells after intravenous, intradermal or subcutaneous injection has been assessed by using different tracing methods, such as membrane or cytosolic fluorescent dyes Austyn et al., 1988, Barratt-Boyes et al., 1997, Ingulli et al., 1997, Lappin et al., 1999 or cells labeled with radionuclides such as Cr-51 Cruz et al., 1990, Saeki et al., 1999 or Indium-111 Kupiec-Weglinski et al., 1988, Larsen et al., 1990, Morse et al., 1999, Thomas et al., 1999. In most of these studies the organs are removed at various times after injection of the label.

The aim of our study was to develop a method to label DC with the positron emitting radionuclide F-18, in order to detect DC migration in vivo by a high-resolution small animal PET camera. Here we describe our labeling method, the effects of labeling on the viability, phenotype and function of BMDC in vitro, and how F-18 labeled cells can be visualized and followed by a high resolution animal scanner.