Rapid Stereology Based Quantitative Immunohistochemistry of Dendritic Cells in Lymph Nodes: A Methodological Study

This study was done to arrive at a fast and reliable protocol for assessment of fractional volumes of immunohistochemically stained dendritic cells in lymph nodes. Twenty axillary lymph nodes of patients with locally advanced breast cancer were immuno‐histochemically stained with an S100 antibody. Fractional volumes of dendritic cells were assessed by stereology based quantitative immunohistochemistry using an interactive video overlay system including an automated microscope. The gold standard percentage of dendritic cells was the fractional volume of S100 stained cells in 500 fields systematically spread over the whole lymph node. Then, in a computer simulation, different sample sizes (1–200 fields of vision) were tested and the coefficient of variation (CV) for each sample size was calculated. The CV dropped with increasing sample size. A sample size of 100 fields of vision appeared to be optimal. Intra‐ and interobserver reproducibility appeared to be good (correlation coefficients of 0.95 and 0.86, respectively) when re‐analyzing the cases with the established protocol. In conclusion, a fast and reliable assessment of the fractional volume of dendritic cells in lymph nodes is possible with semi‐automated quantitative immuno‐histochemistry. This method will form the base for further clinical studies into the immunological response in lymph nodes of patients with locally advanced breast cancer

Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors

Therapeutic tumor vaccination with viral vectors or naked DNA, carrying the genetic code for tumor-associated Ags, critically depends on the in vivo transduction of dendritic cells (DC). Transfection of predominantly nonprofessional APC and only small numbers of DC may hamper proper T cell activation. Aim of this study was, therefore, the targeted, selective, and enhanced in situ transduction of DC. A human skin explant model was used to explore targeted transduction of cutaneous DC after intradermal injection of a bispecific Ab conjugate to link adenoviral (Ad) vectors directly to CD40 on the DC surface. A significantly enhanced transduction efficiency and selectivity, and an increased activation state of migrating DC were thus achieved. Moreover, DC transduced by CD40-targeted Ad maintained their Ag-specific CTL-stimulatory ability for up to 1 wk after the start of migration, in contrast to DC transduced by untargeted Ad, which had lost this capacity by that time. Because DC targeting in vivo might obviate the need for the in vitro culture of autologous DC for adoptive transfer, CD40-targeted Ad vectors constitute a promising new vaccine modality for tumor immunotherapy