Introduction of functional chimeric E/L-selectin by RNA electroporation to target dendritic cells from blood to lymph nodes

Abstract

Background: Inefficient migration of dendritic cells (DC) to regional lymph nodes (LN) upon intracutaneous injection is a major obstacle for effective DC vaccination. Intravenous vaccination is unfavorable, because DC cannot migrate directly from the blood into LN.

Methods: To enable human monocyte-derived (mo)DC to enter LN directly from the blood, we manipulated them by RNA electroporation to express a human chimeric E/L-selectin (CD62E/CD62L) protein, which binds to peripheral node addressin expressed on high endothelial venules.

Results: Transfection efficiency exceeded 95%, and high E/L-selectin surface expression was detected for >48 h. E/L-selectin RNA-transfected DC displayed an identical mature DC phenotype as mock-transfected DC. Furthermore, E/L-selectin-transfected DC maintained their normal CCR7-mediated migration capacity, and their ability to prime and expand functional cytotoxic T cells recognizing MelanA. Most importantly, E/L-selectin-RNA-transfected DC gained the capability to attach to and roll on sialyl-Lewis(X) in vitro.

Outlook: The presented strategy can be readily translated into the clinic, as it involves no stable genetic manipulation or viral transformation, and allows targeting of a large number of LN.

Fig. 1

High E/L-selectin expression, no competition with other RNAs, and high DC yield after E/L-selectin RNA electroporation. a Mature DC were electroporated with or without E/L-selectin (ELS) RNA and cryopreserved after 4 h. ELS expression of ELS-RNA-electroporated DC (black histogram) and mock-electroporated DC (gray histrogram) was determined by FACS analysis 0, 24, or 48 h after thawing. The percentage of ELS-positive cells and mean fluorescence intensity (MFI) are depicted in the histogram. b For comparison, purified CD8⁺ T cells were stained for E-selectin (thick black line) and for L-selectin expression (black histogram); gray histogram: isotype control. c and d Mature DC were electroporated without RNA, with ELS RNA, with RNA encoding the three TAAs MelanA, MAGE-3, and surviving, or with the combination of E/L-selectin RNA and RNA of all three TAAs. Four hours later they were cryopreserved. Directly after thawing, ELS-expression of mock-electroporated (dotted line), and of DC electroporated with ELS RNA only (gray histograms) or with both ELS and TAAs RNA (thick line) was determined by FACS analysis (c). DC transfected without RNA (dotted line), with the TAAs RNA alone (gray histogram), or in combination with the ELS RNA (thick line) were stained intracellularly for the three TAAs and analyzed by FACS (d). The data shown in a, b, c, and d are representative for three independent standardized experiments. DC yield (e) of ELS-RNA-electroporated DC (white square) and mock-electroporated DC (black circle) was determined by trypan-blue staining and cell counting 0, 24, or 48 h after thawing. Yield was expressed as percent surviving cells of input cells before electroporation The average ± standard error of the mean (SEM) of three standardized experiments is shown

Fig. 2

Electroporation with E/L-selectin-encoding RNA does not influence CCR7-mediated migration and LFA-1 expression. a Mature DC were electroporated with or without E/L-selectin (ELS) RNA and cryopreserved after 4 h. After thawing, cells were tested for their CCR7-mediated migratory capacity toward medium containing CCL19 in a standard transwell migration assay. To measure spontaneous migration, cells were incubated in a transwell without CCL19 in the upper or lower compartment (neg.), or with CCL19 in the upper compartment (anti). The average ± standard error of the mean (SEM) of three standardized experiments is shown. b Mature DC were electroporated (EP) with E/L-selectin (ELS) RNA or were only replated (DC no EP). After resting for 4 h, they were cryopreserved, thawed and stained for CD11a and CD18 expression (which form LFA-1 as a heterodimer). As a control for the staining, the LFA-1 expression of CD8⁺ cells was determined. Isotype-controls are depicted in the left panel. The data shown are representative for three independent standardized experiments

Fig. 3

E/L-selectin transfected DC retain mature phenotype and their capacity to stimulate naive CD8⁺ T-cells. a Mature DC were electroporated with (gray histogram) or without (black line) E/L-selectin (ELS) RNA, rested for 4 h, cryopreserved and stained for the characteristic maturation-surface markers CD25, CD80, CD83, CD86, HLA class I, and HLA-DR after thawing. Interrupted lines represent respective isotype controls. b DC were electroporated without RNA, with E/L-selectin RNA (ELS RNA) alone, with MelanA RNA (MelA RNA) alone, or with MelanA in combination with E/L-selectin RNA, and were used to stimulate autologous naive CD8⁺ cells. In addition, mock-electroporated and E/L-selectin RNA-electroporated DC were loaded with MelanA-derived analogue peptide (MelA pep.) and used for stimulation of naïve CD8⁺ T cells. After 1 week of stimulation, the percentage of MelanA/A2-tetramer-binding CD8⁺ T cells, and their phenotype were determined. Assignment of functional T cell phenotype was as follows: lytic effectors (LE): CD45RA⁺/CCR7⁻; effector memory (EM): CD45RA⁻/CCR7⁻; central memory (CM) CD45RA⁻/CCR7⁺; naïve (N): CD45RA⁺/CCR7⁺. Both a and b show one representative out of three independent standardized experiments

Fig. 4

E/L-selectin transfected DC gain the ability to roll on sialyl-Lewis^x/TS-PAA. Human DC (three donors) were electroporated without RNA (No RNA) or with E/L-selectin RNA (ELS RNA) and cryopreserved. Their ability to tether and roll was analyzed in a parallel plate flow chamber using slides coated with sialyl-Lewis^x/TS-PAA directly after thawing. Perfusion was performed at 20°C using a pulse-free pump at a shear rate of 1.04 dyne/s². During perfusion, microscopic phase-contrast images were recorded in real time. After 10 min of perfusion, four different randomly chosen microscopic fields (one representative is depicted) (10× objective) were recorded and the numbers of adhering cells were counted for each field. An average (±SEM) of cell numbers of the four fields and the P-value as determined by unpaired, one-tailed Student’s t-test is shown for each donor